Human adult amino acid requirements: [1-13C]leucine balance evaluation of the efficiency of utilization and apparent requirements for wheat protein and lysine compared with those for milk protein in healthy adults.

BACKGROUND: There is considerable debate about the human lysine requirement and the consequent nutritional value of wheat protein. OBJECTIVE: We used a novel [1-(13)C]leucine balance protocol to examine whether adaptive mechanisms to conserve lysine allow wheat to be utilized more efficiently than expected according to current estimates of lysine requirements and wheat utilization. DESIGN: Wheat and milk proteins were compared in 6 adults infused for 9 h with L-[1-(13)C]leucine in the postabsorptive state (0-3 h), who were fed half-hourly with low-protein (2% of energy, 3-6 h) and isoenergetic higher-protein (12-13% of energy, 6-9 h) meals providing maintenance energy intakes. From acute measurements of [1-(13)C]leucine balance, we predicted nitrogen balance, the metabolic demand for protein, the efficiency of postprandial protein utilization (PPU), and the requirements for wheat protein and lysine. RESULTS: Leucine balance was higher after the milk than after the wheat feeding because of the greater inhibition of proteolysis by milk. PPU, calculated as the ratio of Deltanitrogen balance to Deltanitrogen intake between the low-protein and higher-protein periods, was 0.68 +/- 0.06 for wheat and 1.00 +/- 0.09 for milk (P

Variation in the apparent sensitivity of the insulin-mediated inhibition of proteolysis to amino acid supply determines the efficiency of protein utilization.

1. The variability between normal individuals in the efficiency of postprandial protein utilization (PPU), a determinant of the apparent protein requirement, was examined in relation to the relative responses of protein synthesis and proteolysis to protein feeding by means of [1-13C]leucine turnover and balance studies.2. Twenty-five healthy adults were infused intravenously with L-[1-13C]leucine continuously for 9 h. This was started in the postabsorptive state (PA, 3 h) and followed by low-protein feeding (LP, 3 h), and then by isoenergetic high-protein feeding (HP, 3 h). This allowed protein intake to be varied against a constant postprandial insulin level so that the extent of any amino-acid-mediated responses which were additional to those exerted by insulin could be investigated. Leucine oxidation, O, and balance (intake-oxidation), protein synthesis, S, and degradation, D, were calculated from plasma [1-13C]alpha-ketoisocaproic acid enrichment and 13CO2 excretion.3.PPUprotein, calculated as change in leucine balance/change in intake (HP-LP), varied from 0.58 to 0.99 (mean=0. 81+/-0.10), independently of age or sex. PPUprotein varied directly with the inhibition of D and inversely with the increase in leucine concentration and stimulation of O and S.4. Efficient PPU, as demonstrated by the top quintile of individuals categorized in terms of PPUprotein, involves maximal inhibition of D by protein feeding with minimal increases in free amino acid concentrations, O and S. Lesser inhibition of D and greater stimulation of S and O characterized the lower, less efficient quintile. This indicates that the efficiency of protein utilization in individuals, and a component of their apparent protein requirement, is determined by the sensitivity of the insulin-mediated inhibition of proteolysis to amino acid supply.

Aging, protein requirements, and protein turnover.

Current protein requirements for the elderly derive from 1985 FAO/WHO/UNU recommendations of no change with age in adults: i.e., 0.6 g/kg average and 0.75 g/kg safe allowance. Although concern has been expressed that protein requirements for the elderly may be greater, a review of nitrogen balance data, none of which are entirely satisfactory, indicates little reason for any revision. Furthermore, the 1985 recommendation is generally consistent with reports that the rate of whole-body protein turnover, a commonly assumed determinant of the protein requirement, exhibits minimal change with age per unit fat-free mass. Recent novel tracer studies aimed at evaluating protein requirements and turnover in a systematic way also support the 1985 recommendations. [1-13C]leucine balance studies have allowed measurement of metabolic demand from postabsorptive leucine oxidation and the efficiency of protein utilization from changes in leucine balance with feeding. The apparent protein requirement is metabolic demand divided by efficiency, an indication of protein needs and utilization during a standardized protocol at intakes similar to habitual ones. In healthy, mobile, elderly persons, metabolic demands are reduced by about one-third, with no significant impairment in efficiency of protein utilization. Thus, apparent protein requirements appear to fall with age from 0.98 +/- 0.17 to 0.69 +/- 0.22 g/kg. These changes with age reflect an improved restraint of proteolysis in the postabsorptive state, with little change with age in whole-body protein synthesis. The requirements of frail and immobile elderly and the efficiency of protein utilization of meals as eaten by elderly people remain to be evaluated.

Protein requirements and ageing: metabolic demand and efficiency of utilization.

The protein requirements of the elderly were investigated with [13C]leucine balance studies of metabolic demand, the efficiency of postprandial protein utilization (PPU) and the consequent apparent protein requirement. Ten elderly subjects aged 68-91 years (five men and five women) and ten young adult subjects aged 21-31 years (five men and five women) were infused with L-[1-13C]leucine for 9 h commencing in the postabsorptive state (0-3 h), continuing during the half-hourly feeding of low-protein meals (LP; protein 3% energy, 3-6 h), and during similar feeding of isoenergetic higher protein meals (HP; protein 15% energy, 6-9 h). Leucine oxidation and balance were determined from plasma [1-13C]-alpha-ketoisocaproate enrichment and expired 13CO2 excretion measured during the 3rd hour of each 3 h period. The protein intake during the HP phase was similar to the habitual intake estimated in the subjects from 24 h urinary N excretion. Metabolic demand was defined as equal to twice the body-protein equivalent of measured postabsorptive leucine oxidation. The efficiency of PPU was calculated from the increased leucine oxidation observed during feeding, and the apparent protein requirement was defined as metabolic demand/PPU and calculated in relation to both body weight (BW) and fat-free mass (FFM) determined by densitometry or bioimpedance. Metabolic demand in the young adults was 0.83 g protein/kg per d; in both elderly groups it was 36% lower when expressed per kg BW and 30% lower when expressed per kg FFM. The apparent protein requirement calculated from metabolic demand and PPU was 0.99 g protein/kg per d in the young adults and this was also lower in the elderly, although this was only significant in the men (0.66 g per kg BW, P = 0.013; 0.79 g per kg FFM, P = 0.02). The results show that in this group of healthy elderly adults protein requirements as assessed from leucine balance studies were either similar to or less than those of younger adults.

Post-prandial protein metabolism.

Current post-prandial studies of amino acid metabolism and utilization are consistent with a feeding mechanism mediated primarily by insulin and amino acids, with the balance between protein conservation and net deposition dependent on the amino acid supply [1-13C]leucine post-prandial kinetic tracer studies of leucine oxidation, non-oxidative disappearance and endogenous appearance allow study of the regulation of whole-body amino acid oxidation, protein synthesis and proteolysis. On the basis of these studies it appears that for leucine oxidation, the main determinant of the efficiency of protein utilization, the overriding regulatory influence is substrate availability rather than insulin. Such substrate sensitivity is manifest throughout the physiological range of insulin down to the lowest insulin levels observed suggesting that a basal insulin need is not an important part of regulation of this important catabolic pathway. The key protein turnover response is an inhibition of proteolysis sufficient to limit any increases in amino acid levels thus limiting any increase in amino acid oxidation. It appears that the influences of amino acids and insulin on proteolysis are separate and additive and may both be receptor mediated so that extracellular amino acid levels can regulate intracellular levels. It is likely that protein synthesis is regulated by intracellular amino acid levels but post-prandial stimulation through increases in amino acid levels appears to be unhelpful because of parallel increases in amino acid oxidation. Evidence for any influence of insulin on protein synthesis has yet to be unequivocally identified.

Does moderate aerobic activity have a stimulatory effect on 24 h resting energy expenditure: a direct calorimeter study.

This study was designed to establish whether moderate aerobic exercise (2 h at 30-35% VO2 max) in lean non-athletic male adults had a prolonged stimulatory effect on energy expenditure while at rest. Four weight maintaining male adults had their 24 h energy expenditure measured by direct calorimetry on four separate occasions. During the 24 h in the calorimeter each subject received a diet which in total supplied 35 kcal (146 kJ) per kg body weight. All studies in an individual were completed within four to six weeks during which time body weight remained stable. On two of these 24 h periods, individuals rested throughout while on the other two, they also performed the prescribed exercise. This consisted of cycling for two 1 h sessions; the first while they were fasting while the second period was approximately 45 min after consuming a 800 kcal (3.4 MJ) meal. Total 24 h energy expenditure was greater on exercise (8.3 +/- 1.8 MJ/day) than non-exercising days (6.3 +/- 1.4 MJ/day, P < 0.001) In contrast when the acute effects of the cycling where removed 24 h resting energy expenditure on the exercise day (6.8 +/- 1.7 MJ/day) was not significantly different from that of rest days (95% confidence intervals of the difference ranged from -0.36 to 1.27 MJ/day). This study did not demonstrate a prolonged stimulatory influence on non-exercising resting energy expenditure following physical activity likely to be achieved by non-athletes. These data provide no evidence that such exercise is associated with a greater energy deficit than that due to the activity itself.

Influences of dietary energy and protein on leucine kinetics during feeding in healthy adults.

Ten adult men were infused with L-[1-13C]leucine for 9 h commencing in the postabsorptive state (PA, 0-3 h), during the half-hourly feeding of low-protein meals (LP, protein = 2% calories, 3-6 h), and during feeding isoenergetic high-protein meals (HP, protein = 14% calories, 6-9 h). Leucine oxidation and turnover (protein synthesis and degradation) were determined from plasma alpha-[1-13C]ketoisocaproate enrichment and expired 13CO2 excretion measured during the third hour of each 3-h period. Plasma insulin increased markedly with feeding to a level that was maintained with both diets. The negative postabsorptive leucine balance became less negative during the LP meals (P < 0.01) and was positive with the HP meals (P < 0.01). The significant responses to feeding (all P < 0.01) were for oxidation -13% (PA-LP), +50% (LP-HP), and +29% (PA-HP); for degradation -24% (PA-LP), -30% (LP-HP), and -47% (PA-HP); and for synthesis -14% (PA-LP), +29% (LP-HP), and +11% (PA-HP). These data support a feeding mechanism involving both an insulin-mediated, protein-conserving influence of dietary energy that inhibits degradation, lowers amino acid levels, and reduces oxidation, and amino acid-mediated augmentation of the inhibition of degradation, a stimulation of synthesis, and an increase in oxidation when leucine dietary supply exceeds the capacity for its net deposition.

Body composition measurement in elite heavyweight oarswomen: a comparison of five methods.

The study was designed to evaluate the range of body composition in elite heavyweight oarswomen as well as the level of agreement between various methods used to measure this variable. Percent body fat was determined at the start of the competitive season by densitometry, taken to represent the reference standard, and measurement of total body potassium, skinfold thicknesses, bioelectrical impedance analysis and body mass index. The athletes were studied within a two week period with all measurements in any individual taken during one morning. We demonstrated a surprisingly large range of percent fat between these oarswomen, 13.6 to 29.3% by densitometry, which was a feature common to all methods. Percent body fat by total body potassium was lower (p < 0.05) while from body mass index higher (p < 0.01) than the reference value from densitometry. Similar methodologies generated significantly different estimates of % fat (SFT1 versus SFT2, p < 0.01 and BIAv versus BIAB, p < 0.01) highlighting the potential problems that may arise with the use of different regression equations to convert primary measurements into % fat. The limits of agreement between various methods were wide and reflect the large variability about the estimated mean bias. Practically this negates the correction of "non reference" values by adding or subtracting the mean difference or bias between the techniques in individuals. These methodological problems need to be considered when setting specific body composition targets for an athlete.

The effects of two different periods of weight-reduction on selected performance parameters in elite lightweight oarswomen.

Six successful members of the British Women's Lightweight Rowing Team were assessed before and after two-month (1990) and four-month (1991) periods of weight-reduction controlled by reduced caloric intake, while engaged in their normal physical training. Fat free mass (FFM) was calculated from body weight (BW) by utilising total body potassium measurements. Maximal oxygen intake (VO2max), respiratory anaerobic threshold (Tvent), upper body anaerobic peak power (PP) and mean power (MP) outputs, and knee flexor (KF) and extensor (KE) isokinetic peak torques were among the physiological parameters assessed. No statistical differences were noted between the data obtained prior to the two weight-reduction periods, and both periods resulted in lower BW (p < 0.001) and FFM (p < 0.05); approximately 50% of the lost BW was FFM. At the end of the two-month weight-reduction period Tvent (p < 0.02) and KF (p < 0.02) decreased. In contrast, a similar BW loss during the four-month period was associated with higher VO2max (p < 0.01) and PP (p < 0.05) compared with values prior to weight reduction. Comparisons between the percentage changes pre to post BW loss showed that the longer weight-reduction period was associated with significantly improved VO2max (p < 0.01), Tvent (p < 0.005), PP (p < 0.05) and KF (p < 0.05). We conclude: a) the proportion (50%) of weight lost as FFM in the present elite rowers is higher than the suggested optimal figure of 22%, and b) compared to four months, 6-7% of BW loss over two months may adversely influence fitness-related parameters in international lightweight oarswomen.

Nitrogen homoeostasis in man: diurnal changes in nitrogen excretion, leucine oxidation and whole body leucine kinetics during a reduction from a high to a moderate protein intake.

1. The adaptation of the diurnal cycle of nitrogen (N) homeostasis during a change in protein intake was investigated with diurnal measurements of N and leucine balance and turnover during a reduction from a high to a moderate protein intake in normal adults. 2. In experiment 1, during a 9 day period after a reduction from 1.82 to 0.77 g of protein day-1 kg-1, N excretion fell slowly at a similar rate in fed and fasted states so that the lowered intake was unable to replete any of the postabsorptive losses for 3 days. There was a marked negative N balance, which persisted throughout the study, although with a significant reduction in N losses in both fed and fasted states on day 4, balances during days 4-9 (-32.8 +/- 28.3 mg of N day-1 kg-1) were less negative than during days 1-3 (-79.1 +/- 60.4 mg of N day-1 kg-1). 3. In experiment 2, during a 14 day period after a reduction from 1.89 to 0.77 g of protein day-1 kg-1, [1-13C]leucine oxidation and turnover were measured by primed intravenous infusion, during fasting and feeding in subjects before and on days 3, 7 and 14 after the dietary change. Leucine oxidation fell by 32% (P < 0.05) on day 3 in the fed state and by 12% (P < 0.05) during fasting, falling further in each case by day 7 with improved balance.(ABSTRACT TRUNCATED AT 250 WORDS)

Nitrogen homeostasis in man: the diurnal responses of protein synthesis and degradation and amino acid oxidation to diets with increasing protein intakes.

1. The diurnal changes in whole body protein turnover associated with the increasing fasting body nitrogen (N) losses and feeding gains with increasing protein intake were investigated in normal adults. [13C]Leucine, [2H5]phenylalanine and [2H2]tyrosine kinetics were measured during an 8 h primed, continuous infusion during the fasting and feeding phase together with fed-state N turnover assessed with [15N]glycine after 12 days of adaptation to diets containing 0.36 (LP), 0.77 (MP), 1.59 (GP) and 2.07 (HP) g of protein day-1 kg-1. Measurements were also made of fasting and fed resting metabolic rate and plasma hormone levels. 2. Resting metabolic rate in the fasted and fed state was not influenced by dietary protein intake, but was increased by feeding (11-13%, P < 0.01) with no influence of dietary protein concentration. Fasting plasma insulin levels were not influenced by protein intake and were increased by feeding independent of protein intake. Fasted but not fed values of insulin-like growth factor-1 increased with protein intake, although no feeding response was observed. Thyroid hormones (free and total tri-iodothyronine) did not change in any state. 3. For leucine with increasing protein intake the increasing fasting losses reflected increasing rates of protein degradation, although the changes were small and only significant between GP and MP intakes. The increasing leucine gain on feeding was associated with increasing rates of protein synthesis and falling rates of protein degradation, reflecting a progressive inhibition of degradation with feeding, and a change from inhibition of synthesis (LP diet) to stimulation (GP and HP diets). Mean daily rates of synthesis and degradation did not change with protein intake. 4. Phenylalanine and tyrosine kinetics were calculated from adjusted values based on leucine kinetics and published data of the hepatic/plasma enrichment ratio. With the increased protein intake, the increasing fasting losses of phenylalanine (GP > MP) were mediated by increasing rates of degradation (paired t-tests). The increasing phenylalanine gain (GP > MP > LP) was due to increasing fed-state rates of synthesis and falling rates of degradation, reflecting a progressive inhibition of degradation, a stimulation of hydroxylation and a variable response of synthesis ranging from inhibition at the lowest intake to stimulation at higher intakes. For tyrosine a similar progressive inhibition of degradation with intake was shown. Mean daily rates of synthesis and degradation (phenylalanine) and degradation (tyrosine) did not change with protein intake.(ABSTRACT TRUNCATED AT 400 WORDS)

Nitrogen homeostasis in man: influence of protein intake on the amplitude of diurnal cycling of body nitrogen.

1. The diurnal nature of nitrogen (N) homoeostasis was investigated in adults fed increasing protein intakes. N balance was estimated during a 48 h period of consecutive 12 h periods of feeding hourly meals and fasting, after 12 days of adaptation to diets containing 0.36 +/- 0.01, 0.77 +/- 0.03, 1.59 +/- 0.08 and 2.31 +/- 0.65 g of protein day-1 kg-1. N losses were determined from measured urinary N excretion corrected for changes in the body urea pool, and estimated faecal and miscellaneous losses. [13C]Leucine and [2H5]phenylalanine balances were measured during a primed, continuous infusion of the two amino acids during the fasting and feeding phase on the second day. 2. Increasing fasting N losses were observed (47 +/- 7, 60 +/- 6, 95 +/- 15 and 140 +/- 36 mg day-1 kg-1) on the four intakes, with corresponding increasing fed gains of 8.2 +/- 3.9, 40.2 +/- 7.1, 112 +/- 24 and 180 +/- 56 mg day-1 kg-1. 3. Increasing fed-state amino acid gains with increasing protein intake were observed with both [13C]leucine and [2H5]phenylalanine, whereas increasing fasting amino acid losses were confirmed with [13C]leucine. 4. The N equivalent of the leucine oxidation rate was mostly in the range of 10-50% lower than expected from the N excretion rates. This may reflect the timing of the amino acid balance measurements and non-uniform rates of gain and loss throughout the diurnal cycle. 5. We conclude on the basis of both N and amino acid balances that the amplitude of the diurnal cycling of body protein N in human adults increases with increasing dietary protein intake. Thus one component of the protein requirement for N balance reflects a demand for repletion of fasting losses which increases with increasing habitual protein intake.

Metabolism of apolipoprotein B-100 and of triglyceride-rich lipoprotein particles in the absence of functional lipoprotein lipase.

Cholesterol and triglyceride in the various lipoprotein fractions were determined in five patients without functional lipoprotein lipase (LPL) while on their habitual therapeutic diet of 'low fat' content (20-25 g/day). They were also studied following 3 days on either a 'minimal fat' diet (< 15 g/day) or a 'moderate fat' diet (45-50 g/day). Values obtained were compared with the respective levels measured in five control subjects on a 'normal fat' (70-90 g/day) diet. The patients had hypertriglyceridaemia (type V hyperlipoproteinaemia) under all dietary conditions. Cholesterol and triglyceride levels in plasma and in the chylomicron fraction increased in the patients with increasing dietary fat. In the very low density lipoprotein (VLDL) fraction from the patients, triglyceride levels also increased with the dietary fat intake, but cholesterol levels were similar under all dietary conditions. In the patients, cholesterol concentrations in the low (LDL) and high density (HDL) lipoprotein fractions were significantly lower than the respective levels in controls, but the ratio of cholesterol to triglyceride levels in both of these lipoprotein fractions decreased with the dietary fat intake. VLDL apolipoprotein B-100 (apo B-100) pool size was similar in the patients on the two test diets (P = 0.95) and 3.5-fold higher than in five healthy volunteers on a normal fat diet. Using a stable isotope enrichment method, the kinetics of apo B-100 were investigated in the patients under the last two dietary conditions. The fractional and absolute secretion rates of the apolipoprotein in the patients did not vary with fat intake, but fractional secretion rates were significantly lower and the absolute secretion rates were significantly higher in the patients than the respective values in the controls. These results are consistent with the hypothesis that in the absence of LPL activity the metabolism of chylomicron and VLDL particles in the circulation results in triglyceride-rich LDL and HDL particles that are taken up by the liver at increased rates, thus reducing the plasma LDL and HDL cholesterol concentrations, whereas the products of hydrolysis of these particles induce an increased rate of synthesis of triglyceride and an increased rate of secretion of VLDL apo B-100.

Risk factors for peripheral vascular disease in hypertensive subjects with type 2 diabetes mellitus.

Possible factors predisposing to peripheral vascular disease (PVD) in hypertensive subjects with Type 2 diabetes mellitus were studied. Details of age, sex, duration of diabetes, blood pressure, and smoking habit were recorded in 180 subjects of either White, West Indian Black or Asian ethnic origin. Glycosylated haemoglobin, fasting serum total cholesterol, total high density lipoprotein (HDL), HDL2, low density lipoprotein (LDL-cholesterol), and triglycerides were measured in all subjects. Peripheral vascular disease was defined as an ankle/brachial systolic pressure < 1.0 as measured by the Doppler technique. Multivariate analysis was performed and the following factors were identified as being strongly associated with the presence of PVD with a statistical significance of p < 0.001; LDL-cholesterol, total HDL-cholesterol, age, male sex, diet or oral hypoglycaemic therapy, diastolic blood pressure, and of p < 0.003; systolic blood pressure. When blood pressure was excluded from the analysis the other factors retained their predictive value. We conclude that hypertension and dyslipidaemia are important risk factors for peripheral vascular disease in Type 2 diabetes mellitus.