Selection of Aging Biomarkers in Primates: Caloric Restriction of Rhesus MonkeysAlfred B. Ordman1,2, Ricki J. Colman2 and Joseph W. Kemnitz2

1Biochemistry Program, Beloit College, Beloit, WI 535112Wisconsin 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)

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

I. VITAMIN C AND E

II. CALORIC RESTRICTION

III. BIOMARKERS OF AGING

Table 1: Total Intake of Vitamin C and E inCaloric Restriction Studies

WNPRCOnce a day feedingSemi-purified diet

NIATwice a day feedingSemi-synthetic diet

mg/kg diet Avg dailyintake (mg)

mg/kg diet(9)

Avg dailyintake (mg)

Vitamin C 991 129 3900 787Vitamin E 121 16 72 14.5

Table 2: Rate of Intake of Vitamin C and E inCaloric Restriction Studies

mg Vitamin C/kg body weight in Rhesus dietsFeeding Time NIA WNPRC

700 39.35 12.71330 39.35 3Total 78.8 15.7

Frequency Twice a day Once a day

mg Vitamin E/kg body weight in Rhesus dietsFeeding Time NIA WNPRC

700 0.725 1.551330 0.725 0.40Total 1.45 1.95

Frequency Twice a day Once a day

Table 4: Vitamin C and E Dosa ge & Plasma Monkey

plasma conc. Human Values (7, 8)

Vitamin C

1.3 mg/dl when given

100mg vitamin C daily(6)

Human Dose, mg Vit C b.i.d.

mg Vit C/ dl plasma

30 0.35 100 1.14 500 1.32 1250 1.41

Vitamin E

2 M on 7.3iu vit E daily (10) [CR diet is twice this

level]

Human Dose, mg Vit E

M Vit E in plasma

70 63+/-4 140 72+/-18 560 72+/-18 1050 97+/-18

Table 6: Weight and Caloric Intake of Rhesus Monkeys in Caloric Restriction Project Group 1 Data

n Weight (kg)

Lean Body Wt. (kg)

Food intake (kcal)

Intake/70kg (Cal)

Intake/70kg lean (Cal)

Control 9 13.25 9.00 581 3071 4522 Calorie Restricted

11 9.63 8.14 480 3489

4127

% diff 27.32 9.56 17.44 -13.59 8.72

IIIA. BIOMARKER CRITERIA Criteria for Biomarkers (1) 1. avoid obvious redundancies among biomarkers 2. should be absence of biomarker change in immortal cells 3. factors that modulate aging rate also modulate biomarker change 4. must be assessed nonlethally 5. should be reproducible reflecting physical age External Indices for Biological Aging (2) 1. Biomarkers - BIO 2. Antioxidant status - AOX (3) 3. Sexual behavior - SXB

Criteria for Assigning a Unique Value to the Rate of Biological Aging (3) 1. Content validity-many variables sampled 2. Construct validity-combined rates correlate with individual rates 3. Concurrent validity-correlates with external indices BIO, AOX, SXB

IIIB. BIOMARKER MODELSExample below summarizes Figure 3:Measurement Model For AntioxidantActivity Integrated into the [rate ofbiological aging] Model (2).

Chronological Age

Rate of Biological Aging

Aging Rate Modulator

Vitamin C Vitamin E

(3)

Biomarker 1 Biomarker 2 Biomarker n

Table 8: Clusters of biomarkers Clusters

of biomarkers Measures

in Humans Measures in

Monkeys circulatory 10 42 endocrine 6 13

genetic 6 3 immune 4 7

metabolic 1 12 musculoskeletal 9 17

nervous 5 10 oxidative stress 4 13

reproductive 0 2 respiratory 5 0

sensory 13 6 sleep 3 0

urogenital 1 6

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IA. INTRODUCTIONThe 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 RESTRICTIONFor 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 & ENutritional 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

mg vit C per kg BW proportional scaled allometrically scaled for 70 kg human for 70 kg human

Est. Rhesus Reqt 5 to 25 350 to 1,750 Actual Rhesus Diet for CR study 13 900 4282 Est. Human Rqmnt AGE 200 to 1,000* Est. Human Rqmnt FNB 90 * in divided doses

mg vit E

per kg BW proportional scaled allometrically scaled for 70 kg human for 70 kg human Est. Rhesus Reqt 0.4 to 2.6 28 to 182 Actual Rhesus Diet for CR study 1.5 108 522 Est. Human Rqmnt AGE 200 to 400 Est. Human Rqmnt FNB 15

IIA. CALORIC RESTRICTION IN RHESUS MONKEYSWisconsin National Primate Research CenterBegun in 1991, Initiation of CR at age 8-14

Table 5: Characteristics of MonkeysSex Number Ages Male 46 17-30 Female 30 19-25

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 CR2. CR at risk for diabetes in 1991 have normalized blood glucose3. C-reactive protein is lower in CR compared to Controls4. CR have less arthritis5. 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 DATABASEIn 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 BIOMARKERSAs 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

Cluster Examples

circulatory arterial stiffnesscirculatory DNA microarray -

inflammatory respendocrine growth hormonegenetic gene expression-DNA damagegenetic telomere lengthimmune T-cell subsets(CD4- total,

memory, naïve, etc.)metabolic body temperaturemetabolic insulin sensitivitymusculoskeletal bone densitymusculoskeletal grip strengthnervous alternate button tapping time, 30xnervous dopaminergic receptorsoxidative stress DNA microarray - oxidative stressoxidative stress glycosylated hemoglobinreproductive morpho sex characteristicsreproductive sexual behaviorsrespiratory forced vital capacitysensory hearing- ABR thresholdsensory lens fluorescencesleep sleep disturbances, wave formurogenital serum chloride

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 BIOMARKERS1. not robust across different species2. none are used consistently by different research groups – each has different favorites3. statistical combinations of many markers end up with large standard errors so that the values become meaningless.

IV. CONCLUSIONThe 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.

References1. 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 29:825-317. 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?