Rajiv Gandhi University of Health Sciences s49

Rajiv Gandhi University of Health Sciences, Bangalore, Karnataka.

Proforma for registration of subjects for dissertation

1. NAME : SOWMYA S

2. ADDRESS : DEPARTMENT OF PHYSIOLOGY

ST JOHN'S MEDICAL COLLEGE,

BANGALORE-5600 34

3. COURSE OF STUDY AND SUBJECT : MD [PHYSIOLOGY]

4. DATE OF ADMISSION TO COURSE : 18 MARCH 2009

5. TITLE OF THE TOPIC : ROLE OF VITAMIN B12 IN

MODULATING RESTING AND

NORADRENALINE STIMULATED

ENERGY EXPENDITURE IN

HEALTHY ADULT MEN.

6. BRIEF RESUME OF INTENDED WORK:

6.1 NEED FOR THE STUDY

Vitamin B12 status in Indians across the population is not known, but the extent of the problem is likely to be at least as high if not higher than the prevalence reported in western countries for a variety of reasons related to either reduced availability or malabsorption of vitamin B12.Total homocysteine levels are generally higher in vegans and lacto-ovo vegetarians as compared to omnivores in different parts of the world and this has, in part, been attributed to lower dietary intakes of vitamin B12. In 200 adults in Pune, India, the mean serum vitamin B12 of 154 mol/L was half that of the U.S. mean although only 38% excluded meat, poultry, fish and eggs from their diet. Indeed, there was metabolic evidence of B12 deficiency in 75%. It has been proved in vitamin B12 deficient rat models that efficiency of mitochondria to function at various stages of energy metabolism is affected. Hence it will be interesting to study in humans whether vitamin B12 status is involved in affecting two components of energy expenditure, i.e., resting metabolic rate (RMR) and noradrenaline induced thermogenesis. Studying substrate oxidation (RQ) will be crucial in understanding the role of vitamin B12 in fat oxidation and hence adiposity in Indians which is a correlate for insulin resistance. In summary the proposal seeks to understand the role of vitamin B12 deficiency in adiposity in Indians. Vitamin B12 deficiency is expected to be widespread in Indians than any other population in the world and adiposity is an important factor underlying the epidemic of type 2 diabetes. This work has the potential to lead to new interventions to prevent disease.

6.2 REVIEW OF LITERATURE :

An extensive population in India adheres to a vegetarian diet because of religious convictions. Also, due to the high cost of animal food lower and middle socioeconomic groups can not afford it on a regular basis. So, dietary intake of animal protein foods is much lower in India1, 2. A strict vegetarian diet has been

3 associated with an increased risk of vitamin B12 deficiency and is common in apparently healthy Indian subjects1. There are studies done in India showing higher maternal circulating concentrations of homocysteine due to low vitamin B12 status predicts small size

4 of offspring . Reports have shown that Vitamin B12 deficiency due to consumption of macrobiotic diet from infancy not only causes impaired psychomotor functioning in infancy but also leads to impaired cognition in their adololescence5. Elevated plasma homocysteine levels are associated with dementia and

6 Alzheimer’s disease .Thus, deficiency of vitamin B12 has been identified to be harmful to all stages of the life course. There is a high prevalence of hyperhomocysteinemia and elevated Methyl

1 Malonic Acid associated with vitamin B12 deficiency in India .There is reports linking plasma homocysteine levels to insulin resistance7. India has high number of diabetics and is facing rising epidemic of diabetes. Studies have shown that diabetes has positive and independent association with body composition and reduced physical activity8. Indians characterized by different body pattern, they are more truncally adipose and insulin resistant than age and BMI matched Caucasians9 and central obesity in Indians makes them susceptible to hyperglycemia10 . Obesity is associated with higher energy intake than energy expenditure11. Energy expenditure is a critical factor contributing to successful energy regulation in normal individuals, as well as to the dysregulation of energy balance that characterizes obesity12. Obese children and adolescents have been identified as

13 a risk group for low vitamin B12 concentrations .

Role of Vitamin B12 in Energy Metabolism:

Vitamin B12 (5’-deoxyadenosylcobalamin) functions as a co-enzyme of methyl malonyl CoA mutase, catalyzing the isomerisation reaction of L-methyl malonyl CoA to succinyl CoA in mammalian mitochondria. Odd chain fatty acids and branched chain amino acids are metabolized via methyl malonyl CoA to the tricarboxilic acid cycle intermediates by the action of enzyme14. Activity of the enzyme is hampered in vitamin B12 deficiency [Hydroxycobalamin (c-lactam)], a cobalamin analogue, treated rats show 72% decreased activities of ferrocytochrome c: oxygen oxidoreductase (complex IV) activity along with

15 decreased cytochrome content . Rat models have shown that vitamin B12 deficiency is associated with reduced activities of various enzymes involved in metabolic pathways thus suggesting an effect on mitochondrial energy generation. The accumulation of MMA in rat mitochondria causes mainly inhibition of SDH enzyme leading to inhibition of mitochondrial respiration16. Obesity is due to a positive energy balance. This may be due to too much energy intake or too little energy expenditure. While physical activity is an important component of energy expenditure, the question is whether there could be reduced resting or thermoregulatory energy expenditure when there is a lack of vitamin B12. This is because of mitochondrial dysfunction as discussed above. While the amount of dysfunction may be small, it may be relevant in long term energy expenditure. For example, a positive energy balance of just 100 kcal per day, would lead to fat deposition of about 10 gm/day, or more than about 3 kilograms per year. In turn, fat accumulation is linked to lowered insulin sensitivity. It is therefore relevant to assess whether there is a decrease in resting and stimulated EE during the fasted state, in B12 deficient individuals. In addition, it is relevant to assess the enthusiasm of the subject to actually engage in physical exercise this may also be a relevant outcome of a decreased energy flux in muscle mitochondria, which might then exacerbate the positive energy balance.

6.3 OBJECTIVES OF THE STUDY

- To assess the noradrenaline induced thermogenic response (O2 consumption,

Respiratory Quotient, Energy consumption) in sub clinical vitamin B12 deficient

subjects before and after vitamin B12 supplementation.

7. MATERIALS AND METHODS:

7.1 SOURCE OF DATA: 50 male subjects (20-40 years and BMI between 18.5-

2 25 kg/m ) will be screened for sub clinical vitamin B12 deficiency using a total

plasma vitamin B12 concentration of <148 pmol/L. These subjects will be recruited

from in and around the medical college and from near by villages. We anticipate

that at least 15 subjects will show sub clinical vitamin B12 deficiency at a power of

0.8, and an alpha (α) coefficient of 0.05 expecting a difference between the

groups of around 10 %. Noradrenaline infusion will be performed on 15 vitamin

B12 deficient subjects before and after vitamin B12 supplementation and their

age matched controls.

Inclusion Criteria Exclusion Criteria Vitamin B12 level < 148 pmol/L 1. Presence of chronic diseases (i.e., diabetes, hypertension, tuberculosis, cancer, chronic renal failure etc.)

2. Alcohol intake greater than 2 STD per day

3. Any form of anemia

4. Any form of neuropathy

7.2 METHOD OF COLLECTION OF DATA (INCLUDING SAMPLING

PROCEDURE IF ANY)

Subject Assessment:

A) Questionnaires:

These will aim to evaluate current health status and behavior patterns, and will

include:

1. Structured clinical history and physical examination proforma

2. Food frequency questionnaire and 24 hour recall17

3. Physical activity questionnaire18

B) Measurements: i. Anthropometry: a) Measured parameters:

Height, weight, skin folds thickness (biceps, triceps, sub-scapular), waist, hip and mid upper arm circumferences b) Derived parameters (using standard prediction equations):

Body Mass Index (kg/m2), waist-hip ratios, percent fat, fat mass, arm muscle area, muscle mass, forearm muscle area and volume. ii. Biochemistry:

Vitamin B12 estimation: Serum vitamin B12 will be measured using kit employed on an Automated Chemiluminescence System ACS: 180 (Bayer Diagnostics,

Tarrytown, USA).

Red cell folate estimation: Blood cell folate will be measured using a kit employed on an Automated Chemiluminescence system ACS: 180 (Bayer

Diagnostics, Tarrytown, USA). iii. Blood indices:

All subjects will undergo a complete hemogram along with Wintrobe’s indices (MCV, MCH and MCHC) to exclude the possibility of a frank macrocytic anemia (Beckman Coulter counter, Florida, USA). iv. Biochemical indices: Glucose,free fatty acids, thyroid hormones, insulin and serum catecholamine levels.

7.3 DOES THE STUDY REQUIRE ANY INVESTIGATIONS OR INTERVENTIONS TO BE CONDUCTED ON PATIENTS OR OTHER HUMANS OR ANIMALS? IF SO, PLEASE DESCRIBE BRIEFLY. Yes the study requires investigations and interventions.

PROCEDURE The protocol will be explained to the subject and a written consent will be obtained from the subject by the investigator(s). If the subjects are illiterate then thumb impression will be taken. The consent form will be translated into the regional languages. On the experiment day beginning at 7.00 a.m. in the morning, after a rest period of thirty minutes, subject will undergo measurement of heart rate, blood pressure and metabolic rates after overnight fast. This will be followed by measurement of RMR by using ventilated hood system.

Measurement of RMR RMR will be calculated before infusion of noradrenaline. A ventilated hood system will be used to monitor the O2 consumption. This will incorporate a sealed hood around the subject’s head which will be ventilated at the rate of 50 litres of fresh air/min. Both incoming and outgoing air will be analysed using paramagnetic O2 analyser, an infra red CO2 analyser and a dewpoint hygrometer for vapour pressure. The analysers will be calibrated using previously calibrated standard gases. These variables, along with ambient temperature will be fed into a Solartron minate scanner and voltmeter an interfaced with a computer for immediate on-line analysis. Minute to minute O2, CO2 and respiratory quotient will be thus obtained for each subject. After measurement of basal oxygen consumption for 30 minutes continuously blood samples (0 minute, i.e. basal sample) will be collected for measurement of parameters stated above. After this noradrenaline infusion will be started.

After Noradrenaline Infusion: Cannulated subjects will be given rest for half an hour, following which resting energy expenditure will be recorded for 30 minutes. An infusion of noradrenaline will then be started. It will be procured as ampoules of noradrenaline for injection (Norad, 2mg noradrenaline bitatrate per ml), and freshly diluted to requirement on the morning of the experiment. The infusates will be made up in a buffer (Phosphate Buffered Saline, PBS, pH= 7.2), and the infusates will be protected from light. A Braun Perfusor pump (Braun, UK) will be used for these infusions, because of its very high precision. The dose of noradrenaline will be based on FFM of subjects, measured by the skin fold method. It will be administered as a single dose of 0.1 µg/ kg FFM/ min. for a period of 60 minutes. During infusion subjects will be instructed to remain absolutely still19. The calorimeter will be set for minute to minute recordings and five minute means will be computed for oxygen consumption and RQ’s. The response to noradrenaline will be calculated as the mean increment of O2 (VO2) consumption over the baseline during infusion. The dose of noradrenaline (0.1 µg/ kg FFM/ min. for a period of 60 minutes) is well below the lowest cardiovascular toxic dose20.

Monitoring:

Lead II ECG and blood pressure will be continuously monitored using Nihon

Kohden RM-6000 Polygraph System, Japan and Porta Press, Finland.

Vitamin B12 supplementation: 100 micrograms of cyanocobolamin will be given as a single dose everyday for three months. After the intervention noradrenaline infusion will be repeated.

Statistics: Data in the study group will be assessed for normality Noradrenaline infusion induced changes will be compared in before and after vitamin B12 supplementation using Paired‘t’ test/Wilcoxon Rank ‘t’ test

Strength of association between various parameters will be assessed using Pearson’s or Spearman’s correlation

The null hypothesis will be rejected at P < 0.05.

Study design:

The study will adopt Quasi experimental design with supplementation of Vitamin

B12 to only the deficient group.

7.4 HAS ETHICAL CLEARENCE BEEN OBTAINED FROM YOUR INSTITUTION IN CASE OF 7.3 Ethical clearance has been obtained from the Institutional ethical review board (I.E.R.B), St. John’s Medical college (Copy of the ethical approval form enclosed). 8. LIST OF REFERENCES 1. Mehta BM, Rege DV, Satoskar RS. Serum Vitamin B12 and Folic Acid Activity in Lactovegetarian and Nonvegetarian Healthy Adult Indians. Am J Clin Nutr 1964;15:77-84 2. Refsum H, Yajnik CS, Gadkari M, Schneede J, Vollset SE, Orning L et al. Hyperhomocysteinemia and elevated methylmalonic acid indicate a high prevalence of cobalamin deficiency in Asian Indians. Am J Clin Nutr 2001;74:233-241 3. Herbert V. Staging vitamin B-12 (cobalamin) status in vegetarians. Am J Clin Nutr 1994; 59:1213S-1222S. 4. Yajnik CS, Deshpande SS, Panchanadikar AV, Naik SS, Deshpande JA, Coyaji KJ et al. Maternal total homocysteine concentration and neonatal size in India. Asia Pac J Clin Nutr 2005; 14:179-181 5. Louwman MW, van Dusseldorp M, van de Vijver F, Thomas CMG, Schneede J, Ueland PTA et al. Signs of impaired cognition function in adolescents with marginal cobalamin status. Am J Clin Nutr 2000;72:762-769 6. Seshadri S, Beiser A, Selhub J, Jacques PF, Rosenberg IH, D’Agostino RB et al. Plasma homocysteine as a risk factor for dementia and Alzheimer’s disease. N Engl J Med 2002;346: 476-483 7. Meigs JB, Jacques PF, Selhub J, Singer DE, Nathan DM, Rifai N et al. Fasting plasma homocysteine levels in the insulin resistance syndrome: the Framingham offspring study. Diabetes Care 2001;24:1403-1410 8. Ramachandran A, Snehalatha C, Kapur A, Vijay V, Das AK, Rao PV et.al. High prevalence of diabetes and impaired glucose tolerance in India: National Urban Diabetes Survey. Diabetologia 2001;44:1094- 1101 9. Chandalia M, Abate N, Garg A, Stray-Gendersen J, Grundy SM . Relationship between generalized and upper body obesity to insulin resistance in Asian Indian men. J Clin Endocrinol Metab1999;84:2329-2335 10. Shelgikar KM, Hockaday TD, Yajnik CS. Central rather than generalized obesity is related to hyperglycemia in Asian Indian subjects. Diabet Med 1991:8:712-717 11. Luis GJ, Maria MJ, Montsereat B, Jose C, Pilar A, Jordi S. Physical activity energy balance and obesity. Public Health Nutr 2007; 10:1194-1199 12. Saltzman E, Roberts SB. The role of energy expenditure in energy regulation: findings from a decade of research. Nutr Rev. 1995; 53:209-220 13. Pinhas-Hamiel O, Doron-Panush N, Reichman B, Nitzan-Kaluski D, Shalitin S, Geva-Lerner L . Obese children and adolescents: A risk group for low vitamin B12 concentration. Arch Pediatr Adolesc Med 2006;160:933-936 14. Babior B M. Cobamides as cofactors: adenosylcobamide-dependent reactions. In: Babior B M, editor. Cobalamin: Biochemistry and pathophysiology. New York: John Wiley and sons,1975:148-152 15. Krahenbuhl S, Chang M, Brass EP, Hoppel CL. Decreased activities of ubiquinol: Ferricytochrome c Oxidoreductase (Complex III) and Ferrocytochrome c: Oxygen Oxidoreductase (Complex IV) in liver mitochondria from rats with Hydroxycobalamin [c-lactam]-induced methylmalonic acid urea. The Journal of Biological Chemistry 1991;266:998-1003 16. Toyoshima S, Watanabe F, Saido H, Miyataka K, Nakano Y . Methylmalonic acid inhibits respiration in rat liver mitochondria. J Nutr 1995; 125:2846-2850 17.Bharathi AV, Kurpad AV, Thomas T, Yusuf S, Saraswathi G, Vaz M. Development of food frequency questionnaires and a nutrient database for the Prospective Urban and Rural Epidemiological (PURE) pilot study in South India: Methodological issues.Asia Pac J Clin Nutr 2008; 17:178-185 18. Food and nutrition technical report series. Human Energy Requirements. Report of a joint FAO/WHO/UNU expert consultation, Rome. 2001:17-24 19. Kurpad AV, Kulkarni RN, Rodriguez D and Shetty PS. Characteristics of norepinephrine stimulated thermogenesis in undernourished subjects. J Biosci 1992; 17:293-303 20. Silverberg AB, Shah SD, Haymond MW, Cryer PE. Norepinephrine: hormone and neurotransmitter in man. Am J Physiol 1978; 234: G252-G256

9. SIGNATURE OF CANDIDATE

10. REMARKS OF THE GUIDE

11. NAME AND DESIGNATION OF 11.1 GUIDE: DR. ANURA V. KURPAD PROFESSOR, DEPARTMENT OF PHYSIOLOGY, ST.JOHN’S MEDICAL COLLEGE, BANGALORE DEAN, ST JOHN’S RESEARCH INSTITUTE, ST JOHN’S NATIONAL ACADEMY OF HEALTH SCIENCES, BANGALORE

11.2 SIGNATURE:

11.3 CO GUIDE: DR SUCHARITA S, ASSOCIATE PROFESSOR, DEPARTMENT OF PHYSIOLOGY, ST. JOHN’S MEDICAL COLLEGE, BANGALORE-560034

11.4 SIGNATURE

11.5 HEAD OF DEPARTMENT: DR SANDHYA T AVADHANY

PROFESSOR AND HEAD,

DEPARTMENT OF PHYSIOLOGY ST JOHN’S MEDICAL COLLEGE

BANGALORE-34

11.6 SIGNATURE

12.1 REMARKS OF THE CHAIRMAN AND PRINCIPLAL

12.2 SIGNATURE