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1.
We recently reported that in light exercise (30% VO2max) the oxidation of [1-13C]leucine was significantly increased but the rate of urea production was unchanged (J. Appl. Physiol: Respirat. Environ. Exercise Physiol. 52: 458-466, 1982). We have therefore tested three possible explanations for this apparent incongruity. 1) We infused NaH13CO3 throughout rest and exercise and found that, although altered bicarbonate kinetics in exercise resulted in greater recovery of 13CO2, the difference between rest and recovery was small compared with the increase in the rate of 13CO2 excretion during exercise when [1-13C]leucine was infused. 2) We infused [15N]leucine and isolated plasma urea N to determine directly the rate of incorporation of the 15N. During exercise there was no increase in the rate of 15N incorporation. Simultaneously, we infused [2,3-13C]alanine and quantified the rate of incorporation of 15N in alanine. We found that [15N]alanine production from [15N] leucine more than doubled in exercise, and by deduction, alanine production from other amino acids also doubled. 3) We tested our previous assumption that [1-13C]leucine metabolism in exercise was representative of the metabolism of other essential amino acids by infusing [1-13C] and [alpha-15N]lysine throughout rest and exercise. We found that the rate of breakdown of lysine during exercise was not increased in a manner comparable to that of leucine. Thus, these data confirm our original findings that leucine decarboxylation is enhanced in light exercise but urea production is unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

2.
We compared kinetic indices of pulmonary surfactant metabolism in premature infants (n = 41) with respect to i) tracer ([1-(13)C1]acetate, [U-(13)C6]glucose, and [1,2,3,4-(13)C4] palmitate), ii) phospholipid (PL) pool (total PLs or disaturated PLs), or iii) instrumentation [gas chromatography/mass spectrometry (GC/MS) or GC-combustion-isotope ratio mass spectometry (GC-C-IRMS)]. Tracer incorporation was measured in PLs extracted from serial tracheal aspirates after a 24 h tracer infusion. The fractional catabolic rate (FCR), representing the total fractional turnover from all sources of surfactant production, was independent of tracer. The fractional synthesis rate of surfactant PL from plasma palmitate was significantly higher than that from palmitate synthesized de novo from acetate, and these two sources of palmitate together accounted for only half of the total surfactant production in preterm infants. [U-(13)C6]glucose showed significant recycling of the (13)C label in intermediary metabolism, distinguishable by GC-MS but not by GC-C-IRMS, resulting in a slower apparent FCR when GC-C-IRMS was used. The extracted PL pool did not affect the surfactant metabolic indices. We suggest that FCR should be used as a primary measure of surfactant turnover kinetics and that tracers labeling both de novo synthesis (acetate and glucose) and preformed pathways (plasma palmitate) can be used to partition the fractional contribution of each pathway to total production.  相似文献   

3.
The source of nitrogen (N) for the de novo synthesis of brain glutamate, glutamine and GABA remains controversial. Because leucine is readily transported into the brain and the brain contains high activities of branched-chain aminotransferase (BCAT), we hypothesized that leucine is the predominant N-precursor for brain glutamate synthesis. Conscious and unstressed rats administered with [U-13C] and/or [15N]leucine as additions to the diet were killed at 0-9 h of continuous feeding. Plasma and brain leucine equilibrated rapidly and the brain leucine-N turnover was more than 100%/min. The isotopic dilution of [U-13C]leucine (brain/plasma ratio 0.61 +/- 0.06) and [15N]leucine (0.23 +/- 0.06) differed markedly, suggesting that 15% of cerebral leucine-N turnover derived from proteolysis and 62% from leucine synthesis via reverse transamination. The rate of glutamate synthesis from leucine was 5 micro mol/g/h and at least 50% of glutamate-N originally derived from leucine. The enrichment of [5-15N]glutamine was higher than [15N]ammonia in the brain, indicating glial ammonia generation from leucine via glutamate. The enrichment of [15N]GABA, [15N]aspartate, [15N]glutamate greater than [2-15N]glutamine suggests direct incorporation of leucine-N into both glial and neuronal glutamate. These findings provide a new insight for the role of leucine as N-carrier from the plasma pool and within the cerebral compartments.  相似文献   

4.
Immune cell functions can be evaluated in vivo by measuring their specific protein fractional synthesis rates (FSR). Using stable isotope dilution techniques, we describe a new method allowing simultaneous in vivo assessment of FSR in two leukocyte populations in healthy human subjects, using small blood samples. Peripheral blood mononuclear cell (PBMC) and polymorphonuclear neutrophil (PMN) FSR were measured during primed continuous intravenous infusion of L-[1-13C]leucine. Immune cells from 6 ml of whole blood were isolated by density gradient centrifugation. In a first study, we calculated the FSR using plasma [13C]leucine or -[13C]ketoisocaproate (KIC) enrichments as precursor pools. In a second study, we compared protein FSR in leukocytes, using enrichments of either intracellular or plasma free [13C]leucine as immediate precursor pools. The present approach showed a steady-state enrichment of plasma and circulating immune cell free [13C]leucine precursor pools. The linearity of labeled amino acid incorporation rate within mixed PBMC and PMN proteins also was verified. Postabsorptive protein FSR was 4.09 ± 0.39%/day in PBMC and 1.44 ± 0.08%/day in PMN when plasma [13C]KIC was the precursor pool. The difference between PBMC and PMN FSR was statistically significant, whatever the precursor pool used, suggesting large differences in their synthetic activities and functions. Use of the plasma [13C]KIC pool led to an underestimation of leukocyte FSR compared with the intracellular pool (PBMC: 6.04 ± 0.94%/day; PMN: 2.98 ± 0.30%/day). Hence, the intracellular free amino acid pool must be used as precursor to obtain reliable results. In conclusion, it is possible to assess immune cell metabolism in vivo in humans by using small blood samples to directly indicate their metabolic activity in various clinical situations and in response to regulating factors. peripheral blood mononuclear cells; polymorphonuclear neutrophils; protein metabolism; stable isotopes; leucine  相似文献   

5.
The hypothesis is proposed that the body contains a pool of protein turning over by lifetime rather than traditional first-order kinetics. The basis of the hypothesis is the observation of a step in the labelling curve or urinary ammonia during constant infusion of [15N]glycine. A four pool model has been constructed with different values for the rate of uptake of tracer into the lifetime pool; the calculated curves of tracer concentration show a step quite similar to that observed experimentally. It is concluded that it is possible, from an experimental curve, to derive an approximate estimate of the relative flux to the lifetime protein pool. Some suggestions are proposed for the physiological nature of this pool.  相似文献   

6.
Increased synthesis rate of fibrinogen, an independent risk factor for cardiovascular disease, was recently reported in obese insulin-resistant female adolescents with chronic elevated nonesterified fatty acids (NEFA). It is unknown whether a short-term change of NEFA concentrations controls hepatic fibrinogen synthesis. Therefore, 10 healthy male volunteers (24.5 +/- 3.3 yr, body mass index 23.5 +/- 2.9 kg/m2) were investigated in random order under basal and elevated NEFA for 8 h. Leucine metabolism, the fractional synthesis rates (FSR) of plasma fibrinogen, and endogenous urea production rates were measured during primed, continuous infusion of [1-13C]leucine and [15N2]urea, respectively. Plasma alpha-[13C]ketoisocaproic acid and [15N2]urea enrichment values were measured with GC-MS. Plasma fibrinogen was isolated with the beta-alanine method, and fibrinogen-related [13C]leucine enrichment was analyzed by GC-CIRMS. Lipofundin infusion and subcutaneous heparin tripled NEFA and triglycerides in the tests. Plasma glucose, circulating insulin, human C-peptide, and plasma glucagon were not changed by the study procedure. Fibrinogen FSR were significantly lower in tests with NEFA elevation (18.44 +/- 4.67%) than in control tests (21.48 +/- 4.32%; P < 0.05). Plasma fibrinogen concentrations measured were not significantly different (NEFA test subjects: 1.85 +/- 0.33, controls: 1.97 +/- 0.54 g/l). Parameters of leucine metabolism, such as leucine rate of appearance, leucine oxidation, and nonoxidative leucine disposal, were not influenced by NEFA elevation, and endogenous urea production remained unchanged. NEFA contributes to short-term regulation of fibrinogen FSR in healthy volunteers under unchanged hormonal status, leucine metabolism, and overall amino acid catabolism. Its contribution might be of relevance at least after fat-rich meals, counteracting by reduction of FSR the blood viscosity increase implied by hyperlipidemia.  相似文献   

7.
Little is known about amino acid (AA) and protein metabolism in lactating women. We hypothesized: 1) AA sources other than the plasma acid pool provide substrate for milk protein synthesis in humans and 2) if albumin was one such source, then albumin fractional synthesis rate (FSR) is higher in the lactating women. To test these hypotheses, six healthy exclusively breast-feeding women [27 +/- 3 yr; body mass index (BMI) 26 +/- 2 kg/m2] between 6 wk and 3 mo postpartum and six healthy nonlactating women (28 +/- 2 yr; BMI 22 +/- 1 kg/m2) were studied two times, in random order, during 22 h fasting or 10 h of continuous feeding with a mixed nutrient drink. Protein metabolism was determined using [1-13C]leucine and [15N2]urea. In both the fed and fasted states, a significant portion of milk protein (20 +/- 5 and 31 +/- 6%, respectively) was derived from sources other than the plasma free AA pool. A 70% higher (P < 0.02) FSR of albumin was observed in lactating women during feeding, suggesting that albumin is a likely source of AA for milk protein synthesis. We conclude that plasma free AA contribute only 70-80% of the substrate for milk protein synthesis in humans and that albumin may be a significant source of amino acids for the remainder.  相似文献   

8.
The goal of this study was to discover whether using different tracers affects the measured rate of muscle protein synthesis in human muscle. We therefore measured the mixed muscle protein fractional synthesis rate (FSR) in the quadriceps of older adults during basal, postabsorptive conditions and mixed meal feeding (70 mg protein x kg fat-free mass(-1) x h(-1) x 2.5 h) by simultaneous intravenous infusions of [5,5,5-(2)H(3)]leucine and either [ring-(13)C(6)]phenylalanine or [ring-(2)H(5)]phenylalanine and analysis of muscle tissue samples by gas chromatography-mass spectrometry. Both the basal FSR and the FSR during feeding were approximately 20% greater (P < 0.001) when calculated from the leucine labeling in muscle tissue fluid and proteins (fasted: 0.063 +/- 0.005%/h; fed: 0.080 +/- 0.007%/h) than when calculated from the phenylalanine enrichment data (0.051 +/- 0.004 and 0.066 +/- 0.005%/h, respectively). The feeding-induced increase in the FSR ( approximately 20%; P = 0.011) was not different with leucine and phenylalanine tracers (P = 0.69). Furthermore, the difference between the leucine- and phenylalanine-derived FSRs was independent of the phenylalanine isotopomer used (P = 0.92). We conclude that when using stable isotope-labeled tracers and the classic precursor product model to measure the rate of muscle protein synthesis, absolute rates of muscle protein FSR differ significantly depending on the tracer amino acid used; however, the anabolic response to feeding is independent of the tracer used. Thus different precursor amino acid tracers cannot be used interchangeably for the evaluation of muscle protein synthesis, and data from studies using different tracer amino acids can be compared qualitatively but not quantitatively.  相似文献   

9.
1. A formula is proposed for calculating fractional synthesis rates of liver-produced plasma proteins that dispenses with urinary information or information about the size of the urea pool in the body or the fraction of urea that is endogenously catabolized. 2. Synthesis rates obtained for albumin and fibrinogen agreed well with corresponding catabolic rates for the 131I-labelled proteins except in two of the fibrinogen measurements. 3. Significant reutilization of 14C occurs in some animals after [14C]carbonate injections, giving rise to errors in the calculation of protein synthesis rates. These can best be avoided by using results obtained by injecting [13C]urea simultaneously. [15N]urea is shown not to be satisfactory for this purpose.  相似文献   

10.
To determine whether glutamine affects glutathione (GSH, gamma-glutamyl-cysteinyl-glycine) metabolism, seven healthy beagle dogs received 6-h infusions of [(15)N]glutamate and [(13)C]leucine after a 3-day fast. Isotope infusions were performed during oral feeding with an elemental regimen, supplemented with either l-glutamine or an isonitrogenous amino acid mixture, on two separate days and in randomized order. Timed blood samples were obtained, and a surgical duodenal biopsy was performed after 6 h of isotope infusion. GSH fractional synthesis rate (FSR) was assessed from [(15)N]glutamate incorporation into blood and gut GSH, and duodenal protein synthesis from [(13)C]leucine incorporation into gut protein. Glutamine supplementation failed to alter erythrocyte GSH concentration (2189+/-86 vs. 1994+/-102 micromol L(-1) for glutamine vs. control; ns) or FSR (64+/-17% vs. 74+/-20% day(-1); ns). In the duodenum, glutamine supplementation was associated with a 92% rise in reduced/oxidized GSH ratio (P=.024) and with a 44% decline in GSH FSR (96+/-15% day(-1) vs. 170+/-18% day(-1); P=.005), whereas total GSH concentration remained unchanged (808+/-154 vs. 740+/-127 micromol kg(-1); P=.779). We conclude that, in dogs receiving enteral nutrition after a 3-day fast: (1) glutamine availability does not affect blood GSH, and, (2) in contrast, in the duodenum, the preserved GSH pool, along with a decreased synthesis rate, suggests that glutamine may maintain GSH pool and intestinal redox status by acutely decreasing GSH utilization.  相似文献   

11.
The aims of this study were to compare different tracer methods to assess whole body protein turnover during 6 h of prolonged endurance exercise when carbohydrate was ingested throughout the exercise period and to investigate whether addition of protein can improve protein balance. Eight endurance-trained athletes were studied on two different occasions at rest (4 h), during 6 h of exercise at 50% of maximal O2 uptake (in sequential order: 2.5 h of cycling, 1 h of running, and 2.5 h of cycling), and during subsequent recovery (4 h). Subjects ingested carbohydrate (CHO trial; 0.7 g CHO.kg(-1.)h(-1)) or carbohydrate/protein beverages (CHO + PRO trial; 0.7 g CHO.kg(-1).h(-1) and 0.25 g PRO.kg(-1).h(-1)) at 30-min intervals during the entire study. Whole body protein metabolism was determined by infusion of L-[1-13C]leucine, L-[2H5]phenylalanine, and [15N2]urea tracers with sampling of blood and expired breath. Leucine oxidation increased from rest to exercise [27 +/- 2.5 vs. 74 +/- 8.8 (CHO) and 85 +/- 9.5 vs. 200 +/- 16.3 mg protein.kg(-1).h(-1) (CHO + PRO), P < 0.05], whereas phenylalanine oxidation and urea production did not increase with exercise. Whole body protein balance during exercise with carbohydrate ingestion was negative (-74 +/- 8.8, -17 +/- 1.1, and -72 +/- 5.7 mg protein.kg(-1).h(-1)) when L-[1-13C]leucine, L-[2H5]phenylalanine, and [15N2]urea, respectively, were used as tracers. Addition of protein to the carbohydrate drinks resulted in a positive or less-negative protein balance (-32 +/- 16.3, 165 +/- 4.6, and 151 +/- 13.4 mg protein.kg(-1).h(-1)) when L-[1-13C]leucine, L-[2H5]phenylalanine, and [15N2]urea, respectively, were used as tracers. We conclude that, even during 6 h of exhaustive exercise in trained athletes using carbohydrate supplements, net protein oxidation does not increase compared with the resting state and/or postexercise recovery. Combined ingestion of protein and carbohydrate improves net protein balance at rest as well as during exercise and postexercise recovery.  相似文献   

12.
The production rates of apolipoprotein (apo)B-100 in very low density lipoprotein and in low density lipoprotein and apolipoprotein A-I in high density lipoprotein were determined using a primed-constant infusion of [5,5,5,-2H3]leucine, [4,4,4,-2H3]valine, and [6,6-2H2,1,2-13C2]lysine. The three stable isotope-labeled amino acids were administered simultaneously to determine whether absolute production rates calculated using a stochastic model were independent of the tracer species utilized. Three normolipidemic adult males were studied in the constantly fed state over a 15-h period. The absolute production rates of very low density lipoprotein apoB-100 were 11.4 +/- 5.8 (leucine), 11.2 +/- 6.8 (valine), and 11.1 +/- 5.4 (lysine) mg per kg per day (mean +/- SDM). The absolute production rates for low density lipoprotein apoB-100 were 8.0 +/- 4.7 (leucine), 7.5 +/- 3.8 (valine), and 7.5 +/- 4.2 (lysine) mg per kg per day. The absolute production rates for high density lipoprotein apoA-I were 9.7 +/- 0.2 (leucine), 9.4 +/- 1.7 (valine, and 9.1 +/- 1.3 (lysine) mg per kg per day. There were no statistically significant differences in absolute synthetic rates of the three apolipoproteins when the plateau isotopic enrichment values of very low density lipoprotein apoB-100 were used to define the isotopic enrichment of the intracellular precursor pool. Our data indicate that deuterated leucine, valine, or lysine provided similar results when used for the determination of apoA-I and apoB-100 absolute production rates within plasma lipoproteins as part of a primed-constant infusion protocol.  相似文献   

13.
The branched chain amino acids (BCAA) valine, leucine and isoleucine have been implicated in a number of diseases including obesity, insulin resistance, and type 2 diabetes mellitus, although the mechanisms are still poorly understood. Adipose tissue plays an important role in BCAA homeostasis by actively metabolizing circulating BCAA. In this work, we have investigated the link between BCAA catabolism and fatty acid synthesis in 3T3-L1 adipocytes using parallel 13C-labeling experiments, mass spectrometry and model-based isotopomer data analysis. Specifically, we performed parallel labeling experiments with four fully 13C-labeled tracers, [U-13C]valine, [U-13C]leucine, [U-13C]isoleucine and [U-13C]glutamine. We measured mass isotopomer distributions of fatty acids and intracellular metabolites by GC-MS and analyzed the data using the isotopomer spectral analysis (ISA) framework. We demonstrate that 3T3-L1 adipocytes accumulate significant amounts of even chain length (C14:0, C16:0 and C18:0) and odd chain length (C15:0 and C17:0) fatty acids under standard cell culture conditions. Using a novel GC-MS method, we demonstrate that propionyl-CoA acts as the primer on fatty acid synthase for the production of odd chain fatty acids. BCAA contributed significantly to the production of all fatty acids. Leucine and isoleucine contributed at least 25% to lipogenic acetyl-CoA pool, and valine and isoleucine contributed 100% to lipogenic propionyl-CoA pool. Our results further suggest that low activity of methylmalonyl-CoA mutase and mass action kinetics of propionyl-CoA on fatty acid synthase result in high rates of odd chain fatty acid synthesis in 3T3-L1 cells. Overall, this work provides important new insights into the connection between BCAA catabolism and fatty acid synthesis in adipocytes and underscores the high capacity of adipocytes for metabolizing BCAA.  相似文献   

14.
Exercising for 3.75 h on a treadmill at 50% VO2 max in the fed state induced an increased excretion of 71 mg nitrogen/kg over the 18 h after exercise. However, measurements of the time course of changes in 13CO2 excretion from ingested [1-13C]leucine indicated that all of this increased nitrogen production occurs during the exercise period. Because of the reduced renal clearance and slow turnover of the urea pool, urea excretion lags behind urea production. Measurements of nitrogen flux from the plateau labeling of urinary ammonia achieved by repeated oral doses of 15N-labeled glycine indicated that the nitrogen loss resulted from an increase in protein degradation and a decrease in protein synthesis. Further studies with [1-13C]leucine indicated that a 2-h treadmill exercise induced an increase in the nitrogen loss from 5.4 to 16 mg . kg-1 . h-1 measured with a primed constant infusion of [1-13C]leucine. This resulted from a fall in whole-body protein synthesis. Glucose given at the rate of 0.88 g . kg-1 . h-1 depressed the rate of whole-body protein degradation and appeared to suppress the exercise-induced increase in nitrogen excretion. When leucine oxidation rates were measured at increasing work rates, a linear relationship between percentage of VO2 max and leucine oxidation was observed up to 89% VO2 max when 54% of the flux of leucine was oxidized. These changes may involve nonmuscle as well as muscle tissue. Thus the source of the increased nitrogen losses is probably liver. In muscle, protein degradation is actually decreased judged by methylhistidine excretion, whereas in liver, protein degradation may be increased. Also the fall in whole-body protein synthesis may reflect changes in nonmuscle tissues because in running rats protein synthesis in muscle is maintained. As far as leucine metabolism is concerned, because the increase in leucine oxidation occurs when leucine and its keto acid concentration falls, exercise must specifically activate the 2-oxoacid dehydrogenase.  相似文献   

15.
Glutamine kinetics and its relation to transamination of leucine and urea synthesis were quantified in 16 appropriate-for-gestational-age infants, four small-for-gestational-age infants, and seven infants of diabetic mothers. Kinetics were measured between 4 and 5 h after the last feed (fasting) and in response to formula feeding using [5-(15)N]glutamine, [1-(13)C,(15)N]leucine, [(2)H(5)]phenylalanine, and [(15)N(2)]urea tracers. Leucine nitrogen and glutamine kinetics during fasting were significantly higher than those reported in adults. De novo synthesis accounted for approximately 85% of glutamine turnover. In response to formula feeding, a significant increase (P = 0.04) in leucine nitrogen turnover was observed, whereas a significant decrease (P = 0.002) in glutamine and urea rate of appearance was seen. The rate of appearance of leucine nitrogen was positively correlated (r(2) = 0.59, P = 0.001) with glutamine turnover. Glutamine flux was negatively correlated (r(2) = 0.39, P = 0.02) with the rate of urea synthesis. These data suggest that, in the human newborn, glutamine turnover is related to a high anaplerotic flux into the tricarboxylic acid cycle as a consequence of a high rate of protein turnover. The negative relationship between glutamine turnover and the irreversible oxidation of protein (urea synthesis) suggests an important role of glutamine as a nitrogen source for other synthetic processes and accretion of body proteins.  相似文献   

16.
We have developed a new method to determine the fractional synthesis rate (FSR) and breakdown rate (FBR) of muscle protein. This method involves a pulse tracer injection and measurement of enrichment in the arterial blood and muscle at three time points. The calculations of FSR and FBR are based on the precursor-product principle. To test this method, we gave a pulse injection of L-[ring-(13)C(6)]phenylalanine of 4-6 mg/kg in five rabbits. The measured FBR value (0.233 +/- 0.060%/h) was almost identical (P = 0.35) to that (0.217 +/- 0.078%/h) estimated from a leg arteriovenous balance model (Biolo G, Chinkes D, Zhang X-J, and Wolfe RR. J Parenter Enteral Nutr 16: 305-315, 1992). The measured FSR value tended to be lower than that estimated from the leg model (0.125 +/- 0.036 vs. 0.185 +/- 0.086%/h; P = 0.14), possibly because the new method measures only muscle FSR, whereas the leg balance model also includes skin and bone contributions. The pulse tracer injection did not affect muscle protein kinetics as measured by leucine and phenylalanine kinetics in the leg. In another five rabbits, we demonstrated that sampling could be reduced to either one or two muscle biopsies when multiple pulse injections were used. This method can be completed in 1 h with one muscle biopsy and has technical advantages over currently used methods.  相似文献   

17.
Ketone body kinetics in humans: a mathematical model   总被引:2,自引:0,他引:2  
A model has been developed to account for ketone body kinetics in man based on data following bolus injections of [14C]acetoacetate (A) and [14C]beta-OH butyrate (B) into normal humans in the postabsorptive state. The model consists of separate compartments for blood A and B that are linked by a tissue compartment in which rapid interconversion of the ketone bodies occurs. The probability of movement from blood into this compartment was assumed to be the same for both ketone bodies. Two slowly equilibrating tissue compartments are required to account for the slow components in the tracer data, and thus a five-compartment model is proposed. By modeling the transient tracer data with the tracee in a steady state, ketone body kinetics were defined in terms of the rapid interconversions of A and B, and the slow exchanges of carbon within the tissues. The rates of release of new A and B into blood, (UA and UB) were calculated. These rates were less than the apparent production rates, PRA and PRB, as the PR's included carbon atoms first released as the other ketone body. The exchange constants between the compartments were determined in addition to the fractional catabolic rates (FCR) and metabolic clearance rates (MCR) of A and B. The initial space of distribution was 10 L and the mean values +/- SD (n = 11), normalized to this volume, were UA = 6.4 +/- 5.0, UB = 8.8 +/- 8.0 (mumol L-1 min-1), FCRA = 0.226 +/- 0.142, FCRB = 0.188 +/- 0.124 (min-1), MCRA = 2.26 +/- 1.42, MCRB = 1.87 +/- 1.23 (L min-1) and PRA = 11.1 +/- 7.6, PRB = 12.7 +/- 10.0 (mumol L-1 min-1).  相似文献   

18.
In two groups of five adults, each adapted to two different dietary regimens for 6 days, the metabolic fate of dietary [1-(13)C]leucine was examined when ingested either together with a mixture of free amino acids simulating casein (extrinsically labeled; condition A), along with the intact casein (extrinsically labeled; condition B), or bound to casein (intrinsically labeled; condition C). Fed state leucine oxidation (Ox), nonoxidative leucine disposal (NOLD), protein breakdown, and splanchnic uptake have been compared using an 8-h oral [1-(13)C]leucine and intravenous [(2)H(3)]leucine tracer protocol while giving eight equal hourly mixed meals. Lower leucine Ox, increased NOLD, and net protein synthesis were found with condition C compared with condition A (19.3 vs. 24.9; 77 vs. 55.8; 18.9 vs. 12.3 micromol. kg(-1). 30 min(-1); P < 0.05). Ox and NOLD did not differ between conditions B and C. Splanchnic leucine uptake calculated from [1-(13)C]- and [(2)H(3)]leucine plasma enrichments was between 24 and 35%. These findings indicate that the form in which leucine is consumed affects its immediate metabolic fate and retention by the body; the implications of these findings for the tracer balance technique and estimation of amino acid requirements are discussed.  相似文献   

19.
The preparation of leucine and isoleucine labeled with 15N and of site-specific 13C-labeled isoleucines is described. This method is based on the induction of the biosynthetic pathways specific for branched chain amino acids in glutamic acid producing bacteria, and controlled provision of stable isotope labeled precursors. Corynebacterium glutamicum (ATCC 13032), a glutamic acid overproducer, was incubated in leucine production medium which consisted of a basal medium supplemented with [15N]ammonium sulfate, glucose, and sodium alpha-ketoisocaproate. production of L-[15N]leucine reached 138 mumol/ml at an isotopic efficiency of 90%. It was purified and checked by proton NMR and GC-MS. The electron impact (EI) spectrum showed 95 atom% enrichment. The cultivation of C. glutamicum in a similar medium containing alpha-ketobutyrate yielded L-[15N]isoleucine at a concentration of 120 mumol/ml. The GC-MS EI and chemical ionization (CI) spectra confirmed enrichment of 96 atom% 15N as that of the labeled precursors. The biosynthesis of L-[13C]isoleucine was carried out by induced cells which were transferred to a similar medium in which [2-13C]- or [3-13C]pyruvic acid replaced glucose. 13C NMR of the product isoleucine revealed single-site enrichment at C-3 or at C-3' respective to the precursor [13C]pyruvate; i.e., C-3 was labeled from [2-13C]pyruvate and C-3' from [3-13C]pyruvate. Mass spectrometric analysis confirmed that all molecules were labeled only in one carbon. This site-specific incorporation of [13C]pyruvate is contrasted with the labeling pattern obtained when producing cells were supplied with [2-13C]acetate, instead of pyruvate, when most label was incorporated into carbons 3 and 3' of the same isoleucine molecule.  相似文献   

20.
Combined experiments of an isotope dilution method of [1-(13)C]leucine with open circuit calorimetry and a nitrogen (N) balance test were applied to determine the effect of dietary crude protein (CP) intake on plasma leucine flux and protein synthesis and degradation in four sheep. The experiment was conducted in a 3 x 4 Latin rectangle design of three 3-week periods. Dietary CP intake was 5.6, 7.7, and 10.8 g/(kg(0.75) x d). Metabolizable energy intake was 120% of requirement for all dietary treatments. [1-(13)C]Leucine was intravenously infused for 8 h and blood and breath samples were collected during the latter 2-h period of infusion. Isotopic enrichments of plasma [1-(13)C]leucine, alpha-[1-(13)C]ketoisocaproic acid, and exhaled (13)CO(2) were determined. For the N balance test, N digestibility, N excretion in urine, and protein balance (N x 6.25) increased with increasing dietary CP intake. Rates of plasma leucine turnover, protein synthesis, and degradation changed toward reduction with increased dietary CP intake. It is likely that in sheep, high CP intake enhances protein deposition with reduced protein degradation rather than increased protein synthesis.  相似文献   

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