Ascorbic Acid Mega Compile II 2019

Views:
 
Category: Education
     
 

Presentation Description

a Mega-compile of multiple studies involving ascorbic acid, sodium ascorbate, vitamin C and the mechanics of how this life molecule can restore many dysfunctions. This presentation will be updated

Comments

Presentation Transcript

Slide1:

5/18/2019 1

Slide2:

5/18/2019 2 Fifty Years of Research on Ascorbate and the Genetics of Scurvy: From a Better Flavored Beer To Homo Sapiens Ascorbicus Irwin Stone, D.Sc .

Slide3:

INACTIVATION OF POLIOMYELITIS VIRUS IN VITRO BY CRYSTALLINE VITAMIN C ( ASCORBIC ACID ) BY CLAUS W. JUNGEBLUT, M.D . July 3, 1935 In the course of this work it was observed that the same endocrine secretions ( adrenalin , cortin ) (4) as well as certain antitoxic sera (5) were frequently capable of inactivating not only poliomyelitis virus but also diphtheria toxin . When it was furthermore found that an important constituent of the adrenal gland, i.e. vitamin C or ascorbic acid, possessed the power of inactivating diphtheria toxin in vitro and in vivo in extraordinarily small amounts (6) it became an important problem to determine whether or not poliomyelitis virus is vulnerable, in a like manner, to the injurious effect of this substance . This paper presents experiments bearing on this point. The experimental data described in this report are interesting in several ways. First, they show that extraordinarily small amounts of vitamin C are capable of rendering non-infectious multiple paralytic doses of poliomyelitis virus . Second, they reveal a remarkable similarity in the quantitative aspects of this inactivation when compared with the neutralization of diphtheria toxin by vitamin C . The fact that two such heterogenous substances as diphtheria toxin and poliomyelitis virus , one a lifeless poison and the other a presumably living agent , should prove to be susceptible to the inactivating effect of no less than three highly reducing substances , i.e. adrenalin , cortin and vitamin C , all of which are present in the adrenal gland , seems to us particularly worthy of emphasis since it may serve to provide a common basis for a biochemical explanation of their destruction .

Slide4:

Further investigations will have to show whether the neutralization phenomenon observed when certain normal human and animal sera are brought into contact with poliomyelitis virus , is in any way influenced by the presence of vitamin C in the serum ; also, whether the natural resistance of certain species of animals to intracerebral injection of this virus shows any correlation with the concentration of vitamin C in the brain or cord . Experiments are now under way to determine whether vitamin C possesses any prophylactic or therapeutic properties in the treatment or prevention of experimental poliomyelitis . SUMMARY AND CONCLUSIONS The experimental evidence presented in this paper shows that multiple paralytic doses of poliomyelitis virus , when mixed with very small amounts of crystalline vitamin C ( ascorbic acid ), are rendered non-infectious as determined by intracerebral injection of such mixtures into rhesus monkeys. 4. Jungeblut, C. W., Meyer, K., and Engle, E. T., J. Immunol., 1934, ~/, 43. Zwemer , R. L., and Jungeblut , C. W., Proc. Soc. Exp. Biol. and Med., 1935 , 32 , 1583. Jungeblut, C. W., and Zwemer, R. L., in preparation. 5. Jungeblut, C. W., J. Immunol., 1934, 27, 17. 6. Harde , E., Compt rend. Acad., 1934,199, 618. Jungeblut , C. W., and Zwemer , R . L., Proc. Soc. Exp. Biol. and Med., 1935, 32, 1229.

Slide5:

FURTHER OBSERVATIONS ON VITAMIN C THERAPY IN EXPERIMENTAL POLIOMYELITIS BY CLAUS W. JUNGEBLUT, M.D. June 29, 1937 We have previously presented data to show that the parenteral administration of natural vitamin C during the incubation period of poliomyelitis in experimentally infected monkeys is followed by a distinct change in the severity of the disease (1). It remained also to be determined whether or not synthetic vitamin C possessed resistance-enhancing properties similar to that of the natural preparation . It is therefore evident that the percentage of non-paralytic survivors following treatment with natural vitamin C was about six times as large as that of the untreated controls . In the animals treated with synthetic vitamin C this percentage was only twice that of the controls . In both treated groups the percentage of animals developing atypical paralysis was approximately the same between 5 and 6 times as large as that observed among controls . However the difference between the two treated groups is again clearly demonstrated by the fact that treatment with natural vitamin C had reduced the incidence of typical paralysis by about one-third as compared with the controls More conclusive results are obtained with animals in which vitamin C treatment was not begun until the 5 th day of the disease . At this time distinctly larger doses of vitamin C are apparently required to obtain significant results . Thus the percentage of non-paralytic survivors increases progressively from 11.1 per cent (5 rag.) to 20 per cent (25 mg.), and reaches a maximum of 30 per cent when doses of 50 to 100 rag. are used .

Slide6:

It would appear, therefore, that the measure of therapeutic success depends upon certain variables which, in order of significance , may be listed as follows: First , the kind of vitamin C ; second, the dosage of vitamin C ; third, the rapidity of the infection as determined by the amount of virus and type of strain ; and fourth, the time of first administration of vitamin C . As a fifth factor, equally as important as the other four, we should mention differences in individual response from monkey to monkey which necessitate running large series of animals if significant results are to be obtained. While our data are still too limited to make deductions certain , it would appear that during the first few days of the infection doses of 5 to 25 mg. produce an optimum therapeutic effect and that definitely larger doses, ranging from 50 to 100 mg., are required for treatment which is delayed until shortly before the expected onset of paralytic symptoms . The size of the effective dose of vitamin C therefore seems to be directly proportional to the speed of the infection and the stage at which the infectious process is advanced . From the results of our vitamin C titrations of various monkey tissues it would seem possible that the protected animals may utilize vitamin C more effectively than the unprotected ones (2). Whether the degree of this utilization depends upon an unknown endocrine factor is an open question . The mechanism by which vitamin C accomplishes its therapeutic effect still remains obscure .

Slide7:

Since it is commonly suspected that one of the functions of vitamin C is to regulate the oxidation-reduction potential of cellular respiration ( 14 ), it becomes relatively easy to imagine that an increased supply of the vitamin , which has a tendency to diminish during the infection, serves to maintain the oxidation-reduction system of nerve cells at a level at which the oxylabile virus is restrained from intracellular propagation. The Use of Vitamin C as an Antibiotic (1949) Fred R. Klenner – Paper presented at AAN Convention, May 1953 Measles was singled out more so than the others because of the knowledge that it was a small virus like the one causing poliomyelitis . It was reasonable to assume that if measles could be controlled then poliomyelitis too, would have a drug that could prevent as well as cure the disease. The Use of vitamin C in measles proved to be a medical curiosity. For the first time a virus infection could be handled as if it were a dog on a leash . In this experiment it was found that 1000mg every four hours, by mouth, would modify the attack . Smaller doses allowed the disease to progress . When 1000mg was given every two hours all evidence of the infection cleared in 48 hours . If the drug was then discontinued for a similar period (48 hours) the above syndrome returned . We observed this off and on picture for 30 days at which time the drug ( vitamin C ) was given 1000mg every 2 hours around the clock for 4 days . This time the picture cleared and did not return .

Slide8:

Unless the virus was completely destroyed, as demonstrated in the experiments with the virus using measles, the infection will again manifest itself after a short incubation period. Small, single doses do not even modify the course of the infection Jungeblut ( 1937 ) stated that the parenteral administration of natural vitamin C during the incubation period of poliomyelitis in monkeys is always followed by a distinct change in the severity of the disease; that after the 5 th day of the disease LARGER doses are required . He realised, at that early date, that for fast progressing infection such as results from the R.M strain, very large doses must be given ; for the Aycock virus with its slower infection potential small amounts of vitamin C would suffice Under certain conditions 1000mg. To 3000mg. per day were by Kyhos et al ( 1945 ) to be necessary to keep the body saturated . There is a wide individual variation in the renal threshold for vitamin C . Many patients receive as much as 1500mg. of vitamin C per day without significant urinary loss ( Shaw et al, 1945 ). All of us have witnessed ‘ nose bleeds ’ in certain children sick with measles who prior to taking the disease were apparently healthy. Epitaxis ( nose bleed ) is one of the signs of Scurvy . Is this true Scurvy ? I have, many times, stopped nose bleed in children , sick with measles , with one single dose of 2 grams of vit.C Dolldorf ( 1945 ) reported that many conditions may be present in the body that call for a greater supply of vitamin C . He lists fever , infection , physical stress , gastrointestinal disorders , diarrhea , anorexia , and vomiting along with many others.

Slide9:

Case 1 : Measles in a 10-month old baby – 1000 mg – I.M every 4 hours 1000 mg were continued – Baby made an uneventful recovery and was discharged after 60 hours. No measles rash developed . 4 years have now elapsed and there has been no measles . Case 2: A case of virus pneumonia – 4 grams – I.M along with 1000 c.c dextrose 5 in saline solution . 4 hours later, vitamin C resumed with doses ranging from 2 to 4 grams every two to three hours. Vitamin C was continued for 2 weeks; the frequency was cut to every 12 hours, two grams at a dose . The rational of this continued use of vitamin C was to assist the body to clear up the debris in the right lung . It required 3 months to clear the lung by X-ray Case 3: A case of Encephalitis following measles and mumps – 2000 mg I.V . A 2 nd injection of 2000 mg was given intravenously and 1000 mg, of “C” prescribed every 2 hours by mouth . The next day was fever-and-symptom free . As a precautionary measure a 3 rd 2000 mg, was given with directions to continue the drug by mouth for at least 48 hours . He (8-year old) has experienced no residual pathology as determined by examination 5 years following the episode . (Similar cases seen in the interim have shown more dramatic response when the drug was given by needle every 2 or 4 hours )

Slide10:

Case 4: Poliomyelitis (symptoms of photophobia , conjunctivitis , sore throat , back-of-eyes type headache , nausea and vomiting . Two grams (2000mg,) vitamin C intravenously. 2 nd injection of 2 gram s upon arrival at local hospital then every 4 hours . 6 hours after, neck pain was gone, the headache was completely relieved. Eyes were dry and redness was definitely clearing . Nausea and vomiting disappeared (without receiving any pain medication ). Discharged after receiving 24 grams in 48-hour period . Vitamin C was continued by mouth, 1500 mg. every 2 hours taken with citrus fruit . This Schedule was followed for one week after which time a change was made to Vitamin B1 ( thiamine ), 25 mg . before meals and bed hour, Vitamin B1 was given in view of McCormick ( 1938-39 ) theory that inflammatory and degenerative diseases of the Nervous System [are] due to avitaminosis of this particular vitamin . Vitamin B1 in these cases ( Polio ) should be continued for a period of at least 3 months as nerve tissue is slow in recovering from even mild damage . The usual dose of vitamin C is calculated on the basis of 65 mg. per Kg. of body weight , and given every 2 to 4 hours by needle , Under certain conditions larger single injections can be used to good advantage . In using “ C ” as an adjuvant in the treatment of infections caused by the more common bacteria the single 250 mgm per Kg. of body weight injection behaves like other synergistic drug combinations . Likewise when treating an upper respiratory infection , this one single massive “shot” will precipitate the pathology .

Slide11:

Thewlis Clinic ( 1953 ) is interesting in this respect : “ Upper respiratory tract infections may severely tax the vitamin C reserve . It is usually during or following a cold that patients have epitaxis ( nose bleeds ) or cough up blood-streaked sputum . Local inflammation and depletion of vitamin C may be responsible for this haemorrhagic tendency . On numerous occasions, we have observed a dramatic alleviation of symptoms of a upper respiratory tract infection after an injection of 500 mgm of ascorbic acid ( vitamin C )” Vitamin C response when taken by mouth is not predictable . Wright and Lilenfield ( 1936 ) reported that the scorbutic state could develop even though the patient was taking large doses of vitamin C by mouth . In the opinion of Musser ( 1945 ) poor absorption and equally poor storage are cardinal factors in leading to vitamin C deficiencies . It was our privilege to observe this mechanism in one of our daughters several years ago. She had contracted Chickenpox . Vitamin C was started when the macules first put in their appearance . In spite of the fact she was given 24 grams every 24 hours there was no interruption in the progress of the disease . Itching was intense . One gram administered intravenously stopped the itch within 30 minutes and she went on to peaceful sleep for the n ext 8 hours . Although feeling fine, a second injection was given at this time , following which there were no new macules and recovery was fast and uneventful. In the past few years we have noted that in Chickenpox when massive injections are employed there [ are ] no repeating waves of macules , and the usual 7-9 days required for crusting is reduced to less that 24 hrs

Slide12:

The antibiotic power of vitamin C can also be augmented by other biochemical fractions . One of these is a colloidal solution of denatured proteolytic enzyme called ‘ Protamide ’ In Herpes Simplex and Herpes Zoster this “ enzyme ” proved to be of definitive value, and in Herpes Zoster ( Shingles ) did influence the dorsal nerve root pain . Of course it’s common knowledge that vitamin C, especially when injected intramuscularly, possesses the same anti- neuritic properties . Vitamin C itself can also be called a ‘cousin’ of the proteolytic enzymes. This suggested that vitamin C and Protamide should be used at the same time . The clinical results justified this assumption. Cures were obtained in from one to three days . Vitamin C was given as usual , but protamide was limited to one ampoule per day. The same ‘improved’ results were obtained in influenza and definite synergistic action was seen in one case of poliomyelitis in a boy of 10 years. Calcium too , is also a good adjuvant especially in treating influenza . In vivo calcium duplicates the chemical behaviour of vitamin C in many respects . From our experience it would seem that the inclusion of at least 10 c.c vial of calcium gluconate or calcium levulinate in the treating of a virus infection is good therapeutics Pathologic changes due to excessive amounts of vitamin C are unknown. Plasma concentrations twenty times normal have been obtained without any ill-effects ( Youmans ; 1941 ). We have found that a No.23 G needle 3/4 inch long is ideal for intravenous use and a No.22 G needle one inch long for the intramuscular routes . A needle 1 ½ inches long if the latter is employed in adults

Slide13:

The relatively small store of ascorbic acid maintained by the body even under good conditions, the relatively narrow margin between health and pathological changes and the evidence of a considerable incidence to hyporvitaminosis C combine to emphasize the importance of prevention by an adequate diet . Parents must learn that commercial orange drinks which do not contain freshly prepared orange juice are practically free from ascorbic acid. The trend must be away from the carbonated drinks and back to the “ old fashion ” citrus fruit juices . If parents will make their children drink as many glasses of citrus fruit each day as they now allow them bottles of carbonated drinks , Polio and disease in general will rapidly assume a less important role in our lives (See Aspartame and Sodium Fluoride Studies) SUMMARY Vitamin C possesses abilities which are catagorised by its capacity to antagonise many of the pharmacological effects of Histamine . It should be employed with the antihistamine drugs in all allergic states . It is because of this factor that it serves so well in the treatment of acute Rheumatic fever . Aside from this and the virus disease it is of tremendous value in all diseases which an exotoxin is produced . It neutralise al exotoxins . It is directly concerned with antibody formation and this in turn leads to an increase in gamma globulin of the blood serum . It joins with the virus to form a new compound which is destroyed by oxidation . It makes all body cells more permeable which allows entrance of immune factors otherwise denied . It lessens or prevents tissue damage . It serves as a hydrogen transport in cellular respiration . It is the Key to good health.. Don’t lose THIS key for it might lock or unlock your life

Slide14:

On the Genetic Etiology of Scurvy - Irwin Stone Man’s liver lacks the last enzyme in the series needed to convert glucose to ascorbic acid . The lack of this one enzyme, L- gulonolactone oxidase , completely blocks the synthesis even though the human liver contains the other intermediate enzymes in the series ( Chatterjee   et al .,   1961 ). The loss of the gene for the synthesis of the active enzyme, L- gulonolactone oxidase, probably took place in some remote Primate ancestor of Man by a conditional lethal mutation ( Gluecksohn-Waelsch , 1963 ), possibly some fifty million years ago. It may be possible to better pinpoint where in t ime this mutation occurred if the author’s suggestion for examining various members of the Primate order for this enzyme system , is followed ( Stone, 1965 ). The reason that this unfavorable mutation did not eradicate the mutated animals was the presence of small amounts of ascorbic acid in their available foodstuffs, adequate to insure their survival .

Slide15:

5/18/2019 15 Man has suffered from this genetic defect throughout his entire existence and has been absolutely dependent upon his food supply to provide him with marginal amounts of this important physiological substance that his liver fails to synthesize . He was never able to obtain in his foods amounts of ascorbic acid that his liver should have been producing , if we judge this by the amounts synthesized by another mammal , the rat , endowed with the complete enzyme system . The unstressed normal rat synthesizes ascorbic acid at the rate of 70 mg per Kg. of body weight per day (Salomon and Stubbs, 1961 ) and the stressed rat increases this to 215 mg. per Kg. per day ( Conney   et al., 1961 ). This is equivalent to the production of 4.9 to 15.0 gm. of ascorbic acid per day calculated to the 70 Kg. weight of an adult human . Because of the meager amounts of the unstable ascorbic acid in foods , it is just physically impossible to ingest enough weight of food material to supply these gram quantities of ascorbic acid . Bourne ( 1949 ) also suggested that the currently recommended mg. per day intakes may be wide of the optimal amount and perhaps should be measured in gm. per day instead . He stated “Perhaps it is normal for our blood and tissues always to be saturated with the vitamin and for large quantities to be flushing constantly through our urinary system and excreted in our sweat . We may find that continuous doses of vitamin C at this ( high ) level over a considerable period of time may have pronounced and unequivocal anti-infective action ”.

Slide16:

Due to the lack of L- gulonolactone oxidase , human is one of the few species not capable of synthesizing the chemically instable ascorbic acid ( vitamin C ) ( Chatterjee et al. 1961 ). This mutational loss , which probably took place in a remote primate ancestor of man ( Gluecksohn-Waelsch 1963 ) , causes a dependency on dietary vitamin C sources , but can also be considered as an advantage since ascorbic acid synthesis requires a lot of costly glucose reserves . On average, the human body loses approximately 3% of its vitamin C content per day , which is the percentual daily loss corresponding with the first-order elimination process of vitamin C assuming no intake. This severely limits the disease-free and survival time when subjects are on a diet poor in vitamin C , because this nutrient is a first-line antioxidant acting as a free radical scavenger . The half-life of ascorbic acid is approximately 16 days (Yung et al. 1978 ). In subjects without vitamin C intake , ascorbic acid is no longer detected in blood after 35–40 days (Willet 1998 ). In 1939 , a Harvard surgeon deliberately went on to a C-free diet, and although his blood vitamin level dropped rapidly, it was only after 12 weeks that he began to have feelings of fatigue (Crandon et al. 1940 ). In a larger British trial during World War II, it took 17–20 weeks for any signs to appear among 120 volunteers (No authors listed 1948 ). In a later trial using 4 American prisoners, using a purified liquid diet, skin changes appeared after 8–13 weeks and gum changes in 5–27 weeks (Hodges et al. 1969 ). So, the clinical symptoms due to vitamin C deficiency develop very slowly . In the early history of mankind, a successful mutation took place, which proved to be beneficial in terms of conservation of iron , although it had a major impact on vitamin C stability in vivo ( Kamel and ‘Umar 1975 ).

Slide17:

5/18/2019 17 The first four papers describing the genetics of scurvy and the human "inborn error of carbohydrate metabolism", Hypoascorbemia , appeared in the period 1 965-1967 . Hypoascorbemia is a potentially-fatal genetic liver-enzyme disease , a human birth defect caused by a defective gene in the human gene pool for the synthesis of the active enzyme protein , L- gulonolactone oxidase ( GLO ). This defective gene appears to be present in 100 percent of the human population and is the biochemical reason that the human liver is unable to complete the normal stress-related mammalian biochemical conversion of blood sugar, glucose , into ascorbic acid . This is an extremely vital biochemical pathway that proceeds normally and continuously throughout the lifetime of the non-Primate mammals carrying the intact gene for GLO , producing large stress-related daily amounts of ascorbate in the liver , which is funneled directly into the blood stream to give the necessary high blood and tissue levels of ascorbate required for the maintenance of biochemical homeostasis throughout the body . Mammals with the intact gene for GLO also have an inherited biochemical feedback mechanism that increases the liver synthesis of ascorbate in response to various stresses . This feedback mechanism was a great factor in assuring the survival and dominance of the Earth by the mammals during the past 165 million years of evolution. Homo sapiens have paid a very high cost in deaths, disease and misery during the past few million years of evolution in trying to survive without the benefit of the intact gene for GLO . I regard Homo as our most endangered mammal, scheduled for extinction in the 21st Century from overpopulation, pollution , unless humans take evolutionary destiny into their own hands and convert themselves into the robust human subspecies , Homo Sapiens Ascorbicus I have been taking about 20 grams of ascorbate a day for many years which is over three hundred times the current RDA , enjoying full health during this time. In my 1983 paper on the effect of megascorbate on Aging and Alzheimer's disease , I cite as shining examples of the value of this daily megascorbic regimen in maintaining a long healthy active, disease-free life : they are my long time friends and colleagues, Albert Szent-Gyorgyi , 91; Linus Pauling , 83; Frederick Klenner , 77; and myself, 77; all still working..

Slide18:

McCormick said: “Once the acute febrile or toxic stage of an infectious disease is brought under control by massive ascorbic acid administration , a relatively small maintenance dose of the vitamin will be adequate in most cases to prevent relapses , just as in fire protection small chemical extinguishers may be adequate to prevent fires in their incipiency , whereas when large fires have developed, water from large high-pressure fire hoses becomes necessar y .” The Pioneering Work of William J. McCormick, M.D. McCormick considered vitamin C to be the pivotal therapeutic nutrient “by reason of its chemical action as a reducing agent , and sometimes as an oxidizing agent , vitamin C is also a specific antagonist of chemical and bacterial toxins .” Furthermore, in “ Ascorbic Acid as a Chemotherapeutic Agent ” he stated: Fifty years ago , McCormick wrote: “The writer has found, in clinical and laboratory research , that the smoking of one cigarette neutralizes in the body approximately 25 mg of ascorbic acid , or the equivalent of the vitamin C content of one average-size orange . On this basis, the ability of the heavy smoker to maintain normal vitamin C status from dietary sources is obviously questionable , and this alone may account for the prevalence of vitamin C deficiency in our modern adult population .” This was quite a statement in 1954

Slide19:

The first four papers describing the genetics of scurvy and the human "inborn error of carbohydrate metabolism", Hypoascorbemia , appeared in the period 1 965-1967 . Hypoascorbemia is a potentially-fatal genetic liver-enzyme disease , a human birth defect caused by a defective gene in the human gene pool for the synthesis of the active enzyme protein , L- gulonolactone oxidase ( GLO ). This defective gene appears to be present in 100 percent of the human population and is the biochemical reason that the human liver is unable to complete the normal stress-related mammalian biochemical conversion of blood sugar, glucose , into ascorbic acid . This is an extremely vital biochemical pathway that proceeds normally and continuously throughout the lifetime of the non-Primate mammals carrying the intact gene for GLO , producing large stress-related daily amounts of ascorbate in the liver , which is funneled directly into the blood stream to give the necessary high blood and tissue levels of ascorbate required for the maintenance of biochemical homeostasis throughout the body . Mammals with the intact gene for GLO also have an inherited biochemical feedback mechanism that increases the liver synthesis of ascorbate in response to various stresses . This feedback mechanism was a great factor in assuring the survival and dominance of the Earth by the mammals during the past 165 million years of evolution.

Slide20:

5/18/2019 20 Homo sapiens have paid a very high cost in deaths, disease and misery during the past few million years of evolution in trying to survive without the benefit of the intact gene for GLO . I regard Homo as our most endangered mammal, scheduled for extinction in the 21st Century from overpopulation pollution, unless humans take evolutionary destiny into their own hands and convert themselves into the robust human subspecies , Homo Sapiens Ascorbicus I have been taking about 20 grams of ascorbate a day for many years which is over three hundred times the current RDA , enjoying full health during this time. In my 1983 paper on the effect of megascorbate on Aging and Alzheimer's disease , I cite as shining examples of the value of this daily megascorbic regimen in maintaining a long healthy active, disease-free life : they are my long time friends and colleagues, Albert Szent-Gyorgyi , 91; Linus Pauling , 83; Frederick Klenner , 77; and myself, 77; all still working..

Slide21:

5/18/2019 21

Slide22:

5/18/2019 22 I started publication of my megascorbic leukemia ideas in 1974 . In 1975 , as a result of my collaboration with my good friend, Wendell O. Belfield , D.V.M., we wrote a paper showing the need for and the good results obtained with megascorbic therapy in cats and dogs , even though they had the intact gene for GLO . We found that cats and dogs were poor producers of daily ascorbate in their livers . They made about one-fifth the daily amount produced in other mammals with the intact gene for GLO . For many years, Wendell had been independently using Megascorbics with great success in canine distemper ( viral encephalitis ) and in hip dysplasia in large dogs . He also recently megascorbically solved the problem of cat leukemia , the number one killer of pet cats . He, like his counterparts in human medicine, has had his problems with the veterinary medical establishment. His two books on dogs ( 1981 ) and cats ( 1983 ) are classics in maintaining these pets in good health .

Slide23:

Clinical Guide to the Use of Vitamin C - The Clinical Experiences of Frederick R. Klenner , M.D. abbreviated, sumarized and annotated by Lendon H. Smith, M.D. 2233 SW Market Street, Portland, Oregon 97201 What follows is a review , and abbreviation, a summary and a critique of the 27 scientific papers he ( Fredrick Klenner ) wrote . In the light of the recent developments and research in the use of Vitamin C, it is essential that the roots of its usage be reviewed. Briefly, Vitamin C does attenuate most virus infections by aiding the production of interferon , controls many cancers , relieves some depression , modifies much pain and changes the course of many diseases , like multiple sclerosis , amyotrophic lateral sclerosis , spider bites , the bites of poisonous insects and reptiles . The watchword is, “ If in doubt, give Vitamin C .” He inspired Linus Pauling and Irwin Stone to expand the research on the great benefits of Vitamin C. Dr. Klenner died in 1984 General Remarks He believed in the healing power of nature, but believed that natural remedies could enhance that power and were safer and usualiy more effective than drugs. Hippocrates said , “ Of several remedies the physician should choose the least sensationa l”. Vitamin C fills that criterion. In 1948 , he published his first paper on the use of large doses of Vitamin C in the treatment of virus diseases . In 1960 , he realized, “ Every head cold must be considered as a probable source of brain pathology .” Hold on to this thought; it is significant for the understanding of diseases like multiple sclerosis . He also felt—as do Archie Kalikarinos and Glen Dettman of Australia —that the dreaded Sudden Infant Death Syndrome was basically a Vitamin C deficiency . His maxim: the patient should “ get large doses of Vitamin C in all pathological conditions while the physician ponders the diagnosis .”

Slide24:

5/18/2019 24 We have misled ourselves with the mistaken notion that all C was supposed to do was keep us from scurvy . If, however, we base our needs on the amounts other mammals manufacture with their intact enzyme it comes to 2-4 grams daily in the unstressed condition . Under stress 70 kg of rats make 15 grams of C . [Burns; Salomon; Conney ] Can’t we accept the fact that we all have a genetic deficiency of the enzyme , L- gulonolactone oxidase and have to take Vitamin C for health, even for life ? [ Burns,1959 ] Irwin Stone calls this human genetic lack, this inability, hypoascorbemia . The point that Dr. Klenner is making: “ The physiological requirements in man are no different from other mammals capable of carrying out this syntheses .” If one is anemic due to poor iron intake , is it cheating to swallow iron tablets for a while ? If you are hypoascorbemic because you cannot manufacture Vitamin C from sugar , extra glucose in your diet will not help , you need to take Vitamin C (ascorbic acid ). He reports that one of the Pilgrim Fathers wrote to a friend in England in 1621 : “ Bring juice of lemon, and take it fasting. It is of good use .” Scurvy develops slowly . Crandon (in 1940 ) found that the Vitamin C level of the blood plasma fell to zero for 90 days before there was obvious clinical evidence and that this was as long as *132 days before the first signs appeared . ( *20 weeks is 140 days total)

Slide25:

5/18/2019 25 It works as an oxidizing agent massive amounts , i.e., 5-150 grams, intravenously , for certain pathological conditions , if allowed to run in rapidly (20 gauge needle), acts as a “Flash Oxidizer” and may correct the condition in minutes . It can be a reducing agent . It neutralized toxins , viruses and histamine . The more serious the condition , the more C is required . It appears that Vitamin C acts as a reducing agent , an oxidizing agent , an anticlotting agent , an antihistamine , and as an anti-infective agent . He summarized the function of C in poliomyelitis : 1. Virus destruction . 2. Dehydrates the brain and the spinal cord safely. 3. Supports and normalized the stressed adrenal glands . 4. It preserves the lining of the central canal and maintains more regular spacing and less crowding of ependymal cells (surface cells of the spinal cord). Ascorbic acid enters all cells . It “ proceeds to take up the protein coats being manufactured by the virus nucleic acid , thus preventing the assembly of new virus units .” Cells expand , rupture and die , but there is no virus particles available to enter and infect new cells . If a virus has invaded a cell , the Vitamin C contributes to its breakdown to adenosine deaminase , which converts adenosine to inosine . Purines are formed which are catabolized (broken down) and cannot be used to make more virus nucleic acid . Viral nucleic acid has a protein coat which protects this parasite as it rides the blood or lymph highway to gain specific cell entry . [Larson] it is possible that if the ascorbic acid can remove that protective protein coat in the blood stream or in the cells , the white cell phagocytes and immune globulin could then neutralize these vulnerable virus particles . I like this from Dr. Klenner : “ Ascorbic acid also joins with the available virus protein, making a new macromolecule which acts as the repressor factor .” ( interferon ?) Multiplication of new virus bodies is inhibited .

Slide26:

5/18/2019 26 He summarizes the study of Lojkin , ( 1937 ), who discovered the inactivation of one virus was due to a specific intermediate product formed in the course of the oxidation of C but needed the stimulation of copper ions . It is a peroxide and is decomposed as rapidly as it is formed . This study indicates why Vitamin C works better in the body and not the test tube . Every function of the body requires enzymes , some vitamins and some minerals to act as coenzymes . If enough Vitamin C is supplied, the enzyme system that breaks down invading viruses and bacteria , will be able to do its job properly . Quote: “ Unless the white blood cells are saturated with ascorbic acid, they are like soldiers without bullets .”

Slide27:

The Treatment of Poliomyelitis and Other Virus Diseases with Vitamin C Fred R. Klenner , M.D., Reidsville, North Carolina, July 1949 Poliomyelitis is in most instances an acute febrile disease of sudden onset, with symptoms of a systemic infection which either abruptly abort or develop to hyperesthesia , asymmetry of reflexes and flaccid paralysis or palsies of muscle groups Only slight contact between the carrier of the virus and the susceptible person suffices in some cases for the transfer of the causative organism . In this respect and also in that the virus can be demonstrated in the nasal washings as early as six days before onset of symptoms , poliomyelitis resembles measles . We never have an epidemic of poliomyelitis preceding an epidemic of measles ; the opposite is frequently true . Transmission ( Brodie , 1934 ) is by means of droplets from the mucous membrane of the upper respiratory tract . Experiments show the cerebral cortex to be the most unsatisfactory site for growth, that large amounts of the virus placed in this area are apt to disappear in a short time. Observations in monkeys and in man show that the anterior horn cells , particularly those of the lumbar cord , are the most favourable sites for proliferation of the virus .

Slide28:

The virus can make a direct assault through the olfactory bulb , to the brain , medulla and spinal cord . The virus can enter the blood stream directly or through the lymph channels . Following damage to the natural protective barrier, the choroid plexus , it can make its way to the central nervous system , or it can be excreted back onto the nasal mucous membrane where it will pick up the direct route of the olfactory bulb . Since there is always a period of septicaemia in the first few days of poliomyelitis , it might be that this is the all-important route and that the virus is grown on a living tissue, the blood, and then is deposited out on the surface of the olfactory bulb . From this we conclude that the time to destroy the virus is during this incubation period which varies more with virulence and power of multiplication than with size of initial dose. Poliomyelitis in man is always more severe if exercise is taken at time of the infection . Also, that, by the acceleration of the blood flow caused by greater oxygen demand in physical effort , a marked increase in the percentage of the virus deposited on the nasal mucosa would result . Heaslip found a definite relationship between the severity of the infection and the level of vitamin C nutrition . It is consistent with accepted physiological action of vitamin C to expect and anti- edema effect in any given affected area. It is worthy of note that bacterial toxins can cause losses of from 50 to 85 per cent of the vitamin C normally contained in the adrenals . Jungeblut's investigations seemed to justify the conclusion that vitamin C was the " antibiotic " that would destroy the virus organism .

Slide29:

Diphtheria can be cured in man by the administration of massive frequent doses of hexuronic acid ( vitamin C ) given intravenously and/or intramuscularly . To the synthetic drug, by mouth , there is little response, even when 1000 to 2000 mg. is used every two hours . The only disadvantage of the ascorbic acid therapy is the inconvenience of the multiple injections . This concept of the action of vitamin C against certain toxins has led to treating other diseases producing exotoxins . With the work of Kligler , Warburg and others who believed that the detoxification effected by hexuronic acid is brought about by a direct combination of the vitamin with the toxin or virus , this followed by oxidation of the new compound which destroys both the virus or toxin and the vitamin . Borsook et al . decided that the main chemical action of ascorbic acid is as a powerful reducing agent , and the virus causing poliomyelitis is known to be susceptible to the oxidizing action of various agents . It is in point here to remark that vitamin C is an integral part of the oxidation-reduction system of the body , thus playing a definite part in natural resistance . (1) Flexner and Amoss had warned that " simple lumbar puncture attended with even very slight hemorrhage opens the way for the passage of the virus from the blood into the central nervous system and thus promotes infection ."

Slide30:

In herpes zoster 2000 to 3000 mg. of vitamin C was given every 12 hours , this supplemented by 3 000 mg. in fruit juice by mouth every two hours . Eight cases were treated in this series, all of adults. Seven experienced cessation of pain within two hours of the first injection and remained so without the use of any other analgesic medication . Seven of these cases showed drying of the vesicles within 24 hours and were clear of lesions within 72 hours . They received from five to seven injections In several cases 10 mg. of riboflavin (vitamin b3) by mouth t.i.d . in conjunction with the vitamin C injections appeared to cause faster healing . Chickenpox gave equally good response , the vesicles responding in the same manner as did those of herpes . These vesicles were crusted after the first 24 hours , and the patient well in three to four cays . We interpreted this similarity of response in these three diseases to suggest that the viruses responsible were closely related to one another . The response of virus encephalitis to ascorbic acid therapy was dramatic . Six cases of virus encephalitis were treated and cured with vitamin C injections . Two cases were associated with virus pneumonia ; one followed chickenpox , one mumps , one measles and one a combination of measles and mumps . In the case that followed the measles-mumps complex , definite evidence was found to confirm the belief that massive, frequent injections are necessary in treating virus infections with vitamin C .

Slide31:

Whenever a patient presented a mixed-virus infection , such as receding mumps and developing measles , it was found that double the calculated dose of vitamin C was necessary to obtain the usual results A second boy, aged 11 , was allowed to develop mumps to the point of maximum swelling without any therapy, then given vitamin C , 1000 mg. intramuscularly, every two to four hours . This lad was entirely well in 48 hours . In using vitamin C as an antibiotic no factor of toxicity need be considered . To confirm this observation 200 consecutive hospital patients were given ascorbic acid, 500 to 1000 mg. every four to six hours, for five to ten days . One volunteer received 100,000 mg . in a 12-day period . It must be remembered that 90 per cent of these patients did not have a virus infection to assist in destroying the vitamin . In no instance did examination of the blood or urine indicate any toxic reaction , and at no time were there any clinical manifestations of a reaction to the drug . When vitamin C was given by mouth one per cent of these patients vomited shortly after taking the drug . In half of these cases the vomiting was controlled by increasing the carbohydrate content of the mixture . This reaction was not interpreted as representing a toxic manifestation; rather it was thought to be due to a hypersensitive gastric mucosa . The dose was reduced from 1000 to 100 mg . in young children showing this complex ; vomiting occurred as before . However, in these same patients administration of massive, frequent doses of vitamin C by needle affected a cure of the infection without causing vomiting

Slide32:

Massive doses of Vitamin C destroys A cow is valuable to a farmer not only for her ability to produce milk, but also as a source for organic fertiliser. Vitamin C , likewise, is important, not only as a detoxifying agent , as a catalyst aiding cellular respiration by acting as a hydrogen transport , as a catalyst in the assimilation of iron , and as a conservator of collagen fibres and bundles in tissues of mesenchymal origin ; but, also, because of its function as a reducing agent or the precursor of such response . In this latter capacity , it fulfils the requirements of an antibiotic . A striking phenomenon of vitamin C is the similarity of response, whether to correct pathogenic processes due to deficiency of this compound, acting as a vitamin, or to destroy the ferments of micro-organisms , acting as an antibiotic . Cases 5 and 6 are of pulmonary virus infection . 31 grams ( 31,000 mg ) of vitamin C was injected intravenously over a period of 60 hours. It is to be noted that the same amount of vitamin C (2 grams every four hours) was given to the boy and to the man, disregarding the factor of body weight (255 mgm . Per Kg of body weight). Had the man received four or five grams every four hours, or two grams every two hours, his hospital course would probably have followed the same pattern as the boy. In the case of the boy, the fever curve was of the type showing a fast response to heavy vitamin C injections. The WBC was 4,300, urine sugar ++. 26 grams (26,000 mg.) was given IV to this patient in a 44 hour period. A point of great interest was that at subsequent examinations the urine was consistently negative for sugar. The course in these cases emphasises the necessity of administering massive doses of vitamin C at frequent, regular intervals so as to maintain the proper level of this antibiotic in the tissue. .

Slide34:

In poliomyelitis vitamin C performs three important functions : 1) It destroys the virus 2) acting as a dehydrator and diuretic of first choice, it removes the edema fluid from the brain and the cord ; 3) it preserves the lining of the central canal and maintains more regular spacing and less crowding of the ependymal cells (Altman). The pressure within the bony vault of the Central Nervous System resulting from the inflammatory response excited by the virus ( interleukin release ), acts as a haemostat to cut-off the blood supply to the anterior horn cells . This compression of their vessels denies to the horn cells the essentials for function , for life even . The paralysis which complicates acute poliomyelitis appears to be due to a vitamin B1 avitaminosis . Vitamin C, by removing edema fluid , relieves from pressure these vessels that supply nutrient to the horn cells , thus allowing the normal complement of vitamin B1 to reach these cells . (special note: Vitamin B1 (Thiamine) and reducing potential for Paralysis/Palsy/flaccid limbs Magnesium sulfate compress on injection sites alleviates pain and swelling IV push speeds need careful monitoring due to syncope or fainting –slower IV push) That the adrenal glands and vitamin C are closely allied in the defence of the body has been proven by experimentation and by autopsy . In normal people, any excess of vitamin C is excreted in the urine . In people suffering with an acute infection , particularly virus infection , vitamin C is not only absent from the urine , but it is also missing from the blood serum . This is true even when moderate amounts are given intravenously .

Slide35:

At this time, the anaerobic conditions in the tissues will be relieved by the catalytic action of vitamin C acting as a gas transport to aid this cellular respiration . The abnormal acidity of the blood will be removed and abnormal amounts of free adrenalin will disappear from the blood stream . Following this constriction of the blood vessels will cease , allowing the liver and *pancreatic tissue to return to normal function . Continuance of frequent injections of properly calculated doses of vitamin C will restore the normal physiology of the body . This is not all of the story. ( * As a result of avitaminosis C , liver glycogen is mobilized – glycogenlysis ; and further storing of sugar in the liver is prevented – glycogenesis . To further enhance the hyperglycemia , this vasoconstriction brings about a decrease in the pancreatic secretions by lessening the amount of blood passing through the gland ) Loijkin ( 1937 ), studying the various phases of the inactivation of crystaline tobacco mosaic virus by L-Ascorbic Acid , suggested that the action was not due to reduced vitamin C , nor to the irreversibly oxidised dehydroascorbic acid . Loijkin felt that it was due to a specific intermediate product which is formed in the course of the catalytic auto-oxidation of vitamin C , an action stimulated by the presence of copper ions . This intermediate product must be a peroxide because a peroxide is formed during copper-catalysed oxidation of vitamin C . This action of the copper ion elucidates the findings that vitamin C in massive, frequent doses works better in the body than in a laboratory test tube

Slide36:

The human organism depends on external sources for its supply of vitamin C , a daily intake of about 30—100 mg . being adequate for normal subjects (M.R.C. Report, 1948 ; McIntosh, 1959 ). Stress , anxiety and excitement accelerate the depletion of ascorbic acid , and the results of this trial indicate that these chronic psychiatric patients were suffering from subscurvy . Especially significant was the improvement in the depressed state of the patients. Ascorbic Acid in Chronic Psychiatric Patients — A Controlled Trial G . MILNER, M.B., Ch.B. Registrar in Psychiatry, The Towers Hospital, Leicester British Journal of Psychiatry , 1963 , Volume 109, pp. 294-299 Anxiety and excitement have been shown by Maas ( 1961 ) to increase the rate of breakdown of ascorbic acid , and in schizophrenics this process may be exaggerated by an abnormality of adrenaline metabolism ( Briggs , 1962 ). Schizophrenics receiving “adequate” dietary amounts of vitamin C , as judged by the requirements of the normal population, are commonly found to have low blood ascorbate levels  Harris and Ray ( 1935 ) claimed that patients with scurvy ( Cancer : sic) or on the borderline of scurvy, required 7 to 10 days for saturation . Normal subjects can be saturated with the vitamin within 24—48, hours, by giving them 1 Gram of the vitamin per day . The suggestion from the results is that saturation with vitamin C has brought about an improvement in overall personality functioning .  If the signs of development of cancer and scurvy are similar , could they be fundamentally the same disease under different names ?

Slide37:

SUMMARY (1) A controlled, blind trial of ascorbic acid saturation , involving 40 male, chronic psychiatric patients , is described. (2) Standardized (objective and subjective) psychological techniques were used to assess changes dependent upon ascorbic acid intake . (3) A clinical state of subscurvy was found in these patients . (4) Psychiatric patients are shown to have an unusually high demand for ascorbic acid . (5) Statistically significant improvement in the depressive, manic and paranoid symptom-complexes, together with an improvement in overall personality functioning, was obtained following saturation with ascorbic acid. (6) It is suggested that chronic psychiatric patients would benefit from the administration of ascorbic acid . Ascorbic Acid in Chronic Psychiatric Patients — A Controlled Trial By G. MILNER, M.B., Ch.B . 1963 Registrar in Psychiatry, The Towers Hospit al, Leicester The suggestion from the results is that saturation with vitamin C has brought about an improvement in overall personality functioning . Conversely, the psychological and physical improvement shown by these long-stay psychiatric patients after saturation with ascorbic acid indicates that their diet has been unsatisfactorily low in vitamin C content . This had led to a deficiency state — subscurvy —having quite definite psychiatric symptoms, the most important of which are marked depression and irritability

Slide38:

INDIVIDUALITY IN VITAMIN C NEEDS BY ROGER J. WILLIAMS AND GARY DEASON Read before the Academy, April 26, 1967 Individual human needs vary greatly; intakes of vitamin C which may be regarded as safe for the average person may be far below what is necessary to prevent deficiency in some individuals. In particular circumstances, back injury for example , an individual may benefit from large doses because the presence of an abundant supply of vitamin C speeds the building of supportive tissue necessary for healing . Does high variability of vitamin C needs really exist to a significant degree in the human population ? We have sought to help answer this question by determining the incidence of such variation in guinea pigs .

Slide39:

Why extreme variation exists is a subject for speculation . We suspect from our experiments and other observations that guinea pigs , monkeys , and human beings may have varying abilities hitherto undetected to produce ascorbic acid endogenously but unlike other mammals cannot produce it fast enough to maintain health .

Slide40:

Adenovrisues and Vitamin C The simple structure characteristic of tobacco mosaic virus was soon found to be a basic property of human viruses such as coxsackie virus (which I believe to be the cause of Multiple Sclerosis ), Echoviruses and polioviruses – they all contain only ribonucleic acid and protein . There exists minor variations. Adenoviruses contain deoxyribonuclei c acid ( DNA ) and protein . Other viruses such as that causing influenza contain added lipid and polysaccharides . Deoxyribonucleic acid is used to program the large viruses , like mumps , ribonucleic acid is used to program the small viruses , like measles . The role of the protein coat is to protect the parasitic but unstable nucleic acid as it rides the “ blood highway ” or “ lymphatic system ” to gain specific cell entry . Pure viral nucleic acid without its protein coat can be inactivated by constituents of normal blood Observations on the dose and administration of Ascorbic Acid when employed beyond the range of a Vitamin in Human Pathology , 1971 Bakay (24) reported that the permeability of the blood-brain barrier can be changed by introducing toxic agents into the blood circulation . Chambers and Zweifach (25) emphasized the importance of the intracellular cement of the capillary wall in regulating permeability of the blood vessels of the Central Nervous System (CNS). In this syndrome (Encephalitis) the toxic substance is an adenovirus . Ascorbic Acid will repair and maintain integrity of the capillary wall .

Slide41:

Klasson ( 32 ) although limiting the amount of ascorbic acid to a dose range of 300 mg. to 2000 mg. daily , in divided doses , found that it hastened the healing of wounds ( burns ) by producing healthy granulation tissue and also that it reduced local edema . He rationalised that ascorbic acid used locally as a 2%-3% dressing possessed astringent properties similar to hydrogen peroxide . He also reported that antibiotic therapy was rarely necessary . The Ascorbic Acid kidney stone story is a myth . Methylene Blue will dissolve calcium Oxalate stones giving 65 mg orally 2 to 3 times daily ( Dr. M.J. Vernon Smith: Med. World News, December 4, 1970 ) It is demonstrated principle that the production of histamine and other end products from deaminized cell proteins released by injury to cells are a cause of shock . The clinical value of ascorbic acid in combating shock is explained when we realise that the deaminizing enzymes from the damaged cells are inhibited by vitamin C ( 42 ) It has been shown by Chambers and Pollock ( 43 ) that mechanical damage to a cell results in pH changes which reverse the cell enzymes from constructive to destructive . The pH changes spread to other cells. This destructive activity releases histamine , a major shock-producing substance , The presence of vitamin C inhibits this enzyme transition into the destructive phase . Clark and Rossiter ( 44 ) reported that conditions of shock and stress cause depletion of the ascorbic acid content of the plasma . As with the virus bodies, ascorbic acid also joins with the protein factor of these toxins effecting quick destruction . The answer to these emergencies is simple. Large amounts of ascorbic acid 350 mg to 700 mg per Kg. body weight given intravenously. Ascorbic acid can be given intramuscularly in amounts up to 2 grams at one site .

Slide42:

Large doses of intravenous “vitamin C” has a striking influence on the course of mononucleosis . In one patient who was given the last rites of her church, the girls’ mother took things into her own hands when the attending physician refused to give ascorbic acid . In each bottle of intravenous fluids she would quickly “tap in” 20 to 30 grams of vitamin C . The patient made an uneventful recovery. Her mother has her B.S in Nursing and has been a long-time advocate of massive doses of vitamin C . In Malignancy At least one research team has demonstrated that in Cancer , all available “ C ” is mobilised at the site of the malignancy . Lauber and Rosenfield reported that “ C ” is mobilised from the tissues of the body and selectively concentrated in traumatised areas , Schlegel (from Tulane University) found that even a dose of 1.5 grams a day, by mouth, would prevent bladder cancer . He and biochemist Pipkin have been able to demonstrate that in the presence of ascorbic acid , carcinogenic metabolites will not develop in the urine . They suggest that spontaneous tumour formation is the result of faulty tryptophan metabolism while urine is retained in the bladder . Schlegel termed ascorbic acid “ an Anticancer vitamin ” Along this line, Glick and Hosoda ( 51 ) reported on work by Von Numers and Pettersson that the depletion of Mast Cells from guinea pigs skin was due to ascorbic acid deficiency . The possibilities indicated are that vitamin C is necessary either directly or indirectly for formation of Mast Cells , or for their maintenance once formed, or both . Ascorbic acid will control myelocytic leukemia provided 25 to 30 grams are taken orally each day

Slide43:

In arthritis at least 10 grams daily and those taking 15 to 15 grams daily will experience commensurate benefit . Supportive treatment must also be given. Repair of collagenous tissue is dependent of adequate ascorbic acid . Complications of smallpox vaccination are usually handled by adequate oral ascorbic acid . Several times we found it necessary to give the “C” intravenously along with Adenosine . Twenty percent ichthammol used locally with vaccinia necrosum is good psychology. In Herpes Zoster , 2 grams vitamin C intramuscularly and 50 mg Adenosine 5-Monophosphoric acid , aqueous solution, also intramuscularly every 12 hours. In massive “ shingles ” ascorbic acid should be given by vein. Always as much as can be tolerated . Heavy metal intoxication is also resolved with adequate vitamin C therapy . It has been suggested that ascorbic acid metabolism may be an index of total metabolism and thus serve as a general diagnostic guide. Adults taking at least 10 grams of ascorbic acid daily, and children under 10 at least one gram for each year of life that one will find that the brain will be clearer, the mind more active, the body less wearied and the memory more retentive . 10 grams of vitamin C and 200 mg to 400 mg of vitamin B6, by mouth daily, will shield one from mosquito bites (see Mosquirix , TRS,S/AS01 adjuvant and Vaccines powerpoint )

Slide44:

The simple stress of pregnancy demands supplemental vitamin C. Vitamin C seems especially concerned with mesenchymal tissue . When one considers the demands of the fetus and infant, especially premature babies , it is obvious that high vitamin C intakes are required during pregnancy because this “ parasite ” will drain available “ C ” from the mother . Pregnancy and nursing mothers Greenblatt ( 67 ) reports excellent results following oral administration of vitamin C in the therapy of habitual abortion . The German literature is “ stacked ” with articles recommending high doses of vitamin C during gestation because they believe that this substance is of great benefit in influencing the health of the mother and in preventing infections . The vital contribution of ascorbic acid to the body tissues can be summed up in the formation and maintenance of normal intercellular material , especially the connective tissue, bones, teeth, and blood vessels . Genetic errors might be prevented if prospective mothers were advised to take 10 grams or more of ascorbic acid daily . It is significant that we found simple stress of pregnancy , a normal physiological process, that equivalent requirements paralleled those found in a rat when under stress . Experiments by King et. Al ( 68 ) have shown that the need for supplemental vitamin C begins in the embryo . One can only speculate on what massive therapy would do in all forms of cancer . Many pathologic conditions are cured by giving 5 million to 100,000 million units of penicillin as an intravenous drip over a period of 4 to 6 weeks . How long must we wait for someone to start continuous ascorbic acid drip for 2 months , giving 100 to 300 grams each day, for various malignant conditions ?

Slide45:

Sodium ascorbate potentiates the growth inhibitory effect of certain agents on neuroblastoma cells in culture , 1979 We report that sodium ascorbate at nonlethal concentrations potentiates the growth inhibitory effect of 5-fluorouracil (5-FUra), bleomycin sulfate, sodium butyrate , cyclic AMP stimulating agents , and x-irradiation on neuroblastoma ( NB ) cells , RESULTS Effect of Ascorbic Acid and Glutathione . Sodium L-ascorbate at 500 , ug /ml ( 2.53 mM ) produced >99% lethality in NB cells in culture within 48 hr of treatment , whereas glioma cells required a higher concentration for a similar effect Sodium D- isoascorbate was equally effective , but glutathione , another reducing agent , was more toxic to NB cells than was L- or D-ascorbate DISCUSSION The present study shows that sodium ascorbate produces a cytotoxic effect on NB cells in culture . This confirms previous studies on Ehrlich ascitic carcinoma ( 9 ) and chicken embryo fibroblasts and other mammalian cells in culture ( 10 ). Thus, in vitro studies support Cameron and Pauling's reports ( 3 ) that vitamin C at high doses exhibits antitumor activity .

Slide46:

Because sodium ascorbate inhibits catalase activity in vitro ( 14 ), we suggest that this mechanism could become important in those tumor cells that have predominantly catalase activity with little or no peroxidase because of a mutational event. The inhibition of catalase would lead to an accumulation of H 2 0 2 in such cells and thereby would cause cell death . A complete deficiency of peroxidase in amniotic fluid of pregnant patients with carcinoma of cervix has been noted ( 18 ). In addition to the mechanisms leading to an accumulation of H 2 0 2 , other mechanisms such as an inhibition of superoxide dismutase by sodium ascorbate , leading to an accumulation of free radicals , may account for the cytotoxic effect of this agent . The present study also points out the possibility that sodium ascorbate may modulate the effect of various pharmacological agents .( toxins/exotoxins/pathogens/metals )

Slide47:

Massive Vitamin C as an Adjunct in Methadone Maintenance and Detoxification Jordan Scher , M.D., Harry Rice, M.D., Suck- oo Kim, M.D., Ralph DiCamelli , Ph.D., and Helen O'Connor , R.N.- 1 Presented at the North American Congress on Alcohol and Drug Problems, December 12-18, 1974 , San Francisco , California. Vitamin C has been implicated in cellular respiration . It can be oxidized by cytochrome oxidase plus cytochrome C . Dehydroascorbic acid (DHAA) can be reduced by glutathione . Ascorbic acid is important in maintaining SH-activated enzyme systems in their reduced form, and it can act as a hydrogen donor . Ascorbic acid is also involved in carbohydrate metabolism , since scorbutic animals exhibit hyperglycemia , reduced glucose tolerance , low hepatic glycogen content , and are resistant to insulin . Vitamin C is also important in the conversion of folic acid to folinic acid (Goodman and Gilman, 1970 ). GOODMAN, L. S., and GILMAN, A.: The Pharmacological Basis of Therapeutics. MacMillan, 4th Ed., p. 1666, 1970 Ascorbic acid is highly concentrated in the cortex and medulla of the adrenals . In the latter location it tends to prevent the oxidation of epinephrine . Under the stress of administering ACTH, there is an increased secretion of adrenocorticosteroids , associated with a rapid decrease in the amount of adrenal ascorbic acid and cholesterol .

Slide48:

Professor Roger Williams , Biochemical Individuality ( 1956 ), stated: "Some inbred rats on identical diets excreted eleven times as much urinary phosphate as others . . . some voluntarily consumed consistently sixteen times as much sugar as others . . . some appeared to need about forty times as much vitamin A as others . . . some young guinea pigs required for good growth at least twenty times as much vitamin C as others ." E. S. Wagner ( 1973 ) who found that despite the fact that the usual claim is that excess vitamin C is readily excreted , only about half appears in the urine . There seems , therefore, to be an unknown reservoir for storing the vitamin with which we have not been familiar before . Should he persist in the chronic misuse of the particular agent , or agents , he may discover a number of additional symptoms beginning to develop as well. There will be a tendency toward acute and chronic exhaustion and tiredness despite the use of what had previously been a stimulating and relieving agent . There will be a kind of " brain fog ," in which will occur problems of retention of recent events and experiences , as well as difficulty in holding , conceiving , and developing ideas , as well as finding and using the right words . There will generally be a progressively reduced level of effective functioning intellectually . There may also develop a number of physical symptoms, including reduced libido , impotence , constipation , cold clammy skin , inefficiency in finer movements of limbs, headaches resembling , imitating , or conceived as migraine, pains of chest , stomach , back, and other body parts.

Slide49:

But as in many scientific discoveries, serendipity and accident were to have a role . A friend, perhaps caught up in the then fashionable and still current vitamin faddery , suggested he try some vitamin C , since it might make him feel better on general principles. To his great amazement , the food-allergic individual mentioned above , after scoffing a handful of vitamin C tablets , very shortly noticed a distinct tendency toward alleviation and relief of the exhaustion , brain fog , and tension phenomena . It became possible to overcome for a considerable period of time most, if not all, of the phenomena associated with his particular food-allergic coffee addiction problem . In fact, 20-50,000 mg per day was not unusual . Unfortunately, ultimately the protective effect of vitamin C seemed to diminish and be overcome by the more prominent and pervasive food-allergic symptoms , so that the individual in question was compelled again to completely abstain from the offending agent perhaps for the final time. In a strictly empirical basis we have observed that patients in a state of narcotic withdrawal often show symptoms of irritability , muscular tensions , a tendency toward exhaustion , and other phenomena suggestive of both the food allergic syndrome as well as the magnesium depletion syndrome . Based on the analogy of similar symptoms in the food-allergic patient , it was decided to administer mega-doses of vitamin C with a suggested average of 5,000 mg per day to all methadone-maintained patients

Slide50:

For example, many methadone patients will show low-grade irritability , minor and discomforting emotionality , debility , and mood shifts . After vitamin C, these patients will seem to feel an enhanced sense of comfort and well-being . These symptoms, though minimal and annoying , are discovered to be more disturbing than the patients had originally realized when they are relieved through the use of large doses of vitamin C. In fact, they frequently comment that they had not expected the vitamin C to do anything at all, but are quite surprised by the fact that they feel considerably better in a general way when they are using it . Again, based on the analogy of the use of vitamin C on an empirical basis to relieve the acute and chronic symptoms of food allergy otherwise, it was decided that ascorbic acid might well play an alleviating role in the course of detoxification and methadone withdrawal . Ascorbic acid has a role in oxidative processes , collagen , muscular , vascular , and adrenal metabolism . It was therefore hypothesized that since all of these areas seem to be implicated symptomatically in the process of detoxification , ascorbic acid may very well play a role here, too . Furthermore, it would appear that ascorbic acid seems to have a moderating and tranquillizing influence on behavior and emotional states , so that it is of great assistance in the management of patients who are in the process of detoxification .

Slide51:

In each of these instances, on a clinical basis , it was clearly apparent that vitamin C had a marked effect in relieving the fatigue state , the tension state , muscular pains and cramps , vasoconstriction and cold limbs , constipation and impotence . In all of these areas and conditions, there was relief in 60-70 percent of instances , or more . But restless sleep and the other symptoms mentioned above were distinctly alleviated in a considerable proportion of the cases so compared. It was therefore felt that vitamin C represented a clear addition to the armamentarium of narcotic addiction treatment on a clinical and statistical basis , despite the fact that we could not demonstrate biochemically or pharmacologically what the relief was based on. The tranquillizing effect of vitamin C was again a distinctive plus which had not been anticipated and one to which we feel more attention should be paid . In fact, it may be that many so-called mild anxiety states , as well as mild depressive states , may represent subacute hypovitaminosis , perhaps more specifically the hypoascorbemia to which Stone refers ( 1966 , 1966a ). The other alternative is that many individuals suffering mild symptoms of the kind described above may be really reacting to some kind of environment or food-allergic assault at a relatively low level , and about which they have no real concept . If this is the case, and if ascorbic acid can and may be used in a fashion in which we are describing here , many of these conditions may be somewhat alleviated in their early phases , or controlled for a considerable period of time .

Slide52:

Since ecological and environmental and food allergies are at this point so poorly understood , unfortunately many individuals suffering from them will not be so lucky as to be able to identify and eliminate the source of their problem. Only a much broader educational and awareness program will permit this vital necessity to occur in the future . One other clinical suggestion. We feel also that attention should be paid to the use of trace elements , but have not seriously studied the effects or relationship of these to the problems of addiction. We do, however , recommend to our patients the use of kelp tablets , on general principles, since kelp is well known to have a rather adequate supply of all of the known trace elements . ( vitamin E used as a further antioxidant ). The work of the Shutes and others ( 1956 ) suggests that vitamin E might also be instrumental in alleviating a number of vague symptoms, many referring to the circulatory system . the use of vitamin E in doses of 250 to 400 international units three to four times a day will have no deleterious effect and may well be helpful in the conditions described above . " premum non nocere ," – “first do no harm” We also feel that our practice of megavitamin therapy may be adopted usefully by those who are involved in methadone maintenance and detoxification programs elsewhere . We have not found any of the side effects others have claimed , such as kindey stone formation, or any involvement at all with oxalic acid in the course of this use of ascorbic acid or the other vitamins cited.

Slide53:

The Hypoascorbemia -Kwashiorko r Approach to Drug Addiction Therapy : A Pilot Study, 1977 Alfred F. Libby  1  and Irwin Stone  2 Ascorbate injected into rats at the rate of 100 mg per kg body weight attenuated and abolished the narcotic effects of morphine ( Ghione , 1958 ). Ascorbate’s detoxification of a wide variety of inorganic and organic poisons was reviewed (Stone, 1972 ) and included Klenner’s work on the successful megascorbic treatment of barbiturate poisoning , snakebite , and Black Widow spider bites . It was also suggested in this review that megadoses of ascorbate be used in drug addiction (Stone, pp. 157-158, 1972 ) (see study earlier – “ Massive Vitamin C as an Adjunct in Methadone Maintenance and Detoxification .”) These authors realized that scurvy played a large part in the drug abuse problem , but they only saw ascorbate as a means to reduce some of the side effects of methadone administration like constipation , loss of libido , and restless sleep . For this they used about 5 g of ascorbic acid a day . It apparently never occurred to them that by switching to sodium ascorbate and increasing their dosage by a factor of 10 , they could completely eliminate the ill-conceived Methadone Program with all its problems and at the same time have a simple, nontoxic, and elegant solution to the drug abuse problem . Also the presence of high levels of sodium helps dislodge the narcotic from the brain opiate receptor sites (located in the brain-limbic system).

Slide54:

Nutritional treatments for opioid addiction By Alan R. Gaby, M.D. In 2015 , approximately 2 million Americans were addicted to prescription pain relievers (obtained legally or illegally) and 591,000 people were addicted to heroin . In that year, 20,101 drug overdose deaths were related to prescription pain relievers , and 12,990 overdose deaths were related to the use of heroin . Intravenous heroin use is also associated with an increased risk of viral hepatitis , HIV infection , and crime . Problems related to opioid misuse cost the U.S. economy an estimated $78 billion per year . Refined sugar The diet of opioid addicts frequently contains large amounts of refined sugar . In one study, mean daily intake of sucrose was more than twice as high in opioid addicts as in healthy controls (107 vs. 52 g per day; p = 0.001). The majority of addicts reported having intense cravings for sweets , which were sometimes as powerful as their craving for opioids . If consumption of refined sugar contributes to the addictive state , then elimination of sugar from the diet could make it easier for opioid addicts to withdraw from their drug . . Such an approach would include avoidance of caffeine and alcohol ; consumption of small, frequent meals that are high in protein; and supplementation with nutrients such as chromium , other trace minerals , magnesium , and B vitamins .

Slide55:

Vitamin C-based regimens Administration of vitamin C suppressed the development of morphine tolerance and physical dependence in mice ( 3 ) and inhibited morphine withdrawal in guinea pigs ( 4 ). Of 31 patients addicted to various drugs , 58% had laboratory evidence of vitamin C deficiency .( 5 ) In uncontrolled studies of patients withdrawing from opioids , supplementation with large doses of vitamin C in combination with other nutrients decreased the severity of withdrawal symptoms or eliminated them entirely, and frequently reduced cravings for the drug .( 6 , 7 , 8 ) Acetyl-L- carnitine Thirty patients with methadone dependence were randomly assigned to receive, in double-blind fashion, acetyl-L- carnitine (ALC; 2 g per day in 3 divided doses per day) or placebo during a 3-week detoxification period. Withdrawal symptoms such as muscle tension , muscle cramps , and insomnia were significantly less in the ALC group than in the placebo group. No adverse effects were reported .( 9 ) Intravenous nutrient therapy One practitioner reported in 1954 that intravenous administration of B vitamins and vitamin C was beneficial for patients undergoing heroin withdrawal and, to a lesser extent, for patients withdrawing from morphine . The infusions contained thiamine (200-400 mg), niacinamide (100-300 mg), riboflavin (10 mg), pyridoxine (100-200 mg), pantothenic acid (0-25 mg), vitamin C (500 mg), and glucose (0-1 g), and were administered 2-3 times per day during the withdrawal period and less frequently thereafter (12). 3 Khanna NC, Sharma SK . Megadoses of vitamin C prevent the development of tolerance and physical dependence on morphine in mice . Life Sci. 1983 ;33( Suppl 1): 401-404 4 Johnston PA, Chahl LA. Chronic treatment with ascorbic acid inhibits the morphine withdrawal response in guinea-pigs . Neurosci Lett . 1992 ;135:23-27 .. 5 Croft LK, et al. Ascorbic acid status of the drug addict patient . Am J Clin Nutr . 1973 ;26:1042

Slide56:

Oxalic acid excretion after intravenous ascorbic acid administration Metabolism. 2009 February ; 58(2): 263–269. doi:10.1016/j.metabol.2008.09.023. Does the excretion of 80 mg of oxalic acid over the 6 hours after a 100-g intravenous dose of ascorbic acid substantially increase the risk of calcium oxalate stone formation ? Oxalate nephrocalcinosis and calcium oxalate stones develop over months to years in primary hyperoxaluria , a disease in which oxalic acid excretion exceeds 100 mg/d and can reach 400 mg/d [20]. The relatively slow natural history of primary hyperoxaluria suggests that a time-limited course of ascorbic acid infusions 3 times per week would not create a severe or immediate risk of oxalate stone accumulation . We found that only approximately 0.2% ( mol / mol ) of large doses of ascorbic acid appeared in the urine as oxalic acid 6 hours post-infusion , and as explained above, this is an overestimation . Nevertheless, oxalate stones occur commonly in the general population; for people already at high risk of oxalate stone, one may assume that even intermittent high-dose ascorbic acid infusions could further increase this risk. The present data are important because they indicate a remarkable lac k of severe hyperoxaluria after massive intravenous doses of ascorbic acid in people with normal renal function .

Slide57:

Vitamin C Transforms Mouse Stem Cells Into Heart Muscle Cells American Heart Association, April 1, 2003 Vitamin C's beneficial activity has been attributed to its ability to neutralize oxidants , which are damaging substances produced naturally by the body. However, other antioxidant compounds tested, including vitamin E , did not trigger the development of heart cells. " This suggests that the effect of vitamin C on cardiac differentiation is independent of its antioxidant effect ," Lee says He and his colleagues repeated their experiment many times , always with the same results . "The real significance of the study is that it indicates that we will be able to find other ways to generate heart cells from stem cells more efficiently," Lee says. "It also raises interesting questions about the role of vitamin C in the development of the embryo's heart ." Lee and his colleagues tested 880 bioactive substances – including drugs and vitamins – approved by the U.S. Food and Drug Administration ( FDA ) to see if they stimulated the mouse stem cells to become heart muscle cells . The cells were genetically altered to give off a fluorescent bright green colour when viewed under a microscope if they had become heart muscle cells " We only got 1 out of the 880 to light up, and that was from ascorbic acid, the chemical commonly known as vitamin C ," says Lee, an associate professor of medicine at Harvard Medical School and Brigham and Women's Hospital in Boston , and a lecturer in biological engineering at the Massachusetts Institute of Technology in Cambridge , Mass.

Slide58:

In cultured human skin fibroblasts , ascorbic acid stimulates collagen production with no apparent change in the intracellular degradation of newly synthesised procollagen . To understand the basis for this effect, we measured the steady-state levels of type I and type II procollagen mRNA’s in cells with ascorbic acid . A three-to-fourfold increase in collagen synthesis was associated with a two-to-threefold increase in levels of mRNA’s for both type I and type II procollagens . These effects of ascorbic acid are explained by a translational control linked to either procollagen gene transcription or mRNA degradation .

Slide60:

Case Study: High-Dose Intravenous Vitamin C in the Treatment of a Patient with Adenocarcinoma of the Kidney Hugh D. Riordan, M.D.1, James A. Jackson, MT(ASCP)CLS, Ph.D., BCLD2 and Mavis Schultz, A.R.N.P.3 The patient decided not to undergo chemotherapy , hormone therapy or cytotoxic treatment of any kind . He requested and was started on vitamin C intravenous treatment . He was started on 30 grams of vitamin C Ascorbic Acid Injection , Sodium Ascorbate equivalent to 250 mg/mL , in 250 mL of Ringer's Lactate given by intravenous injection ( 60 drops per minute ) twice a week. In April 1986 , about six weeks after the x-ray and CT. scan studies, the oncologist’s report showed " the patient returns feeling well . His exam is totally normal . His chest x-ray shows a dramatic improvement in pulmonary nodules compared to six weeks ago. The periaortic lymphadenopathy is completely resolved . ...either he has had a viral infection with pulmonary lesions with lymphadenopathy that has resolved or (two) he really did have recurrent kidney cancer which is responding to your vitamin C therapy ." In June 1986 , the oncologist reported the patient "has been receiving vitamin C shots now twice weekly , feeling well and playing golf. On exam day , his weight is up a couple of pounds and he looks well. He has absolutely no evidence of progressive cancer ."... " I recommend you continue your vitamin C shots until he returns in six weeks time for a repeat chest x-ray and CT.scan of his abdomen ."

Slide61:

The oncologist's report in July 1986 stated " the patient has been feeling well with no symptoms of cancer ... there is no evidence of progressive cancer . He looks well... chest x-ray today is totally normal . The pulmonary nodules are completely gone . There is no evidence of lung metastasis , liver metastasis or lymph node metastasis today , whatsoever ." The report of September 1986 stated "... Over all , the patient is totally well , golfing and having no symptoms from his cancer . On exam today , there is absolutely no evidence of recurrent cancer and we have opted to continue our observation . I suggest he continue with you the vitamin C shots ..." During and after the treatments , the patient showed no toxic, or unusual side effects from the high-dosage I.V. vitamin C therapy . Periodic blood chemistry profiles and urine studies were normal . In March 1987 , 15 months after surgery , the report stated "... is feeling well, and on exam today there is absolutely no evidence of recurrent cancer . We thus thought (this patient) has no evidence of recurrent cancer and opt to continue his follow-up. The patient wishes to continue his vitamin C shot once weekly as well, which seems reasonable to me .“. To date ( 1990 ), after 3'/2 years the patient remains cancer free . He will continue to be followed both at our centre and by the oncologist.

Slide62:

Vitamin B3 appears to protect patients against tardive dyskinesia . Hawkins in a personal communication reported that over fifteen years he has not seen any cases among many thousands treated . In my own practice I have not seen any develop in twenty years . It may well be some of the protective effect is due to the lower doses of tranquilizer required but there is a direct protective effect as well, probably at the synapse . The Adrenochrome Hypothesis of Schizophrenia Revisited A. Hoffer, M.D., Ph.D.1 There the medulla , containing noradrenaline and adrenaline , is surrounded by the adrenal cortex which is very rich in ascorbic acid . This stabilizes the amines as do other natural anti oxidants . Manganese may have a protective effect by inhibiting aminochrome formation But since ascorbic acid is as active as Haldol ( one of the most powerful dopamine receptor Blockers ) it would be worthwhile to determine whether pregnant women given optimum doses of ascorbic acid would have children who would eventually be less apt to develop schizophrenia . Galzigna ( 1970 ) suggested a relationship between acetylcholine, a neurotransmitter , and catecholamines . Acetylcholine interacts with oxidized noradrenaline , yielding a complex which does not change to adrenolutin in ascorbic acid medium . It reacts similarly with dopamine . Both acetylcholine and nicotinamide increase the auto-oxidation of noradrenaline but the complex reacts differently with ascorbic acid . ORTHOMOLECULAR PSYCHIATRY, VOLUME 10, NUMBER 2, 1981, Pp. 98-118

Slide64:

The active agent, the enolic form of 3-keto-L-gulofuranlactone , christened ascorbic acid or vitamin C , was isolated in the late 1920's by Albert Szent-GyOrgyi Ascorbic Acid and Cancer : A Review Ewan Cameron, Linus Pauling, and Brian Leibovitz , March 1979 Our own interest arose from the realization that ascorbic acid , known to be required for collagen synthesis , might be required in increased amounts for the protective encapsulation of tumors ( 253 ) and from the independent simultaneous conclusion that the ascorbate molecule (or some residue thereof) must be involved in the feedback inhibition of lysosomal glycosidases (60) responsible for malignant Invasiveness . Two points deserve early emphasis: (a) although most animals can synthesize ascorbic acid , a human cannot , and he is totally dependent upon dietary intake to satisfy all his requirements ; (b) ascorbic acid , known to be essential for the structural integrity of the intercellular matrix , is closely related to glucuronic acid , an essential building block of the principal matrix structures . Any measure that can protect the integrity of the intercellular matrix will (a) effectively retard malignant infiltration , (b) restrict the selective nutrition of tumors , (c) protect pre-existing collagen barriers from neoplastic erosion , and (d) facilitate protective collagen encapsulation . This is one important function of ascorbic acid .

Slide65:

Increased utilization of ascorbate , as measured by depletion of ascorbate reserves , is by no means confined to cancer ; it is a characteristic feature of many other “ cell-proliferative disorders ”, such as inflammation (17, 91), and of the reparative processes after surgical trauma (88, 89 ), myocardial infarction (160, 161), and thermal burns (16). The last 3 studies demonstrate that ascorbate is removed from the circulating reserves and concentrated at the site of the reparative process . Dodd and Giron-Conland (101), using electron spin resonance, have demonstrated that the ascorbyl radical is present in a wide variety of tumors in higher concentration than in the corresponding normal tissues. Human cancer tissue also contains elevated levels of ascorbate . Goth and Littman (139) demonstrated this for a variety of human tumors . Chinoy (69) reported that certain human tumors contain far greater ascorbate levels than does their tissue of origin , and using histochemical techniques demonstrated that the greatest concentration of ascorbate was deposited at the periphery of the tumor , against the actively growing invasive margin . “All tissue cells have an inherent tendency to divide but this tendency is normally restrained by the viscous nature of their intimate extracellular environment of high-molecular-weight ground-substance Glycosaminoglycans . Proliferation is initiated by the cellular release of hyaluronidase , which permits the cell local freedom to divide and to migrate within the limits of the altered field . Proliferation will continue as long as hyaluronidase is being released ; proliferation will cease and normal tissue restraint and organization will be restored when the production of hyaluronidase returns to normal .”

Slide66:

Hyaluronidase inhibition by bioflavonoids and related compounds has been reported (125, 207, 239). Furthermore, in Vitro studies by Kojima and Hess (182) demonstrated that ascorbic acid functions as a noncompetitor inhibitor of N-acetylglucosaminidase . Research papers on the inhibition of f3-glucuronidase have been reviewed by Dutton (108). Collagen catabolism results in an increased level of UHP , and, in the human, measurement of UHP is useful to the clinician, rising levels reflecting increasing spread and activity of the disease . A loading dose of 1 g of ascorbic acid produces a sharp decrease in UHP excretion (18). Ascorbic acid has been shown to increase collagen synthesis by fibroblasts in vitro (99, 280) and to maintain collagen synthesis in non-mitotic fibroblasts for extended periods (99). Prolyl hydroxylase , the enzyme hydroxylating prolyl and lysyl residues of procollagen , requires ascorbate to function in vitro (181) Thus , there is no doubt that a sufficiency of ascorbate is essential for collagen fibrillogenesis , both by stabilizing the matrix and protecting it against the erosive effects of lysosomal glycosidases and by facilitating the hydroxylation of prolyl residues in procollagen .

Slide67:

Lewis Thomas (321) pictures the immune system as a police force (more like an army) , constantly patrolling the body cells, keeping an eye open for cells becoming neoplastic and, upon recognition , destroying them . For such a system to work, cancer cells must display a surface antigen for “ recognition ' different from their non-neoplastic compatriots . Relative to other cells, lymphocytes contain substantially higher concentrations of ascorbate , and there are strong indications that this characteristic feature is “ purposeful ” and related to their active role in cell-mediated immuno-competence Yonemoto et al. (356) demonstrated in healthy young subjects that a high loading dose of ascorbate ( 5 g/day for 3 days ) evokes a significant increase in lymphocyte blastogenesis as measured by response to phytohemagglutinin challenge and, furthermore, that this increase in immuno-competence was further significantly enhanced by larger doses ( 10 g ). These observations support the common sense view that lymphocytes rich in ascorbate should be able to conduct their protective business more efficiently than those that are not , a characteristic feature of established cancer . The highest concentrations of ascorbate are found in the adrenal and pituitary glands , and the terminal stages of scurvy are just preceded by complete depletion of adrenal ascorbate , leading, it has been frequently stated , to “ scurvy death ” from adrenocortical failure .

Slide68:

Numerous studies have demonstrated that ascorbate is required for active phagocytosis both in vivo and in vitro (71, 87, 128, 137, 210, 240, 270). In Japan , Morishige and Murata (227) have used supplemental ascorbate in the successful prevention and treatment of measles , mumps , viral orchitis , viral pneumonia , herpes zoster , and viral encephalitis . The most striking effect recorded by these Japanese workers has been the virtually complete prevention of post-transfusion hepatitis in a country where such a complication is common (227). Ascorbate has also been shown to enhance interferon production ( 92 , 298-301 ), as well as to enhance the phagocytic properties of the reticuloendothelial system , both potent in vivo defenses against viruses . The interaction of ascorbate with L-lysine in rats (67) may also contribute to the effectiveness of ascorbate . Whatever the mechanisms may be, there is clinical and experimental evidence that supplemental ascorbate possesses some antiviral activity . Thus, if a viral etiology of human cancer is ever proved , ascorbate might be expected to exert some prophylactic and therapeutic effect . Ascorbic acid is also a requirement of the mixed-function oxidases that hydroxyl ate tryptophan (83, 233, 307), tyrosine (187, 233, 307, 324), dopamine (193), and phenylalanine (314).

Slide69:

Ascorbic acid deficiency in guinea pigs has been shown to decrease rnicrosomal cytochrome P-450 activity to about 50% of its normal value (96, 97, 192, 203, 268). Repletion experiments with scorbutic guinea pigs have shown that supplementation with ascorbic acid returned cytochrome P-450 and demethylation activities to normal within 48 hr (97, 192, 203). It has also been shown, using the Ames test (148), that ascorbate inhibits bacterial mutagenesis by N-methyl- Nnitrosoguanidine , and Marquardt , Rugino , and Weisburger , using the same test procedure, have shown mutagenic (and presumably carcinogenic ) activity in nitrite-treated foods and have suggested that human stomach cancer might well be related to this dietary factor , which to some extent might be reduced by ascorbate (204). Urine from patients with bladder cancer oxidizes 3-HOA faster than does control urine in Vitro, and the addition of ascorbate inhibits this reaction (259, 286). 3-HOA implanted directly into the mouse bladder is carcinogenic , and this effect can be prevented by ascorbate (259, 284). The whole concept that supplemental ascorbate has a protective and inhibitory value in bladder cancer has been developed by Schlegel (284) and his associates in the Department of Urology of Tulane University . There is considerable evidence that smoking depletes ascorbic acid reserves (4, 142, 156, 255,-257, 269), as shown by diminished whole blood , serum , and leukocyte ascorbate levels in smokers relative to non-smoker controls.

Slide70:

Mouse neuroblastoma ( NB ) cells in culture were more sensitive to sodium L-ascorbate than were rat glioma cells by the criterion of growth inhibition (due to cell death and reduction in cell division). Glutathione , a reducing agent , was more toxic to NB cells in comparison to D- or L-ascorbate Sodium ascorbate potentiates the growth inhibitory effect of certain agents on neuroblastoma cells in culture – November 27 , 1978 (vitamin C/X-ray/Chemotherapeutic agents) Effect of Ascorbic Acid and Glutathione. Sodium L-ascorbate at 500 , ug /ml ( 2.53 mM ) produced > 99% lethality in NB cells in culture within 48 hr of treatment , whereas gliomacells required a higher concentration for a similar effect R. M. Mulligan (University of Colorado Medical Center ) has observed that sodium L-ascorbate at 1 ,g/ml ( 5.05 uM ) causes cytotoxic effect on human melanoma cells in culture (personal communication). These studies reveal that the concentration requirements for a cytotoxic effect differ from one cell line to another . Thus, in vitro studies support Cameron and Pauling's reports (3) that vitamin C at high doses exhibits antitumor activity . The mechanism of the cytotoxic effect of sodium ascorbate is unknown ; there may be more than one.

Slide71:

Discussion 12 Ascorbic acid is most well known as Vitamin C , the nutritional supplement essential for preventing scurvy . The recommended daily dose has varied over time, but it is presently about 75 to 90 mg/day . However, several authors, Linus Pauling among them, have suggested that higher daily doses might prevent cancer [ 12 ]. We have recently demonstrated that ascorbic acid is a competitive inhibitor of adenylate cyclase activity , resulting in a decrease of intracellular cAMP concentration . However the mechanism underlying the regulation of tRNA synthetase and ieF subunits expression in mammalian cells are still unknown. In particular, the role of cAMP remains to be clarified . We may note that positive trials involved IV injection , in contrast with negative results that involved oral administration . From data presented in this manuscript, it is obvious that treatment with increasing doses of AA induces a specific down regulation of expression of a selected set of genes , resulting in an arrest of cell proliferation and , at higher doses , in cell death . Xenografted mice treated with the highest AA concentration survive after 40 days (30 days of treatment plus 10 days of grafting). We have no explanation but we observed numerous carcinogenic invasion, in placebo treated animals and in animals treated with lower doses of AA . In contrast, animals treated with 1000 mg/days did not present carcinogenic invasion . 12 . Pauling L, Moertel C ( 1986 ) A proposition: megadoses of vitamin C are valuable in the treatment of cancer . Nutr Rev 44: 28–32.

Slide73:

Stromal Cell Oxidation: A Mechanism by Which Tumors Obtain Vitamin C Article in Cancer Research · October 1999 – Researchgate : Source: PubMed Vitamin C enters through the facilitative glucose transporters ( GLUTs or Hexose Transporters ) in the form of dehydroascorbic acid , which is then reduced intracellularly and retained as ascorbic acid Dietary vitamin C is critical for humans and other primates because we cannot synthesize the vitamin in the liver as do most other animals (1). The mechanism by which cancers accumulate vitamin C in vivo, however, is unknown. Ascorbic acid , the form of vitamin C in the bloodstream, does not readily cross the blood-brain barrier , whereas dehydroascorbic acid crosses the blood-brain barrier and accumulates in the brain as ascorbic acid . Thus, the uptake of vitamin C in the form of dehydroascorbic acid through the GLUTs appears to be a general mechanism for vitamin C uptake . The increased intracellular concentration of vitamin C may have effects on tumor growth and the tumor’s ability to respond to oxidative stress associated with chemotherapy and radiation therapy . Although studies evaluating the role of vitamin C supplementation in cancer patients have generally shown no benefit with respect to survival or tumor regression (22), it is not known whether high concentrations of vitamin C in human tumors afford the malignant cells with a metabolic advantage .

Slide75:

Cytotoxicity of ascorbate , lipoic acid, and other antioxidants in hollow fibre in vitro tumours – British Journal of Cancer 2001 JJ Casciaril , NH Riordanll ', TL Schmidt1, XL Mengl , JA Jackson2 and HD Riordan It plays roles in collagen and camitine synthesis and may be important in maintaining proper immune cell function (Goldschmidt, 1991; Penn et al, 1991). It can also act as a pro-oxidant by converting free radicals into hydrogen peroxide , a molecule that can damage cell membranes and DNA if not neutralized by the cellular enzyme catalase ( Koch and Biaglow , 1978). At sufficient concentrations , vitamin C can produce cytotoxic levels of hydrogen peroxide . Tumour cells are often catalase deficient and therefore more sensitive than normal to hydrogen peroxide ( Benade et al, 1969). Vitamin C accumulates in solid tumours at concentrations higher than those in surrounding normal tissue ( Langemann et al, 1989; Agus et al, 1999). This has raised concerns that vitamin C may provide tumours with antioxidant protection from traditional therapeutic modalities ( Raloff , 2000). However , vitamin C may be useful as an anti-cancer agent if cytotoxic ascorbate concentrations can be achieved in tumours (* Riordan et al, 1995 ).

Slide76:

In 2 Scottish studies , terminal cancer patients given intravenous vitamin C ( I0 g day- ') showed longer survival times than historical controls ( Cameron and Campbell, 1974 ; Cameron and Pauling, 1976 ). A Japanese study yielded s imilar results ( Murata et al, 1982 ). Oral vitamin C supplementation is unlikely to produce plasma ascorbate levels sufficient to kill tumour cells directly (* Riordan et al, 1995 ). Intravenous vitamin C at higher doses has been effective in individual cases (* Riordan et al, 1990 , 1998; Jackson et al, 1995 ). Ascorbate concentrations on the order of 10 mM (-200 mg dl-I) were required to kill a significant percentage of the tumour cells . To determine if cytotoxic ascorbate concentrations (on the order of I0 mM ) could be obtained in vivo , we measured plasma concentrations with time in a volunteer during 9 intravenous ascorbate Infusions Plasma vitamin C concentrations for 60 and 30 gram infusions , each over 80 minutes . With 60 grams infused , plasma concentrations reached a maximum value of 21.8 mM (432 mg dl-,) and then slowly decreased as the vitamin was cleared or absorbed . An infusion of 30 grams over the same time period resulted in a maximum ascorbate concentrations of 6.6 mM (130 mg dl-'). Lipoic acid ( sodium salt ) enhances the anti-tumor efficacy of ascorbate to the point where significant tumour cell killing can occur at concentrations achievable by intravenous infusion . vitamin K 3 is also an anticancer agent as a producer of hydrogen peroxide

Slide77:

The ability of ascorbate to kill tumour cells preferentially through hydrogen peroxide - generation (H 2 0 2 ) has been confirmed in several in vitro studies (Bram m I et al. 1980 ; Leung et at, 1993 ) (Benade et al. 1969 ; *Riordan et al. 1995 ). Rodent studies indicate that ascorbate supplementation can inhibit tumour growth in vivo ( Tsao et al, 1988 ; Varga and Airoldi , 1983 ). Lipoic acid is a lipophilic antioxidant that, among other things, inhibits hydrogen peroxide generation by ascorbate in erythrocytes ( Ou et al, 1995 ). We were thus surprised to find that not only was lipoic acid toxic to tumour cells at millimolar concentrations , but that it enhanced the cytotoxicity of ascorbate in a synergistic fashion . The reason for this synergy is unknown First, vitamin C concentrations in tumours are, on the average, 3 times higher than those in surrounding normal tissues ( Langemann et al, 1989 ), presumably because tumour cells use membrane glucose transporters to internalize ascorbate (Agus et al, 1999 ). While vitamin C has shown no major side effects in published clinical trials , the doses used in these trials have not exceeded 10 grams per day ( Cameron and Campbell , 1974 ; Creagan et al. 1979 ). Studies at higher doses are needed .

Slide78:

A recent study (manuscript in preparation. Casciari JJ , Tempeso MA , Riordan NH . Rodrigues G. Taylor P . Jackson JA et al ) at the University of Nebraska indicates that blood count and chemistry parameters are relatively stable in terminal cancer patients given continuous infusions of up to 50 grams per day for up to 8 weeks . We are not at this time aware of any clinical trials testing the safety of lipoic acid at high doses The accumulation of ascorbate in tumours has raised fears among some that it may protect tumour cells from oxidative damage associated with traditional therapeutic modalities ( Raloff , 2000 ). However , studies in cell culture suggest that vitamin C can enhance the efficacy of chemotherapy ( Kurbacher et al, 1996 ) and in vivo data suggest the same for radiation (Taper et al, 1996 ).

Slide79:

Stromal Cell Oxidation: A Mechanism by Which Tumors Obtain Vitamin C Article in Cancer Research · October 1999 – Researchgate : Source: PubMed Vitamin C enters through the facilitative glucose transporters ( GLUTs or Hexose Transporters ) in the form of dehydroascorbic acid , which is then reduced intracellularly and retained as ascorbic acid Dietary vitamin C is critical for humans and other primates because we cannot synthesize the vitamin in the liver as do most other animals (1). The mechanism by which cancers accumulate vitamin C in vivo, however, is unknown. Ascorbic acid , the form of vitamin C in the bloodstream, does not readily cross the blood-brain barrier , whereas dehydroascorbic acid crosses the blood-brain barrier and accumulates in the brain as ascorbic acid . Thus, the uptake of vitamin C in the form of dehydroascorbic acid through the GLUTs appears to be a general mechanism for vitamin C uptake . The increased intracellular concentration of vitamin C may have effects on tumor growth and the tumor’s ability to respond to oxidative stress associated with chemotherapy and radiation therapy . Although studies evaluating the role of vitamin C supplementation in cancer patients have generally shown no benefit with respect to survival or tumor regression (22), it is not known whether high concentrations of vitamin C in human tumors afford the malignant cells with a metabolic advantage .

Slide80:

Pilot study of ascorbic acid for the treatment of refractory immune thrombocytopenic purpura , 1993 Abstract The treatment of corticosteroid- and/or splenectomy -refractory immune thrombocytopenic purpura ( ITP ) includes vinca alkaloids, immunosuppressives , Danazol , intravenous gammaglobulin , and alpha-interferon . However, these treatments have often been associated with toxic side effects . Brox et al . (Br J Haematol 70:341-344 , 1988 ) reported the efficacy of ascorbic acid in the treatment of ITP ; the platelet count normalized in seven of 11 patients studied, and tolerance was excellent . However, other investigators have reported less impressive results. These conflicting reports prompted a pilot study of ascorbic acid in 12 patients with refractory ITP. Patients were given 2 g every morning for at least 10 weeks . All have received glucocorticoids , three had undergone splenectomy , and six received other treatments. The maximal increase in platelet count above baseline (i.e., the pre-study platelet count) achieved was < 20,000 in 11 patients and 25,000 in one patient. Therapy was well tolerated , with only two patients complaining of dyspepsia . The results of this study suggest that ascorbic acid is not very effective in patients with refractory ITP Opinion , I don’t believe these doses were anywhere near as high as they should h ave been, considering the stress of operations upon the body. 2 grams seems too low

Slide81:

Sodium Ascorbate induces apoptosis in neuroblastoma cell lines by interfering with iron uptake Accepted: 30 August 2007 It has been recently reported that vitamin C is effective in a large panel of tumor cell lines [ 3,4 ]. Sodium Ascorbate ( vitamin C ) has a controversial history in cancer treatment . The discrepancy obtained in research and analysis by many authors was principally due to the different dose and route of administration of vitamin C and consequently to its plasma concentration [ 5-7 ]. 3. Chen Q, Espey MG, Krishna MC, Mitchell JB, Corpe CP, Buettner GR Shacter E, Levine M: Pharmacologic ascorbic acid concentrations selectively kill cancer cells: action as a pro-drug to deliver hydrogen peroxide to tissues. Proc Natl Acad Sci U S A 2005 , 102(38): 13604-13609. 4. Kang JS, Cho D, Kim YI, Hahm E, Kim YS, Jin SN, Kim HN, Kim D , Hur D, Park H, Hwang YI, Lee WJ: Sodium ascorbate (vitamin C ) induces apoptosis in melanoma cells via the down-regulation of transferrin receptor dependent iron uptake. J Cell Physiol 2005 , 204(1): 192-197. 5. Riordan NH Riordan H. D., Casciari J. J.: Clinical and experimental experiences with intravenous vitamin C. J Orthomol Med 2000:201. 6. Riordan HD, Riordan NH, Jackson JA, Casciari JJ, Hunninghake R , Gonzalez MJ, Mora EM, Miranda- Massari JR, Rosario N, Rivera A : Intravenous vitamin C as a chemotherapy agent: a report on clinical cases. P R Health Sci J 2004, 23(2):115-118. 7. Padayatty SJ, Sun H, Wang Y, Riordan HD, Hewitt SM, Katz A, Wesley RA , Levine M: Vitamin C pharmacokinetics: implications for oral and intravenous use. Ann Intern Med 2004, 140(7):533-537. furthermore , it was reported that melanoma cells were more susceptible to ascorbate toxicity than any other tumor cells . Ascorbate seems to induce apoptosis by inducing disequilibrium of iron uptake due to down-regulation of transferrin receptor ( TfR ) [ 4 ].

Slide82:

Morphological inspection of treated cells , suggested that cell death took place through apoptosis rather than necrosis. This was confirmed by cytofluorimetric analysis of DNA content. There was a dose- and time-dependent increase of cells in the sub-G1 phase of the cell cycle (typical of apoptotic cells ) upon treatment with sodium ascorbate Intracellular iron level In order to find the possible mechanism of neuroblastoma cells apoptosis induced by vitamin C , we investigated whether this phenomenon was correlated with change in the intracellular iron levels ; therefore we treated HTLA-230 and SH-SY5Y for 24 hours with 1.5, and 2 mM sodium ascorbate and iron level was then measured . As shown in fig. 3 vitamin C significantly reduced intracellular iron levels in a dose-dependent manner . Treatment with vitamin C induced depolarization of the mitochondrial transmembrane potentia l in all neuroblastoma cell lines investigated Sodium Ascorbate-induced apoptosis is mediated by caspases activation Apoptosis arises from proteolytic activation of the cysteine proteases called caspases [ 17,18 ]. 17 . Nicholson DW, Thornberry NA: Caspases : killer proteases . Trends Biochem Sci 1997, 22(8): 299-306. 18 . Thornberry NA: Caspases : key mediators of apoptosis. Chem Biol 1998, 5(5): R97-103

Slide83:

To understand which caspases was mainly involved in the apoptotic mechanism , we used all the commercially available kits for single caspases ( i.e , caspase-1 , caspase-2 , caspase-3 & 7 , caspase-6 , caspase-8 , caspase-9 , and caspase-10 ) and measured the increase in fluorescence after 16 hours in SH-SY5Y and HTLA-230 cell lines . As depicted in fig. 7, all the above caspases showed a similar pattern of activation , in particular in SH-SY5Y caspases were already activated at 1 mM ascorbate . Although these experiments did not allow us to individuate a hierarchy of activation , they clearly confirmed the apoptotic nature of cell death induced by sodium ascorbate in neuroblastoma cell lines . Thirty years ago Cameron , Campbell and Pauling reported that high-dose vitamin C had beneficial effect for patient with terminal cancer . Subsequently, double-blind, randomized clinical trials, conducted by Moertel of the Mayo clinic , failed to show any benefit and the use of sodium ascorbate in cancer treatment was abandoned [ 19-24 ]. However, Moertel's results were not comparable to those of Cameron , as ascorbate was given orally and not intravenously . It was not recognized that the route of administration might produce large difference in plasma concentration . Recent clinical data show that when given i.v . , ascorbate plasma concentration is 25 fold higher respect that of the same oral dose [ 7 ]. 7 . Padayatty SJ, Sun H, Wang Y, Riordan HD, Hewitt SM, Katz A, Wesley RA , Levine M: Vitamin C pharmacokinetics: implications for oral and intravenous use. Ann Intern Med 2004, 140(7): 533-537.

Slide84:

Our data show that sodium ascorbate killed neuroblastoma cells , using lower concentrations compared to those active in other tumor cell lines ; in fact vitamin C, in the range of 0.5 to 3 mM turned out to be strongly cytotoxic. Sodium ascorbate-induced cell death was apoptosis , as documented by our experiments regarding the decrease of mitochondrial membrane potential , the phosphatidylserine externalization , the increase of cells in the sub-G1 phase of the cell cycle and caspases activation In this study we show that all the caspases are activated by the incubation with vitamin C , in fact, not only caspases related to the mitochondrial pathway were found to be activated but also those related to the death receptor pathway . The two pathways, extrinsic and intrinsic, are probably interconnected by caspase-8 mediated cleavage of the pro-apoptotic Bcl-2 family member Bid , producing a truncated Bid ( tBid ) fragment that promotes the mitochondrial release of proteins from the intermembrane space . In particular , it is known, that the release of cytochrome c induces the activation of caspase proteases through the induction of apoptosome formation [ 25 ]. Many neuroblastomas produce very large amount of ferritin Furthermore the mechanism by which vitamin C decreased intracellular iron level seems to be correlated with the down-regulation of transferrin receptor, Our data are consistent with the fact that tumor cells express high level of transferrin receptor to meet the increase in iron required by growing tumor tissue [ 11 ]. 11 . Richardson DR, Ponka P: The molecular mechanisms of the metabolism and transport of iron in normal and neoplastic cells . Biochim Biophys Acta 1997, 1331(1): 1-40.

Slide85:

Some in vitro studies showed that ascorbate causes toxicity to cancer cells at concentrations that do not affect normal cells [ 3 , 9 ]; this phenomenon is probably due to a tumor-specific intracellular transport of ascorbate . Extracellular ascorbate is oxidized, transported as dehydroascorbic acid , and reduced intracellularly to ascorbate [ 32 ]. Many cell types transport ascorbate only in its oxidized form , through facilitated glucose transporters [ 33 ]. Tumor cells have an increased requirement for glucose [ 34 ] and to compensate for this they increase the expression of glucose transporters . This allows ascorbate to act as a selective , non toxic to normal cells, chemotherapeutic . 3. Chen Q, Espey MG, Krishna MC, Mitchell JB, Corpe CP, Buettner GR , Shacter E, Levine M: Pharmacologic ascorbic acid concentrations selectively kill cancer cell s : action as a pro-drug to deliver hydrogen peroxide to tissues . Proc Natl Acad Sci U S A 2005 , 102(38):13604-13609 9 . Leung PY, Miyashita K, Young M, Tsao CS: Cytotoxic effect of ascorbate and its derivatives on cultured malignant and non-malignant cell lines . Anticancer Res 1993 , 13(2):475-480 . 32 . Wilson JX : Regulation of vitamin C transport. Annu Rev Nutr , 2005 , 25:105-125. 33 . Astuya A, Caprile T, Castro M, Salazar K, Garcia Mde L, Reinicke K , Rodriguez F, Vera JC, Millan C, Ulloa V, Low M, Martinez F, Nualart F : Vitamin C uptake and recycling among normal and tumor cells from the central nervous system. J Neurosci Res 2005 , 79(1-2 ): 146-156 . 34. Spielholz C, Golde DW, Houghton AN, Nualart F, Vera JC : Increased facilitated transport of dehydroascorbic acid without changes in sodium-dependent ascorbate transport in human melanoma cells . Cancer Res 1997, 57(12):2529-2537 Conclusion This study demonstrates that sodium ascorbate is highly toxic to neuroblastoma cell lines and the specific mechanism of vitamin C -induced apoptosis is due to a perturbation of intracellular iron levels ensuing TfR ( Transferrin Receptor ) down-regulation

Slide86:

CELL LINES SH-SY5Y and HTLA-230 cell lines

Slide87:

Erythrocyte Glut-1 Triggers Dehydroascorbic Acid Uptake in Mammals Unable to Synthesize Vitamin C, 2008 Here, we report that glucose transport actually decreases during human erythropoiesis despite a >3-log increase in Glut-1 transcripts . In contrast , Glut1-mediated transport of L- dehydroascorbic acid ( DHAA ), an oxidized form of ascorbic acid ( AA ), is dramatically enhanced . We identified stomatin , an integral erythrocyte membrane protein, as regulating the switch from glucose to DHAA transport . Notably though , we found that erythrocyte Glut-1 and associated DHAA uptake are unique traits of humans and the few other mammals that have lost the ability to synthesize AA from glucose . Accordingly, ,we show that mice , a species capable of synthesizing AA , express Glut-4 but not Glut-1 in mature erythrocytes . Thus, erythrocyte-specific co-expression of Glut-1 with stomatin constitutes a compensatory mechanism in mammals that are unable to synthesize vitamin C . Fourteen Glut isoforms have now been identified in the human genome but Glut-1 is the main functional transporter of glucose in most transformed cells as well as in various hematopoietic cell lineages ( Mueckler , 1994, 1985 ). The Glut-1 transporter is also crucial for facilitating glucose transport in the brain ; a haplo insufficiency of Glut-1 results in infantile seizures, delayed development, and microcephaly ( Seidner et al., 1998 ).

Slide88:

Once transported into the cell, DHAA is immediately reduced to AA allowing a recycling of ascorbate (May, 1998 ). The uptake of AA into cells is mediated by a distinct class of transporters; sodium-dependent vitamin C transporters ( SVCT1 and SVCT2 ) whose expression profiles differ from those of Glut family members ( Tsukaguchi et al., 1999 ). AA is essential for maintaining plasma and tissue reductive capacity , removing superoxide via its own oxidation into DHAA . Here, we show that in erythrocytes , Glut-1 preferentially transports DHAA rather than glucose . The switch from glucose to DHAA transport is associated with an induction of stomatin , an integral erythrocyte membrane protein . Erythrocyte-specific Glut-1 expression and DHAA transport are specific traits of the few vitamin C-deficient mammalian species, encompassing only higher primates , guinea pigs and **fruit bats . (Note: Refer to Interleukin-3 [ IL-3 ] and Interleukin-6 [ IL-6 ] cytokines ) As shown above Glut1-mediated DHAA uptake in human erythrocytes is distinctive in that it is not competed by glucose . Thus , either differences in Glut-1 itself and/or differences in Glut-1 partners account for the specific inability of glucose to inhibit DHAA uptake in erythrocytes .

Slide89:

We hypothesized that DHAA uptake by human erythrocytes could be linked to the inability of humans to synthesize the reduced form of DHAA , AA , from glucose . Of the > 4000 species of mammals , it appears that only man , other higher primates , guinea pigs and fruit bats are unable to synthesize AA from glucose . Importantly , we detected Glut-1 expression on human , guinea pig and fruit bat erythrocytes but not on any other mammalian RBC tested including rabbit , rat , cat , dog and chinchilla AA is a vital substance that is produced in the livers of most mammals ( Chatterjee et al., 1961 ). The absence of AA production in humans , due to an inactive L- gulono -y-lactone oxidase ( GLO ), the enzyme that catalyzes the terminal step of L-ascorbic acid biosynthesis (Burns, 1957 ), has been described by some scientists as a ‘‘ species inborn metabolic error ’’ (Stone, 1966 ). Indeed , supplementation of human diets with exogenous vitamin C has significantly reduced the incidence of scurvy . There has been much debate spurred by several scientists, including Linus Pauling , as to the appropriate daily recommended vitamin C allowance . May and colleagues have shown that erythrocyte uptake of DHAA and its rapid intracellular conversion to AA is crucial for ascorbate recycling , providing a critical export source of this reducing agent (May, 1998 ). The rapid uptake and reduction of DHAA by erythrocytes is also likely to account for the extremely low plasma concentration of DHAA Chatterjee , I.B., Kar , N.C., Ghosh , N.C., and Guha , B.C. ( 1961 ). Biosynthesis of L-ascorbic acid : missing steps in animals incapable of synthesizing the vitamin . Nature 192, 163–164 Stone, I. ( 1966 ). Hypoascorbemia , the genetic disease causing the human requirement for exogenous ascorbic acid. Perspect . Biol. Med. 10, 133–134 .- Unable to read or access

Slide90:

The persistence of erythrocyte Glut-1 expression is a unique trait of vitamin C-defective mammals , resulting in a massive expression of DHAA transporters on the most abundant cell in the circulation . Our experiments, showing that Glut-1 is highly expressed on differentiating erythroblasts , suggest that DHAA can be effectively removed from the bone marrow by erythroid cells that have progressed to the basophilic stage . The high DHAA uptake by circulating erythrocytes would then allow the AA redox molecule to be efficiently transported throughout the body .

Slide91:

Fruit Bats and Ascorbic Acid

Slide92:

Recent evidence shows that oral administration of the maximum tolerated dose of vitamin C ( 18 grams/day ) produces peak plasma concentrations of only 220 μmol /L , whereas intravenous administration of the same dose produces plasma concentrations about 25-fold higher . Larger doses ( 50–100 g ) given intravenously may result in plasma concentrations of about 14 000 μmol /L . At concentrations above 1000-5000 μmol /L , vitamin C is toxic to some cancer cells . In light of recent clinical pharmacokinetic findings and in vitro evidence of anti-tumour mechanisms , these case reports indicate that the role of high-dose intravenous vitamin C therapy in cancer treatment should be re-assessed . Intravenously administered vitamin C as cancer therapy : three cases Sebastian J. Padayatty , Hugh D. Riordan, Stephen M. Hewitt, Arie Katz, L. John Hoffer, Mark Levine - 2006 Concentrations achieved only by the intravenous route may function as a pro-drug for hydrogen peroxide delivery to tissues . (20) 1. Cameron E, Campbell A. The orthomolecular treatment of cancer. II. Clinical trial of high-dose ascorbic acid supplements in advanced human cancer. Chem Biol Interact 1974;9:285-315. 2. Cameron E, Campbell A, Jack T. The orthomolecular treatment of cancer. III. Reticulum cell sarcoma: double complete regression induced by high-dose ascorbic acid therapy. Chem Biol Interact 1975;11:387-93. 3. Cameron E, Pauling L. Supplemental ascorbate in the supportive treatment of cancer: prolongation of survival times in terminal human cancer. Proc Natl Acad Sci US A 1976;73:3685-9. 4. Cameron E, Pauling L. Supplemental ascorbate in the supportive treatment of cancer: reevaluation of prolongation of survival times in terminal human cancer. ProcNatl Acad Sci U S A 1978;75:4538-42. 20. Chen Q, Espey MG, Krishna MC, et al. Pharmacologic ascorbic acid concentrations selectively kill cancer cells: Action as a pro-drug to deliver hydrogen peroxide to tissues. Proc Natl Acad Sci U S A 2005 Thirty years ago Cameron , Campbell and Pauling reported beneficial effects of high-dose vitamin C ( ascorbic acid / AA ) therapy for patients with terminal cancer (1–4)

Slide93:

Patients with untreated stage III diffuse B-cell lymphoma have a dismal prognosis. This case, like the preceding one, is unusual in that the patient refused chemotherapy, which might have produced a long-term remission. It appears, nonetheless, that a cure occurred in connection with intravenous vitamin C infusions Patient 3: She received 15 g of vitamin C twice per week for about 2 months , 15 g once to twice per week for about 7 months , and then 15 g once every 2–3 months for about 1 year . This began in mid-January 1995 concurrently with the radiation therapy. With maximally tolerated oral doses of 3 g every 4 hours , peak plasma concentrations are estimated to not exceed 220 μmol /L. Intravenous administration of vitamin C bypasses tight control for several hours , until homeostasis is restored by renal excretion . Ascorbic acid is metabolized to oxalate Vitamin C may serve as a pro-drug for hydrogen peroxide delivery to extravascular tissues , but without the presence of hydrogen peroxide in blood. These data are consistent with clinical pharmacokinetics of vitamin C administered intravenously Patient 2: He received 30 g of vitamin C twice per week for 3 months , followed by 30 g once every 1–2 months for 4 years , interspersed with periods of 1–2 months during which he had more frequent infusions. Patient 1: She received 65 g twice per week starting in October 1996 and continuing for 10 months . More than 4 years after stopping intravenous vitamin C therapy and with the renal cell cancer in complete remission , primary small-cell lung cancer was diagnosed in this patient, who was a long-standing cigarette smoker . The second cancer did not respond to high-dose vitamin C therapy .

Slide94:

5/18/2019 94 Cancer and Oncogenesis

Slide95:

Discussion In brain, sepsis is characterized by increased production of inflammatory cytokines , iNOS and free radicals ; depletion of ascorbate ; oxidative modification of lipids and proteins ; and elevation of extracellular glutamate concentration to excito - toxic levels (Yousef et al. 1994; Koedel and P®ster 1999; Hirst et al. 2000; Papadopoulos et al. 2000; Willard et al. 2000).

Slide96:

Sepsis inhibits reduction of dehydroascorbic acid (DHAA) and accumulation of ascorbate in astroglial cultures: intracellular ascorbate depletion increases nitric oxide synthase induction and glutamate uptake inhibition Journal of Neurochemistry, 2002 , 81, 185-193 Sepsis is a frequent cause of inflammatory encephalopathy and neurologic complication is often fatal ( Papadopoulos et al. 2000). Bacterial endotoxins [e.g. lipopolysaccharide ( LPS ) from the cell wall of Gram-negative bacteria ] and cytokines induce the reactive phenotype in brain astrocytes and up-regulate inducible nitric oxide synthase ( iNOS ) NOS enzymes use NADPH , oxygen and arginine to produce nitric oxide ( NO ) and superoxide ( Pou et al. 1999; Tolias et al . 1999 ). These products kill invading organisms but also evoke oxidative stress in host cells . ( LPS + IFNy ) induces iNOS , increases intracellular levels of reactive nitrogen and oxygen species, and sensitizes astrocytes to killing by oxidative stressors ( Noack et al. 1999). Infammatory encephalopathy also involves excitotoxicity by glutamate . Septicemia elevates glutamate concentration fivefold in the brain interstitial fluid (Guerra-Romero et al . 1993 ). LPS elicits cardiovascular responses that can be prevented by blockade of NMDA-type glutamate receptors in brain ( Yousef and Lang 1994).

Slide97:

However, these in vitro experiments were performed in the absence of an important antioxidant, ascorbate ( vitamin C ). This antioxidant normally is present at millimolar concentrations in the brain . But because brain cells cannot synthesize vitamin C de novo , and it is very unstable in culture medium, extracellular and intracellular ascorbate concentrations fall rapidly to non-detectable levels in vitro ( Siushansian et al. 1997). Our results showed that LPS + IFNy impairs the transport and metabolic systems responsible for intracellular ascorbate accumulation . Additionally , LPS + INFy induces iNOS expression and inhibits glutamate uptake by mechanisms sensitive to intracellular ascorbate . Taken together , these results indicate that LPS + IFNy did not decrease cell viability during the experimental period . The above experiments with Asc -p ( ascorbate analogue ) showed that astrocytic responses to septic insult are modified by the intracellular antioxidant , ascorbate . These results are consistent with the view that ascorbate depletion may contribute to inflammatory encephalopathy . These signs of sepsis were prevented by prior ascorbate loading . Depletion of intracellular ascorbate may be characteristic of sepsis because LPS + IFNy also impaired ascorbate uptake Microscopic examination of six random fields revealed iNOS expression in 89% of the cells exposed to LPS + IFNy compared with expression in 32% of cells that had been treated with Asc -p prior to and during LPS + IFNy exposure .

Slide98:

The high NO levels inhibit mitochondrial respiration , at least in part by decreasing the affinity of cytochrome c oxidase for oxygen (Brown 2000 ). Additionally , NO combines with superoxide to form peroxynitrite , which impairs respiration irreversibly . Inhibition of respiratory and glycolytic enzymes by peroxynitrite may lead to energy depletion in brain cells ( Bolanos et al. 1997 ). This cellular energy depletion may explain why sepsis-induced elevation of NO production is accompanied by increases in extracellular hydrogen peroxide and glutamate ( McNaught and Jenner 2000 ). Furthermore , administration of a NOS inhibitor prevents the inhibition of astrocytic glutamate uptake by another inducer of iNOS , interleukin-1b (Hu et al. 2000 ). Millimolar concentrations of ascorbate reduce superoxide and thereby prevent peroxynitrite formation from superoxide and NO (Jackson et al. 1998 ; Kirsch and de Groot 2000 ). Ascorbate also chemically reduces peroxynitrite and other reactive oxygen and nitrogen species (Kirsch and de Groot 2000 ). Both ascorbate and antioxidant enzymes ( superoxide dismutase and catalase ) are capable of decreasing the induction by inflammatory cytokines of NOS activity in a human vascular endothelial cell line (Galley et al. 1996c).

Slide99:

Because brain cells cannot synthesize vitamin C de novo from glucose , they rely on blood and cerebrospinal fluid . This extracellular supply is diminished in sepsis because ascorbate concentrations in blood and cerebrospinal fluid fall below normal (Galley et al. 1996a ; Schorah et al. 1996 ; Block and Schwarz 1997 ; Metnitz et al. 1999 ; Armour et al. 2001 ). Additionally, cellular mechanisms of vitamin C transport and metabolism may be defective , as LPS depletes ascorbate from adrenal glands (Garcia et al. 1990 ), lung (Benito and Bosch 1997 ), and heart (Rojas et al. 1996 ). One way that ascorbate becomes concentrated in cells is through Na-ascorbate co-transporters ( SVCT ). The SVCT2 isoform mediates the uptake of authentic ascorbate (but not DHAA ) by astrocytes ( Korcok et al. 2000 ). The present experiments demonstrate that SVCT2-mediated transport is inhibited in septic astrocytes In sepsis , ascorbate may be oxidized by reactive oxygen species at rates that overwhelm the ability of cells to regenerate the vitamin . The observation that the concentration of ascorbyl radical is increased in septic patients ( Galley et al. 1996b ) is consistent with an imbalance in the redox cycling of vitamin C . We found that LPS + IFNy inhibits accumulation of intracellular ascorbate from extracellular DHAA . However , after DHAA is taken into cells it is reduced to ascorbate by cytosolic DHAA reductase or other enzymes , using reducing equivalents from glutathione and NADPH

Slide100:

Enhancement of Nitric Oxide and Superoxide Generations by a- Tocopheryl Succinate and Its Apoptotic and Anticancer Effects April 30, 2003 In VSMC ( vascular smooth muscle cells) , NO ( Nitric Oxide ) is known to play a critical role in vasodilatory function and atherosclerosis processes [38], and inducible NO production in the VSMC system has been well studied [39]. In atherosclerotic plaque, cytokines such as tumor necrosis factor-a ( TNF-a ) and interleukin-1 ( IL-1 ) secreted from macrophages and from cells have been implicated in the pathogenetic events [40]. We used the system containing lipopolysaccharide ( LPS ) known as a stimulant of iNOS expression through similar signalling cascade to these cytokines [40, 42]. Interferon-y ( IFNy ), which was also reported to be secreted from T cells in the lesions of atherosclerosis , was further used together with LPS to strengthen LPS function . It has been found that TS ( Tocopheryl Succinate ) induces apoptosis in various cell lines such as human breast cancer cells , neuroblastoma cells , and lymphoblastoid cells [12, 14-27]. Multiple signalling pathways have been suggested to play a role in the TS-induced apoptosis . These may involve transforming growth factor-b ( TGF-b ), PKC, Fas, and the mitogen-activated protein kinase ( MAPK ) signallings [12, 14-19, 21, 26-30]. However, the trigger event of TS-induced apoptosis is still obscure .

Slide101:

Human pharmacokinetics data indicate that i.v. ascorbic acid ( ascorbate ) in pharmacologic concentrations could have an unanticipated role in cancer treatment . Our goals here were to test whether ascorbate killed cancer cells selectively , and if so, to determine mechanisms, using clinically relevant conditions . Pharmacologic ascorbic acid concentrations selectively kill cancer cells: Action as a pro-drug to delive r hydrogen peroxide to tissues, Communicated by J. E. Rall , National Institutes of Health, Bethesda, MD, August 2, 2005 Taken together, these data indicate that ascorbate at concentrations achieved only by i.v. administration may be a pro-drug for formation of H 2 O 2 , and that blood can be a delivery system of the pro-drug to tissues . These findings give plausibility to i.v. ascorbic acid in cancer treatment , and have unexpected implications for treatment of infections where H 2 O 2 may be beneficial . Recent pharmacokinetics studies in men and women show that 10 g of ascorbate given i.v. is expected to produce plasma concentrations of nearly 6 mM , which are 25-fold higher than those concentrations from the same oral dose (7–9). As much as a 70-fold difference in plasma concentrations is expected between oral and i.v. administration, depending on dose . Despite inconsistencies , some in vitro studies showed that ascorbate killed cancer cells , although mechanisms and physiologic relevance were unclear

Slide102:

Our goals were to address the following: Does ascorbate in pharmacologic concentrations kill cancer cells , but not normal cells , using conditions that mimic i.v. use and a clinically relevant time course? Is action dependent on extracellular ascorbate , intracellular ascorbate , or both ? If effective, what are the mechanisms ? Can ascorbate be delivered to tissues without harm ? Are there implications for other diseases ? The data showed that pharmacologic concentrations of ascorbate killed cancer but not normal cells , that cell death was dependent only on extracellular but not intracellular ascorbate , and that killing was dependent on extracellular hydrogen peroxide ( H 2 O 2 ) formation with ascorbate radical as an intermediate . Ascorbate generated detectable levels of H 2 O 2 in extracellular medium in the presence of trace serum protein but not in whole blood. The findings indicate that ascorbate at pharmacologic concentrations in blood may be a pro-drug for H 2 O 2 delivery to tissues , with major therapeutic implications . The roles of intracellular versus extracellular ascorbate in causing cell death were examined, using ascorbate and its oxidized product dehydroascorbic acid . Ascorbate is transported into cells as such by sodium-dependent transporters , whereas dehydroascorbic acid is transported into cells by glucose transporters and then immediately reduced internally to ascorbate (29). Despite similar intracellular ascorbate concentrations under both conditions , cells died only when ascorbate was present externally

Slide103:

Taken together , these data indicate that ascorbate cannot generate sustainable H 2 O 2 concentrations in whole blood . The data are consistent with the hypothesis that ascorbate in pharmacologic concentrations is a pro-drug for H 2 O 2 generation in the extracellular milieu but not in blood . Discussion Our data show that ascorbic acid selectively killed cancer but not normal cells , using concentrations that could only be achieved by i.v . Ascorbate-mediated cell death was due to protein-dependent extracellular H 2 O 2 generation , via ascorbate radical formation from ascorbate as the electron donor . The mechanism of this previously unexplained observation is now straightforward , based on the results here. H 2 O 2 generated in blood is normally removed by catalase and glutathione peroxidase within red blood cells , with internal glutathione providing reducing equivalents . The electron source for glutathione is NADPH from the pentose shunt, via glucose-6-phosphate dehydrogenase . If activity of this enzyme is diminished , the predicted outcome is impaired H 2 O 2 removal causing intravascular hemolysis , the observed clinical finding.

Slide104:

H 2 O 2 as the product of pharmacologic ascorbate concentrations , has potential therapeutic uses in addition to cancer treatment , especially in infections . H 2 O 2 is a potent mammalian antimicrobial defence mechanism (54 ). Neutrophils generate H 2 O 2 from superoxide , in turn formed by NADPH oxidase- catalyzed reduction of molecular oxygen . Old observational animal experiments , although uncontrolled, suggest that i.v. ascorbate is effective in some viral infections (56, 57). This finding is also consistent with in vitro experiments , in which H 2 O 2 is toxic to hepatitis C (58). Use of ascorbate as an H 2 O 2 - delivery system against sensitive pathogens , viral or bacterial , has substantial clinical implications that deserve rapid exploration . Complementary and alternative medicine practitioners worldwide currently use ascorbate i.v. in doses as high as 70 g over several hours (14, 15, 59). Because i.v. ascorbate is easily available to people who seek it , a phase I safety trial in patients with advanced cancer is justified and underway . Ascorbate administered i.v. is likely to be safe in most patients, with virtually no toxicity compared to most currently available cancer chemotherapeutic agents . 14. Riordan, N. H., Riordan, H. D. & Casciari, J. J. (2000) J. Orthomol. Med. 15, 201–203. 15. Riordan, H. D., Hunninghake , R. B., Riordan, N. H., Jackson, J. J., Meng , X., Taylor, P . Casciari , J. J., Gonzalez, M. J., Miranda-Massari, J. R.,Mora, E. M., et al. (2003) P. R. Health Sci . J. 22, 287–290 . 59. Riordan, N. H., Riordan, H. D., Meng , X., Li, Y. & Jackson, J. A. (1995) Med. Hypotheses 44 , 207–213.

Slide105:

Mechanisms and Applications of Ιnterleukins in Cancer Immunotherapy The goal of this review is to evaluate the major cytokines-interleukins involved in cancer immunotherapy and project their basic biochemical and clinical applications.  1 Interleukin-6 is a pleiotropic, pro-inflammatory cytokine that can promote both innate and adaptive immune responses. In humans with respiratory virus infections, such as Respiratory Syncytial Virus ( RSV ), elevated concentrations of IL-6 are associated with more severe disease . In contrast the polymorphisms in the  Il6  promoter which favour lower IL-6 production are associated with increased risk of both RSV and Rhinovirus infections 1 Early IL-6 signalling promotes IL-27 dependent maturation of regulatory T cells in the lungs and resolution of viral immunopathology We have found that the early production of IL-6 after infection promotes the production of the regulatory mediator Interleukin-27 by lung resident immune cells , which in turn drives suppression of otherwise damaging inflammation (ROS) . Removal of either IL-6 or IL-27 enhances disease during viral infection , while restoration of IL-27 is sufficient to allow faster recovery . IL-6 also mediates regulatory T cells ( Treg ) suppression as a function The question arises: How to promote IL-6 (leading to IL-27 activation) naturally, as it promotes CD4 + & CD8 + T h 17 ( T-Helper ) cells and is up-regulated by Cancer and Vaccines

Slide106:

Attenuating the p53 Pathway in Human Cancers : Many Means to the Same End Inactivation of the p53 pathway is a defining feature of human cancers , with nearly all cancers evolving a way to circumvent this essential tumor-suppressive mechanism . Although a large number of human cancers directly inactivate p53 through mutations or deletions of the TP53 ( p53 ) locus (Hainaut and Pfeifer 2016), there are a vast number of other molecular alterations that can functionally serve to attenuate the pathway p53 is widely acknowledged as the guardian of the genome (Lane 1992). In normal, unstressed cells, p53 levels and transcriptional activity are kept in check by important negative regulators. In response to a variety of cellular stresses , including DNA damage , oncogene activation , and oxidative stress , posttranslational mechanisms stabilize and activate p53 . As a transcription factor , p53 can then bind specific promoters and regulate the expression of genes that drive cell-cycle arrest , apoptosis , senescence , and several other cellular functions discussed in this collection.

Slide107:

The p53 tumor suppressor plays a critical role in the cellular response to stress . 6, 7 Apart from its important role in tumor suppression, p53 is also implicated in diabetes-associated complications , 8, 9   endothelial dysfunction 10  and arthrosclerosis . 11  Recent studies have shown that p53 might play a critical role in regulating ROS production in cultured cells. 12  Under normal growth conditions , p53 is required for maintaining basal transcription of antioxidant genes SESN1 , SESN2 , and GPX1 . 12, 13   Suppression of p53 significantly decreases the basal transcriptions of those genes , 13  which increases cellular ROS levels and subsequently leads to oxidative damage of DNA . Thus regulating antioxidant defence to reduce ROS levels may represent one of the important tumor suppressing and cardiovascular protecting mechanisms of p53 . High glucose-induced p53 phosphorylation contributes to impairment of endothelial antioxidant system 2017 Jun 30.  doi : 10.1016/j.bbadis.2017.06.022 Intracellular ATP level was measured by a luciferin /luciferase test ROS levels generate more production of Hydrogen Peroxide  catabolised by GPXs and catalase to convert H 2 O 2 into water Parallel reduction in GPX1 ( Glutathione Peroxidase-1 ) protein level High glucose inhibits p53 , which leads to suppression of GPX1 expression and accumulation of ROS in HAECs (Human Aortic Endothelial cells)

Slide108:

In addition to the foregoing mechanism, prolonged HG exposure (6 days) has been shown to reduce SIRT1 protein expression and increase p53 acetylation , which leads to p21 activation and endothelial senescence (36, 37) It is thus possible that, upon HG exposure , cells undergo a transit p53 inactivation due to increased Thr55 phosphorylation (6–16 hours) and subsequent GPX1 down-regulation , which contributes to generation of ROS and oxidative stress in the cell. If the stress prolongs , endothelial cells will likely call upon additional cell defence system, including SIRT1 , which results in sustained hyperacetylation of p53 , and potentially SIRT1 / p53 / p21 - mediated endothelial senescence High glucose-induced p53 phosphorylation contributes to impairment of endothelial antioxidant system 2017 Jun 30.  doi : 10.1016/j.bbadis.2017.06.022 To protect themselves from oxidative damage , cells have developed a sophisticated antioxidant enzyme defence system . In this system, superoxide dismutases ( SODs ) convert O2 •− into H 2 O 2 , whereas GPXs and catalase convert H 2 O 2 into water (44) In endothelial cells , H 2 O 2 detoxification is primarily mediated by GPX1 as catalase is reportedly not expressed or expressed at very low levels in these cells.

Slide109:

Inhibiting the system x C − / glutathione axis selectively targets cancers with mutant-p53 accumulation 2017 Mar 28. doi: 10.1038/ncomms14844 We show that accumulated mutant- p53 protein suppresses the expression of  SLC7A11 , a component of the cystine /glutamate antiporter , system x C − , through binding to the master antioxidant transcription factor NRF2 . This diminishes glutathione synthesis , rendering mutant- p53 tumours susceptible to oxidative damage We demonstrate that high levels of mutant- p53 , through binding to NRF2 and impairing its canonical antioxidant activities , directly promote ROS accumulation in cancer cells … normal cells can mount an optimal NRF2-mediated response to oxidative stress  Seminal studies have shown that the master antioxidant transcription factor , NRF2 , intimately regulates  SLC7A11  transcription 14,15 and cells that are p53 null or wild-type have relatively higher levels of SLC7A11 expression , It may be prudent to seek supply or elements for cystine to enable GSH synthesis This protein ( SLC7A11 ) has been identified as the predominant mediator of Kaposi sarcoma-associated herpes virus fusion and entry permissiveness into cells

Slide110:

A continual cystine supply is crucial for  de novo synthesis of glutathione and thioredoxin antioxidant peptides , highlighting CD44v9's role in redox regulation  (4, 5). The transcription factor NRF2 (encoded by the  NFE2L2  gene ), a master regulator against oxidative stress Dual pharmacological inhibition of glutathione and thioredoxin systems synergizes to kill colorectal carcinoma stem cells 2016 Aug 3. doi: 10.1002/cam4.844 NRF2 , as a redox sensor and feedback regulator , integrates three groups of enzymes (those associated with the glutathione and thioredoxin systems and NADPH production ) to reduce reactive oxygen species ( ROS ) (9). Hence, NRF2 stabilizes intracellular redox potential and ensures robust cellular systems against potential harmful effects of ROS . For example, once the glutathione system is disrupted (e.g., by pharmacological inhibition), increased ROS activates NRF2 , and subsequently, the other thioredoxin system is augmented for compensation NRF2 up‐regulates   GCLC  and  GCLM  genes , components of glutathione system . NRF2 up‐regulates PRDX1 , TXN , and TXNRD1 genes , components of thioredoxin system .

Slide111:

For IL‐1 β‐ stimulated chondrocytes , significant down‐regulation of IL‐1 β , tumor necrosis factor‐alpha ( TNF‐ α ), MMP‐3 , and MMP‐9 mRNA expression was found when cells were cultured in HA‐supplemented media. Moreover, HA  +  AA supplementation further significantly decreased MMP‐3 (matrix metalloproteinase -3 ) and MMP‐9 mRNA expression . The protein production of MMP‐3 was decreased , with a significant difference between the HA  +  AA group and HA group . The antioxidant capacity and superoxide dismutases activity were also partially restored in stimulated chondrocytes . HA supplemented with AA modulates MMPs expression and antioxidant function in chondrocytes . AA may enhance the anti-catabolic effects of HA on OA chondrocytes . Synergistic effect of   l ‐ascorbic acid and hyaluronic acid on the expressions of matrix metalloproteinase‐3 ( MMP-3 ) and −9 i n human chondrocytes First published: 15 September 2017 Abstract Pro-inflammatory cytokines and reactive oxygen species ( ROS ) are known to be involved in the initiation and progression of osteoarthritis (OA). New evidence clarifying the correlation between ROS and inflammation has indicated that oxidative stress can up‐regulate inflammatory cytokines .  l ‐Ascorbic acid ( AA ), an antioxidant , has been shown to have anti‐inflammatory effects and improve matrix deposition in chondrocytes. Caspases are specific proteases responsible for the regulation and the execution of apoptotic cell death.

Slide112:

Connecting terms to investigate causality p 53 ( TP53 )  Oxidative Stress and Oocytes  Gametes and Sperm Fusion JUNO and IZOMITOL  Acetylcholine Receptors , Muscaritic Acid , Serotonin , Melatonin , Norepinephrine , Dopamine Receptors D1-5 Sodium Fluoride  Affects Antioxidant Defence Enzymes  Reduces Glutathione levels , SOD , Catalase , IgA , IgG , IgM , Apoptosis and Cell-mediated Death  Promotes Pro-inflammatory Responses ( Cytokines ) Helicobactor Pylori  Lowers Glutathione  Adrenal Glands  TP53/p53 GLUT-1 and GLUT-4 transporter used by Ascorbic Acid  Aspartame  Sodium Fluoride Helicobacter pylori induces somatic mutations in TP53 via overexpression of CHAC1 in infected gastric epithelial cells 7 February 2018 - doi:10.1002/2211-5463.12402 Infection with Helicobacter pylori is known to decrease the level of glutathione (GSH) in gastric epithelial cells and increase the production of reactive oxygen species ( ROS ), which can lead to DNA damage and the development of gastric carcinoma (cancer)

Slide113:

1 The two main defects of mice homozygous for the nude spontaneous mutation (Foxn1 nu , formerly Hfh11 nu ) are abnormal hair growth and defective development of the thymic epithelium . Homozygous nude mice lack T cells and suffer from a lack of cell-mediated immunity . Because of a defect in helper T-cell activity , responses to thymus-dependent antigens when detectable are primarily limited to IgM . Homozygous nude mice show partial defect in B cell development probably due to absence of functional T cells . Other endocrine and neurological deficiencies have been reported Since the nude mouse lacks a thymus , it also lacks the T-cell-mediated rejection reflex . Because of this, human cancer cells can be injected into the nude mouse , where they grow into full tumors . some types of tumors, like melanomas , colon and lung cancers , were able to take and grow very easily in the nude mouse, other types were much more difficult to grow (breast cancers 6%. No Leukemias) 1 https://www.jax.org/strain/002019

Slide114:

Effects of smoking and tumor process on the contents of key proteins of apoptosis and activity of antioxidant enzymes in blood . [ 2012 Jan-Feb;(1):19-26 - Article in Russian] Abstract The effects of smoking on the contents of the apoptosis markers Bcl-2 and p53 proteins in blood plasma; the activity of the antioxidant ( AO ) enzymes , Cu , Zn, superoxide dismutase ( SOD ), glutathione peroxidase ( GP ), glutathione reductase ( GR ), glutathione S- transferase ( GST ), and catalase ; and the content of malondialdehyde ( MDA ) in erythrocytes from healthy donors and cancer patients were studied. Two groups of donors were revealed among healthy smokers: one with high SOD and GP activities and high Bcl-2 protein levels and the other with lower Bcl-2 levels compared with those found in non-smokers . In the group of cancer patients (both smokers and non-smokers ), significantly increased p53 protein levels and increased activity of GST were found . A negative correlation between MDA and GST in the group of smokin g healthy donors and a positive correlation between MDA and p53 in cancer patients were found. The results suggest a relationship between the components of enzymatic defence and lipid peroxidation and the content of apoptosis regulator proteins in healthy smokers and cancer patients .

Slide115:

1 We found that AA inhibited the expression of two categories of genes necessary for cell cycle progression, tRNA synthetases and translation initiation factor subunits . In in vitro assays, we demonstrated that AA ( Ascorbic Acid ) induced the S-phase arrest of proliferative normal and tumor cells . Highest concentrations of AA lead to necrotic cell death . Ascorbic Acid, an antioxidant , act on the PMP22 gene expression and AA , but not other antioxidants, to down-modulate cAMP intracellular concentration by a competitive inhibition of the adenylate cyclase enzymatic activity. 1 Antiproliferative Effect of Ascorbic Acid Is Associated with the Inhibition of Genes Necessary to Cell Cycle Progression, February 2009 , Volume 4, Issue 2, AA has an antiproliferative activity , potentially due to the inhibition of expression of genes involved in cell division progression and may be related to its action as a ‘‘global regulator’’ of intracellular cAMP pool At moderate concentration (0,3 mM ), AA partially inhibited cell proliferation . Higher concentrations (0.6 and 2mM) respectively resulted in a cell proliferation arrest or cellular death . We then tested the effect of AA on cell lines derived from human cancers . Cell growth was affected by AA treatment in all of the cell lines , although sensitivity varied This difference is not understood at present..

Slide116:

HISTORY OF CANCER TREATMENT Several decades ago, McCormick , Cameron and Rotman , without supporting data, postulated two hypotheses regarding the use of ascorbate for cancer therapy ( 8-10 ). The Prospects of Vitamin C in Cancer Therapy DOI 10.4110/in. 2009 .9.5.147

Slide117:

BIOLOGICAL ROLE One biochemical function of ascorbate is to enhance hydroxylation in a large number of biosynthetic reactions ( 19 , 20 ). In a majority of these biosynthetic processes, ascorbate provides necessary electrons to participating enzymes and is required to achieve full enzymatic activity ( 19 ). The characteristic role of ascorbate is as a cofactor for prolyl and lysyl hydroxylase enzymes ( 20 ). Ascorbate is also necessary for cholesterol metabolism , cytochrome p450 activity ( 21 ), neurotransmitter synthesis ( 20 ) and the synthesis of carnitine from lysine ( 22 , 23 ). Importantly, ascorbate has dual properties in oxidative processes, acting as both an antioxidant and a prooxidant . Ascorbate is considered to be an important antioxidant in extracellular fluid ( 24 ); it also guards against aqueous radicals in blood ( 25 ) and protects plasma lipids from peroxidative damage caused by peroxyl radicals (26). Thus, in this capacity , ascorbate protects a number of cells and tissues throughout the body from oxidative stress . Conversely, ascorbate also accelerates oxidative metabolism by preventing the use of pyruvate for glycolysis (27). This property helps to inhibit the proliferation of tumor cells , but not normal cells ( 28 - 30 ). (See GLUT-1 receptor and SVCT transport ) In a great number of malignant cancer cell lines , it is quite interesting that the cytotoxic effect of ascorbate is correlated with its prooxidant activity ( 31 - 35 ).

Slide118:

Ascorbate enhances resistance against pathogens by stimulating the immune system ( 36 - 38 ). Recently, we reported that ascorbate suppresses production of IL-18 , a key regulator in malignant skin tumors, including melanomas and squamous cells carcinomas (Fig. 3) (39). IL-18 is known as an interferon-γ-inducing factor , and is capable of stimulating interferon-γ production by natural killer ( NK ) cells , activated macrophage , and T cells (40). Importantly, it has been recently reported that IL-18 expression is positively correlated with various tumors ( 41 ).

Slide119:

In gastric cancer cells , IL-18 production is enhanced by vascular endothelial growth factor ( VEGF ), resulting in increased IL-18-mediated tumor cell migration ( 42 ). In breast cancer cells , IL-18 induces the expression of transferrin receptor ( 43 ), which is a positive regulator of cell growth and proliferation ( 44 ). Thus , one mechanism by which ascorbate may be effective against cancer is through down-regulation of IL-18 , which plays an important role in controlling the escape of various cancer cells , including melanomas , gastric, and breast cancer cells , from immune surveillance (Fig. 4). Importantly, dosage is a key to the effectiveness of ascorbate as an immune-modulator . On the basis of the above reports , we recently postulated that a dose of ascorbate, 100∼ 250 μM may help prevent the immune escape of cancer cells . These dosages can be achieved by daily oral supplements of ascorbate . Ascorbate is the reduced form of vitamin C , which also exists physiologically in the oxidized form, dehydroascorbic acid ( DHAA or DHA ). DHA is taken up into cells by glucose transporters ( 45 , 46 ). Inside the cell, it is reduced to ascorbic acid ( 45 , 46 ) and decreases intracellular ROS levels , thus acting initially as an antioxidant ( 47 - 49 ). Ascorbate produced hydrogen peroxide-dependent cytotoxicity in various cancer cells without affecting normal cells . More importantly , Levine suggested that ascorbate-induced formation of hydrogen peroxide preferentially occurs in extracellular fluid compared with blood (Fig. 5) (60). These studies provide a mechanistic basis for applying ascorbate as a prooxidant therapeutic agent for cancer treatment .

Slide121:

8. McCormick WJ: Cancer: the preconditioning factor in pathogenesis ; a new etiologic approach. Arch Pediatr 71;313-322 , 1954 9. McCormick WJ: Cancer: a collagen disease, secondary toa nutritional deficiency. Arch Pediatr 76;166-171, 1959 10. Cameron E, Rotman D: Ascorbic acid, cell proliferation, and cancer . Lancet 1;542, 1972 19. González MJ, Miranda- Massari JR, Mora EM, Guzmán A, Riordan NH, Riordan HD, Casciari JJ, Jackson JA, Ráman -Franco A: Orthomolecular oncology review: ascorbic acid and cancer 25 years later. Interg cancer therapies 4;32-44, 2005 20. Levine M: New concepts in the biology and biochemistry of ascorbic acid. N Engl J Med 314;892-902, 1986 21. Block G: Vitamin C and cancer prevention: the epidemiologic evidence . Am J Clin Nutr 53;s270-282, 1991 22. Rebouche CJ: Ascorbic acid and carnitine biosynthesis. Am J Clin Nutr 54;s1147-1152, 1991 23. Englard S, Seifter S: The biochemical functions of ascorbic acid . Annu Rev Nutr 6;365-406, 1986 24. Sies H, Stahl W, Sundquist AR: Antioxidant functions of vitamins . Vitamins E and C, beta-carotene and other carotenioids . Ann N Y Acad Sci 669;7-20, 1992 25. Niki E: Action of ascorbic acid as a scavenger of active and stable oxygen radicals. Am J Clin Nutr 54;1119-1124 , 1991 28. Mikino Y, Sakagami H, Takeda M: Induction of cell death by ascorbic acid derivatives in human renal carcinoma and glioblastoma cell lines. Anticancer Res 19;3125-3132, 1999 29. Poydock ME, Reikert D, Rice J, Aleandri L: Inhibiting effect of dehydroascorbic acid on cell division in ascites tumors in mice. Exp Cell Biol 50;34-38, 1982 30. Tomita Y, Eto M, Lio M: Antitumor potency of 3-methyl-3,4-dihydroxytetrone . Sci Bull Fac Agr Kyushu Univ 28;131-137, 1974 38. Jeng KC, Yang CS, Siu WY, Tsai YS, Liao WJ, Kuo JS:Supplementation with vitamin C and E enhances cytokine production by peripheral blood mononuclear cells in heathly adults. Am J Clin Nutr 64;960-965, 1996 39. Cho D, Hahm E, Kang JS, Kim YI, Yang YH, Park JH, Kim D , Kim S, Kim YS, Hur D, Park H, Pang S, Hwang YI, Lee WJ : Vitamin C downregulates interleukin-18 production by increasing reactive oxygen intermediate and mitogen-activated protein kinase signaling in B16F10 murine melanoma cells . Melano Res 13;549-554, 2003

Slide122:

40. Okamura H, Tsutsi H, Komatsu T, Yutsudo M, Hakura A,Tanimoto T, Torigoe K, Okura T, Nukada Y, Hattori K, et al : Cloning of a new cytokine that induces IFN-gamma production by T cells . Nature 378;88-91, 1995 41. Park HU, Byun DG, Kim TG, Kim YI, Kang JS, Hahm ES , Lee WJ: Enhanced IL-18 expression in common skin tumors. Immunol Lett 79;215-219, 2001 42 . Kim KE, Song H, Kim TS, Yoon D, Kim CW, Bang SI, Hur DY , Park H, Cho DH: Interleukin-18 is a critical factor for vascular endothelial growth factor-enhanced migration in human gastric cancer cell lines. Oncogene 26;1468-1476 , 2007 43. Park S, Yoon SY, Kim KE, Lee HR, Hur DY, Song H, Kim D , Bang SI, Cho DH: Interleukin-18 induces transferrin expression in breast cancer cell line MCF-7. Cancer Lett 2009 Jun 15 44. Park S, Cheon S, Cho D: The dual effects of interleukin-18 in tumor progression. Cell & Mol Immunol 4; 329-335, 2007 45. Vera JC, Rivas CI, Fischbarg J, Golde DW: Mammalian facilitative hexose transports mediate the transport of dehydroascorbic acid . Nature 364;79-82, 1993 46. Vera JC, Rivas CI, Zhang RH, Farber CM, Golde DW : Human HL-60 myeloid leukemia cells transport dehydroascorbic acid via the glucose transporters and accumulate reduced ascorbic acid. Blood 84;1628-1634 47. Guaiquil VH, Farber CM, Golde DW, Vera JC: Efficient transport and accumulation of vitamin C in HL-60 cells depleted of glutathione. J Biol Chem 272;9915-9921, 1997 48. Guaiquil VH, Vera JC, Golde DW: Mechanism of vitamin C inhibition of cell death induced by oxidative stress in glutathione-depleted HL-60 cells. J Biol Chem 276;40955-40961 , 2001 49. Galleano M, Aimo L, Puntarulo S: Ascorbyl radical/ascorbate ratio in plasma from iron overloaded rats as oxidative stress indicator. Toxical Lett 133;193-201, 2002

Slide123:

Chen and colleagues have hypothesized that pharmacological ascorbate may deplete ATP three mechanisms which would lead to cell death in tumors (6). First, DNA damage induced by H 2 O 2 can activate PARP . The activated PARP catabolizes NAD+, thereby depleting substrate for NADH formation and subsequent ATP synthesis (7,8). Secondly, H 2 O 2 can be catabolized by concurrent oxidation of glutathione ( GSH ) to glutathione disulfide ( GSSG ). In reducing GSSG back to GSH , glutathione reductase utilizes NADPH, which is provided by the pentose shunt from glucose . Glucose used to reduce NADP+ to NADPH cannot be used for glycolysis or NADH-production so that ATP generation is decreased ( 9 , 10 ). Finally, H 2 O 2 could directly damage mitochondria , especially ATP synthase, so that ATP production decreases ( 11 , 12 , 13 ). MECHANISMS OF ASCORBATE-INDUCED CYTOTOXICITY IN PANCREATIC CANCER Clin Cancer Res. 2010 January 15; 16(2): 509–520. doi:10.1158/1078-0432.CCR-09-1713 The goal of our study was to determine whether ascorbate is cytotoxic to pancreatic cancer cells and if so , to characterize the mechanism of ascorbate-induced cytotoxicity . Our results demonstrate that ascorbate decreased clonogenic survival and cell viability . Although ascorbate decreased ATP levels in pancreatic cancer cell lines in a dose-dependent manner , this depletion may not play a role in ascorbate-induced cytotoxicity . Overexpression of extracellular and intracellular catalase reversed ascorbate-induced cytotoxicity .

Slide124:

ATP levels demonstrated a dose-dependent decline with ascorbate treatment , while pre-treatment of catalase prevented the decrease in ATP seen with ascorbate 2 mM or 5 mM . In the parental cell line , there is a significant decrease in clonogenic survival with doses of ascorbate of 2 or 5 mM , while in the rho° cells there is no significant decrease in clonogenic survival. However, the rho° medium was supplemented with 100 μg /mL pyruvate , a known scavenger of peroxide , to compensate for the respiratory metabolism deficit as described ( 20 , 29 ). When pyruvate was removed from the rho° media , clonogenic survival decreased following ascorbate treatment . This result suggests that the direct scavenging of H 2 O 2 by pyruvate may explain the resistance of the rho° cells to ascorbate . (Or Pyruvate requires down-regulation to stop glucose conversion to Pyruvate as a source of nutrient for Cancer cells more acidic microenviroment ) Ascorbate may lead to death through a unique caspase -independent autophagy pathway ( 14 ) and is characterized by accumulation of autophagosomes that fuse with lysosomes to form auto- phagolysosomes . 14. Ohtani S, Iwamaru A, Deng W, et al. Tumor suppressor 101F6 and ascorbate synergistically and selectively inhibit non-small cell lung cancer growth by caspase -independent apoptosis and autophagy . Cancer Res 2007 ;67:6293–303. [PubMed: 17616688] 20. Du J, Daniels DH, Asbury CA, et al . Mitochondrial production of reactive oxygen species mediate dicumarol -induced cytotoxicity in cancer cells . J Biol Chem 2006;281:37416–26. [PubMed : 17040906 ] 29 . Cloos C, Daniels DH, Kalen A, et al. Mitochondrial DNA depletion induces radioresistance by suppressing G2-checkpoint activation in human pancreatic cancer cells . Radiat Res 2009;171:581–7 . [PubMed: 19580493]

Slide125:

DISCUSSION Ascorbate is one of the early unorthodox therapies for cancer ( 33 – 35 ). This approach was subsequently promoted by Cameron and Pauling ( 36 , 37 ). Cameron and Campbell initially published case reports of 50 patients, some of whom seemed to have benefited from high dose ascorbate treatment ( 38 ). To test whether ascorbate was effective, Moertel conducted two randomized placebo controlled studies using ascorbate given orally; neither study showed any benefit ( 40 , 41 ). However, oral and intravenous ascorbate have strikingly different pharmacokinetic properties ( 27 ). Cameron gave patients ascorbate intravenously as well as orally , while Moertel’s patients received only oral ascorbate . Our study suggests that the role of ascorbate in pancreatic cancer treatment should be further examined . 27 . Padayatty SJ, Sun H, Wang Y, et al. Vitamin C pharmacokinetics : implications for oral and intravenous use . Ann Intern Med 2004 ;140:533–7. [PubMed: 15068981 ] 33 . McCormick WJ. Cancer: the preconditioning factor in pathogenesis . Arch Pediatr 1954 ;71:313–22 . [ PubMed: 13208373] 34 . McCormick WJ. Cancer : a collagen disease, secondary to a nutritional deficiency ? Arch Pediatr 1959 ;76:166–71 . [PubMed: 13638066] 35 . Cameron E, Rotman D. Ascorbic acid, cell proliferation, and cancer . Lancet 1972;299:542. [PubMed : 4110043] 36 . Cameron E, Pauling L. Ascorbic acid and the glycosaminoglycans . An orthomolecular approach to cancer and other diseases. Oncology 1973 ;27:181–92. [PubMed: 4267127] 40 . Creagan ET, Moertel CG, O’Fallon JR, Schutt AJ, O’Connell MJ, Rubin J, Frytak S. Failure of high dose vitamin C ( ascorbic acid ) therapy to benefit patients with advanced cancer . A controlled trial . N Engl J Med 1979 ;301:687–90. [PubMed: 384241] 41 . Moertel CG, Flemin TR, Creagan ET, Rubin J, O’Connell MJ, Ames MM . High-dose vitamin C versus placebo in the treatment of patients with advanced cancer who have had no prior chemotherapy . A randomized double-blind comparison. N Engl J Med 1985 ;312:137–41. [PubMed : 3880867 ]

Slide126:

Adverse effects Adverse events reported by survey respondents were minor (Table 3). No side effects were reported for 9227 patients while 59 were reported to have lethargy or fatigue . A single practitioner listed change in mental status in 10% of his patients ( 20 patients ) but provided no details The most common of these side effects were lethargy or fatigue (reported by 27 practitioners), vein irritation (by 9 practitioners), and nausea and vomiting (by 9 practitioners ). Vitamin C : Intravenous Use by Complementary and Alternative Medicine (CAM) Practitioners and Adverse Effects Published July 7, 2010 We searched the Adverse Events Reporting System , a database of drug side effects maintained by the Food and Drug Administration ( FDA ). Data from 20 consecutive quarters available from 2004–2008 were queried. We also searched for side effects in publications on therapeutic use of high dose IV vitamin C . We searched Medline , Web of Science ( ISI Thompson ) and Scopus databases for papers in English that reported IV vitamin C administration in humans . Several different search terms and possible variants of each search term were used to capture the maximum number of papers However, all patients either had serious or life-threatening systemic illnesses , and/or were receiving many potentially toxic drugs (i.e. cancer therapeutics ) in addition to IV vitamin C (for details, see Table S3).

Slide127:

There were minimal adverse effects reported , which was also the case in the published literature . Exceptions were for patients with pre-existing renal insufficiency/failure or glucose 6-phosphate dehydrogenase ( G6PD ) deficiency , both known to predispose to vitamin C toxicity [ 30 ].

Slide128:

1 Tumor growth was clearly reduced in animals receiving the highest concentration of AA ( 1000 mg/kg/d ) when compared to the placebo group. All of the seven grafted mice in the 1000 mg/kg/d group were alive at the end of 1 month . On the contrary , tumor growth was not affected in the 15 mg/kg/d group . Moreover, only three of the seven grafted mice were still alive at the end of the assay the others having died abruptly during the experiment due to peritoneal and peri hepatic carcinosis . This suggests that AA could also protect from metastatic invasion , although this hypothesis should be confirmed in further experiments. 1 Antiproliferative Effect of Ascorbic Acid Is Associated with the Inhibition of Genes Necessary to Cell Cycle Progression, February 2009 , Volume 4, Issue 2, Key Words and Terms: cAMP : cyclic AMP stimulating agents/intracellular signalling SLC23A2 Sodium-dependent Vitamin C Transporter 2 : ( SVCT2 ) mRNA Neoplasia : uncontrolled, abnormal growth of cells or tissues in the body

Slide129:

Vitamin C : A Concentration-Function Approach Yields Pharmacology and Therapeutic Discoveries Molecular and Clinical Nutrition Section, Digestive Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases , National Institutes of Health, Bethesda, MD 20892-1372, Adv. Nutr. 2: 78–88, 2011 At least 3 mechanisms were responsible for tight control : absorption ( bioavailability), tissue transport , and renal reabsorption and excretion . Not explored in these studies is a potential 4th mechanism , utilization , which is discussed below Ascorbate transport and its transporters , SLC23A1 ( SVCT1 ) and SLC23A2 ( SVCT2 ), play a key role in tight control (23–25). The widely distributed tissue transporter SLC23A2 is responsible for ascorbate accumulation against its concentration gradient in many tissues . Knockout mice for this transporter have generalized severe ascorbate deficiency and die at birth ( 24 ), indicating that ascorbate transport , as ascorbate itself , is necessary for tissue accumulation . Nevertheless, these data provide clear evidence that ascorbate transport as such is the primary mechanism of ascorbate accumulation and that dehydroascorbic acid ( DHAA ) and ascorbate cannot be considered equivalent . The epithelial ascorbate transporter SLC23A1 plays a central role in tight control by mediating ascorbate renal reabsorption .

Slide130:

The upper limit vitamin C dose in the Dietary Reference Intakes guidelines is 2 g and maximally tolerated oral doses are in the range of 3–4 g ( 22 ). Oral administration is limited by osmotic diarrhea and saturation of intestinal absorption and by currently available means could never produce peak plasma concentrations approaching 1 mmol /L ( 58 ). Analyses of all data showed that i.v. pharmacologic doses of ascorbate could produce plasma concentrations 70-fold higher than those possible with maximally tolerated oral doses ( 58 ). Plasma concentrations of 10 mmol /L ( 10,000 mmol /L ) were predicted to be attainable for >3 h with infusion rates of 0.5–1 g/min . In contrast to data from i.v . ( parenteral ) administration , maximal peak plasma concentrations from oral dosing were predicted , and subsequently found by others, to be < 0.25 mmol /L ( 58 , 62 , 63 ). Of the normal cells tested , none were affected by 20 mmol /L ascorbate . In contrast , more than three-quarters of the 43 cancer lines tested showed sensitivity of < 10 mmol /L , defined as the effective ascorbate concentrations for killing 50% of a cancer cell type ( 60 , 64 ). 58 . Padayatty SJ, Sun H, Wang Y, Riordan HD, Hewitt SM, Katz A, Wesley RA , Levine M. Vitamin C pharmacokinetics: implications for oral and intravenous use . Ann Intern Med. 2004;140:533–7 . 60 . Chen Q, Espey MG, Sun AY, Pooput C, Kirk KL, Kirshna MC, Khosh DB , Drisko J, Levine M. Pharmacologic doses of ascorbate act as a prooxidant and decrease growth of aggressive tumor xenografts in mice . Proc Natl Acad Sci USA. 2008;105:11105–9.

Slide131:

Addition of the enzyme catalase to the cell culture media , which catalyzes decomposition of hydrogen peroxide ( H 2 O 2 ) to oxygen and water , ameliorated the cytotoxicity of pharmacologic ascorbate . Based on these and other in vitro experiments, the killing of cancer cells was proposed to be mediated by H 2 O 2 formation , which in the presence of reduced transition metal catalysts is classically thought to produce the highly reactive hydroxyl radical ( OH ·) species. This is commonly referred to as Fenton chemistry , Transition metals such as iron ( and Cu, copper ) are easily reduced by ascorbate . These reduced metal centers can donate their electron to molecular oxygen to produce the species known as superoxide H 2 O 2 was proposed to achieve effective steady-state concentrations of <25–50 mmol /L to elicit cell death ( 60 , 64 ). We observed that these concentrations could be achieved in extracellular fluid but not whole blood . RBC contain large quantities of catalase and peroxidases , which efficiently quell Fenton chemistry to protect hemoglobin from oxidative damage . 62 . Park CH, Kimler BF, Yi SY, Park SH, Kim K, Jung CW, Kim SH, Lee ER , Rha M, et al. Depletion of L-ascorbic acid alternating with its supplementation i n the treatment of patients with acute myeloid leukemia or myelodysplastic syndromes . Eur J Haematol . 2009;83:108–18. 63 . Micallef J, Attarian S, Dubourg O, Gonnaud PM, Hogrel JY, Stojkovic T , Bernard R, Jouve E, Pitel S, et al. Effect of ascorbic acid in patients with Charcot-Marie-Tooth disease type 1A : a multicentre, randomised , double-blind , placebo-controlled trial. Lancet Neurol. 2009;8:1103–10. 64 . Chen Q, Espey MG, Krishna MC, Mitchell JB, Corpe CP, Buettner GR , Shacter E, Levine M. Pharmacologic ascorbic acid concentrations selectively kill cancer cells : action as a pro-drug to deliver hydrogen peroxide to tissues. Proc Natl Acad Sci USA. 2005;102:13604–9.

Slide132:

Peak plasma concentrations approaching 30 mmol /L are produced by i.v. infusion of pharmacologic ascorbate in humans, at a dose of ~1.5 g/kg administered at rates of 0.5–1.0 g/min ( 60 , 61 ). With these parameters , plasma concentrations > 10 mmol /L are maintained in humans for at least 3 h ( 61 ). The concentration of H 2 O 2 induced by pharmacologic ascorbate is far higher , as much as 2 orders of magnitude , than those concentrations that regulate normal cellular processes ( 74 ). Whether transient changes in H 2 O 2 concentration have long-term effects are unknown, although available clinical data do not indicate pharmacologic ascorbate produces long-term adverse effects or complications . It is apparent both from in vitro and clinical studies that adverse effects of pharmacologic ascorbate are few . It is possible that as clinical studies increase and/or as dose frequency increases , more adverse effects will emerge . Nevertheless, the absence of toxicities is striking compared with many chemotherapeutic agents . One potential explanation as a metabolic untoward consequence of constant pharmacologic ascorbate concentrations is chronic hyperoxalemia , which could occur from constant catabolism of excess ascorbate . Although oxalate urine excretion increased acutely following administration of pharmacologic ascorbate concentrations , concentrations were not dramatically elevated ( 75 ). Perhaps constant elevations in H 2 O 2 concentrations have untoward effects on cell repair and growth , but available data do not support these concerns .

Slide133:

Apparent benefit outweighed apparent harm and the absence of Information about the mechanism of drug effects . Pharmacologic ascorbate can be viewed similarly . Already available are data about mechanisms in vitro and in vivo and pharmacology and safety in vivo . In light of new knowledge, what is lacking is evidence of efficacy in people and this is what merits proper and rigorous testing. Patients currently do not have choices of many treatments that provide benefit with minimal toxicity , and pharmacologic ascorbate potentially offers this possibility ( 57 , 83 ). Clinical investigation of pharmacologic ascorbate should be considered as an addition to existing cancer treatments . Its mechanism of action as a pro-drug for H 2 O 2 generation is distinct from most currently used agents . For this reason , there is potential for synergy, or at least an additive effect , in combination with other drugs . This strategy is similar to that used for treatment of many cancers , tuberculosis , serious bacterial infections , hepatitis , and HIV . Emerging data indicate that there are additive effects of ascorbate with other neoplastic agents ( 76 ). Finally, pharmacologic ascorbate as a treatment can be applied to conditions where H 2 O 2 and/or ROS could be beneficial . Obvious candidates are infectious agents , including viruses , bacteria , and other human pathogens . Particularly attractive candidates are infectious agents for which few or no treatments currently exist . As in cancer treatment , pharmacologic ascorbate also has the potential to be added to existing therapies for synergy .

Slide134:

Vitamin K3 and vitamin C alone or in combination induced apoptosis in leukemia cells by a similar oxidative stress signalling mechanism Bonilla- Porras et al. Cancer Cell International 2011 , Accordingly, the use of vitamin K3 ( VK3 , also known as menadione [ 3 ]) and vitamin C ( VC, also known as sodium ascorbate [ 4 ]) alone or in combination ( VK3 : VC [ 5 ]) is highly promising in cancer treatment . Yet, the precise pathway(s) by which VK3 and/or VC induce leukemia cell death are not well established . Several observations suggest that vitamin K3 might induce apoptosis - a type of cell death - [ 6 ] through different biochemical routes including severe depletion of glutathione and sulfhydryl-containing proteins and alteration of intracellular Ca2 + homeostasis [ 7 ], activation of c-Jun NH2-terminal kinase ( JNK, [8]), activation of Fas/Fas ligand system independently of the pro-apoptotic p53 protein [ 9 ], activation of Fas-dependent and Fas-independent pathways [10] and NF- xB activation [ 11 ]. Vitamin C is a water-soluble vitamin effective as antioxidant compound under normal conditions [12]. However , Chen and collaborators [ 13 , 14 ] have shown that pharmacological concentrations of ( 5-15 mM ) vitamin C was prooxidant , generating H 2 O 2 -dependent cytotoxicity toward a variety of cancer cells in vitro and in vivo without adversely affecting normal cells .

Slide135:

Interestingly, it has been shown that ( 10 μM ) VK3 in combination of ( 2 mM ) VC kill leukemia cells (e.g., K562 cells) by oxidative stress independent of caspase-3 , with a minor percentage of cell displaying mitochondrial depolarisation and DNA fragmentation without chromatin condensation consistent with a necrosis-like cell death [15]. Since it is known that the transcription factor NF- xB [ 16], p53 [17], c-Jun [18] and caspase-3 [19] are involved in apoptosis signalling , we hypothesized that VK3 and VC might induce cell death in leukemia cells through activation of such factors by oxidative stress . Remarkably, K562 cells were the most sensible to VC , whereas both cell lines were more sensible to VK3 than lymphocytes Recently, it has been shown that mM concentrations of ascorbate were cytotoxic to neuroblastoma cells mediated by H 2 O 2 [ 23 ]. 13 . Chen Q, Espey MG, Krishna MC, Mitchell JB, Corpe CP, Buettner GR , Shacter E, Levine M: Pharmacologic ascorbic acid concentrations selectively kill cancer cells : action as a pro-drug to deliver hydrogen peroxide to tissues . Proc Natl Acad Sci USA 2005, 102(38):13604-13609. 14 . Chen Q, Espey MG, Sun AY, Pooput C, Kirk KL, Krishna MC, Khosh DB , Drisko J, Levine M: Pharmacologic doses of ascorbate act as a prooxidant and decrease growth of aggressive tumor xenografts in mice. Proc NatlAcad Sci USA 2008, 105(32):11105-11109. 23 . Deubzer B, Mayer F, Kuçi Z, Niewisch M, Merkel G, Handgretinger R, Bruchelt G: H(2)O(2)-mediated cytotoxicity of pharmacologic ascorbate concentrations to neuroblastoma cells : potential role of lactate and ferritin . Cell Physiol Biochem 2010, 25(6):767-774.

Slide136:

To confirm the participation of NF- xB , p53 , c-Jun and caspase-3 in VK3 , VC and VK3 plus Vc induced apoptosis , we performed immunocytochemical assessment . In the present study, we provide for the first time in vitro evidence supporting a causative role for oxidative stress in VK3-, and VC-induced apoptosis in Jurkat and K562 cells in a domino-like mechanism involving O2.-/ H2O2 , mitochondrial depolarization, transcription factor activation such as NF-, p53, and c-Jun converging in caspase-3 activation and apoptotic morphology. Most importantly , it is shown that high concentration of VC ( e.g. 10 mM ) alone or in combination with VK3 (e.g. 10 μM ) in a ratio 1000:1 and 100:1 induced apoptosis in Jurkat and K562 cells, respectively by a comparable mechanism to VK3 (e.g. 10 μM ) and VC (e.g. 10 mM ) alone . Moreover, by using antioxidant compounds, we demonstrated that O2 -/ H2O2 production are essential in VK3-and VC-induced cytotoxicity.

Slide137:

Ascorbic Acid Has Superior Ex Vivo Antiproliferative , Cell Death-Inducing and Immunomodulatory Effects over IFN-a in HTLV-1-Associated Myelopathy Pathogenesis remains poorly understood and attention has been particularly focused on the cellular and humoral immune response to HTLV-1 ( H uman T - L ymphotropic V i rus type 1 ) infection [2,3]. It is assumed that the HTLV-1-specific CD8 + cytotoxic T cell (CTL) response may lead to bystander neural tissue damage when recruited in the CNS through release of pro-inflammatory cytokines [ 4 ]. We demonstrate superior antiproliferative , cell death-inducing and immunomodulatory effects of high-dose AA compared to IFN-a treatment , which are confirmed by microarray and pathway analysis . AA is an essential nutrient acting as an antioxidant and co-factor for various enzymes [ 9 ]. Both immunomodulatory as well as antiproliferative effects have been described for AA , although controversy still exists [ 10 – 13 ].(?) – What is this ‘controversy ?) In parallel, IFN-a has been reported to exert antiviral , immunomodulatory and antiproliferative effects in several types of human cancer and viral infections [ 14–16 ]. In contrast, in vitro studies exploring the potential effects of AA and IFN-a in the context of HAM/TSP are limited , although antiproliferative effects have been described for high-dose AA in HTLV-1-infected cell lines [ 17 ]. We demonstrate superior antiproliferative , cell death-inducing and immunomodulatory effects of high-dose AA compared to IFN-a treatment , which are confirmed by microarray and pathway analysis . 11 . Fromberg A, Gutsch D, Schulze D, Vollbracht C, Weiss G, et al. (2011 ) Ascorbate exerts anti-proliferative effects through cell cycle inhibition and sensitizes tumor cells towards cytostatic drugs . Cancer chemotherapy and pharmacology 67: 1157–1166.

Slide138:

IFN-y production was significantly reduced by high-dose AA with 25% in comparison to untreated cells , when expressed as % of control. Furthermore, high-dose AA significantly reduced TNF-a levels with 42% in comparison to untreated cells , when expressed as % of control. Variable effects of high-dose AA were observed on IL-2 and IL-6 levels , respectively. Altogether, high-dose AA significantly reduced IFN-y and TNF-a pro-inflammatory cytokine levels , whereas IFN-a exerted variable effects , demonstrating differential immunomodulatory effects of high-dose AA in comparison to IFN-a . AA significantly reduced IL-6 secretion with 37% in comparison to untreated cells , whereas IFN-a significantly increased IL-6 secretion In contrast, intermediate-dose and low-dose AA exerted no effect on IL-6 levels Neither high-, intermediate- or low-dose AA , nor IFN-a exerted an effect on IL-10 levels In the case of low-dose AA, eleven significantly modulated molecular networks could be identified, of which the principal network contained 10 genes, both up- and down-regulated , and again represented cell death . Taken together , the most relevant pathway dose-dependently modulated by AA treatment was related to cell death , whereas IFN-a up-regulated antiviral pathways . ..the majority of HAM/TSP patients produced IL-2 , IL-6, TNF-a and IFN-c pro-inflammatory cytokines . Although I FN-a exerted variable effects on pro-inflammatory cytokine levels, it non-significantly reduced IL-2 levels in HAM/TSP patients .

Slide139:

Furthermore, we were able to confirm the antiproliferative, cell death-inducing and immunomodulatory effects of high-dose AA in both HTLV-1-infected cell lines , although MT-4 cells appear to be more sensitive to AA treatment than MT-2 cells . Whereas AA dose-dependently induced cell death in HTLV-1-infected cell lines , only high-dose AA exerted antiproliferative and immunomodulatory effects We hypothesize that the cell death-inducing and immunomodulatory effects of high-dose AA in HTLV-1-infected cell lines were most probably a direct consequence of programmed cell death , with morphological evidence of apoptosis . However, active caspase 3 activation was not detected, suggesting that the effect of high-dose AA on DNA degradation and cell death might not be mediated by classical, caspase -dependent apoptosis Another possible explanation for the anti-HTLV-1 effect of AA , might be through the binding of its oxidized form, dehydroascorbic acid ( DHA ), to the ubiquitous HTLV-1 receptor GLUT-1 [ 42 ] and thereby blocking cell-to-cell viral spread or through interactions of DHA with cellular pathways involved in cell proliferation or surviva l , such as NF- kB . Through the promotion of the development of inflammatory T cells, including the IFN-y producing Th1 , and the IL-17-producing Th17 cell subsets, and T cell-dependent tissue inflammation , miR-155 could be a key player in various autoimmune diseases [ 50 , 51 ]. In addition, miR-155 has also been shown to function as a positive regulator of IFN-y production in natural killer cells [54]. In HTLV-1-transformed cells , miR-155 has been reported to be up-regulated when compared to HTLV -negative control cells [ 55 , 56 ].

Slide140:

Our results , revealing high-dose AA-induced down-regulation of miR-155 in MT-2 cells , suggest that this microRNA could represent a novel therapeutic target in HAM/TSP ( HTLV-1 ). Moreover, as IFN-a has a higher cost price and more severe side effects in comparison to high-dose AA treatment, the therapeutic potential of high-dose AA should be further explored , in parallel with widely used treatments such as corticosteroids and IFN-a , in future clinical studies with a biomarker discovery design . In conclusion , high-dose AA treatment has superior ex vivo and in vitro cell death-inducing, antiproliferative and immunomodulatory anti- HTLV-1 effects , as compared to IFN-a . However , differential pathway activation by both drugs opens up avenues for targeted treatment in specific patient subsets . Our findings reveal molecular mechanisms of action as well as candidate biomarkers for both IFN-a and high-dose ascorbic acid therapy and provide a rational basis for their use in HAM/TSP treatment . 16. Trinchieri G ( 2010 ) Type I interferon : friend or foe? The Journal of experimental medicine 207: 2053–2063. 42 . Manel N, Kim FJ, Kinet S, Taylor N, Sitbon M, et al. ( 2003 ) The ubiquitous glucose transporter GLUT-1 is a receptor for HTLV . 115: 449–459 . 50 . O’Connell RM, Kahn D, Gibson WS, Round JL, Scholz RL, et al. ( 2010 ) MicroRNA-155 promotes autoimmune inflammation by enhancing inflammatory T cell development . Immunity 33: 607–619 . 51 . Murugaiyan G, Beynon V, Mittal A, Joller N, Weiner HL ( 2011 ) Silencing microRNA-155 ameliorates experimental autoimmune encephalomyelitis . Journal of immunology 187: 2213–2221 . 55 . Pichler K, Schneider G, Grassmann R ( 2008 ) MicroRNA miR-146a and further oncogenesis -related cellular microRNAs are dysregulated in HTLV-1-transformed T lymphocytes . Retrovirology 5: 100. 56 . Bellon M, Lepelletier Y, Hermine O, Nicot C ( 2009 ) D eregulation of microRNA involved in hematopoiesis and the immune response in HTLV-I adult T-cell leukemia . Blood 113: 4914–4917.

Slide141:

Effect of high-dose intravenous vitamin C on inflammation in cancer patients Mikirova et al. Journal of Translational Medicine 2012 Vitamin C has been suggested as having both a preventative and therapeutic role in a number of pathologies when administered at much higher-than-recommended dietary allowance levels . Since in vitro studies demonstrated inhibition of pro-inflammatory pathways by millimolar concentrations of vitamin C , we decided to analyse the effects of high dose IVC therapy in suppression of inflammation in cancer patients . Our test of the effect of IVC on pro-inflammatory cytokines demonstrated that inflammation cytokines IL-1α , IL-2 , IL-8 , TNF-α , chemokine eotaxin and CRP ( C-Reactive Protein ) were r educed significantly after treatments . Key features of cancer-related inflammation ( CRI ) include leukocyte infiltration , cytokine build-up , tissue remodelling , and angiogenesis . These leukocytes secrete pro-inflammatory cytokines such as IL-1 , IL-6 , TNF- α , TGF- β , FGF , EGF and HGF21 , as well as chemokines such as CCL2 and CXCL8 [ 9 ]. Several studies indicate that inflammation is a marker of high cancer risk and poor treatment outcome [ 18-22 ]. In response to systemic inflammation , and in particular in response to elevated IL-6 levels , the liver produces CRP [ 23 ], a protein that binds to dead or dying cells to activate the complement system . CRP can be used as a marker of systemic inflammation . It correlates with disease progression and can be used to monitor infection [ 18 , 24-27 ]. 9 . Joyce JA, Pollard JW: Microenvironmental regulation of metastasis. Nat Rev Cancer 2009 , 9:239–252

Slide142:

For example , cancer patients with highly elevated CRP showed increased mortality by a factor of 28 . Thus, CRP concentration data confirm a correlation between cancer progression and inflammation . Experiments by Hartel et al . [ 43 ] indicate that 20 mM ascorbate inhibited production of IL-6 and TNF-α in monocytes without affecting IL-1 or IL-8 levels . For lymphocytes , the same ascorbate concentrations inhibited IL-2 production without affecting TNF-α or IFN-γ levels . Ascorbate, at milli molar concentrations , may also inhibit NF- κB activation in endothelial cells [ 44 ]. NF- κB is an important transcription factor that mediates changes in gene expression during inflammation . Other studies show that ascorbate inhibits TNF-α activation of NF- κB ( NF- kappaB ) in human cell lines in vitro in a concentration-dependent fashion , and can also inhibit GM-CSF , IL-3 , and IL-5 production [ 46 ]. While millimolar ascorbate concentrations are not usually considered ‘physiological ’, they can be achieved if the vitamin is administered intravenously at high dose. Intravenous vitamin C therapy has been used in the treatment of cancer [ 47 , 48 ]. Rationales for IVC therapy include preferential toxicity of ascorbate toward cancer cells [ 49 , 50 ], potential benefits of ascorbate for immune cells [ 48 ], and ascorbate inhibitory effect on angiogenesis [ 51 , 52 ]. In a study with guinea pigs , tumour growth was significantly reduced in cases where intra-tumour ascorbate concentrations reached the millimolar level [ 53 ]. In addition , inflammation and oxidative stress can cause down-regulation of immune system associated with T cell dysfunction , which has been described in cancer , infectious , and autoimmune diseases .

Slide143:

treatment by ascorbic acid may augment T-cell and NK (Natural Killer) cell immunity . To analyse the effect of IVC on the level of pro-inflammatory cytokines , IVC was given to 11 new coming cancer patients at dose of 15 g, 25 g, 50 g, 50 g, 50 g, and 50 g each time for 6 times . All 11 patients were at relative clinical stable status after the conventional cancer treatments ( surgery , chemo , or radiation therapies ). Patients had different types of cancers ( breast , colon , lung , pancreatic , renal and prostate cancer ). Six patients were tested further for the effect of 50 g IVC infusion on C-reactive protein . Most of the subjects, 76 ± 13% (95% confidence ) showed a reduction in CRP values during IVC therapy . If only subjects who had initially elevated CRP (> 10 mg/L ) levels are considered, 86 ± 13% (95% confidence) showed a reduction in CRP values during IVC therapy . This is also indicated by the fact that twenty-eight subjects had elevated CRP prior to therapy , but only 14 had elevated CRP levels after treatment . Among the twenty eight subjects who started with elevated CRP levels , the median CPR reduction was 80% with an IQR ( inter-quartile range) of 39 % to 94%. 18. McMillan DC: Systemic inflammation, nutritional status and survival in patients with cancer . Curr Opin Clin Nutr Metab Care 2009 , 12:223–226 . Review. 23 . Hirschfield GM, Pepys MB: C-reactive protein and cardiovascular disease : new insights from an old molecule. QJM 2003 , 96:793–807 . 46 . Bowie AG, Carcamo JM, Pedraza A, Borquez-Qjeda O, Golde DW: Vitamin C suppresses TNFa -induced NF- kB activation by inhibiting Ik αB phosphorilation . Biochemistry 2002, 41:12995–13002 . 47 . Cameron E, Pauling L : Supplemental ascorbate in the supportive treatment of cancer : Prolongation of survival times in terminal human cancer . Proc. Natl. Acad. Sci. USA 1976, 73:3685–3689. 48 . Riordan HD, Casciari JJ, González MJ, Riordan NH, Miranda- Massari JR, Taylor P , Jackson JA: A pilot clinical study of continuous intravenous ascorbate in terminal cancer patients . P R Health Sci J 2005, 24(4):269–276 . https://www.researchgate.net/publication/7207940_A_pilot_clinical_study_of_continuous_intravenous_ascorbate_in_terminal_cancer_patients

Slide144:

personal note : It is unfortunate there is no further expanded data on when surgeries took place, and the time in between treatments , chemo , radiotherapy , ect

Slide146:

After six treatments , patients had noticeably lower levels of IL-2 , TNF-α , and eotaxin . All cytokine levels were reduced after the last IVC injection . For six of these subjects , CRP levels were measured before and after the last injection of IVC. In all six cases , CRP concentrations decreased, typically by ten percent, after the infusion. Several mechanisms of action for ascorbate efficacy against cancer have been proposed over the years, but to our knowledge its potential role in mediating inflammation has not been previously addressed. In the research described in this manuscript, we used CRP as a clinical inflammation marker .

Slide147:

A potential effect of IVC in reducing inflammation is also supported by our serum cytokine data, which demonstrate that levels of pro-inflammatory cytokines decrease during IVC therapy . IL-2 , TNF-α , and eotaxin appeared to be chronically reduced in patients getting IVC therapy , while all six cytokines studied ( IL-1α , I FN-γ , and IL-8 , in addition to IL-2 , TNF-α , and eotaxin ) were acutely reduced after ascorbate infusions of 50 g . Average depression of IL-1 was 20% for six patients and the average decrease for eotaxin was 25%. Interleukin-1 is known to promote inflammatory processes and augment metastasis [ 74 ]. It is abundant at tumour sites , where it affects the process of carcinogenesis , tumour growth and invasiveness , and the patterns of tumour-host interactions [ 75 , 76 ]. IL-1 induces uPA expression and NF- kB activation . TNF-alpha , another key inflammatory cytokine , plays a central role in the tumour progression .

Slide148:

In summary, our analysis of data from cancer patients given IVC, along with our tests of cytokine levels , suggest that IVC may reduce inflammation in cancer patients , and that this reduction in inflammation is correlated with reductions in the tumour markers PSA . Further research in this area and clinical studies of the efficacy of intravenous high dose vitamin C are warranted Pancreatic cancer is s ensitive to pharmacologic ascorbate both in vitro and in animal models [ 15 ]. Emerging evidence in both model systems indicates that ascorbate has synergistic effects with gemcitabine [20]. When pharmacologic ascorbate was combined with gemcitabine , synergy was observed in all eight cell lines tested in vitro . In mouse models, ascorbate- gemcitabine combinations were more effective at inhibiting tumor growth compared to gemcitabine alone and also produced gemictabine dose-sparing effects . Phase I Evaluation of Intravenous Ascorbic Acid in Combination with Gemcitabine and Erlotinib in Patients with Metastatic Pancreatic Cancer 15 . Chen Q, Espey MG, Sun AY, Pooput C, Kirk KL, et al. ( 2008 ) Pharmacologic doses of ascorbate act as a prooxidant and decrease growth of aggressive tumor xenografts in mice. Proc Natl Acad Sci U S A 105: 11105–11109. Millimolar concentrations were achieved as expected, particularly among those receiving 100 g per infusion . For these patients the plasma ascorbate level was between 25.3 and 31.9 millimoles /L . They had no increase in adverse events compared to the other dosage tiers or to what would be expected from gemcitabine and erlotinib alone or from progression of disease .

Slide149:

For a frame of reference , the usual plasma ascorbic acid concentrations in people are 0.010–0.080 millimoles /L and are dependent on dietary and supplement intake . Even with massive oral supplementation of many grams daily taken every few hours , plasma ascorbic acid concentrations in people do not exceed 0.25 millimoles /L [ 13 ]. The data from this trial indicate that pharmacologic ascorbic acid concentrations were achievable in patients who received intravenous ascorbic acid in combination with gemcitabine and erlotinib

Slide150:

There were other potential issues with properly capturing the efficacy signal of ascorbic acid plus gemcitabine and erlotinib in this trial design. Ascorbic acid may act differently than classic cytotoxic chemotherapy . In particular, unlike many cancer therapies , ascorbate does not appear to have toxicity on rapidly dividing normal cells such as those in intestine cells , hair follicle cells , and bone marrow . Because of the absence of apparent tissue toxicity , effects of ascorbic acid treatment on human tumors might be expected to be more gradual, and as a corollary to require longer treatment . This possibility is consistent with observations from case reports of patients who received intravenous ascorbic acid as treatment for several types of cancers [ 20 , 32 , 33 ]. Given the possibility that longer ascorbic acid treatment is necessary to see disease improvement by RECIST 1.0 criteria, and the somewhat encouraging findings in the nine subjects in this trial, studying a longer treatment period at the 100 gram dosage seems warranted. Our primary goal was to evaluate safety of the combination treatment and provide a preliminary assessment of treatment effect . Because ascorbic acid appears to be safe with concomitant gemcitabine and erlotinib , a next reasonable step would be a phase II study with patients randomized to ascorbic acid plus gemcitabine/ erlotinib versus gemcitabine/ erlotinib alone for a longer treatment duration and to assess for progression free and overall survival .

Slide151:

Malignant cells exhibit altered metabolic profiles and bioenergetic needs which reflect the shift from more efficient metabolism of glucose and mitochondrial adenosine triphosphate ( ATP ) production to aerobic glycolysis , a wasteful glucose consumption leading to increased lactate production and other bioenergetic demands despite accessibility to oxygen ( 1 ) Glutamate , a metabolic biomarker of aggressiveness and a potential therapeutic target for prostate cancer Asian Journal of Andrology , January 14, 2013 The biological relevance of glutamate in PCa ( prostate cancer aggressiveness) cells ) was established by testing the effect of glutamate deprivation or pharmacological antagonists in PCa cells . Glutamate deprivation or blockade with metabotropic glutamate receptor 1 ( GRM1 )- antagonists (i.e ., Riluzole , BAY 36-7620) decreased PCa cell growth , migration and invasion , and led to apoptotic-cell death as demonstrated by dose-dependent increases in cleaved caspases-3 , - 7 and - 9 The above report on biological and clinical relevance of glutamate and its receptor GRM1 has provided proof of concept for circulatory metabolites that could serve not just as intermediate or endpoint metabolites , but also as prognostic or diagnostic biomarkers for clinical discrimination of aggressive tumors from indolent ones and as potential therapeutic targets for PCa .

Slide152:

The Riordan IVC Protocol for Adjunctive Cancer Care Intravenous Ascorbate as a Chemotherapeutic and Biological Response Modifying Agent Riordan Clinic Research Institute, February 2013 Vitamin C was first suggested as a tool for cancer treatment in the 1950’s : its role in collagen production and protection led scientists to hypothesize that ascorbate replenishment would protect normal tissue from tumor invasiveness and metastasis ( McCormick, 1959 ; Cameron, et al., 1979 ). Also , since cancer patients are often depleted of vitamin C ( Hoffman, 1985 ; Riordan, et al., 2005 ), replenishment may improve immune system function and enhance patient health and well-being ( Henson, et al., 1991 ). Cameron and Pauling observed fourfold survival times in terminal cancer patients treated with intravenous ascorbate infusions followed by oral supplementation ( Cameron & Pauling, 1976 ). • Plasma ascorbate concentrations in the millimolar range can be safely achieved with IVC infusions . • At millimolar concentrations , ascorbate is preferentially toxic to cancer cells in vitro and is able to inhibit angiogenesis in vitro and in vivo . • Vitamin C can accumulate in tumors , with significant tumor growth inhibition seen (in guinea pigs) at intra-tumor concentrations of 1 mM or higher . • Published case studies report anti-cancer efficacy , improved patient well-being, and decreases in markers of inflammation and tumor growth . • Phase I clinical studies indicate that IVC can be administered safely with relatively few adverse effects . While ascorbate tends to accumulate in adrenal glands , the brain , and in some white blood cell types , plasma levels stay relatively low ( Hornig , 1975 ; Keith & Pelletier, 1974 ; Ginter , et al., 1979 ; Kuether , et al., 1988 ).

Slide153:

Deficiency ( below 10 μM ) was correlated with elevated CRP ( c-reactive protein, an inflammation marker ) levels and shorter survival times . Given the role of vitamin C in collagen production , immune system functioning, and antioxidant protection , it is not surprising that subjects depleted of ascorbate would fare poorly in mounting defences against cancer . This also suggests that supplementation to replenish vitamin C stores might serve as adjunctive therapy for these patients . Physicians at the Riordan Clinic have observed that ( a ) peak plasma concentrations attained after IVC infusions tend to be lower in cancer patients than in healthy volunteers , suggesting their depleted tissues act as a “sink” for the vitamin ; and ( b ) in cancer patients given multiple IVC treatments, baseline plasma ascorbate concentrations tend to increase to normal levels slowly over time as reserves are restored with adequate IVC dosing . Vitamin C , at normal physiological concentrations ( 0.1 mM ), is a major water-soluble antioxidant ( Geeraert , 2012 ). At concentrations on the order of 1 mM , however, continuous perfusion of ascorbate at doses that trigger “ redox cycling ” can cause a build-up of hydrogen peroxide , which is preferentially toxic toward tumor cells ( Benade , et al ., 1969; Riordan, et al ., 1995; Casciari , et al ., 2001 ; Chen, et al., 2005 ; Frei & Lawson, 2008 ), often leading to autophagy or apoptosis . The cytotoxic threshold could be reduced significantly (LC50 = 4 mM ) by using ascorbate in combination with alpha- lipoic acid . Other reports suggest that ascorbate cytotoxicity against cancer cells can be increased by using it in combination with menadione ( Verrax , et al., 2004 ) or copper containing compounds ( Gonzalez, et al., 2002 ).

Slide154:

The Riordan clinic researchers evaluated angiogenesis inhibition using four different experimental models. In all cases , there is an inhibitory effect on angiogenesis at ascorbate concentrations of 1 to 10 mM ( Mikirova , et al., 2008 ; Mikirova , et al., 2012 ). Tumor angiogenesis is the process of new blood vessel growth toward and into a tumor . It is considered to be critical in tumor growth and metastasis . Reports in the literature suggest that ascorbate’s effect on collagen synthesis can act to inhibit formation of new vascular tubules ( Ashino , et al., 2003 ), that ascorbate can inhibit genes necessary for angiogenesis ( Berlin, et al., 2009 ), and that it might influence angiogenesis through its effect on hypoxia inducable factor ( Page, et al., 2007 ). Analysis of clinical data from the Riordan Clinic suggests that inflammation is an issue for cancer patients , and that it can be lessened during IVC therapy ( Mikirova , et al., 2012 ). C-reactive protein was used as a marker of inflammation , as reports in the literature indicate that elevated CRP is correlated with poor patient prognosis ( St. Sauver , et al., 2009 ). Over sixty percent of analysed Riordan Clinic cancer patients had CRP levels above 10 mg/L prior to IVC therapy . In 76 ± 13% of these subjects, IVC reduced CRP levels . This improvement was more prevalent, 86 ± 13%, in subjects with elevated ( above 10 mg/L ) CRP . Since many of the subjects in this database were prostate cancer patients, we examined prostate specific antigen ( PSA ) levels before and after therapy. Most of the prostate cancer patients showed reductions in PSA levels during the course of their IVC therapy . This is consistent with observations from the literature showing a correlation between CRP levels and PSA levels in prostate cancer patients ( Lin, et al., 2010 ).

Slide155:

The potential effect of IVC in reducing inflammation is also supported by cytokine data : serum concentrations of the pro-inflammatory cytokines IL-1α , IFN-γ , IL-8 , IL-2 , TNF-α and eotaxin were acutely reduced after a fifty gram ascorbate infusion , and in the case of the last three cytokines listed , reductions were maintained throughout the course of IVC therapy ( Mikirova , et al., 2012 ). A variety of laboratory studies suggest that, at high concentrations, ascorbate does not interfere with chemotherapy or irradiation and may enhance efficacy in some situations ( Fujita, et al., 1982 ; Okunieff & Suit, 1987 ; Kurbacher , et al., 1996 ; Taper, et al., 1996 ; Fromberg , et al., 2011 ; Shinozaki, et al., 2011 ; Espey , et al., 2011 ). This is supported by meta-analyses of clinical studies involving cancer and vitamins ; these studies conclude that antioxidant supplementation does not interfere with the toxicity of chemotherapeutic regiments ( Simone, et al., 2007 ; Block, et al., 2008 ). A 49 year old male with a bladder tumor ( invasive grade 3/3 papillary transitional cell carcinoma ) and multiple satellite tumors declined chemotherapy and instead chose to receive intravenous ascorbate . He received 30 grams twice weekly for three months, followed by 30 grams monthly for four years . Patient supplementation included botanical extract , chondroitin sulfate , chromium picolinate , flax oil , glucosamine sulfate , alpha- lipoic acid , lactobacillus acidophilus , L. rhamnosus , and selenium . Nine years after the onset of therapy , patient is in good health with no signs of recurrence or metastasis ( Padayatti , et al., 2006 ). Note: Other supplementation cited is the use of supplementation included coenzyme Q10 , magnesium , beta-carotene , parasidal , vitamin B and C supplements , vitamin E , vitamin A , Parex and n- acetylcysteine

Slide156:

In a recent German study, breast cancer patients receiving IVC along with standard therapy were compared to subjects receiving standard therapy alone ( Vollbracht , et al., 2011 ). Patients given IVC benefited from less fatigue , reduction in nausea , improved appetite , reductions in depression and fewer sleep disorders . Overall intensity scores of symptoms during therapy and aftercare was twice as high in the control group as the IVC group . No side effects due to ascorbate were observed, nor were changes in tumor status compared to controls reported . The study by Monti and coworkers ( Monti , et al., 2012 ), fourteen patients received IVC in addition to nucleoside analogue gemcitabine and the tyrosine kinase inhibitor erlotinib . Observed adverse events were attributable to the chemotherapeutic agents , but not to the ascorbate, but no added efficacy due to the ascorbate was observed . It is thus recommended that treatment start at a low dose and be carried out using slow “ drip ” infusion . Fatal Hemolysis can occur if a patient has glucose-6-phosphate dehydrogenase (G6PD) deficiency . It is thus recommended that G6PD levels be assessed prior to the onset of therapy. IVC should only be given by slow intravenous drip at a rate of 0.5 grams per minute . ( Rates up to 1.0 gram/minute are generally tolerable , but close observation is warranted . Patients can develop nausea, shakes, and chills.) As noted above (Scientific Rational), research and experience has shown that a therapeutic goal of reaching a peak-plasma concentration of ~20 mM ( 350- 400 mg/ dL ) is most efficacious . ( No increased toxicity for post IVC plasma vitamin C levels up to 780 mg/ dL has been observed .) The first post IVC plasma level following the 15 gram IVC has been shown to be clinically instructive: levels below 100 mg/ dL correlate with higher levels of existent oxidative stress, presumably from higher tumor burden, chemo/radiation damage, hidden infection, or other oxidative insult, such as smoking .

Slide157:

In our experience , the majority of cancer patients require 50 gram IVC infusions 2-3x/week to maintain therapeutic IVC plasma levels . All patients reaching therapeutic range should still be monitored monthly with post IVC plasma levels to ensure that these levels are maintained long term . We advise patients to orally supplement with at least 4 grams of vitamin C daily, especially on the days when no infusions are given , to help prevent a possible vitamin C “rebound effect.” Oral alpha lipoic acid is also recommended on a case by case basis. CONCLUSIONS Vitamin C can be safely administered by intravenous infusion at maximum doses of one-hundred grams or less , provided the precautions outlined in this report are taken . At these doses, peak plasma ascorbate concentrations can exceed 20 mM . There are several potential benefits to giving IVC to cancer patients that make it an ideal adjunctive care choice: • Cancer patients are often depleted of vitamin C, and IVC provides an efficient means of restoring tissue stores. • IVC has been shown to improve quality of life in cancer patients by a variety of metrics. • IVC reduces inflammation (as measured by c-reactive protein levels) and reduces the production of pro-inflammatory cytokines. • At high concentrations, ascorbate is preferentially toxic to tumor cells and is an angiogenesis inhibitor. Ashino , H. et al., 2003. Novel function of ascorbic acid as an angiostatic factor. Angiogenesis, Volume 6, pp. 259-69 . Cameron, E. & Pauling, L., 1976. Supplemental ascorbate in the supportive treatment of cancer: Prolongation of survival times in terminal human cancer. PNAS USA, Volume 73, pp. 3685-9.

Slide158:

The Effect of Ascorbic Acid and Garlic Administration on Lead-Induced Apoptosis in Rat Offspring's Eye Retina - 25 May 2013 ( Wistar rats) High levels of lead exposure result in an increase in lysosomal inclusions in the retinal pigment epithelium , swollen photoreceptor mitochondria , photoreceptor disorientation , and necrosis [ 8 ]. Antioxidants such as vitamin C may decrease injurious activity of reactive oxygen species ( ROS ), especially on the newly formed neurons [9, 10] and may clear ROS and free radicals [ 11 , 12 ] to protect lead toxicity . In addition , vitamin C may decrease reaction between lead and critical biomolecule , modification of genomic protection through suppression of intracellular ROS , and apoptosis inhabitation [ 13 ]. Ninety percent of lead accumulates in the bones of the body and has a half-life of more than twenty years . Bone releases lead during periods of increased bone turnover in women’s lives , such as pregnancy , lactation , and menopause [ 1 ]. It is well known that lead can cross the placenta during pregnancy and can cause intrauterine fetal death , preterm delivery , low birth weight , and abnormalities on brain and eyes [ 1 , 2 ]. Lead toxicity may have an effect on the eye lens and retina by loss of lens transparency [ 3 ] and induce retinal cell apoptosis [ 5 ]. It has been indicated that the water-soluble organosulfur and Cysteine S- allyl compounds of garlic extract have strong antioxidant potential that can cause free radical scavenging in lead poisoning comparable with the vitamin C antioxidant properties [ 19 , 20 ].

Slide159:

A previous study has shown that the lead exposure induces cell apoptosis in rods and bipolar retinal cells [ 5 ]. Our results also revealed that lead exposure during pregnancy and lactation induces and increases apoptosis in rat offspring's retinal photoreceptor layer The process of apoptosis is controlled by a diverse range of cell signals , which may originate either extracellular , such as toxins , hormones , growth factors , nitric oxide or i ntracellula r. These signals may affect apoptosis positively or negatively Based on the lead-induced toxicity mechanisms, we used vitamin C as a known antioxidant, and garlic juice as an antioxidant candidate to protect lead-induced toxicity during pregnancy and lactation on rat's eye retina . Vitamin C, a water-soluble vitamin, is necessary for growth and repair of tissues in all parts of the body , and as a highly effective antioxidant protects vital molecules from damage by free radicals and ROS [ 12 , 25 ]. Vitamin C is used to prevent and treat heart diseases , lead poisoning , cancer , the common cold , stroke , and osteoarthritis [ 26 , 27 ] It can be suggested that the ameliorative potential of garlic juice is perhaps due to its combined effects both on metal absorption and on excretion from the body . Allicin and Alexin are major components of garlic organosulfur , and their antioxidant properties in neutralizing several types of ROS have been confirmed [ 19 ]. In summary, this study indicates that lead exposure during pregnancy and lactation can cause apoptosis in photoreceptor retinal layer in rat offspring. Moreover , the fresh garlic juice as well as ascorbic acid showed preventive and beneficial effects in lead-induced apoptosis in eye retina .

Slide160:

11. Shalan MG, Mostafa MS, Hassouna MM, El- Nabi SE , El- Refaie A. Amelioration of lead toxicity on rat liver with Vitamin C and silymarin supplements . Toxicology . 2005 Jan; 206(1):1-15 . 12. Vissers MC, Lee WG, Hampton MB. Regulation of apoptosis by vitamin C. Specific protection of the apoptotic machinery against exposure to chlorinated oxidants . J Biol Chem. 2001 Dec; 276(50): 46835-40 13. Han JM, Chang BJ, Li TZ, Choe NH, Quan FS, Jang BJ, et al. Protective effects of ascorbic acid against leadinduced apoptotic neurodegeneration in the developing rat hippocampus in vivo. Brain Res. 2007 Dec; 1185:68-74 . 25 . Nishikawa Y, Dmochowska B, Madaj J, Xue J, Guo Z , Satake M, et al. Vitamin C metabolomic mapping in experimental diabetes with 6-deoxy-6-fluoro-ascorbic acid and high resolution 19F-nuclear magnetic resonance spectroscopy. Metabolism. 2003 Jun ; 52(6 ):760-70 . 27 Zandi PP, Anthony JC, Khachaturian AS, Stone SV , Gustafson D, Tschanz JT, et al. Reduced risk of Alzheimer disease in users of antioxidant vitamin supplements : the Cache County Study. Arch Neurol . 2004 Jan; 61(1):82-8 . 1 Ameliorating effects of garlic, calcium, and vitamin C on chronic lead toxicity in albino rats , September 2014 , Volume 23, Issue 5, pp 1215–1223 2 Protective effect of L- carnitine on experimental lead toxicity in rats: A clinical, histopathological and immunohistochemical study ,   Biotechnic & Histochemistry 86(6):436-43 · November 2010 1 In conclusion, vitamin C has a greater protective effect than garlic or calcium against lead toxicity   2 These results show that CA (L- carnitine ) as a food additive reduced the severity of tissue damage caused by PbAc . ( Lead acetate ) 27 RESULTS: Analyses of prevalent and incident AD ( Alzheimer’s Disease ) yielded similar results. Use of vitamin E and C (ascorbic acid) supplements in combination was associated with reduced AD prevalence . A trend toward lower AD risk was also evident in users of vitamin E and multivitamins containing vitamin C , but we saw no evidence of a protective effect with use of vitamin E or vitamin C supplements alone , with multivitamins alone , or with vitamin B-complex supplements . CONCLUSIONS : Use of vitamin E and vitamin C supplements in combination is associated with reduced prevalence and incidence of AD . Antioxidant supplements merit further study as agents for the primary prevention of AD .

Slide161:

More slides to follow

Slide164:

Role of Vitamin C in the Function of the Vascular Endothelium James M. May and Fiona E. Harrison - ANTIOXIDANTS & REDOX SIGNALING Volume 19, Number 17, 2013 Ascorbate donates a single electron in all its redox reactions , generating the ascorbate radical . This radical is not very reactive with anything but itself ( 17 ). Dismutation of two ascorbate radicals forms a molecule each of ascorbate and dehydroascorbate ( Fig. 1 ). Ascorbate uptake Since humans cannot synthesize their own vitamin C , it should be absorbed in the intestine and carried through the circulation to the various organs ( Fig. 2 ). The vitamin is taken up as ascorbate into intestinal cells on a dedicated sodium and energy-dependent transporter , termed the Sodium-dependent Vitamin C Transporter 1 ( SVCT1 ). Dehydroascorbate uptake on the intestinal Sodium-dependent Glucose Transporter-1 ( SGLT1 ) may also contribute to absorbed ascorbate ( 16 ). Cellular uptake of ascorbate occurs largely on the Sodium-dependent Vitamin C Transporter 2 ( SVCT2 ), which is closely related to the SVCT1 in structure and function ( 161 ), but is located mostly in non-epithelial cells (Fig. 2). It is the only known ascorbate transporter in endothelial cells ( 141 ). This transporter generates a sharp gradient of ascorbate from the blood to human white blood cells , resulting in intracellular concentrations of the vitamin as high as 3 mM in monocytes ( 14 ) and 2 mM in neutrophils ( 173 ) and platelets ( 53 ).

Slide167:

Perhaps, of significance , such low millimolar concentrations are required for optimal synthesis of type IV collagen ( 103 ). Ascorbate can also enter cells via its two-electron-oxidized form , dehydroascorbate (Fig. 3). As noted earlier, the hemiketal of dehydroascorbate (Fig. 1) is transported into cells by facilitated diffusion on the ubiquitous GLUT-type glucose transporters . Once inside the cell, dehydroascorbate is rapidly reduced back to ascorbate , However , given that plasma dehydroascorbate concentrations are usually 2 lM or less ( 42 ), and that the normal circulating level of 5mM glucose will compete for uptake with dehydroascorbate , it is unlikely that ascorbate concentrations in cells are bolstered much by dehydroascorbate , except perhaps in erythrocytes or in conditions of high oxidative stress in blood , such as during the respiratory burst of phagocytic cells ( 124 , 172 ). Additional evidence suggesting that the SVCT2 may be the only route of ascorbate entry in most cells is that brains of embryonic mice lacking both copies of the SVCT2 have undetectable ascorbate levels ( 146 ), as do primary culture macrophages lacking the SVCT2 (8 ).

Slide168:

Ascorbate recycling Once inside cells , ascorbate is essentially trapped due to its hydrophilic nature and negative charge . Intracellular ascorbate can then scavenge a variety of radicals , with superoxide being one of the most important . Superoxide might be generated within the cell as a result of receptor activation , such as by advanced glycation end products or by thrombin (Fig. 3). The major scavenger of superoxide in cells is likely to be superoxide dismutase , which has been noted to react in vitro with superoxide 10 5 times faster than ascorbate ( 78 ). In the endothelial cell, there will also be plentiful nitric oxide , which will react at diffusion-limited rates with superoxide , generating the strong oxidant peroxynitrite ( 13 ). Nonetheless, ascorbate at concentrations of 1–10 mM does scavenges superoxide and enhances arterial relaxation to acetylcholine in rabbit thoracic aorta (78), suggesting that the presumed low millimolar ascorbate concentrations in endothelial cells may allow ascorbate to aid in scavenging both superoxide ( 78 ) and peroxynitrite ( 78 , 83 ). Ascorbate can also reduce enzyme-bound ferric to ferrous iron in enzymes (Fig. 3), As noted earlier in Figure 1, two ascorbate radicals can also dismutate to form a molecule each of ascorbate and dehydroascorbate . The latter is reduced directly via a two-step mechanism by reduced glutathione , or enzymatically by GSH-dependent thiol transferases ( 174 , 179 ) or by NADPH-dependent reductases ( 41 ), the latter again including thioredoxin reductase ( 100 ) (Fig. 3).

Slide169:

Ascorbate has long been considered a key cellular antioxidant , serving as a primary antioxidant by detoxifying exogenous radical species that have entered cells or which have arisen within cells due to excess superoxide generation by mitochondrial metabolism , by NADPH oxidase , xanthine oxidase , or by uncoupled nitric oxide synthase ( NOS ).

Slide170:

GSH is often thought of as the major low-molecular-weight antioxidant in cells, both scavenging radicals and serving as the electron donor for antioxidant enzymes such as glutathione peroxidase . It is certainly a stronger reducing agent than ascorbate ( 24 ) and even recycles ascorbate , as noted earlier. However, GSH is more than 100-fold less reactive with superoxide than is ascorbate . only ascorbate will effectively remove superoxide . Further, ascorbate concentrations decrease at lower levels of oxidative stress than do GSH concentration s when these are generated by several oxidants , including menadione (110), ferricyanide (101), and oxidized low-density lipoprotein ( LDL ) ( 99 ). Although these results might suggest that ascorbate ‘‘takes the first hit ’’ in the defence against reactive oxygen or nitrogen species , ascorbate could also appear to be more sensitive to oxidative stress than GSH , because the latter has amore robust recycling mechanism and as GSH can be synthesized by the cell . Ascorbate also recycles and, thus , preserves several readily oxidized molecules in the cell . It repairs both protein ( 45 ) and lipid radicals ( 55 ). The latter occurs indirectly as ascorbate recycling of a- tocopherol in cell membranes . When a- tocopherol reacts with lipid peroxyl radicals in the lipid phase, it is oxidized to the a- tocopheroxyl radical , which ascorbate subsequently reduces to a- tocopherol ( 24 , 122 ). Although the mechanism by which ascorbate stimulates collagen synthesis is often considered to involve procollagen hydroxylation , many studies suggest that the major effect of ascorbate is to stimulate de novo collagen synthesis .

Slide171:

Hydroxylation of the oxygen-sensitive HIF-1a ( hypoxia-inducible transcription factor-1 alpha ) subunit on specific proline or lysine residues targets it for ubiquitination and proteosomal destruction ( 29 ). Lack of ascorbate prevents this hydroxylation and allows HIF-1a to accumulate ( 21 , 63 ). The surviving HIF-1a then forms a nuclear transcription complex that activates diverse pathways , including those for glucose transport and metabolism , angiogenesis , inflammation , and cell survival ( 22 , 68 , 168 ). However, it has been shown in a randomized , double-blind , placebo-controlled study that long-term moderate oral ascorbate supplements improved endothelial dependent flow-mediated brachial artery dilation in people with atherosclerosis ( 60 ). In this study , 46 subjects with coronary artery disease were administered 500 mg/day of ascorbate orally for 30 days . The results showed that the already normal plasma ascorbate concentrations were doubled by the supplement and that flow-mediated brachial artery dilation increased by 50 %. Although it has not been possible to show that ascorbate or related antioxidants decrease event frequency in established atherosclerosis (69, 142), these results suggest that ascorbate might prevent or delay the early endothelial dysfunction that is associated with atherosclerosis . Indeed , it may even require megadoses of the vitamin to replete the vitamin and improve endothelial function in some of these conditions . For example, taking 800mg a day of oral vitamin C for 4 weeks doubled low vitamin C plasma levels in subjects with insulin-resistant type 2 diabetes , but not to levels expected from this dose of the vitamin . Moreover, this treatment did not improve forearm blood flow in response to acetylcholine or insulin resistance. A similar difficulty in repleting low vitamin C levels was found in critically injured or infected patients , even with intravenous infusions of approximately 1000mg of vitamin C a day ( 90 ).

Slide173:

As doses exceed 200 mg , absorption decreases , urine excretion increases and ascorbate bioavailability is reduced ( 42 , 43 ). In contrast, when 1.25 grams of ascorbate are administered intravenously , plasma concentrations as high as 1 mM are achieved . Some clinicians have infused more than 10 grams of ascorbate in cancer patients and achieved plasma concentration of 1 to 5 mM ( 27 , 28 ). Both intravenous and intraperitoneal administration of ascorbate achieved serum ascorbate concentrations up to 20 mM ( 44 ). Thus, it is clear that intravenous administration of ascorbate can yield very high plasma levels , while oral treatment does not . Our study demonstrates that ascorbate induces cytotoxicity in pancreatic cancer cells at levels achievable with intravenous infusions . Additionally , systemic administration of ascorbate inhibited pancreatic tumor growth in mice . In summary , ascorbate, in doses achievable in humans, decreased viability in all pancreatic cancer cell lines via a H 2 O 2 –mediated mechanism . Treatment with pharmacological ascorbate induced a non- caspase -mediated cell death consistent with autophagy . In mice with pre-established pancreatic tumors , treatment with ascorbate inhibited tumor growth and prolonged survival . Pharmacological doses of ascorbate, achievable in humans may have potential for therapy in pancreatic cancer . 28 . Hoffer LJ, Levine M, Assouline S, et al. Phase 1 clinical trial of i.v. ascorbic acid in advanced malignancy . Ann Oncol 2008;19: 1969–74 . [PubMed: 18544557] 44 . Verrax J, Calderon PB. Pharmacologic concentrations of ascorbate are achieved by parenteral administration and exhibit antitumoral effects . Free Rad Biol Med 2009 ;47:32–40. [PubMed : 19254759 ]

Slide174:

* 1 Synergistic Killing of Ehrlich Ascites Carcinoma Cells by Ascorbate and 3-Amino-1,2,4,-triazole Benade L. · Howard T. · Burk D.  * Clinical and experimental Experiences with Intravenous Vitamin C Neil H. Riordan , PA-C, Hugh D.Riordan , M.D., Joseph P. Casicari , Ph.D. Vitamin C has potential as a Chemotherapeutic agent. Vitamin C has side benefits such as increasing collagen production , and enhancing the immune system . We found that Vitamin C was preferentially toxic to tumour cells – it killed tumour cells before killing normal cells . This phenomenon first came to our attention through the work of 1 Benade et al in 1991 Cancer at the cellular level is caused by very unique and common microbes which are inside of the cancer cells . These microbes do many amazing things to help cancer cells do their damage and protect themselves . Cancer cells create and excrete large amounts of lactic acid , as they process large amounts o f glucose i nefficiently . This lactic acid goes into the bloodstream and gets to the liver . The liver converts the lactic acid to glucose . This “ ping pong ball ” cycle, where the cancer cells convert glucose into lactic acid and the liver converts lactic acid into glucose , is called the lactic acid cycle or cachexia cycle . The lactic acid also blocks many key nutrients from getting to the cancer cells . Proteins we are interested in are the proteins needed for the conversion of glucose to pyruvate ,

Slide175:

* 1 Synergistic Killing of Ehrlich Ascites Carcinoma Cells by Ascorbate and 3-Amino-1,2,4,-triazole Benade L. · Howard T. · Burk D.  DMSO , MSM , honey , maple syrup or molasses as carriers (i.e. Trojan Horses ) to get microbe-killing substances inside of cancer cells . Vitamin D3 . Tumeric kills Helicobacter Pylori (combine with vitamine c , ( Vitamin C creates Hydrogen peroxide causing release of Iron ) Curcumin , ginger , honey , cinnamon , Zeolite ( heavy metals interfere with immune system ( bentonite clays and all nut and seed oils and alkaline foods and high protein diets . Laetrile ( Vitamin B17 ) clean, flush and restore liver function. Cancer is a systemic disease. It is caused by microbes in the organs (which weaken the immune system ), microbes inside the cancer cells (which block the production of ATP energy ), microbes in the bloodstream (which block the immune system ) The immune system creates two key anti-cancer molecules called interleukin and interferon . Interleukin and interferon , and likely several other neuropeptides are absolutely deadly to cancer cells . The problem is that the human body generally does not produce an optimal amount of these neuropeptides . In fact, the body only creates a very small number of these neuropeptides . When the body has no microbes in the bloodstream the immune system becomes supercharged . The body makes the neuropeptides in “large” amounts (this is a relative term, not an absolute term) when the immune system is supercharged by getting rid of the microbes , quickly destroy the cancer cells .

Slide176:

1

Slide177:

The cells of our body cannot just help themselves to blood sugars ( glucose , galactose or fructose ). They need to have a special protein within the cell itself in order to absorb the sugar and then use it in a chemical process inside the cell . As a family, these proteins are called glucose transporters , or GLUTs for short. Thankfully biochemists are a pragmatic lot and rather than name each of them after their favourite Greek or Latin word, they just numbered each of the GLUTs . I found that the best way to think of a GLUT is that it exposes an indent on the outside of a cell into which only one type of sugar molecule will fit . Sort of like a jigsaw puzzle looking for a missing piece . So far, biochemists have identified 13 different members of the GLUT family. They are not entirely sure what all of them are for, but GLUTs 1 to 5 , the important ones from our perspective , are well settled. GLUTs 1 , 3 and 4 are for glucose only , GLUT5 is only for fructose , and GLUT2 is for both glucose and fructose

Slide179:

For IL‐1 β‐ stimulated chondrocytes , significant down‐regulation of IL‐1 β , tumor necrosis factor‐alpha ( TNF‐ α ), MMP‐3 , and MMP‐9 mRNA expression was found when cells were cultured in HA‐supplemented media. Moreover, HA  +  AA supplementation further significantly decreased MMP‐3 (matrix metalloproteinase -3 ) and MMP‐9 mRNA expression . The protein production of MMP‐3 was decreased , with a significant difference between the HA + AA group and HA group. The antioxidant capacity and superoxide dismutases activity were also partially restored in stimulated chondrocytes . HA supplemented with AA modulates MMPs expression and antioxidant function in chondrocytes. AA may enhance the anticatabolic effects of HA on OA chondrocytes . Synergistic effect of  l ‐ascorbic acid and hyaluronic acid on the expressions of matrix metalloproteinase‐3 and −9 in human chondrocytes First published: 15 September 2017 Abstract Pro-inflammatory cytokines and reactive oxygen species ( ROS ) are known to be involved in the initiation and progression of osteoarthritis (OA). New evidence clarifying the correlation between ROS and inflammation has indicated that oxidative stress can up‐regulate inflammatory cytokines .  l ‐Ascorbic acid ( AA ), an antioxidant , has been shown to have anti‐inflammatory effects and improve matrix deposition in chondrocytes. Caspases are specific proteases responsible for the regulation and the execution of apoptotic cell death .

Slide180:

Investigation of selective cytotoxicity was expanded to 43 cancer cell lines ( 19 ). The effective concentration of ascorbate required to kill 50% of the cells (Effective dose required to kill 50 % of cells ) was less than 10mM for 34 of 43 cancer cell lines tested; normal cells remained insensitive to concentrations in excess of 20mM Verrax and Calderon also reported that parenteral administration of ascorbate in mice transiently produced pharmacologic concentrations in plasma and that these concentrations were cytotoxic to several cancer cell lines , regardless of mutations in p53 and caspase-3 ( 77 ). Earlier reports indicating that cellular copper levels are increased in several cancers combined with the co-localization of copper and chromatin led these authors to hypothesize that the interaction between ascorbate and copper contributes to the selective killing of cancer cells . ..a dose-dependent decrease in transferrin receptor ( TfR ) expression with a corresponding decrease in intracellular iron , and increased apoptosis after treatment with 0.5–3mM ascorbate (13 ). Apoptosis was indicated by depolarization of the mitochondrial trans-membrane potential and global caspase activation . The loss of an electron results in the formation of the ascorbate radical , with the electron donated to a transition metal ion currently thought to be protein bound . The reduced metal is then available to react with molecular oxygen resulting in the generation of superoxide and subsequently H 2 O 2 .

Slide181:

After ingestion or injection , ascorbate is equally distributed between blood (left) and the extracellular fluid (right). In the extracellular fluid , the oxidation of ascorbate concomitantly forms the ascorbate radical and reduces a protein- centered metal (In this example, Fe 3 + is reduced to Fe 2 + ). Fe 2 + then interacts with oxygen to form superoxide , which undergoes dismutation to H 2 O 2 . H 2 O 2 then interacts with another transition metal to generate ROS , including the highly reactive hydroxyl radical , via Fenton chemistry. The generation of H 2 O 2 is largely inhibited in blood by catalase and GSH peroxidase , both of which are primarily found in erythrocytes

Slide182:

These reactions likely occur in the blood as well, but H 2 O 2 generated in that compartment is thought to be instantly neutralized by the catalase and/or glutathione peroxidase present in erythrocytes (Fig. 5). In the presence of pharmacologic ascorbate concentrations , H 2 O 2 is then likely to interact with another transition metal ion, such as ferrous iron in the Fenton reaction , to generate the highly reactive and damaging hydroxyl radical ( 39 ). High-dose ascorbic acid treatment resulted in dose-dependent cytotoxicity in four human mesothelioma cell lines ( 74). This was associated with increased apoptosis , decreased expression of the anti-apoptotic protein B-cell lymphoma 2 ( Bcl-2 ), an increased number of cells with a low mitochondrial membrane potential , and increased mitochondrial superoxide production . Consistent with prior reports , cytotoxicity was associated with a dose-dependent increase in H 2 O 2 formation ; both apoptotic and necrotic cell death pathways were involved with necrosis prevailing at higher concentrations . Not surprisingly, ascorbate treatment generated differential gene expression as a function of p53 status , with decreased transcription of antioxidant genes including catalase in the HCT116 + / + ( human colorectal carcinoma cell line) cells .

Slide183:

RESULTS: Both MMP-1 and MMP-13 levels were significantly highest in the massive full-thickness group. MMP-13 levels were significantly different between groups, but pro-inflammatory cytokine IL-1β and IL-6 levels were not . However, IL-1β levels were significantly positively correlated with VAS (r = 0.66; P <.01) and functional scores (r = 0.61; P <.01), but IL-6 , MMP-1, and MMP-13 levels were not. CONCLUSIONS: IL-1β levels in  shoulder synovial fluid   correlated positively with  shoulder pain and functional scores in patients with chronic RCTs. Both MMP-1 and MMP-13 levels were altered and increased with cuff tear severity .

Slide184:

Results IL‐8 level was correlated with IL‐6 ( r  =   0.434,  P  =   0.006) and IL‐1β ( r  =   0.575,  P  <   0.001) levels. The cuff tear size was inversely correlated with the VAS score at rest. A multiple stepwise linear regression analysis revealed that the VAS (V isual A nalogue S cale) score at rest could be explained by the VAS score at night, the VAS score during movement and the intra‐articular IL‐8 level (adjusted  R ² = 0.544,  P  <   0.001). The intra‐articular IL‐8 level is associated with resting pain in rotator cuff tear patients . Conclusion These results suggest that an increased concentration of IL‐8 is a ssociated with resting pain in rotator cuff tear patients Melatonin Plays a Role as a Mediator of Nocturnal Pain in Patients with Shoulder  Disorders RESULTS: MTNR1A, MTNR1B, and ASIC3 expression was significantly increased in both the rotator cuff tear and frozen shoulder groups compared with the control group of patients with shoulder instability. Interleukin-1 βeta ( IL-1β ) and tumor necrosis factor-α lpha ( TNF-α ) significantly stimulated the expression of MTNR1A and MTNR1B in primary cultured fibroblast-like synoviocytes treated with pro-inflammatory cytokines . Melatonin treatment at a physiological concentration ( 10 nM ) induced ASIC3 expression and IL-6 production . Treatment with luzindole , a melatonin-receptor antagonist, reversed melatonin-stimulated ASIC3 expression and IL-6 production . CONCLUSIONS: Our study suggests that melatonin may play a role as a mediator of nocturnal pain with a rotator cuff tear or frozen shoulder, and this effect may be mediated via melatonin receptors .

Slide186:

Parenteral Ascorbate As a Cancer Therapeutic: A Reassessment Based on Pharmacokinetics (16 page PDF) High dose ascorbate is selectively cytotoxic to cancer cell lines through the generation of extracellular hydrogen peroxide ( H 2 O 2 ). The Canadian physician William J. McCormick is largely credited with being the first to postulate that ascorbate might limit the spread of cancer ( 52 ) Depletion-repletion studies in healthy volunteers showed that oral doses of 30–100mg daily produced *60 lM fasting plasma concentrations ( 44). About 1000mg ascorbate orally / day produced fasting plasma concentrations approaching saturation at 75–80 lM , with minimal additional increases resulting from doses as high as 2500 mg . Leukocyte ascorbate concentrations were saturated at oral doses of 100 mg/day . Ascorbate absorption and tissue accumulation are principally controlled by the sodium-dependent transporters SLC23A1 and SLC23A2 , also denoted sodium-dependent vitamin C transporters ( SVCT ) 1 and SVCT2 , respectively. SVCT2 is the primary transporter in the brain , pituitary , adrenals , and pancreas and responsible for a portion of the ascorbate normally found in muscle , liver , and kidney (73). Uptake of the oxidized product of ascorbate , dehydroascorbic acid , is mediated by glucose transporters 1, 3, and 4 (GLUT-1) .

Slide187:

Riordan et al . reported that plasma concentrations of greater than 5mM could be sustained for several hours by continuous intravenous infusion of up to 115 g over the course of 8 h in cancer patients and concentrations of ~1.7mM were selectively cytotoxic against four tumor cell lines in vitro ( 69 ). To summarize, both animal and human data indicate that pharmacologic ascorbate concentrations , where ascorbate is present in sufficient quantities to act as a drug rather than a vitamin , are only achievable by parenteral administration . These observations suggest a credible reason for differential results in clinical studies of ascorbate in cancer . Animal studies and clinical ascorbate pharmacology data have fuelled renewed interest in the potential therapeutic effect of high dose ascorbate in cancer treatment. Exposure to ascorbate concentrations of up to 20mM ( millimolars ) for 1 h did not affect survival in the normal human cells tested , whereas more than half of the cancer cell lines tested showed a 50% decrease in survival after exposure to 5mM or less ( 17 ). Cell death was dependent on hydrogen peroxide ( H 2 O 2 ) production mediated by extracellular ascorbate oxidation . Both apoptotic and necrotic pathways were implicated , with necrosis more prevalent at increasing doses or longer post-exposure times . These studies show that pharmacological ascorbate is a precursor for H 2 O 2 generation, which is preferentially cytotoxic to cancer cells H 2 O 2 formation required ascorbate radical concentrations attainable only with pharmacologic doses of ascorbate .

Slide188:

Decreased cyclooxygenase-2 (COX-2) expression may have a role in the molecular mechanism of pharmacologic ascorbate through various signalling mechanisms . The cyclooxygenases , COX-1 and COX-2 , catalyze the production of prostaglandins from arachidonic acid ( 80 ). COX-1 is constitutively expressed , whereas COX-2 expression only occurs in response to certain stimuli, such as growth factors and cytokines Apoptosis was linked to loss of mitochondrial membrane permeability and release of cytochrome c , but was insensitive to inhibition of caspase-8. transcription of the genes encoding the angiogenic proteins basic fibroblast growth factor ( bFGF ) , VEGF (Vascular Endothelial Growth Factor) , and matrix metalloproteinase-2 ( MMP-2 ) was decreased in tumor-bearing mice treated with ascorbate Ascorbate treatment was also associated with a dose-dependent decrease in nitric oxide production Findings showed differential expression of most of the genes examined. In response to 30mM ascorbate , both cell lines showed decreased transcription of the gene encoding the angiostatic protein C-X-C motif chemokine ( CXCL ) 10 and increased transcription of the SOD2 gene , encoding superoxide dismutase ( SOD ). The genes encoding the angiogenic proteins , C-C motif chemokine ( CCL2 ), CXCL6 , and interleukin-8 , and a second gene involved in the oxidative stress response , vanin 3 ( VNN3 ), were differentially regulated by ascorbate dose , cell line, or both.

Slide190:

Pharmacologic ascorbate doses ranging from 2 to 16mM resulted in a dose-dependent decrease in cell viability resulting from a combination of apoptotic and necrotic mechanisms . Apoptosis occurred via a mitochondria-dependent pathway , as evidenced by dose-dependent increases in cytosolic Ca2 + , combined with decreases in mitochondrial membrane potential , non-cleaved caspase-3 , and Bcl-2 protein expression Further investigation of combination therapy using higher doses of ascorbate is warranted. Ascorbate was synergistic in combination with either gemcitabine or the natural green tea extract epigallocatechin-3-gallate . These two combinations also resulted in increased lactate dehydrogenase release , indicative of necrosis , and increased caspase-3 activity , suggesting the synergistic effects were mediated by more than one cell death pathway . Low ( 100 mg/kg ascorbate plus 1 mg/kg vitamin K3 ) and high ( 1000 mg/kg ascorbate plus 10 mg/kg vitamin K3) doses decreased tumor volume , lung metastases , and protein expression levels of MMP-2 and - 9 in the lungs of tumor-bearing mice relative to untreated controls in a dose-dependent manner . A primary human cell line showed a 4-fold to 10-fold increase in cell death when treated with radiation doses ranging from 3 to 6 Gy plus 5mM ascorbate relative to the level obtained with radiation alone

Slide191:

Cell killing was facilitated, in part , by ascorbate-mediated H 2 O 2 production and occurred by necrosis rather than apoptosis or autophagy . Cell death in response to radiation or 5mM ascorbate was associated with apoptotic mechanisms , whereas necrotic cell death resulted from 20mM ascorbate treatment . Caspase-3 and - 9 were activated by all treatment regimens ; caspase-8 activity increased in response to ascorbate treatment or ascorbate combined with radiation , but not in response to radiation alone . Riordan et al . reported on cases of renal cell carcinoma , breast cancer , colorectal cancer , pancreatic cancer , and non-Hodgkin’s lymphoma where intravenous ascorbate was administered in doses ranging from 15 to 100 g up to three times per week without evidence of toxicity ( 68 ). Experimental evidence indicates that N- acetylecysteine , like catalase , can inhibit the activity of pharmacologic ascorbate in vitro (38). Consistent with the data obtained in the previous trial , plasma ascorbate levels exceeded 25mM in patients treated with 100 g per infusion . Eight of nine patients who completed 8 weeks of treatment had decreased primary tumor size after treatment Several important scientific questions remain. It is not fully understood why there is selective toxicity against cancer but not normal cells , nor is there clarity about molecular mechanisms downstream of H 2 O 2 that produce cancer cell death . Identification of clinical biomarkers indicating sensitivity would be extremely useful in selecting patients who might benefit from ascorbate treatment .

Slide193:

* 1 Synergistic Killing of Ehrlich Ascites Carcinoma Cells by Ascorbate and 3-Amino-1,2,4,-triazole Benade L. · Howard T. · Burk D.  * Clinical and experimental Experiences with Intravenous Vitamin C Neil H. Riordan , PA-C, Hugh D.Riordan , M.D., Joseph P. Casicari , Ph.D. Vitamin C has potential as a Chemotherapeutic agent. Vitamin C has side benefits such as increasing collagen production , and enhancing the immune system . We found that Vitamin C was preferentially toxic to tumour cells – it killed tumour cells before killing normal cells . This phenomenon first came to our attention through the work of 1 Benade et al in 1991 Cancer at the cellular level is caused by very unique and common microbes which are inside of the cancer cells . These microbes do many amazing things to help cancer cells do their damage and protect themselves . Cancer cells create and excrete large amounts of lactic acid , as they process large amounts o f glucose i nefficiently . This lactic acid goes into the bloodstream and gets to the liver . The liver converts the lactic acid to glucose . This “ ping pong ball ” cycle, where the cancer cells convert glucose into lactic acid and the liver converts lactic acid into glucose , is called the lactic acid cycle or cachexia cycle . The lactic acid also blocks many key nutrients from getting to the cancer cells . Proteins we are interested in are the proteins needed for the conversion of glucose to pyruvate ,

Slide194:

* 1 Synergistic Killing of Ehrlich Ascites Carcinoma Cells by Ascorbate and 3-Amino-1,2,4,-triazole Benade L. · Howard T. · Burk D.  DMSO , MSM , honey , maple syrup or molasses as carriers (i.e. Trojan Horses ) to get microbe-killing substances inside of cancer cells . Vitamin D3 . Tumeric kills Helicobacter Pylori (combine with vitamine c , ( Vitamin C creates Hydrogen peroxide causing release of Iron ) Curcumin , ginger , honey , cinnamon , Zeolite ( heavy metals interfere with immune system ( bentonite clays and all nut and seed oils and alkaline foods and high protein diets . Laetrile ( Vitamin B17 ) clean, flush and restore liver function. Cancer is a systemic disease. It is caused by microbes in the organs (which weaken the immune system ), microbes inside the cancer cells (which block the production of ATP energy ), microbes in the bloodstream (which block the immune system ) The immune system creates two key anti-cancer molecules called interleukin and interferon . Interleukin and interferon , and likely several other neuropeptides are absolutely deadly to cancer cells . The problem is that the human body generally does not produce an optimal amount of these neuropeptides . In fact, the body only creates a very small number of these neuropeptides . When the body has no microbes in the bloodstream the immune system becomes supercharged . The body makes the neuropeptides in “large” amounts (this is a relative term, not an absolute term) when the immune system is supercharged by getting rid of the microbes , quickly destroy the cancer cells .

Slide195:

The cells of our body cannot just help themselves to blood sugars ( glucose , galactose or fructose ). They need to have a special protein within the cell itself in order to absorb the sugar and then use it in a chemical process inside the cell . As a family, these proteins are called glucose transporters , or GLUTs for short. Thankfully biochemists are a pragmatic lot and rather than name each of them after their favourite Greek or Latin word, they just numbered each of the GLUTs . I found that the best way to think of a GLUT is that it exposes an indent on the outside of a cell into which only one type of sugar molecule will fit . Sort of like a jigsaw puzzle looking for a missing piece . So far, biochemists have identified 13 different members of the GLUT family. They are not entirely sure what all of them are for, but GLUTs 1 to 5 , the important ones from our perspective , are well settled. GLUTs 1 , 3 and 4 are for glucose only , GLUT5 is only for fructose , and GLUT2 is for both glucose and fructose

Slide196:

1

Slide197:

The Riordan IVC Protocol for Adjunctive Cancer Care Intravenous Ascorbate as a Chemotherapeutic and Biological Response Modifying Agent Riordan Clinic Research Institute February 2013 Vitamin C ( ascorbate , ascorbic acid ) is a major water-soluble antioxidant that also increases extra cellular collagen production and is important for proper immune cell functioning (Hoffman, 1985; Cameron, et al., 1979). It also plays key roles in L- carnitine synthesis , cholesterol metabolism , cytochrome P-450 activity , and neurotransmitter synthesis ( Geeraert , 2012 ). The rationales for using intravenous ascorbate infusions (IVC) to treat cancer , can be summarized as follows: • Plasma ascorbate concentrations in the millimolar range can be safely achieved with IVC infusions . • At millimolar concentrations, ascorbate is preferentially toxic to cancer cells in vitro and is able to inhibit angiogenesis in vitro and in vivo . • Vitamin C can accumulate in tumors , with significant tumor growth inhibition seen (in guinea pigs) at intra-tumor concentrations of 1 mM or higher. • Published case studies report anti-cancer efficacy , improved patient well-being , and decreases in markers of inflammation and tumor growth . • Phase I clinical studies indicate that IVC can be administered safely with relatively few adverse effects

Slide198:

While ascorbate tends to accumulate in adrenal glands , the brain , and in some white blood cell types , plasma levels stay relatively low ( Hornig , 1975; Keith & Pelletier, 1974; Ginter , et al., 1979; Kuether , et al., 1988). Cancer patients tend to be depleted of vitamin C : fourteen out of twenty-two terminal cancer patients in a phase I study were depleted of vitamin C , with ten of those having zero detectable ascorbate in their plasma (Riordan, et al., 2005). Deficiency (below 10 µM) was correlated with elevated CRP (C -Reactive Protein , an inflammation marker ) levels and shorter survival times. Physicians at the Riordan Clinic have observed that peak plasma concentrations attained after IVC infusions tend to be lower in cancer patients than in healthy volunteers, suggesting their depleted tissues act as a “sink” for the vitamin 50 gram, 1 hr. infusion would yield a peak plasma concentration of roughly 18 mM and an integral average of roughly 2.6 mM , a reasonable target for producing anti-cancer effects . Reports in the literature suggest that ascorbate’s effect on collagen synthesis can act to inhibit formation of new vascular tubules ( Ashino , et al., 2003), that ascorbate can inhibit genes necessary fo r angiogenesis (Berlin, et al., 2009), and that it might influence angiogenesis through its effect on hypoxia inducable factor (HIF-1) (Page, et al., 2007). Asocrbate also reduced ATP production in these endothelial cells by twenty perceont , but did not affect cell viability.

Slide199:

C-reactive protein was used as a marker of inflammation , as reports in the literature indicate that elevated CRP is correlated with poor patient prognosis (St. Sauver , et al., 2009). The potential effect of IVC in reducing inflammation is also supported by cytokine data : serum concentrations of the pro-inflammatory cytokines IL-1α , IFN-γ , IL-8 , IL-2 , TNF-α and eotaxin were acutely reduced after a fifty gram ascorbate infusion , and in the case of the last three cytokines listed , reductions were maintained throughout the course of IVC therapy ( Mikirova , et al., 2012) It should also be noted that the goal of IVC is to attain millimolar intra-tumor concentrations (for the reasons described above) and thus the accumulation of ascorbate in tumors is considered an advantage . Other substances to be used in conjunction: Selenium , CoQ10 ( ubiquinone ), Magnesium , Glutathione , Niacinamide , Beta Carotene , bioflavinoids , sweet wormwood , Glucosamine sulfate 500 mg , Chromium picolinate , Vital Dophilus ( Lactobacillus acidophilus , Lactibaccilus.rhamnosus )

Slide200:

A variety of laboratory studies suggest that, at high concentrations , ascorbate does not interfere with chemotherapy or irradiation and may enhance efficacy in some situations (Fujita, et al., 1982; Okunieff & Suit, 1987; Kurbacher , et al., 1996; Taper, et al., 1996; Fromberg , et al., 2011; Shinozaki, et al., 2011; Espey , et al., 2011). This is supported by meta-analyses of clinical studies involving cancer and vitamins; these studies conclude that antioxidant supplementation does not interfere with the toxicity of chemotherapeutic regiments (Simone, et al., 2007; Block, et al., 2008). Note: Supplementations with vitamin E , coenzyme Q10 , vitamin C , beta-carotene , and vitamin A , lactobacillus acidophilus , Lactobacilus rhamnosus , selenium , magnesium , Glutathione Patients given IVC benefited from less fatigue, reduction in nausea, improved appetite, reductions in depression and fewer sleep disorders. There have been some reports of iron overload with vitamin C therapy.

Slide201:

IVC should only be given by slow intravenous drip at a rate of 0.5 grams per minute . ( Rates up to 1.0 gram/minute are generally tolerable , but close observation is warranted. Patients can develop nausea , shakes , and chills .) Administration of IVC Having taken all precautions listed above and having obtained informed consent from the patient, the administering physician begins with a series of three consecutive IVC infusions at the 15, 25, and 50 gram dosages followed by post IVC plasma vitamin C levels in order to determine the oxidative burden for that patient so that subsequent IVCs can be optimally dosed . Research and experience has shown that a therapeutic goal of reaching a peak plasma concentration of ~20 mM (350- 400 mg/ dL ) is most efficacious . ( No increased toxicity for post IVC plasma vitamin C levels up to 780 mg/ dL has been observed .) The first post- IVC plasma level following the 15 gram IVC has been shown to be clinically instructive : levels below 100 mg/ dL correlate with higher levels of existent oxidative stress , presumably from higher tumour burden , chemotherapy/radiation damage , hidden infection , or other oxidative insult , such as smoking

Slide202:

If higher dosages are not tolerated, or there is tumour progression in spite of achieving the therapeutic range, lower dosages can still augment the biological benefits of IVC , including enhanced immune response, reduction in pain, increased appetite, and a greater sense of well-being . In our experience, the majority of cancer patients require 50 gram IVC infusions 2-3 times a week to maintain therapeutic IVC plasma levels . We advise patients to orally supplement with at least 4 grams of vitamin C daily CONCLUSIONS Vitamin C can be safely administered by intravenous infusion at maximum doses of one-hundred grams or less , provided the precautions outlined in this report are taken. At these doses, peak plasma ascorbate concentrations can exceed 20 mM

Slide204:

Vitamin B3 appears to protect patients against tardive dyskinesia . Hawkins in a personal communication reported that over fifteen years he has not seen any cases among many thousands treated . In my own practice I have not seen any develop in twenty years . It may well be some of the protective effect is due to the lower doses of tranquilizer required but there is a direct protective effect as well, probably at the synapse . The Adrenochrome Hypothesis of Schizophrenia Revisited A. Hoffer, M.D., Ph.D.1 There the medulla , containing noradrenaline and adrenaline , is surrounded by the adrenal cortex which is very rich in ascorbic acid . This stabilizes the amines as do other natural anti oxidants . Manganese may have a protective effect by inhibiting aminochrome formation But since ascorbic acid is as active as Haldol ( one of the most powerful dopamine receptor Blockers ) it would be worthwhile to determine whether pregnant women given optimum doses of ascorbic acid would have children who would eventually be less apt to develop schizophrenia . Galzigna ( 1970 ) suggested a relationship between acetylcholine, a neurotransmitter , and catecholamines . Acetylcholine interacts with oxidized noradrenaline , yielding a complex which does not change to adrenolutin in ascorbic acid medium . It reacts similarly with dopamine . Both acetylcholine and nicotinamide increase the auto-oxidation of noradrenaline but the complex reacts differently with ascorbic acid . ORTHOMOLECULAR PSYCHIATRY, VOLUME 10, NUMBER 2, 1981, Pp. 98-118

Slide205:

Vitamin B3 ( Niacinamide ) and B6 ( pyridoxine ) dependency should be accounted for and the hypothesis should try to account for the therapeutic value of other orthomolecular treatments such as ascorbic acid For excess adrenochrome for any reason whatever would flow into adrenolutin which like adrenochrome is an hallucinogen . Leukoadrenochrome is not an hallucinogen . Any quantity of adrenochrome could be neutralized by diverting it into its leuko derivative . The same reasoning applies to every amine which is oxidized in the body into an aminochrome . If there is an increased production of adrenochrome , properties of adrenochrome should be conferred upon the patient . Thus it is known that adrenochrome has antihistamine properties ; schizophrenia should then impart antihistaminic properties , i.e. they should be less subject to physical expressions of allergy such as asthma , hay fever ; adrenochrome has antimitotic properties . This suggests schizophrenics should be less subject to attack by cancer and should have lesser growth rates of rapidly growing tissues such as hair and nails . A few studies suggest that both these conclusions are true . The role of stress : Stress is harmful for two reasons. The increase in the production of noradrenaline and adrenaline will lead to an increase in adrenochrome and in those genetically disposed to reaction 7 instead of 6 this will increase the amount of adrenolutin which is toxic . Secondly any stress decreases the amount of ascorbic acid in the body . Most people are on very low ascorbic acid intake and can ill afford any losses . A decrease in ascorbic acid will increase reaction 5 .

Slide208:

5/18/2019 208 INTRODUCTION Vitamins are organic compounds which have to be obtained from the diet, either because an organism does not have the enzymes necessary to synthesize them or because it cannot produce them in sufficient quantities . Humans cannot synthesize vitamins A , B1 ( thiamine ), B2 ( riboflavin ), B5 ( pantothenic acid ), B6 ( pyridoxine ), B7 (biotin), B9 ( folate ), B12 ( cobalamin ), E and K but are able to synthesize some vitamin B3 ( niacin ) and D . The last vitamin required by humans, vitamin C ( ascorbic acid ), is a special case in that this organic compound is synthesized by the large majority of vertebrate and invertebrate species . Interestingly, the vertebrate organ used to synthesize vitamin C changed twice from the kidney to the liver during evolution , once in birds and once in mammals . Whereas vitamin C is produced by the kidneys of fishes , amphibians , reptiles , and older bird orders , it is produced by the liver of more recent bird orders and of mammals . This switch to a larger organ in more active species has been interpreted as being the result of selective pressures to maintain biochemical homoeostasis under more stressful conditions . This is reflected by the fact that the human daily recommended intake for vitamin C ( 60 mg ) is the highest among all vitamins

Slide209:

Early in our research we found that adrenochrome injected intravenously into known epileptics greatly worsened the EEG abnormality . One young patient with no previous psychotic episodes was given adrenochrome . Within a few minutes her EEG became more pathological , she became morose and quiet . A few days later she had to be admitted to a psychiatric ward for treatment of her first psychosis . It is clear nicotinic acid can quickly reverse the adrenochrome -induced EEC pathology Pyridoxine ( vitamin B6 ) was established as an important treatment for autism by Rimland ( 1978 ); Rim land , Callaway and Dreyfus ( 1978 ) and by Lelord et al . ( 1978 , 1979 ) It is essential for the conversion of tryptophan into nicotinamide adenine dinucleotide ( NAD ). A deficiency of NAD is present in the Vitamin B3 deficiency disease pellagra. Pellagra is caused by a diet deficient in Vitamin B3 , by a diet which is too low in tryptophan , too low in absorbable Vitamin B3 and too rich in leucine . Adequate amounts of NAD at the same receptor would decrease the formation of the aminochrome and thus protect the receptor . This idea has not been tested . This hypothesis suggests that ascorbic acid and Vitamin B-3 in adequate quantities should protect dopamine receptors against TOPA The well-known toxicity of 6 hydroxy dopamine (also called tri- hydroxyindole or TOPA ) is due to its conversion to an aminochrome

Slide210:

S ome patients will be psychotic because they require large amounts of Vitamin B3 and B6 to keep the adrenochrome/ adrenolutin reaction under control , others will require an allergy-free environment ( including food ) while many will require both . My experience suggests that patients free of cerebral allergies can seldom tolerate more than 6 grams per day of nicotinic acid However, ascorbic acid which in the brain is as active as Haldol (which is not a tranquilizer). It has been valuable in controlling anxiety in some schizophrenics and has " cured " a few schizophrenics when 20 grams per day was used . A deficiency of serotonin would increase the conversion of dopamine to aminochrome . Smythies ( 1976 ) elaborated the dopamine hypothesis by involving serotonin . He suggests there is an imbalance between dopamine , which is too active , and serotonin , which is not active enough Glutathione protects the enzyme monoamine Oxidase ( Klemm and Baumgarten , 1978). Inhibiting monoamine oxidase will drive more of the catecholamines into other pathways including the aminochrome pathway . The adrenochrome hypothesis immediately called attention to Vitamin B3 and ascorbic acid as potential treatments for schizophrenia . The fact that we have indeed found these substances so helpful is a plus for the usefulness of the hypothesis . Osmond and I did not foresee that these vitamins would have a specific reaction with brain receptors .

Slide211:

Conclusion The adrenochrome hypothesis accounts for the syndrome schizophrenia more accurately than do any of the competing hypotheses . It helped originate the use of large doses of Vitamin B3 for treating schizophrenia patients and for alleviating the symptoms created by LSD . It also predicted the therapeutic use of ascorbic acid , again in large doses . Both these vitamins are important components in orthomolecular treatment as applied to schizophrenics . Finally it helped point to the catecholamines as significant factors in the etiology of schizophrenia .

Slide212:

Any viewer reading this should feel slightly relieved that there is a NATURAL remedy for any possible Ebola infection by using Vitamin C – Sometimes intravenously in high dosages is or may be required . This will be discussed further on through the presentation. Vitamin C IS a solution for pathogens and viruses , but the media won’t tell you this.

Slide213:

“to date, not a single virus has been tested that is not inactivated (killed) by a large enough dose of Vitamin C (Ascorbic Acid or Ascorbate). Many other antioxidants have similar virucidal (virus-inactivating/killing) effects, but Vitamin C appears uniquely to be of greatest potency and clinical efficacy ..”

Slide215:

 In addition, fluoride can also inhibit the protein secretion and/or synthesis involved in signalling pathways (mitogen-activated protein kinase ( MAPK ), p53 , activator protein-1 ( AP-1 ), and nuclear factor kappa B ( NF- κ B ) [37–39] and antioxidant enzymes ( SOD , glutathione levels , and CAT ) [40]. Thus, the inhibition of antioxidant enzymes results in the excessive production of ROS at the mitochondrial level, leading to cell damage . 1 Chronic Exposure to Sodium Fluoride Triggers Oxidative Biochemistry Misbalance in Mice: Effects on Peripheral Blood Circulation The CAT activity was significantly decreased upon treatment with fluoride , regardless of the dose. However, only the highest fluoride dose significantly decreased GSH levels compared to control, denoting that fluoride can act as inhibitor of enzymatic antioxidants ( CAT ) or nonenzymatic antioxidants ( GSH – Glutathione ).   Other studies show that fluoride (F) reduces CAT activity and GSH levels , but not the activity of SOD activity in human erythrocytes [49]  1 https ://www.ncbi.nlm.nih.gov/pmc/articles/PMC6129794/

Slide216:

[49] 1 In Vitro Effect of Sodium Fluoride on Malondialdehyde Concentration and on Superoxide Dismutase ( SOD ), Catalase ( CAT ), and Glutathione Peroxidase ( GSHPx ) in Human Erythrocytes The Scientific World Journal, vol. 2013 Oxidative stress is characterized by an excess of reactive oxygen species ( ROS ) that react with the main cellular macromolecules, causing alterations in cellular homeostasis [16, 17]. The main damage caused by ROS is lipid peroxidation of the polyunsaturated fatty acids of cellular membranes , from which malondialdehyde ( MDA ) is produced as a product . Moreover, the activity of the main antioxidant enzymes , such as superoxide dismutase ( SOD ), catalase ( CAT ) and glutathione peroxidase ( GlPx ), is also affected [16, 17] NaF ( Sodium Fluoride ) increases ROS , which in turn increases MDA , as well as decreasing the activity of the major antioxidant enzymes such as SOD , CAT , and GlPx

Slide217:

1 https ://www.hindawi.com/journals/tswj/2013/864718/  In this regard, in addition to vitamin E , other well-known natural products (e.g., curcumin , N- acetylcysteine , and Ginkgo biloba ), with antioxidant properties , have been used in in vivo and in vitro experiments to prevent or counteract the damage caused by free radicals generated by the intoxication with NaF and other toxic compounds that frequently contaminate the environment, mainly aquifers [41–45]. Our results show that the presence of Vit -E partially prevented the inhibition of the activity of antioxidant enzymes caused by NaF . Nonetheless, the mechanism by which Vit -E prevents the inhibition of SOD , CAT , and GlPx antioxidant enzymes induced by NaF must be investigated in more detail.

Slide218:

* Sodium Fluoride , Aspartame and Organophosphates – A common denominator of Inflammatory , Oxidative Stress ( ROS ) responses and pain pathways 1 Main toxic effect is * Sodium Fluoride and interaction with Enzymes Modifies cellular processes which lead to oxidative stress (or ROS generation ) Dysfunction of the male reproductive system, CNS , reduction of humoral cell immunity Interferes with signalling pathways MAPK , p53 , antioxidant enzymes SOD , Glutathione ( GSH ) levels Reduction of Catalase , Activator Protein-1 ( AP-1 ) Nuclear Factor Kappa causing ROS elevation by inhibiting enzymatic antioxidants but not always SuperOxide Dismutase ( SOD ) and can modify cellular processes such as respiration and metabolism C auses lipid peroxidation , protein carbonyls and Nitric Oxide ( NO ) reduction As more antioxidant enzyme are impaired , increase of Malondialdehyde ( MDA ) observed Alters TEARS , Nitric Oxide ( NO ) Catalase Activity and Glutathione levels indicating the ion is a toxicant, inducting metabolic alterations to the antioxidant syste m 1 Chronic Exposure to Sodium Fluoride Triggers Oxidative Biochemistry Misbalance in Mice: Effects on Peripheral Blood Circulation [ 9 ] Permanent damage of all brain structures , impaired learning ability , memory dysfunction and behavioural problems C hanges the concentration of non-enzymatic advanced glycation end products ( AGEs ), M etabolism of neurotransmitters (influencing mainly glutamatergic neurotransmission ) and the energy metabolism of neurons by the impaired glucose transporter—GLUT1

Slide219:

[ 9 ] The Influence of Fluorine on the Disturbances of Homeostasis in the Central Nervous System - Biological Trace Element Research Volume 177, Issue 2, 1 June 2017, Pages 224-234 CD3* T- Lymphocyte Cell significantly decreased as were CD19* B Lymphocytes The protein expression levels of TGF-β ( Transforming Growth Factor Beta ), TNF-α ( Tumour Necrosis Factor Alpha ), and Cytokine IL-2 ( Interleukin-2 ) were significantly decreased , Interleukin-10 ( IL-10 ) protein expression levels were significantly higher IgA (Immunoglobulin secreted by B-Cells ) contents were significantly decreased IgG contents were significantly lower IgM contents were significantly decreased , as were Interferon Gamma ( IFN-y ) IL-10 and TGF-β are inhibitory cytokines and are synthesized by T cells and B cells . Glutathione ( GSH ) protected the cells from loss of mitochondrial membrane potential and there was no cytochrome c exit or Bcl-2 down-regulation , and we suggest that these antioxidants prevent apoptosis induced by NaF ( fluoride ) Other changes include : Sodium fluoride  ( NaF ) administered orally to adult male rats at a dose level of 4.5 ppm and 9.0 ppm for 75 days caused significant decrease in the body weight ( Cachexia ) brain index and testicular index (see Cachexia/ Anorexia Nervosa in Cancer patients)

Slide220:

Vitamin E which promotes antioxidant enzymes (Alpha- Tocapherol found in hazelnuts, dry roasted peanuts, sunflower seeds/oil, curcumin / tumeric ) Research Ginseng Extract [GE] and Banaba Leaf Extract [BLE]) to off-set Sodium Fluoride toxicity Potential remedies and natural adjuvants 1 1 the presence of Vit -E partially prevented the inhibition of the activity of antioxidant enzymes caused by NaF (fluoride) 1 https ://www.hindawi.com/journals/tswj/2013/864718/ The mechanism by which Vit -E p revents the inhibition of SOD , CAT , and GlPx antioxidant enzymes induced by NaF must be investigated in more detail. [17] Effects of chronic fluoride intake on the antioxidant systems of the liver and kidney in rats - Journal of Fluorine Chemistry, vol. 168, no. 0, pp. 212–217, 2014. In the kidneys , the SOD, GPx , GSH and antioxidants levels significantly decreased, while the fluoride and LH levels significantly increased. In the liver , the CAT and TBARS levels significantly decreased , while the fluoride , SOD , LH and antioxidants levels significantly increased . In summary, these results show that chronic fluoride administration alters the antioxidant system of rats.

authorStream Live Help