The effects of intermittent or continuous energy restriction on weight loss and metabolic disease risk markers: a randomized trial in young overweight women.

BACKGROUND: The problems of adherence to energy restriction in humans are well known. OBJECTIVE: To compare the feasibility and effectiveness of intermittent continuous energy (IER) with continuous energy restriction (CER) for weight loss, insulin sensitivity and other metabolic disease risk markers. DESIGN: Randomized comparison of a 25% energy restriction as IER (∼ 2710 kJ/day for 2 days/week) or CER (∼ 6276 kJ/day for 7 days/week) in 107 overweight or obese (mean (± s.d.) body mass index 30.6 (± 5.1) kg m(-2)) premenopausal women observed over a period of 6 months. Weight, anthropometry, biomarkers for breast cancer, diabetes, cardiovascular disease and dementia risk; insulin resistance (HOMA), oxidative stress markers, leptin, adiponectin, insulin-like growth factor (IGF)-1 and IGF binding proteins 1 and 2, androgens, prolactin, inflammatory markers (high sensitivity C-reactive protein and sialic acid), lipids, blood pressure and brain-derived neurotrophic factor were assessed at baseline and after 1, 3 and 6 months. RESULTS: Last observation carried forward analysis showed that IER and CER are equally effective for weight loss: mean (95% confidence interval ) weight change for IER was -6.4 (-7.9 to -4.8) kg vs -5.6 (-6.9 to -4.4) kg for CER (P-value for difference between groups = 0.4). Both groups experienced comparable reductions in leptin, free androgen index, high-sensitivity C-reactive protein, total and LDL cholesterol, triglycerides, blood pressure and increases in sex hormone binding globulin, IGF binding proteins 1 and 2. Reductions in fasting insulin and insulin resistance were modest in both groups, but greater with IER than with CER; difference between groups for fasting insulin was -1.2 (-1.4 to -1.0) µU ml(-1) and for insulin resistance was -1.2 (-1.5 to -1.0) µU mmol(-1) l(-1) (both P = 0.04). CONCLUSION: IER is as effective as CER with regard to weight loss, insulin sensitivity and other health biomarkers, and may be offered as an alternative equivalent to CER for weight loss and reducing disease risk.

Associative and predictive biomarkers of dementia in HIV-1-infected patients.

BACKGROUND: Infection with HIV can result in a debilitating CNS disorder known as HIV dementia (HIV-D). Since the advent of highly active antiretroviral therapy (HAART), the incidence of HIV-D has declined, but the prevalence continues to increase. In this new era of HIV-D, traditional biomarkers such as CSF viral load and monocyte chemotactic protein 1 levels are less likely to be associated with dementia in patients on HAART and biomarkers that can predict HIV-D have not yet been identified. OBJECTIVE: To identify biomarkers that are associated with and can predict HIV-D. METHODS: We grouped patients with HIV based on changes in cognitive status over a 1-year period and analyzed sphingolipid, sterol, triglyceride, antioxidant, and lipid peroxidation levels in CSF. RESULTS: We found that increased levels of the vitamin E and triglyceride C52 predicted the onset or worsening of dementia. Elevated levels of sphingomyelin were associated with inactive dementia. Elevated levels of ceramide and the accumulation of 4-hydroxynonenals were associated with active dementia. CONCLUSIONS: We interpret these findings to indicate that early in the pathogenesis of HIV dementia, there is an up-regulation of endogenous antioxidant defenses in brain. The failure of this attempted neuroprotective mechanism leads to the accumulation of sphingomyelin and moderate cognitive dysfunction. The breakdown of this enlarged pool of sphingomyelin to ceramide and the accumulation of highly reactive aldehydes are associated with declining cognitive function. Thus, elevations in endogenous protective mechanisms may identify patients who are at increased risk of the development of HIV dementia.

Differential response induced by exposure to low-dose ionizing radiation in SHSY-5Y and normal human fibroblast cells.

Radiation therapy has been used in the treatment of a wide variety of cancers for nearly a century and is one of the most effective ways to treat cancer. Low-dose ionizing radiation (IR) can interfere with cell division of cancer and normal cells by introducing oxidative stress and injury to DNA. The differences in the response to IR-induced DNA damage and increased reactive oxygen species between normal human fibroblasts (NHFs) and cancerous SHSY-5Y cells were considered. H2AX staining and comet assays revealed that NHF cells responded by initiating a DNA repair sequence whereas SHSY-5Y cells did not. In addition, NHF cells appeared to quench the oxidative stress induced by IR, and after 24 h no DNA damage was present. SHSY-5Y cells, however, did not repair their DNA, did not quench the oxidative stress, and showed characteristic signs that they were beginning to undergo apoptosis. These results indicate that there is a differential response between this cancerous and normal cell line in their ability to respond to low-dose IR, and these differences need to be exploited in order to treat cancer effectively. Further study is needed in order to elucidate the mechanism by which SHSY-5Y cells undergo apoptosis following radiation and why these normal cells are better equipped to deal with IR-induced double-strand breaks and oxidative stress.

Dysregulation of sphingolipid and sterol metabolism by ApoE4 in HIV dementia.

BACKGROUND: Polymorphisms in apolipoprotein E have been associated with worse prognoses in numerous neurodegenerative conditions, including HIV dementia (HIVD). Despite these correlative observations, there has been little evidence suggesting a mechanism whereby the expression of ApoE4 renders neurons susceptible to insult. METHODS: Electrospray ionization tandem mass spectrometry was used to quantify levels of sphingolipids and sterols in brains of HIVD patients. Data were stratified according to APOE genotype. RESULTS: The authors found evidence of dysregulated lipid and sterol metabolism in HIVD patients with an APOE4 genotype. They also found elevations of sphingomyelin, ceramide, and cholesterol in the medial frontal cortex, parietal cortex, and cerebellum of HIVD patients with an APOE3/4 or APOE4/4 genotype compared with HIVD patients with an APOE3/3 genotype. There was no difference in the number of astrocytes or activated microglia in any brain region of the two patient populations, suggesting that modification of lipid metabolism in HIVD patients with an APOE4 genotype was not the result of increased CNS inflammation. CONCLUSIONS: HIV dementia patients with an APOE4 genotype may be sensitized to neural insults because of dysregulations in lipid metabolism.

Sphingomyelin and ceramide as regulators of development and lifespan.

Sphingomyelin (SM) is a prominent phospholipid component of cell membranes that has evolved diverse functions in cells beyond its role in membrane structural organization. Cleavage of SM by acid or neutral sphingomyelinase results in the liberation of ceramide, an intracellular messenger that regulates the activities of an array of kinases, phosphatases and transcription factors. Signals that activate sphingomyelinases range from growth factors and cytokines, to neurotransmitters, hormones and reactive oxygen species. Studies of experimental cell culture and animal models, and of patients with inherited defects in sphingomyelin metabolism suggest important roles for SM-ceramide signaling in the regulation of cell proliferation, differentiation and survival. At low concentrations SM and ceramide can stimulate cell proliferation and survival, whereas higher levels can induce cell dysfunction or death. Analyses of development and aging suggest a major role for SM metabolism in regulating development rate and lifespan. Several factors that alter the metabolism of sphingolipids, including oxidative and metabolic stress, also increase risk and progression of age-related diseases. In addition, recent findings have linked alterations in SM metabolism to the pathogenesis of several age-related diseases including cancers and neurodegenerative disorders. The emerging data suggest the possibility that dietary and pharmacological manipulations of SM metabolism might prove effective in extending lifespan and treating various age-related diseases.

Uric acid and serum antioxidant capacity: a reaction to atherosclerosis?

BACKGROUND: the evidence of a potential beneficial role of antioxidants in preventing atherosclerotic disease is not entirely consistent. OBJECTIVE: to assess the longitudinal association of serum total antioxidant capacity and serum antioxidants with the presence of subclinical carotid atherosclerosis. METHODS: Prospective case-control study nested within an historical cohort. Cases were 150 individuals with elevated carotid intimal-medial thickness measured by B-mode ultrasound at the first two examinations of the Atherosclerosis Risk in Communities Study (1987-92). Controls were 150 age-gender-matched individuals with low carotid intimal-medial thickness. Serum antioxidant vitamins, uric acid, and serum total antioxidant capacity were measured in frozen serum samples collected from the same individuals in 1974 (13-15 years prior to the determination of case-control status). RESULTS: Compared to controls, atherosclerosis cases had significantly higher levels of serum total antioxidant capacity in 1974 than controls. This difference was almost entirely explained by increased serum concentration of uric acid in cases. In contrast with cross-sectional results, uric acid serum concentration in 1974, was significantly higher in cases than in controls, even after adjusting for the main cardiovascular risk factors. Cases had significantly lower levels of alpha-carotene in the 1974 sera than controls, but no other differences in serum antioxidant vitamin concentrations were observed. CONCLUSIONS: The higher serum uric acid concentration seemed associated with elevated total serum antioxidant capacity among individuals with atherosclerosis. This finding is consistent with experimental evidence suggesting that hyperuricemia may be a compensatory mechanism to counteract oxidative damage related to atherosclerosis and aging in humans.

Mouse embryonic stem cells carrying one or two defective Msh2 alleles respond abnormally to oxidative stress inflicted by low-level radiation.

Chronic oxidative stress may play a critical role in the pathogenesis of many human cancers. Here, we report that mouse embryonic stem (ES) cells deficient in DNA mismatch repair responded abnormally when exposed to low levels of ionizing radiation, a stress known to generate oxidative DNA damage. ES cells derived from mice carrying either one or two disrupted Msh2 alleles displayed an increased survival following protracted exposures to low-level ionizing radiation as compared with wild-type ES cells. The increases in survival exhibited by ES cells deficient in DNA mismatch repair appeared to have resulted from a failure to efficiently execute cell death (apoptosis) in response to radiation exposure. For each of the ES cell types, prolonged low-level radiation treatment generated oxidative genome damage that manifested as an accumulation of oxidized bases in genomic DNA. However, ES cells from Msh2(+/-) and Msh2(-/-) mice accumulated more oxidized bases as a consequence of low-level radiation exposure than ES cells from Msh2(+/+) mice. The propensity for normal cells with mismatch repair enzyme deficiencies, including cells heterozygous for inactivating mismatch repair enzyme gene mutations, to survive promutagenic genome insults accompanying oxidative stresses may contribute to the increased cancer risk characteristic of the hereditary nonpolyposis colorectal cancer syndrome.

A spin trap, N-tert-butyl-alpha-phenylnitrone extends the life span of mice.

To characterize the pharmacological effects of N-tert-butyl-alpha-phenylnitrone (PBN) on life span, we administered PBN in drinking water to 24.5-month-old mice, and the survivors were counted. Their water consumption and body weights were measured as biological markers. PBN-treated animals as compared with control animals had prolonged mean and maximum life spans. Their water consumption decreased but no significant change was found in their body weights, indicating that the metabolism was improved. Results showed that PBN indeed affects physiological functions and extends life span. We propose that nitric oxide release from PBN may be involved in altering the aging process.

Release of nitric oxide from a spin trap, N-tert-butyl-alpha-phenylnitrone, under various oxidative conditions.

Nitric oxide (NO) generation from a spin trap, N-tert-butyl-alpha-phenylnitrone (PBN) under various oxidative conditions was examined. The absorbance of PBN at 295 nm decreased with time of UV-irradiation, showing that PBN was decomposed by UV irradiation. The hydroxyl radical formed from a Fenton reagent also decomposed PBN, but there was little effect by a peroxyl radical and a superoxide. Nitrite, an oxidative product of NO, in PBN solution was determined using a NOx analyzer based on Griess reaction. UV-irradiation and the hydroxyl radical also formed nitrite. Direct detection of NO from the sample on reaction with hydroxyl radical was successful using a GC/MS/SIM on the UV-irradiated sample. NO generated in PBN solutions activated guanylate cyclase. From these results, PBN is viewed as a new kind of medicine which acts as an antioxidant and as an NO donor in vivo.

The risk of developing lung cancer associated with antioxidants in the blood: ascorbic acid, carotenoids, alpha-tocopherol, selenium, and total peroxyl radical absorbing capacity.

Lung cancer cases diagnosed during the period 1975 through 1993 and matched controls were identified in the rosters of Washington County, Maryland residents who had donated blood for a serum bank in 1974 or 1989. Plasma from participants in the 1989 project was assayed for ascorbic acid; serum or plasma was assayed for participants in either project for alpha- and beta-carotene, cryptoxanthin, lutein/zeaxanthin, lycopene, alpha-tocopherol, selenium, and peroxyl radical absorption capacity. Among the total group of 258 cases and 515 controls, serum/plasma concentrations were significantly lower among cases than controls for cryptoxanthin, beta-carotene, and lutein/zeaxanthin with case-control differences of -25.5, -17.1, and -10.1%, respectively. Modest nonsignificant case-control differences in a protective direction were noted for alpha-carotene and ascorbic acid. There were only trivial differences for lycopene, alpha-tocopherol, selenium, and peroxyl radical absorption capacity. Findings are reported for males and females and for persons who had never smoked cigarettes, former smokers, and current smokers at baseline. These results and those from previous studies suggest that beta-carotene is a marker for some protective factor(s) against lung cancer; that cryptoxanthin, alpha-carotene, and ascorbic acid need to be investigated further as potentially protective factors or associates of a protective factor; and that lycopene, alpha-tocopherol, selenium, and peroxyl radical absorption capacity are unlikely to be associated with lung cancer risk. Until specific preventive factors are identified, the best protection against lung cancer is still the avoidance of airborne carcinogens, especially tobacco smoke; second best is the consumption of a diet rich in fruits and vegetables.

Preferential use of less toxic detoxification pathways by long-lived species.

This study was undertaken to determine whether the detoxification pathways producing the least oxidative stress appear to be favored in longer-lived mammalian species. Firstly, we focused on the cytochrome P-450 monooxygenase system. Although this system is an important component of the defenses that protect living organisms against toxic chemicals, some reactions catalyzed by the cytochrome P-450 system result in the formation of products that are highly reactive as well as active oxygen species. Our results suggest that the lower amount of hepatic cytochrome P-450 content found in longer-lived species may have evolved to reduce the toxic side-effect of this detoxification system. Support of the idea that the cytochrome P-450 system is an important source of oxidative stress is the positive correlation between cytochrome P-450 content and the amount of oxidized proteins found in liver of different human individuals. Secondly, we have measured the specific activity of other detoxification enzymes as a function of life span. Instead of a direct comparison of detoxification capabilities of the species, the approach used in this study was: (1) to select those detoxification enzymes which utilize the same substrate but differ in toxicity of the intermediate compounds formed in the reaction, and (2) to measure the levels of these enzymes in the two pathways to determine which pathway is dominant for each species. Our results suggest that the detoxification pathways producing the least oxidative stress do appear to be favored in longer-lived species.

Effect of dietary restriction on serum antioxidant capacity in rats.

The effects of dietary restriction on serum antioxidant capacities were studied in male Fischer 344 rats. Dietary restriction was started at the age of 6 weeks and consisted of 60% of the mean daily food intake of the ad libitum fed controls. They were killed at 7 and 18 months of age. The antioxidant capacities of whole serum and the non-protein fraction of serum were assessed using the oxygen radical absorbance capacity (ORAC) assay with a peroxyl radical generator. Rats that consumed a diet restricted by 40% in calories had significantly lower ORAC activities in whole serum and the non-protein fraction of serum. The decreased serum ORAC activity is seemingly an organism's physiologically appropriate response to reduced oxidative stress through a down-regulation mechanism.

Calorie restriction lowers body temperature in rhesus monkeys, consistent with a postulated anti-aging mechanism in rodents.

Many studies of caloric restriction (CR) in rodents and lower animals indicate that this nutritional manipulation retards aging processes, as evidenced by increased longevity, reduced pathology, and maintenance of physiological function in a more youthful state. The anti-aging effects of CR are believed to relate, at least in part, to changes in energy metabolism. We are attempting to determine whether similar effects occur in response to CR in nonhuman primates. Core (rectal) body temperature decreased progressively with age from 2 to 30 years in rhesus monkeys fed ad lib (controls) and is reduced by approximately 0.5 degrees C in age-matched monkeys subjected to 6 years of a 30% reduction in caloric intake. A short-term (1 month) 30% restriction of 2.5-year-old monkeys lowered subcutaneous body temperature by 1.0 degrees C. Indirect calorimetry showed that 24-hr energy expenditure was reduced by approximately 24% during short-term CR. The temporal association between reduced body temperature and energy expenditure suggests that reductions in body temperature relate to the induction of an energy conservation mechanism during CR. These reductions in body temperature and energy expenditure are consistent with findings in rodent studies in which aging rate was retarded by CR, now strengthening the possibility that CR may exert beneficial effects in primates analogous to those observed in rodents.

Longevity and the genetic determination of collagen glycoxidation kinetics in mammalian senescence.

A fundamental question in the basic biology of aging is whether there is a universal aging process. If indeed such a process exists, one would expect that it develops at a higher rate in short- versus long-lived species. We have quantitated pentosidine, a marker of glycoxidative stress in skin collagen from eight mammalian species as a function of age. A curvilinear increase was modeled for all species, and the rate of increase correlated inversely with maximum life-span. Dietary restriction, a potent intervention associated with increased life-span, markedly inhibited glycoxidation rate in the rodent. On the assumption that collagen turnover rate is primarily influenced by the crosslinking due to glycoxidation, these results suggest that there is a progressive age-related deterioration of the process that controls the collagen glycoxidation rate. Thus, the ability to withstand damage due to glycoxidation and the Maillard reaction may be under genetic control.

The utilization of 5-hydroxyl-2-amino valeric acid as a specific marker of oxidized arginine and proline residues in proteins.

Alteration of cellular proteins by oxidative modification could represent an important mechanism leading to cellular dysdifferentiation and age-related diseases. There is difficulty in testing this hypothesis because of a lack of specific assays that can measure the extent proteins are oxidized in nonpurified tissue preparations. Some methods used to measure carbonyl groups in nonpurified samples have serious limitations because of interference from other sources of carbonyl groups not being a product of oxidation-mediated damage. Oxidation of arginine and proline residues has been reported to produce gamma-glutamyl semialdehyde, which on reduction and acid hydrolysis, was predicted to form 5-hydroxy-2-amino valeric acid (HAVA). In this article we confirm this prediction using a GC/MS/SIM technique, and carry out additional experiments to determine if HAVA may be a useful marker of oxidative damage in proteins. These experiments utilized purified preparations of arginine, proline, histidine, and lysine amino acid homopolymers and six different purified proteins preparations in nonoxidized and oxidized states. Results demonstrate that HAVA compares well with the carbonyl group formation as a specific marker of oxidized protein, and that the GC/MS/SIM technique can detect HAVA reliably to 150 femtomoles per injection. Thus, HAVA as a specific marker of oxidized arginine and proline could prove to be a useful assay in pure and nonpurified samples.

Comparison of 5-hydroxy-2-amino valeric acid with carbonyl group content as a marker of oxidized protein in human and mouse liver tissues.

Previous studies indicate that 5-hydroxy-2-amino valeric acid (HAVA) is an excellent marker of oxidized arginine and proline in purified proteins. We report here experiments testing the specificity of the HAVA assay technique using the unpurified 100,000 x g supernatant fraction prepared from mice and human liver tissue. Results are compared to carbonyl group analysis on the same tissue samples. Mice at ages 3, 12, and 30 months were exposed to 100% oxygen. Results showed a significant increase of HAVA content in each age group. No significant changes were found in carbonyl group content. Because it has been reported that carbonyl group content increases with age, we applied the HAVA assay to reexamine this question. Using mice of 1 to 30 months of age, we failed to detect any significance difference in either HAVA or carbonyl group content. However, on using human liver samples a significant decrease from age 16 to 40 years and then an increase to 85 years of age was found for both HAVA and carbonyl groups. Liver proteins may be oxidized from hydrogen peroxide produced from the cytochrome P450 detoxification system. This possibility was supported by a significant positive correlation found between HAVA and cytochrome P450 content in 18 human individuals of different ages.

Energy balance in rhesus monkeys (Macaca mulatta) subjected to long-term dietary restriction.

Male rhesus monkeys of various age groups representative of the species life span were fed ad libitum amounts (controls) or 30% less food than control monkeys of comparable age and body weight. Despite significantly lowered energy intake and body weight, the amount of energy lost in the feces, and fecal energy density (concentration) were not altered in diet-restricted (DR) monkeys, compared to age- and weight-matched controls. Absolute energy expenditure (EE; 24-hr) was consistently lower in DR monkeys, but this trend was not statistically significant. Expressed as a function of metabolic mass (body weight, metabolic body size, lean mass), 24-hr EE was not different in monkeys subjected to long-term DR, compared to controls. Calculations of net energy (intake-loss), as an index of energy balance, revealed that energy expenditure generally exceeded energy intake in all juvenile and adult group monkeys. However, this discrepancy was not statistically different from zero, suggesting that most animals were in energy balance. Also, there was no difference between control and DR animals with respect to energy balance. Diet restriction induced significant reductions in the absolute amount of lean body mass; however, percent (of total weight) lean and fat mass did not differ from controls.

Protein oxidation and aging. I. Difficulties in measuring reactive protein carbonyls in tissues using 2,4-dinitrophenylhydrazine.

A current hypothesis explaining the aging process implicates the accumulation of oxidized protein in animal tissues. This hypothesis is based on a series of reports showing an age-dependent increase in protein carbonyl content and an age-dependent loss of enzyme function. This hypothesis is also supported by the report of a novel effect of N-tert-butyl-alpha-phenylnitrone (PBN) in reversing these age-dependent changes. Here we specifically study the method that was used to measure reactive protein carbonyls in tissues. This method uses 2,4-dinitrophenylhydrazine (DNPH) and includes a washing procedure. Our results indicate that reactive protein carbonyls in normal crude tissue extracts cannot be reliably measured by this method, although it does reliably measure reactive carbonyls in purified proteins which have been oxidatively modified in vitro. The nucleic acids in tissues could be a major problem encountered in the assay. Using the streptomycin sulfate treatment combined with a dialysis step, we were successful in removing most nucleic acids from a crude tissue extract, but then the reactive carbonyl level in the crude tissue extract was too low to be reliably measured. This streptomycin sulfate treatment procedure, however, had no effect on the reactive carbonyl measurement of an oxidized protein sample. The unwashed free DNPH was another major problem in the assay because of its very strong absorption around 370 nm, where reactive carbonyls were quantitated. Nevertheless, on using the procedure described in the literature to measure total "reactive carbonyls" in rat liver and gerbil brain cortex, no change with age or PBN treatment was found. Then, we investigated a HPLC procedure which uses sodium dodecyl sulfate in the mobile phase but this was also found to be unsuitable for the reactive protein carbonyl assay in tissues.

Protein oxidation and aging. II. Difficulties in measuring alkaline protease activity in tissues using the fluorescamine procedure.

A current hypothesis explaining the aging process implicates the accumulation of oxidized protein in animal tissues. This is primarily based on a series of reports showing an age-dependent increase in protein carbonyl content and an age-dependent decrease in the activities of enzymes, especially of alkaline proteases, which preferentially degrade oxidatively modified protein. Recently, this hypothesis was strongly supported by the report of a novel effect of the spin-trapping compound N-tert-butyl-alpha-phenylnitrone (PBN) in reversing these age-dependent changes. However, we found that the reactive protein carbonyls could not be reliably measured in tissues by using the 2,4-dinitrophenylhydrazine procedure described in the PBN study. We now focus on the alkaline protease activity assay and show that alkaline protease activity cannot be reliably measured in crude tissue extracts by using the fluorescamine procedure also described in the PBN study. We were, however, able to reliably measure a protease activity in crude tissue extracts at alkaline pH by using a synthetic fluorogenic peptide substrate, but no effect of aging or PBN treatment was found on the protease activity in rat brain cortexes. Thus, the reported age-dependent changes in protein carbonyl formation and alkaline protease activity remain to be confirmed.

Aging and food restriction alter some indices of bone metabolism in male rhesus monkeys (Macaca mulatta).

Food restriction increases life span, reduces aging rate and affects a wide variety of biological functions. In rats, food restriction delays bone growth and reduces bone density and mineral content. We report the effects of aging and long-term (> 6.0 y) food restriction on several indices of bone growth and metabolism in rhesus monkeys (Macaca mulatta). Food allotments for controls approximated free access consumption, whereas food-restricted monkeys received 30% less food on a body weight basis. Cross-sectional and longitudinal age effects on serum alkaline phosphatase paralleled those reported for humans. Food restriction induced a significant delay in the developmental decline (to adult levels) in total alkaline phosphatase and significantly suppressed serum interleukin 6 concentrations, particularly in younger monkeys. Also, food restriction slowed skeletal growth, as reflected by shorter crown-rump length, and significantly reduced total body bone mineral content, but not bone mineral density, measured by dual energy X-ray absorptiometry. Analyses of serum parathyroid hormone, calcium, phosphate and osteocalcin concentrations suggested that the effects on skeletal growth were not related to alterations in calcium and phosphate homeostasis or a primary defect in bone formation. These findings suggest that long-term food restriction delays skeletal development in male rhesus monkeys while allowing the development of a reduced but otherwise normal skeleton.