1. Selection of Aging Biomarkers in Primates: Caloric Restriction of Rhesus Monkeys Alfred B. Ordman 1,2, Ricki J. Colman 2 and Joseph W. Kemnitz 2 1 Biochemistry Program, Beloit College, Beloit, WI 53511 2 Wisconsin National Primate Research Center, Madison, WI 53715 ABSTRACT Human aging and oxidation biomarkers are a subject of controversy. Possible aging biomarkers include telomere length, cell division potential, and cellular concentrations of lipoic acid, N-acetyl-carnitine, and S-adenosyl-methionine. Biomarkers for oxidative stress include plasma antioxidant concentrations, LDL oxidizability, urinary isoprostanes, oxidized nucleotides, malondialdehyde, protein carbonyls, and Heinz bodies. Macroscopic biomarkers like memory ability, sensory function, muscle strength and endurance can be measured. Caloric restriction (CR) has been proven to delay physiologic aging and extend lifespan in all animals tested. Comparison of CR and age-matched controls may distinguish chronologic, physiologic, and useless markers of aging and oxidative stress. Started in 1989, the University of Wisconsin caloric restriction project with rhesus monkeys can provide preserved and fresh samples to evaluate proposed biomarkers. Based on individually tracked intake, restricted primates are fed a chow diet containing 30% fewer calories than matched controls. A 70 kg human on a similar diet would consume approx. 940 mg vitamin C and 110 iu vitamin E per day. Taken at least annually are samples of whole serum, plasma, urine, and biopsies of fat, muscle and skin. Biomarkers of aging currently being measured include advanced glycosylation endproducts, lipid peroxdation, mitochondrial DNA deletions, and glucose tolerance testing. Gene expression profiling and proteomic anaylsis are conducted. However, overall individual health profiles are now preferred as an indication of physiological age compared to any available biomarker. Discussion of candidate biomarkers to test and collaboration for sample evaluation are invited. Many collaborations are underway and samples may be obtained under specific conditions. (Supported by NIH Grants P01 AG11915 and P51 RR000167) Table 2: Clusters of biomarkers 12.1 yrs. 14.3 yrs. 27.7 yrs. 25.5 yrs. 8.5 yrs. 9.0 yrs. 22.0 yrs. 22.4 yrs. Control Restricted IA. INTRODUCTION The National Institute of Aging (NIA) has been supporting caloric restriction of non-human primate studies since before 1990. One purpose of the studies has been to identify biomarkers of aging. These would distinguish maximal lifespan potential from chronological age. In primates, the impact of antioxidant supplementation on maximum and median lifespan remains unclear. The Rhesus monkey studies could provide biological samples to measure antioxidant biomarkers and determine their relevance to aging in primates. IB. VITAMIN C & E INTAKE DURING CALORIC RESTRICTION For more than 15 years ongoing studies of NIA and the Wisconsin National Primate Research Center (WNPRC) have maintained two colonies of Rhesus monkeys on calorically-restricted diets. A major aspect of the project is to identify biomarkers of aging. The study also provides an opportunity to examine the long- term consequences of antioxidant vitamin C and E consumption in primates. The following analysis indicates the feeding of the two colonies: IC. PRIMATE REQUIREMENTS FOR VITAMINS C & E Nutritional requirements for non-human primates have been set at levels substantially higher than those chosen by the U.S. Food and Nutrition Board (FNB) in 2001 for humans. However, the American Aging Association (AGE) held a Consensus Conference in 1999 that chose recommended human intakes comparable to those determined for other primates. Table 3: Choices for Vitamin C and E Requirements in Human and Rhesus Primates IIA. CALORIC RESTRICTION IN RHESUS MONKEYS Wisconsin National Primate Research Center Begun in 1991, Initiation of CR at age 8-14 Table 5: Characteristics of Monkeys Diet: Synthetic Diet with 10% fat and premixed vitamin supplement CR paradigm: 30% of individual baseline caloric intake with complete nutrition Feeding: Breakfast chow is given about 8 am. A piece of fruit is given about 4 pm. Calories consumed/kg body weight is nearly the same in controls as in restricted, because weight falls when restriction starts. This is also the case in rodent studies. When they eat, monkeys eat food quickly if restricted. Controls eat mainly one meal at 8 am, but nibble as the day goes on. Food is removed at night to provide a feeding pattern similar to in the wild. NIA also has a caloric restriction project, where restricted monkeys are fed twice a day. Data is being recorded for weight and caloric intake based on feeding with vitamin-enriched monkey chow. Here is an example of the data produced, and how that would translate for a monkey of average human weight of 70 kg. Results as of 2004: 1. Diabetes incidence: 3 of 38 controls vs 0 of 38 CR 2. CR at risk for diabetes in 1991 have normalized blood glucose 3. C-reactive protein is lower in CR compared to Controls 4. CR have less arthritis 5. CR have less sarcopenia - controls lose muscle mass; in general Rhesus are very active until age 3, sendentary by age 8 to 9. 6. Age-related deaths are 50% lower in CR. If you would like further information about the Caloric Restriction Project, please contact Ricki Colman rcolman@primate.wisc.edu. The Caloric Restriction Project is supported by Grant #PO1 AG11915. IIB. PRIMATE AGING DATABASE In an effort to determine biomarkers that meet the criteria above, a Primate Aging Database is being established that contains information about primates in a standardized format. This will allow analysis as is done with genomic and nutritional databases to select effective biomarkers. The database contains data from 5 Old World monkey species, 7 New World monkey species and 5 ape species. Data include basic hematology and body weights. Twelve sites have contributed in excess of 500,000 data points. The database is not currently available to the public, but hopefully it will be soon. The Database will include information about the NIA Caloric Restriction Project where monkeys obtain food and vitamins twice a day, vs. the WNPRC where vitamins are mostly obtained once a day. Standardized antixoxidant biomarker data from these two studies could demonstrate the impact on health and lifespan of divided doses. How to Contact: The Primate Aging Database is supported by a contract from NIA/NCRR #N01 AG31014. For information please contact Wendy Newton wschliew@primate.wisc.edu. IIIC. ACTUAL BIOMARKERS As shown in Table 8, a review of recent literature provided total of 198 biomarkers that have been proposed or measured. Please ask to see handout with complete lists of biomarkers and references or contact ordman@beloit.edu Table 7: Biomarker examples IIID. Oxidative Stress/Antioxidant status biomarkers: -glycation, glycoxidation, -DNA microarray analysis for oxidative stress response proteins, -measures of oxidative damage including DNA(8-oxo-7,8-dihdroguanine), protein carbonyls, lipofuscin, and Heinz bodies, -levels of antioxidants including carotenoids, vitamins C and E, urate, bilirubin, albumin, and ceruloplasmin. IIIE. PROBLEMS WITH BIOMARKERS 1. not robust across different species 2. none are used consistently by different research groups – each has different favorites 3. statistical combinations of many markers end up with large standard errors so that the values become meaningless. IV. CONCLUSION The Dietary Restriction and Aging Project possesses a longitudinal set of samples that may be used to examine the effect of antioxidant intake and dose rate on biomarkers of antioxidant status. These data can also be compared to other biomarkers of aging, and to maximum and mean lifespan of primates living in uniform environments. References 1. Butler, R.N. et al, 2004. "Biomarkers of aging: from primitive organisms to humans", J Gerontol Biol Sci 59(6): B560-7. 2. Short, R.A., Williams, D.D., Bowden, D.M., 1994. "Modeling biological aging in a nonhuman primate", In: Balin, A.K., Ed. Practical Handbook of Human Biologic Age Determination. CRC Press, Boca Raton, FL. 3. Short, R., Williams, D.D., Bowden, D.M., 1997. "Circulating antioxidants as determinants of the rate of biological aging in pigtailed macaques (Macaca nemestrina)", J Gerontol Biol Sci 52A: B26-38. 4. Bucci, T.J., Johnsen, D.O., Baker, E.M., and Canham, J.E., 1975. "Nutritional Requirement for vitamin C", Fed Proc 34: 883. 5. Committee on Animal Nutrition, 2003. Nutrient Requrements of Nonhuman Primates, 2nd revised edition, National Academies Press, Washington, DC. 6. Machlin, L.J. et al, 1976. "Lack of antiscorbutic activity of ascorbate 2- sulfate in the rhesus monkey (Macaca mulatta)", Am J Clin Nutr 29:825-31 7. Levine, M. et al, 1996. "Vitamin C pharmacokinetics in healthy volunteers: Evidence for a recommended dietary allowance", PNAS USA 93: 3704-9 8. Brown, K.M., Morrice, P.C., and Duthie, G.G., 1997. "Erythrocyte vitamin E and plasma ascorbate concentrations in relation to erythrocyte peroxidation in smokers and nonsmokers: Dose response to vitamin E supplementation", Am J Clin Nutr 65: 496-502. 9. Ingram, DK et al, 1990. "Dietary Restriction and Aging: The Initiation of a Primate Study," J Gerontol 45: B148-163. 10. Crabtree, DV et al, 1996. "Vitamin E, retinyl palmitate, and protein", Invest Ophthalmol Visual Sci 37:47-60. This presentation was sponsored by BeloitCollege PPDC. HOW MUCH VITAMIN C AND VITAMIN E TO SUPPLEMENT TWICE A DAY OR ONCE A DAY?