Effect of vitamins C and E on endogenous synthesis of N-nitrosamino acids in humans: precursor-product studies with [15N]nitrate.

The endogenous formation of nitrosoproline (NPRO) following administration of nitrate and proline is reported in ten healthy young adults. There was a relatively constant basal excretion of NPRO, 26 +/- 10 (SD) nmol/day, in excess of amounts found in the diet. This basal synthesis of NPRO was not reduced by ascorbic acid (2 g/day) or alpha-tocopherol (400 mg/day). A significant rise in the excretion of NPRO was observed following the administration of nitrate and proline, ranging from 29 to 318 nmol/24 h with a mean of 100 nmol/24 h. [15N]Nitrate was used as a tracer to study the observed excess excretion of NPRO in urine. The data revealed that urinary NPRO excretion as a result of endogenous synthesis is not totally derived from ingested nitrate as its precursor. The ingestion of ascorbic acid and alpha-tocopherol inhibited the incorporation of [15N]nitrate into NPRO by 81 and 59%, respectively. An additional nitrosamino acid, N-nitrosothiazolidine-4-carboxylic acid, was present in the urine. It was found that N-nitrosothiazolidine-4-carboxylic acid increased 6-fold upon ingestion of nitrate. Ascorbic acid and alpha-tocopherol blocked this nitrate induced synthesis.

Modulation of endogenous synthesis of N-nitrosamino acids in humans.

Endogenous production of N-nitrosoproline (NPRO) was demonstrated in human subjects ingesting a diet low in nitrate and NPRO. The daily endogenous synthesis of NPRO was 26 +/- 10 nmol/day (mean +/- SD). Upon administration of nitrate and proline, the NPRO excreted in urine ranged from 50-318 nmol/24 h. It was found that ascorbic acid and alpha-tocopherol did not lower the background endogenous synthesis of NPRO; however, ascorbic acid was very effective in preventing the nitrate-induced synthesis of NPRO in all subjects, while alpha-tocopherol was less effective in some subjects. Furthermore, it was demonstrated that administered 15N-nitrate could be incorporated into NPRO, suggesting clearly that nitrosation reactions indeed occur in humans. Ascorbic acid significantly inhibited the incorporation of 15N-nitrate into NPRO. The presence of another N-nitrosamino acid, N-nitrosothiazolidine-4-carboxylic acid (NTCA), was detected in the urine of many subjects even without nitrate intake. However, upon ingestion of nitrate, there was a six-fold increase in mean NTCA synthesis (25 +/- 16 nmol/24 h), and all subjects had detectable levels of NTCA in the urine. Ascorbic acid completely blocked the nitrate-induced synthesis of NTCA, while alpha-tocopherol was not as effective.

The changing pattern of whole body protein metabolism in aging humans.

Dynamic aspects of whole body protein (nitrogen) metabolism were explored in healthy young adults and elderly men and women. Measurements were made of the rate of whole body protein breakdown, with the aid of 15N-glycine, and the rate of muscle protein breakdown, as estimated from urinary N tau-methylhistidine excretion. The results also were evaluated in relation to obligatory (endogenous) urinary nitrogen losses, previously determined in this laboratory for the two age groups. Rates of whole body and muscle protein breakdown, per unit body weight, were lower in elderly subjects than in young adults. Muscle accounted for a mean of 27% of whole body protein breakdown in young adults and 20% or less (p less than 0.01) in elderly subjects. Daily obligatory N loss was positively correlated (p less than 0.01) with whole body protein breakdown. It was calculated that muscle contributed less to the obligatory N output in elderly subjects than in young adults. These results indicate a change in the distribution of whole body protein metabolism during aging in human subjects, with muscle making a lower contribution to total body protein metabolism in elderly subjects compared with young adults.

Musle protein breakdown rates in humans based on Ntau-methylhistidine (3-methylhistidine) content of mixed proteins in skeletal muscle and urinary output of Ntau-methylhistidine.

Samples of psoas muscle from nine infants (aged 1 day to 14 mo) and of several skeletal muscles from seven adult males (age 19-74 yr) were analyzed for content of protein-bound Ntau-methylhistidine (3-methylhistidine; 3-Mehis). The mean content of 3-Mehis (expressed as mumoles/g mixed protein) was 3.2 (range 2.4-3.7) in infants and 4.2 (range 3.7-4.6) in adults. The daily urinary excretion of 3-Mehis was measured in four young adult males receiving an egg-protein, flesh-free diet. Mean excretion of 3-Mehis was 211 (range 167-252) mumoles/day. From these two sets of data the mean rate of muscle protein breakdown in adult males was estimated to be 50 g/day, or 0.7 +/- 0.1 g/kg body weight/day. These results are compared with reported values for the 3-Mehis content of mixed proteins in muscle of various species, and with published estimates, computed by other techniques, of the rate of muscle protein breakdown in human subjects.

Regulatory role of dietary leucine on plasma branched-chain amino acid levels in young men.

Four healthy young men were studied to explore the effects of dietary leucine intake on plasma levels of leucine,isoleucine and valine. Leucine-, valine-, leucine-valine-, and leucine-isoleucine-valine-free amino acid mixtures were studied during four-day experimental diet periods. The effects of giving amino acid mixtures with high-carbohydrate, low-fat or low-carbohydrate, high-fat diets were also studied. Deficient leucine intake increased plasma levels of valine and isoleucine, but a valine-free diet did not affect plasma levels of the other branched-chain amino acids. Leucine influence was evident during the postprandial and fasted phases. Valine and isoleucine levels were less markedly reduced when diets devoid of these amino acids were also leucine-free, as compared with a diet providing adequate leucine. Changes in the major dietary energy source failed to influence the qualitative effects of dietary leucine adequacy on the branched-chain amino acid levels in plasma. The results suggest that leucine facilitates both tissue uptake of branched-chain amino acids and their intracellular metabolism.

Myofibrillar protein turnover and urinary N-tau-methylhistidine output. Response to dietary supply of protein and energy.

The urinary excretion of total N-tau-methylhistidine by the growing rat was measured to evaluate the effects of dietary protein and energy restriction on muscle protein turnover in vivo. 2. Young male rats (about 100 g initial wt.) were fed on one of three diets. Group I (controls) received an adequate 18% lactalbumin diet for 28 days, on which they sustained maximum growth. Group II (protein-depleted) was fed for 14 days on 0.5 lactalbumin diet, which caused loss of weight; this was followed by repletion for 14 days with the control diet. Group III (protein-energy restricted) received a 1% lactalbumin diet at one-half the food intake of group II for 14 days, and this was also followed by 14 days of repletion with the control diet. 3. The controls showed a progressive rise in the daily urinary output of N-tau-methylhistidine, which was proportionally slightly less rapid than the body-weight increase. 4. The protein-depleted group II showed a marked and progressive decrease in N-tau-methylhistidine excretion, which was proportionally greater than the fall in body weight; during repletion, N-tau-methylhistidine output rose in parallel with body-weight increase, but it did not reach the value attained by the control group. 5. Group III, restricted in both dietary protein and energy, showed an initial small increase in daily N-tau-methylhistidine output, which contrasted with the sharp loss of body weight during this period. After 11 days on this restricted diet, group III then underwent a decrease in N-tau-methylhistidine output, which persisted into the first 4 days of the repletion period, after which output of the methylated amino acid became the same as for group II. 6. Creatinine output, used as an additional metabolic measure of muscle metabolism, showed a fairly constant relationship to body weight in groups I and II during depletion and repletion. However, rats with protein-energy deficiency (group III) underwent a marked increase in output of creatinine per unit of body weight, which also persisited into the repletion period before it fell to more normal values relative to body weight. 7. Analysis of the N-tau-methylhistidine content of actin isolated from a group of protein-depleted rats revealed a small (5%) but significance (P less than 0.02) decrease relative to well-nourished controls. 8. Hence, the rate of muscle protein degradation, as indicated by changes in urinary N-tau-methylhistidine output, appears to respond sensitively and in opposite directions to insufficiency of protein of energy in the diet.