Oxygen: mandatory but dangerous

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Oxigen: Vital but Dangerous...about oxidative stress in the dog Dominique GRANDJEAN DVM, PhD, HDR Colonel, Chief veterinarian, Paris Fire Brigade Professor, Alfort National Veterinary School Head of Canine Breeding and Sport Medicine Unit

OXIDATIVE STRESS : 

OXIDATIVE STRESS State within oxidative reactions overpass the anti-oxidant defenses of the organism Disturbances of cell functions Pathology Acute Chronic

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Acute and temporary oxidative stress Long term disbalance or chronic oxidative stress Base Level Level of Oxidative Stress Acute pathological problems [Sport Medicine] Chronic diseases Muscle Heart Intestin Cancer Nervous syst Eye Heart Joints

Oxidative stress in racing dogsConsequences and Prevention : 

Free Radicals Production; consequences Main related pathological problems Dogs working in extreme conditions: a model Nutritional and environmental consequences Oxidative stress in racing dogsConsequences and Prevention

Free Radicals(toxic reactive species of oxigen) : 

Cellular lesions Lipoperoxidations Degradation of proteins Destruction of DNA Free Radicals(toxic reactive species of oxigen) ’O2 O2- H2O2 OH . Antiradicals defense systems Enzymes Chelators of minerals Vitamins

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Who are they? Highly unstable toxic compounds, very short life span, generated by oxigen Free Radicals

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Examples of free radicals H• Unstable hydrogen CCl•3 Trichloromethyle O• - Superoxide 2 OH• Hydroxyle RS• Thiol

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Normal conditions: Redox reactions in each cell Induced or increased by environment factors X rays Polluants (pesticides,…) Radiations Tobacco How are they produced ? Free Radicals

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Free Radicals What are their biological actions? Inactivations of enzymes Destruction of cell membranes Destruction of mitochondrias Hemolysis of red cells Impairment of protein synthesis

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FREE RADICALS ACTIVATE INFLAMMATORY CELLS

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CELLULAR AND ORGANS LESIONS INDUCED BY FREE RADICALS

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Free Radicals Chain reaction responsible for the destruction of lipids in membranes, of structure proteins, of nucleic acids Directly responsible of lesions leading to: aging poor performance cancers muscle diseases tendons and joints diseases heart dysfunctions neurological dysfunctions What are their consequences?

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Free Radicals Is the organism able to defend itself? Superoxide dismutase Uric acid Betacarotene Polyphenols Catalase Vitamines E and C Glutathion peroxidase Coenzime Q10 Ceruloplasmine Transferrine

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ANTI OXIDANT SYSTEMS

Structure of a normal cell membrane : 

Structure of a normal cell membrane Fatty acids Glycoproteines

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Structure of a cell membrane destryed by free radicals Malondialdehide Pentane - Ethane Oxidation of aminoacides Peroxidation Rupture

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NORMAL MUSCLE FIBERS OXIDATIVE STRESS Destruction of muscle fibers Dysfunction of muscle vessels

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Muscle lesions induced by Cellular Oxidative Stress

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Future stress fracture

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Production of ROS Oxidative stress Waste (parts of destoyed compounds) Initiation Functional abnormalities Pathological consequences Antioxidants Fixing process

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Production of free radicals O2- ., H2 O2 , OH. Antioxidants Vit E, Vit C, Glutathion, SOD... Pathology Health

Oxidative stress in racing dogsConsequences and Prevention : 

Oxidative stress in racing dogsConsequences and Prevention Free Radicals Production; consequences Main related pathological problems Dogs working in extreme conditions: a model Nutritional and environmental consequences

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Oxidative Stress induced pathologies

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Aging Neurological degenerations Diabetis Arthrosis Chronic Kidney Failure Stress Diarrheas Rhabdomyolysis Ophtalmological Diseases …

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EMOTIONAL STRESS AND LIPOPEROXYDATION 150% 200% 1 2 3 Pentanes in outcoming air days [Philipko, 1982] 15 mn 4 5 Emotional Stress

Oxidative stress in racing dogsConsequences and Prevention : 

Free Radicals Production; consequences Main related pathological problems Dogs working in extreme conditions: a model Nutritional and environmental consequences Oxidative stress in racing dogsConsequences and Prevention

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Stamina in aerobiosis  oxidation processes High level of oxygen consumption  free radicals Source of energy  oxidation of lipids Extreme environments  environmental stress Intense work, motivation  psychological stress WHY??? Conditions for an intense cellular oxidative stress

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Intense stamina in cold weather Intense stamina in altitude HOW?

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Intense Exercise Aerobic Power Hypoxia Ischemia-Reperfusion Inflammation Pathophysiology ?

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Oxydative stress and Stamina in dogs Bardlay, 1991 ROS are a cause of fatigue in racing dogs Jerome, 1994 Ischemia induces production of ROS in the muscle of racing dogs Mo, 1997 Hypoxia generates production of ROS in the muscle Weinstock, 1997 Physical exercise induces the production of inflammatory molecules in the muscle of racing dogs

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• Dogs on a treadmill • 40 km/d, 5 days/week, 55 weeks speed : 6-7 km/h, slope : 15 p.100 • Total Glutathion in Muscle and Liver Endurance training improves anti-oxidant reserves of the organism Oxidative Stress and Stamina in Dogs

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Destruction of cell membranes Lesions Death ± muscle hypoxia ± muscle ischemia-reperfusion High intensity exercise  Free Radicals

« Chiens des cimes » : the concept : 

Increase capacities of « dogs who save lives « Chiens des cimes » : the concept

« Chiens des cimes » : the concept : 

Increase capacities of « dogs who save lives » Increase working time Prevention of injuries « Chiens des cimes » : the concept Resistance to stress

« Chiens des cimes » : the concept : 

Limited practical working time 15 min in 1990 40 min in 2004 ? Selection Intense physical work Concentration Motivation Extreme environment Environment Nutrition Ergogenic aids « Chiens des cimes » : the concept

« Chiens des cimes » : the concept : 

Intense cellular oxidative stress  prevention  Motivation Intense physical work Concentration Motivation Extreme environment Education Antioxidant substances Dedicated exercises Handler’s stress Outsiders Dual Nutritional Ergogenic « Chiens des cimes » : the concept

« Chiens des cimes » : the concept : 

Dedicated pathological problems - SDDS - Rhabdomyolysis - Water diabetis - … Nutrition Ergogenic aids Traumatology Propioception Stress pathology « Chiens des cimes » : the concept Intense physical work Extreme environments Handler’s stress Outsiders Concentration Motivation

« Chiens des cimes » : the concept : 

Why extreme environments ? Real operations can occur everywhere Very high levels of oxidative stress Larger biological modifications « Chiens des cimes » : the concept

« Chiens des cimes » : the concept : 

O2 O2 O2 Stress UV … Polluants Toxic free radicals « Chiens des cimes » : the concept

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«Chiens des cimes - Licancabur» - From sea level  4 500 m in less than 24 hours - Inca rubbles between 2 500 m and 5 500 m - No snow - « Easy » climbing 6000 m Licancabur (Chile)

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 Cynotechnic teams . Sapeurs Pompiers de Paris (France) . Carabinieros de Chile . UMES . Royal Canin  Scientific teams  Logistic team Members of the expedition «Chiens de cimes - Licancabur»

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France: 35/20 Dry food + Vitamin E (500 mg/d) Vitamin C (500 mg/d) Omega 3 (300 mg/d) Chili : 35/20 Dry food  Two groups of dogs Scientific protocole «Chiens des cimes - Licancabur»

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Paris (France) Santiago (Chile) Antofagasta 2500 m San Pedro de Atacama 4500 m Base camp (Bolivia) 6000 m «Chiens des cimes - Licancabur»

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1- Knowledge about « acute mountain sickness » 2- Efficiency of the nutritional approach  Dry food super premium  Anti oxidant protection  Quality of fatty acids in the food «Chiens de cimes - Licancabur» Conclusions

Licancabur ; Chile ; 6000 meters : 

Group 2 >> Group 1 Biological and nutritional consequences of worrk at high altitude in search and rescue dogs : The scientific expedition Chiens des Cimes-Licancabur The journal of nutrition, Vol. 128, No 12S, December 1998, 2694S-2697S  Plasma vitamin E   Peroxidation Resistance Index   Oxygen transfer to working cells   Clinical problems   - stress diarrhea - muscle stiffness and rhabdomyolysis - acute pulmonary oedema Licancabur ; Chile ; 6000 meters

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Chiens des cimes : Mont Blanc 2004

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Mont-Blanc 2004 Test level 0

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Mont-Blanc 2004

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Mont-Blanc 2004 : Results 1/ Heart function 2/ Cellular oxygenation 3/ Cellular oxidative stress

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Mont-Blanc 2004 : Results Cardiac function OXYGEN DEBT ? PROPENTOFYLLINE PLACEBO

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ECG Sinusal Respiratory Arythmia (SRA) cyclic Exists in the 2 groups = sporting dogs (trained) Sinusal rythm OK - Dog : Tarun Mont-Blanc 2004 : Results

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ECG Under segment ST : Base line / segment ST => Whitnesses an heart muscle hypoxia 0,2 mV < NORM < + 0,15 mV Intense altitude work Treated group : OK P P Q Q S S R R T T Dog : Patcho PQRS complex – Work in altitude – Treated group Base line Mont-Blanc 2004 : Results

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ECG PQRS complex – Work in altitude – Placebo group Dog : Malouk P P Q Q S R R T S T Under segment ST : Segment ST => Whitnesses a heart muscle hypoxia 0,2 mV < NORM < + 0,15 mV Intense altitude work Placebo group: severe hypoxia of the heart muscle -0,4 mV Base line Mont-Blanc 2004 : Results

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ECG % of dogs with no myocardial hypoxia in the 2 groups Mont-Blanc 2004 : Results

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1/ Heart function 2/ Cellular oxygenation 3/ Cellular oxidative stress Mont-Blanc 2004 : Results

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Oxygen saturation Evolution of the rate of oxygen saturation of hemoglobin (plaine vs altitude) Mont-Blanc 2004 : Results

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1/ Heart function 2/ Cellular oxygenation 3/ Cellular oxidative stress Mont-Blanc 2004 : Results

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Evolution of plasma vitamin E levels Sea level vs Altitude Placebo Propentofylline Vitamin E (mol/l)

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Total Plasma Anti-Oxidant) Sea level vs Altitude Placebo Propentofylline TPAO (mmol/l)

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ANTIOXIDANT STATUS OF RACING SLED DOGS

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DOGS : Vitamin E deficiency found in necropsics “... the important thing is all three of the dogs with muscle inflammation or degeneration had critically low values of vitamin E...” VITAMIN E : Magic Pill or red herring “... Nutrient may be key to dog health...” FAIRBANKS DAILY NEWS-MINER, SUNDAY, MAY 4, 1997

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Exams find vitamin deficiencies “... three of the five dogs that died running the Iditarod Trail Sled Dog Race last month suffered from vitamin E deficiencies...” Ulcers killed Iditarod dogs “... Iditarod needs to pay attention to how much vitamin E dogs need...” Dead dogs : Veterinary pathologists find deficiencies in vitamin E “... A buildup of toxic oxidants in the blood, or the stress placed on the dog’s heart by widespread cellular and organ failures triggered by the vitamin E storage...” FAIRBANKS DAILY NEWS-MINER, SATURDAY, APRIL 19, 1997

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Vitamin E Supplement (mg/kg) 0 5 15 45 Sudden Death [heart lesion] +++ +++ ++ 0 Muscle Degeneration +++ ++ ++ 0 Gastric ulcer ++ ++ ++ 0 [Bengtsson, 1978] Influence of vitamin E supplementatio on clinical symptoms observed in vitamin E deficient pigs

ALASKA COME BACK RACE 1997 RESEARCH PROTOCOLE UMES-ROYAL CANIN : 

Jacques PHILIP Helmuth PEER No supplement 800mg Vitamin E / day ALASKA COME BACK RACE 1997 RESEARCH PROTOCOLE UMES-ROYAL CANIN Monique BENE

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40 30 20 10 0 Plasma vit. E (µmol/l) Before [Alaska Come Back 97] After 31,6 41,5 30,9 24,7 Suppl. [1g Vit. E / dog/day] no suppl. EVOLUTION OF PLASMA VITAMIN E IN J. PHILIP’S TEAM DURING THE ALASKA COME BACK RACE 1997

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40 30 20 10 0 PRI [s] Before [Alaska Come Back 97] After 41,4 42,3 41,0 35,1 Suppl. [1g Vit. E / dog/day] no suppl. EVOLUTION OF THE PEROXIDATION RESISTANCE INDEX IN J. PHILIP’S TEAM DURING THE ALASKA COME BACK RACE 1997

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Drop in blood vitamin E in 16 sled dogs racing 57 kms per day for 3 days in a row (Hinchcliff, 1998) 10 (% of contrôl) 20 30 40 50 60 70 80 Control Day 1 Day 3 90 100

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Plasma concentration in isoprostanes in 16 sled dogs racing 57 kms per day 3 days in a row (Hinchcliff, 1998) 100 (% of control) 200 300 400 500 600 700 800 Control Day 1 Day 3

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Vitamin E : 400 to 800 mg / dog / day  correlated to lipidic caloric intake  Alaska Come Back, Wyoming, IRSSDR… Super Oxide Dismutase : 600 g / dog / day  natural gliadins protected sources  not cheep… Polyphenols, lignanes, flavonoïds…  actual trials (La Grande Odyssée) Sled dogs in Alaska and Wyoming

Results (IRMSSR) : 

Results (IRMSSR) Clinical Datas Distance Dogs dropped Definitely Dogs dropped temporarely Group E # 250/dog 0 2** Group P # 120/dog 3* 12*** * - 1 stress fracture - 2 acute pumonary oedema ** - stress diarrheas *** - muscle pain, stress diarrheas

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1/ DOES IT INDUCE ANY SIGN OF OXIDATIVE STRESS ? 2/ IS THERE AN INFLUENCE OF THE SIZE OF THE CAGE ? AIR TRANSPORTATION OF RACING SLED DOGS

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1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 AIR TRANSPORTATION OF RACING SLED DOGS UMES-AIR FRANCE RESEARCH PROTOCOLE

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2 weeks supplement [1g vitamin E/day] DOG 1 2 3 4 5 6 7 8 Kennel S S S S L L L L CDG  FBK Depart. 30,5 22,5 27,8 31,3 24,6 28,3 31,5 35,2 Arrival 30,5 24,6 27,9 34,4 22,5 27,9 29,2 36,3 Kennel L L L L S S S S FBK  CDG Depart. 38,6 45,7 40,9 41,5 33,9 41,0 36,8 31,4 Arrival 36,2 40,2 30,1 40,2 33,8 39,9 36,1 30,4 -2,75 -0,72 PLASMA VITAMIN E [µmol/l]

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2 weeks supplement [1g vitamin E/day] DOG 1 2 3 4 5 6 7 8 Kennel S S S S L L L L CDG  FBK Depart. 33 35 31 38 30 38 34 38 Arrival 37 33 36 40 31 36 30 34 Kennel L L L L S S S S FBK  CDG Depart. 41 46 39 43 35 36 37 45 Arrival 32 38 32 36 34 35 41 44 -7,75 -0,75 PEROXIDATION RESISTANCE INDEX (s)

Oxidative stress in racing dogsConsequences and Prevention : 

Free Radicals Production; consequences Main related pathological problems Dogs working in extreme conditions: a model Nutritional and environmental consequences Oxidative stress in racing dogsConsequences and Prevention

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‘70s ‘80s ‘90s 1997 Rhabdomyolisis on Dunlap’s dogs Kronfeld moves to high fat diets Grandjean : High fat diets enhance endurance Kronfeld : Protein / Energy Ratio Reynolds : Fat adaptation in racing dogs Reinhart : Quality of fatty acids Grandjean : L carnitine, antioxidants Back to rhabdomyolisis related to high fat diets BIOLOGIC IS IRONIC

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Life in Kennel Musher’s Stress Perception competition / training Psychological Intensive Training Performance (races) Repetition of competitions Physical Nutritional Prevention OXIDATIVE STRESS Celles and Organs Lesions

Nutritional Anti oxidants : 

Nutritional Anti oxidants

Nutritional Anti oxidants : 

Nutritional Anti oxidants TARGETS DNA Intracell memb Cell memb hydro soluble liposoluble both 1 2 3

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