Deamination Of Leucine Research Articles

A High-Fiber Diet Decreases Postabsorptive Protein Turnover but Does Not Alter Insulin Sensitivity in Men with Type 1 Diabetes Mellitus

High-fiber diets (HFDs) are recommended in the diet of persons with diabetes, yet such diets can impair macronutrient digestion and/or absorption, modify insulin sensitivity, and reset metabolism. We studied the effects of a HFD on the kinetics of whole-body protein, a macronutrient that could be affected by dietary fiber, in type 1 diabetes mellitus (T1DM), under both basal-low insulinemic and hyperinsulinemic conditions. Eight men with T1DM (body mass index range: 21.8-27.8kg/m2) were studied twice - before and after the addition of guar gum (∼15 g/d) to their usual diet for ∼4 mo. Whole-body protein degradation (i.e., the rate of appearance [Ra] of endogenous leucine), leucine disposal to protein synthesis (PS), deamination, and reamination, were determined before and after the HFD, both in the postabsorptive state and following a euglycemic, hyperinsulinemic, hyperaminoacidemic clamp, using isotope dilution methods. After the HFD, mean values (± SEs) for postabsorptive leucine Ra decreased by ∼20%: from 2.52 (0.15) to 2.03 (0.16) μmol x kg-1 x min-1, P<0.049, after vs. before the HFD respectively. PS also decreased, by ∼25%: from 2.03 (0.15) to 1.57 (0.15), P<0.045. Leucine concentration (P=0.1) and reamination (P=0.095) decreased moderately, whereas deamination was unchanged. Following the clamp, plasma amino acid concentrations (P<0.001), leucine deamination (+ ∼50%, P<0.00002), reamination (+ ∼30%, P<0.0007), and PS (+ ∼35%, P<0.00001) were all increased compared with postabsorptive state values, whereas endogenous leucine Ra was suppressed (by 15%, P<0.00001, and by 25%, P<0.001, with the primary or the reciprocal pool models, respectively). No significant differences in these insulin effects before compared with after the HFD were observed. Metabolic control (glycated hemoglobin), daily insulin requirement, and insulin-mediated glucose disposal were unchanged after the HFD. A HFD downregulates postabsorptive protein turnover in men with T1DM, by decreasing both protein degradation and synthesis, possibly due to a subtle decrease and/or delay in amino acid absorption. It does not significantly affect the insulin (and amino acid sensitivity) to protein turnover, glucose disposal, and metabolic control.

Leucine Transamination Is Lower in Middle-Aged Compared with Younger Adults

Background: Insulin and age affect leucine (and protein) kinetics in vivo. However, to our knowledge, leucine transamination and the effects of insulin have not been studied in participants of different ages.Objective: The aims of the study were to measure whole-body leucine deamination to α-ketoisocaproate (KIC) and KIC reamination to leucine in middle-aged and younger healthy adults, both in the postabsorptive state and after hyperinsulinemia.Methods: Younger (mean ± SE age: 26 ± 2 y) and middle-aged (54 ± 3 y) healthy men and women were enrolled. Isotope dilution methods with 2 independent leucine and KIC tracers, a dual isotope model and the euglycemic, hyperinsulinemic clamp technique, were used.Results: Leucine deamination [expressed as μmol/(kg × min)] was consistently greater than KIC reamination. In middle-aged adults, postabsorptive leucine deamination (0.77 ± 0.05), reamination (0.49 ± 0.04), and net deamination (0.28 ± 0.04) were ∼30% lower than in the younger group (deamination: 1.12 ± 0.07; reamination: 0.70 ± 0.09; net deamination: 0.42 ± 0.04) (P < 0.002, P < 0.05, and P < 0.015, respectively). After the hyperinsulinemic clamp, plasma leucine and KIC concentrations were reduced by ∼50% in both groups. Deamination and reamination also were suppressed by ∼40-50% in both groups (P < 0.001); however, they remained lower [-35% (P = 0.02) and -25% (P = 0.036), respectively] in the middle-aged than in the younger participants. The leucine rate of appearance and its suppression by insulin were similar in the middle-aged and in the younger subjects. By using both the basal and the clamp data, deamination was directly correlated with the plasma leucine concentration (r = 0.61, P < 0.0025) and reamination to that of plasma KIC (r = 0.79, P < 0.00002). Expressing the data relative to lean body mass did not substantially alter the results.Conclusions: Leucine deamination and reamination are lower in middle-aged than in younger adults, both in the postabsorptive and in the insulin-stimulated state. In middle age, a decreased net leucine transamination may represent a mechanism to spare this essential amino acid.

The contribution of muscle, kidney, and splanchnic tissues to leucine transamination in humans.

The first steps of leucine utilization are reversible deamination to α-ketoisocaproic acid (α-KIC) and irreversible oxidation. Recently, the regulatory role of leucine deamination over oxidation was underlined in rodents. Our aim was to measure leucine deamination and reamination in the whole body, in respect to previously determined rates across individual organs, in humans. By leucine and KIC isotope kinetics, we determined whole-body leucine deamination and reamination, and we compared these rates with those already reported across the sampled organs. As an in vivo counterpart of the "metabolon" concept, we analysed ratios between oxidation and either deamination or reamination. Leucine deamination to KIC was greater than KIC reamination to leucine in the whole body (p = 0.005), muscles (p = 0.005), and the splanchnic area (p = 0.025). These rates were not significantly different in the kidneys. Muscle accounted for ≈60% and ≈78%, the splanchnic bed for ≈15% and ≈15%, and the kidney for ≈12% and ≈18%, of whole-body leucine deamination and reamination rates, respectively. In the kidney, percent leucine oxidation over either deamination or reamination was >3-fold greater than muscle and the splanchnic bed. Skeletal muscle contributes by the largest fraction of leucine deamination, reamination, and oxidation. However, in relative terms, the kidney plays a key role in leucine oxidation.

Kinetics and Mechanistic Approach to Palladium (II)-Catalyzed Oxidative Deamination and Decarboxylation of Leucine and Isoleucine by Anticancer Platinum (IV) Complex in Perchlorate Solutions

Oxidations of two aliphatic α-amino acids (AA), namely, leucine and isoleucine by hexachloroplatinate (IV) as an anticancer platinum (IV) complex has been studied using a spectrophotometric technique in perchlorate solutions in the presence of palladium (II) catalyst at a constant ionic strength of 1.0 mol dm-3 and at 25°C. The reactions did not proceed in the absence of the catalyst. The reactions of both amino acids showed a first order dependence on both [PtIV] and [PdII], and less than unit order dependences with respect to both [AA] and [H+]. Increasing ionic strength and dielectric constant of the reactions medium increased the rates of the reactions. A probable oxidations mechanism has been suggested and the rate law expression has been derived. Both spectral and kinetic evidences revealed formation of 1:1 intermediate complexes between AA and PdII before the rate-controlling step. The oxidation products of the investigated amino acids were identified as the corresponding aldehyde, ammonium ion and carbon dioxide. The activation parameters of the second order rate constants were evaluated and discussed

Diverse functional evolution of serine decarboxylases: identification of two novel acetaldehyde synthases that uses hydrophobic amino acids as substrates.

BackgroundType II pyridoxal 5′-phosphate decarboxylases are an important group of phylogenetically diverse enzymes involved in amino acid metabolism. Within plants, this group of enzymes is represented by aromatic amino acid decarboxylases, glutamate decarboxylases and serine decarboxylases. Additional evolutionary divergence of plant aromatic amino acid decarboxylases has resulted in further subcategories with distinct substrate specificities and enzymatic activities. Despite shared homology, no such evolutionary divergence has been characterized within glutamate decarboxylases or serine decarboxylases (SDC).ResultsComparative analysis of two previously characterized serine decarboxylase-like (SDC-like) enzymes demonstrates distinct substrate specificities despite their highly conserved primary sequence. The alternate substrate preference of these homologous SDC-like proteins indicated that functional divergence might have occurred with in SDC-like proteins. In an effort to identify additional SDC-like functional divergence, two uncharacterized SDC-like enzymes were recombinantly expressed and characterized.ConclusionsAn extensive biochemical analysis of two serine decarboxylases-like recombinant proteins led to an interesting discovery; both proteins catalyze the formation of acetaldehyde derivatives from select hydrophobic amino acids substrates. Specifically, Medicago truncatula [GenBank: XP_003592128] and Cicer arietinum [GenBank: XP_004496485] catalyze the decarboxylation and oxidative deamination of phenylalanine, methionine, leucine and tryptophan to generate their corresponding acetaldehydes. The promiscuous aldehyde synthase activity of these proteins yields novel products of 4-(methylthio) butanal, 3-methylbutanal (isovaleraldehyde) and indole-3-acetaldehyde from methionine, leucine and tryptophan respectively. A comparative biochemical analysis of the Medicago truncatula and Cicer arietinum enzymes against two previously characterized SDC-like enzymes further emphasizes the unusual substrate specificity and activity of these novel aldehyde synthases. Due to the strong substrate preference towards phenylalanine, it is likely that both enzymes function as phenylacetaldehyde synthesis in vivo. However, due to their significant sequence divergence and unusual substrate promiscuity these enzymes are functionally and evolutionary divergent from canonical phenylacetaldehyde synthesis enzymes. This work further elaborates on the functional complexity of plant type II PLP decarboxylases and their roles in secondary metabolite biosynthesis.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-014-0247-x) contains supplementary material, which is available to authorized users.

Catabolism of leucine to branched-chain fatty acids in Staphylococcus xylosus.

Staphylococcus xylosus is an important starter culture in the production of flavours from the branched-chain amino acids leucine, valine and isoleucine in fermented meat products. The sensorially most important flavour compounds are the branched-chain aldehydes and acids derived from the corresponding amino acids and this paper intends to perspectivate these flavour compounds in the context of leucine metabolism. GC and GC/MS analysis combined with stable isotope labelling was used to study leucine catabolism. This amino acid together with valine and isoleucine was used as precursors for the production of branched-chain fatty acids for cell membrane biosynthesis during growth. A 83.3% of the cellular fatty acids were branched. The dominating fatty acid was anteiso-C(15:0) that constituted 55% of the fatty acids. A pyridoxal 5'-phosphate and alpha-ketoacid dependent reaction catalysed the deamination of leucine, valine and isoleucine into their corresponding alpha-ketoacids. As alpha-amino group acceptor alpha-keto-beta-methylvaleric acid and alpha-ketoisovaleric acid was much more efficient than alpha-ketoglutarate. The sensorially and metabolic key intermediate on the pathway to the branched-chain fatty acids, 3-methylbutanoic acid was produced from leucine at the onset of the stationary growth phase and then, when the growth medium became scarce in leucine, from the oxidation of glucose via pyruvate. This paper demonstrates that the sensorially important branched-chain aldehydes and acids are important intermediates on the metabolic route leading to branched-chain fatty acids for cell membrane biosynthesis. The metabolic information obtained is extremely important in connection with a future biotechnological design of starter cultures for production of fermented meat.

Crystallization and preliminary X-ray analysis of substrate complexes of leucine dehydrogenase from Thermoactinomyces intermedius.

Leucine dehydrogenase is an octameric enzyme which belongs to the superfamily of amino-acid dehydrogenases and catalyses the reversible oxidative deamination of leucine to 2-ketoisocaproate, with the corresponding reduction of the cofactor NAD(+). Catalysis by this enzyme is thought to involve a large-scale motion of the enzyme's two domains between an 'open' and 'closed' form, with the latter representing a conformation of the enzyme in which the partners involved in the hydride-transfer reaction are appropriately positioned for catalysis. Whilst a structure for the open form of the enzyme has been determined, the nature of the closed form has yet to be observed. In order to trap a closed form, crystals of the complexes of leucine dehydrogenase from Thermoactinomyces intermedius with 2-ketoisocaproate and with 2-ketoisocaproate and NAD(+) have been obtained by the hanging-drop vapour-diffusion method using PEG 4000 as a precipitant. The crystals of the binary complex with 2-ketoisocaproate belong to space group P2(1)2(1)2(1), with approximate unit-cell parameters a = 106, b = 118, c = 320 A and an octamer in the asymmetric unit, corresponding to a V(M) of 3.1 A(3) Da(-1). The crystals of the non-productive ternary complex belong to space group P6(1) or P6(5), with approximate unit-cell parameters a = b = 117, c = 502 A and an octamer in the asymmetric unit, corresponding to a V(M) of 3.0 A(3) Da(-1). These crystals diffract X-rays on a synchrotron-radiation source to at least 2.8 and 3.3 A resolution, respectively, and are suitable for a full structure determination.

Pregnancy, insulin resistance and nitrogen accretion.

Metabolic adaptation to pregnancy in humans and animals is aimed at provision of nutrients for the growth and energy metabolism of the growing conceptus, as well as for the mother. Kinetic studies in human pregnancy have shown that fluxes of energy-yielding substrates, i.e. glucose, fatty acids and glycerol, increase in parallel with the increasing demands of the fetus and the mother. Resistance to insulin action, measured by hyperinsulinemic euglycemic clamp, appears early in gestation and is correlated with the infant's birth weight. Adaptive responses in nitrogen metabolism, decreased plasma urea concentration and decreased rate of urea synthesis, are apparent early in pregnancy, much before any significant increase in fetal demands. Recent studies of branched chain aminoacid (leucine) kinetics show a lower flux of leucine nitrogen and an unchanged flux of leucine carbon in gestation. A linear correlation between rate of deamination of leucine and rate of urea synthesis was observed in pregnant women. It is speculated that decreased anaplerotic carbon flux in the tricarboxylic acid cycle, as a consequence of insulin resistance, may have an important role in the down-regulation of transamination of leucine during pregnancy, and may contribute to the conservation and accretion of nitrogen by the mother and the fetus.

Effects of branched-chain-enriched amino acids and insulin on forearm leucine kinetics

Although amino acid mixtures enriched in branched-chain amino acids (BCAA) and deficient in aromatic amino acids (AAA) are often used together with insulin and glucose in clinical nutrition, their physiological effects on muscle protein anabolism are not known. To this aim, we studied forearm leucine kinetics in post-absorptive volunteers, before and after the systemic infusion of BCAA-enriched, AAA-deficient amino acids along with insulin and the euglycaemic clamp. The results were compared with the effects of insulin infusion alone. A compartmental leucine forearm model was employed at steady state. Hyperaminoacidaemia with hyperinsulinaemia (to approximately 80-100 micro-units/ml) increased the leucine plasma concentration (+70%; P<0.001), inflow into the forearm cell (+150%; P<0.01), disposal into protein synthesis (+100%; P<0.01), net intracellular retention (P<0.01), net forearm balance (by approximately 6-fold; P<0.01) and net deamination to alpha-ketoisocaproate (4-methyl-2-oxopentanoate) (+9%; P<0.05). Leucine release from forearm proteolysis and outflow from the forearm cell were unchanged. In contrast, hyperinsulinaemia alone decreased plasma leucine concentrations (-35%; P<0.001) and leucine inflow (-20%; P<0.05) and outflow (-30%; P<0.01) into and out of forearm cell(s), it increased net intracellular leucine retention (P<0.03), and it did not change leucine release from forearm proteolysis (-20%; P=0.138), net leucine deamination to alpha-ketoisocaproate, leucine disposal into protein synthesis or net forearm protein balance. By considering all data together, leucine disposal into protein synthesis was directly correlated with leucine inflow into the cell (r=0.71; P<0.0001). These data indicate that the infusion of BCAA-enriched, AAA-deficient amino acids along with insulin is capable of stimulating forearm (i.e. muscle) protein anabolism in normal volunteers by enhancing intracellular leucine transport and protein synthesis. These effects are probably due to hyperaminoacidaemia and/or its interaction with hyperinsulinaemia, since they were not observed under conditions of hyperinsulinaemia alone.

Decreased pancreatic islet response to L-leucine in the spontaneously diabetic GK rat: enzymatic, metabolic and secretory data.

Pancreatic islets from hereditarily non-insulin-dependent diabetic Goto-Kakizaki (GK) rats have a deficient insulin response not only to D-glucose but also to L-leucine. Our aim was to explain the cellular mechanism(s) underlying the beta-cell unresponsiveness to this amino acid. Freshly collagenase isolated islets from GK rats and healthy Wistar control rats matched with them for sex and age were compared. Leucine uptake, metabolic fluxes and insulin secretory capacity were investigated on batch incubated-islets. Enzymatic activities were measured on sonicated islets. In GK rat islets, neither leucine transport nor leucine transaminase activity was disturbed. By contrast, 14CO2 production from either L-[U-14C]leucine or L-[1-14C]leucine was decreased. The L-[U-14C]leucine oxidation: L-[1-14C]leucine decarboxylation ratio was unaffected, indicating that the acetyl-CoA generated from leucine undergoes normal oxidation in the Krebs cycle. The leucine non-metabolizable analogue 2-amino-bicyclo[2,2,1]heptane-2-carboxylic acid induced insulin release and enhanced the secretory response to leucine as in controls, whereas leucine failed to amplify the response to the leucine analogue. Moreover, the potentiating action of L-glutamine on leucine-mediated insulin release was preserved. This coincided with normal glutamate dehydrogenase activity and L-[U-14C]glutamine oxidation. Finally, the secretory response to the leucine deamination product 2-ketoisocaproate was decreased, as was the 2-keto[1-14C]isocaproate oxidation. In islet beta cells from GK rats, the defective secretory response to leucine cannot be ascribed to a deteriorated leucine-stimulated glutamate metabolism but rather to an impaired leucine catabolism. A reduced generation of acetyl-CoA from 2-ketoisocaproate, due to the defective oxidative decarboxylation of this keto-acid by the mitochondrial branched-chain 2-ketoacid dehydrogenase, is incriminated.

Relation between transamination of branched-chain amino acids and urea synthesis: evidence from human pregnancy.

Protein and nitrogen (N) accretion by the mother is a major adaptive response to pregnancy in humans and animals to meet the demands of the growing conceptus. Quantitative changes in whole body N metabolism were examined during normal pregnancy by measuring the rates of leucine N (QN) and carbon (QC) kinetics with the use of [1-13C,15N]leucine. Rate of synthesis of urea was measured by [15N2]urea tracer. Pregnancy-related change in total body water was quantified by H2[18O] dilution, and respiratory calorimetry was performed to quantify substrate oxidation. A significant decrease in the rate of urea synthesis was evident in the 1st trimester (nonpregnant 4.69 +/- 1.14 vs. pregnant 3.44 +/- 1.11 micromol . kg-1 . min-1; means +/- SD, P < 0.05). The lower rate of urea synthesis was sustained through the 2nd and 3rd trimesters. QN was also lower in the 1st trimester during fasting; however, it reached a significant level only in the 3rd trimester (nonpregnant 166 +/- 35 vs. 3rd trimester 135 +/- 16 micromol . kg-1 . h-1; P < 0.05). There was no significant change in QC during pregnancy. A significant decrease in the rate of transamination of leucine was evident in the 3rd trimester both during fasting and in response to nutrient administration (P < 0.05). The rate of deamination of leucine was correlated with the rate of urea synthesis during fasting (r = 0.59, P = 0.001) and during feeding (r = 0.407, P = 0. 01). These data show that pregnancy-related adaptations in maternal N metabolism are evident early in gestation before any significant increase in fetal N accretion. It is speculated that the lower transamination of branched-chain amino acids may be due to decreased availability of N acceptors such as alpha-ketoglutarate as a consequence of resistance to insulin action evident in pregnancy.

Why Does Clostridium acetireducens Not Use Interspecies Hydrogen Transfer for Growth on Leucine?

Clostridium acetireducens is the first reported anaerobic bacterium that is dependent on acetate as an electron acceptor for growth on branched-chain amino acids and alanine. The fermentation pathway of leucine and its deamination product α-ketoisocaproate were studied in this organism. Addition of Methanobacterium formicicum to pure cultures of C. acetireducens stimulated the degradation of α-ketoisocaproate but not the degradation of leucine, indicating that the electrons produced during the oxidative deamination of leucine were not transferred to hydrogen. This conclusion is supported by the observed low NAD(P)H ferredoxin reductase activity. Not only acetate but also crotonate proved to be an appropriate electron sink for the regeneration of NAD(P)+ in this bacterium. Interestingly, C. acetireducens was shown to form polyhydroxybutyrate during growth on leucine plus acetate.

Splanchnic versus whole-body production of α-ketoisocaproate from leucine in the fed state

The extent to which dietary branched-chain amino acids are deaminated by the splanchnic tissues (ie, the liver and gut) in the fed state and released as ketoacids into the systemic circulation is not known. To determine this, we combined the oral (L-[1- 13C]-leucine, [ 13C]-Leu) and intravenous ( l-[5,5,5- 2H 3]leucine, [ 2H 3]-Leu) leucine tracer infusion with the intravenous administration of an independent isotope of the leucine ketoanalog α-ketoisocaproic acid (KIC) ([4,5- 3H]KIC). The study was conducted during constant administration of a complete mixed meal. We found that 26% ± 5% of the orally administered leucine was taken up by the splanchnic organs at first pass, whereas 74% ± 5% appeared in the systemic circulation. The rate of splanchnic KIC release from deamination of dietary leucine accounted for 3% ± 0.2% of the oral leucine administration rate and 13% ± 2% of leucine splanchnic uptake (fractional splanchnic deamination). The fraction of whole-body total leucine uptake that was deaminated to KIC was 41% ± 5% ( P < .05 v fractional splanchnic deamination of dietary leucine uptake). We conclude that (1) the release of KIC from leucine deamination within splanchnic tissues constitutes a minimal fraction of first-pass dietary leucine uptake, and (2) splanchnic tissues are relatively less efficient than the whole body in KIC production from leucine deamination.

Fetoplacental deamination and decarboxylation of leucine

Fetal and placental metabolism of leucine (Leu) and ketoisocaproic acid (KIC) were studied in seven fetal lambs at 132 +/- 1.3-days gestation. Fetal infusions of [1-13C]Leu, [1-14C]Leu, and antipyrine were carried out for 4 h. Uterine and umbilical blood flows were measured using the antipyrine steady-state diffusion technique. Leu and KIC concentrations, [14C]Leu-specific activities, 14CO2, [13C]Leu, and [13C]KIC enrichment (mole percent enrichment) were measured in the maternal artery, uterine vein, and umbilical artery and vein to calculate net fluxes of tracee and tracer molecules between fetus and placenta and between the uteroplacenta and the maternal circulation. There were net Leu and KIC fluxes into the fetus from the placenta with the KIC flux equal to approximately 19% of the combined Leu plus KIC flux. In addition, there was a net KIC flux into the uterine circulation. The fraction of infused tracer Leu escaping the placenta into the mother was small (approximately 6%). By contrast, there was a rapid exchange of tracer Leu carbon between placenta and fetus resulting in a significant flux of labeled KIC from placenta to fetus. Approximately 20% of the infused tracer carbon was converted to CO2 within the fetus. This rate of conversion was greater than 80% of the total fetoplacental conversion rate and significantly higher than the flux of KIC tracer carbon from placenta to fetus. Fetal KIC decarboxylation rate, calculated from the fetal KIC enrichment data, was 2.83 +/- 0.40 mumol.min-1.kg fetus-1 and approximately 60% of the combined net Leu and KIC flux into the fetus from the placenta.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of Alanine Production After L-Leucine and AMP Deamination in an Enzymatic Model and in Muscle Specimens

The amination of 2-oxoglutarate to glutamate by the deamination of leucine followed by the transamination of pyruvate to alanine in skeletal muscle is generally accepted. However, alanine formation following AMP deamination by AMP deaminase is still questionable even though it is theoretically possible. For this reason, we investigated in an enzymatic model the dependence of alanine yield both on the increasing concentration of AMP and leucine as amino group donors as well as on AMP deaminase and leucine dehydrogenase augmentation. Up to a concentration of 375 microM neither of the amino group donors produced a difference in the glutamate nor alanine yield. At a concentration of 500 microM ammonia formation was less, but alanine production was higher when leucine was present as a starting material. However, in muscle samples obtained from trained or untrained rats we did not find an increase in alanine yield when AMP was added to the muscle sample, even though NH3 production was significantly higher in samples with than in those without AMP. This discrepancy might be further elucidated by hindquarter perfusion experiments, in which alanine release would be determined after AMP deamination enhanced by electrostimulation of these muscle groups.

Leucine degradation in sheep.

1. In vitro leucine catabolism in adipose tissue, skeletal muscle, kidney and liver homogenates was studied in sheep. 2. In Expt 1, Suffolk X Targhee ram lambs were slaughtered at 1, 28, 56, 84, 112, 140, 168, 196, 224 and 365 d of age. In Expt 2, 5-month-old crossbred wethers were fed on 80, 120 or 180 g crude protein (nitrogen X 6.25)/kg diets for 4 weeks or fed on 120 g crude protein/kg for 4 weeks and then fasted for 48 or 96 h before slaughter. Leucine catabolism was assayed in tissue homogenates for Expts 1 and 2. 3. Leucine deamination (per unit protein) was highest in skeletal muscle at day 1 and then declined; liver exhibited an activity pattern akin to muscle while kidney activity tended to rise over the duration of the study. Adipose tissue in vitro leucine deamination was higher at all ages studied and 9- to 50-fold higher than all other tissues at 365 d. Leucine decarboxylation (per unit protein) was highest at day 1 in muscle and declined to low levels (P less than 0.01) after 28 d; liver and kidney decarboxylation activities were higher than muscle at all ages with kidney showing the highest activity. Whilst adipose had high initial activity it declined significantly (P less than 0.01) by day 28 and remained low. 4. Dietary protein intake had no effect on leucine deamination in any tissue. Leucine decarboxylation tended to increase with protein intake for all tissues except kidney.(ABSTRACT TRUNCATED AT 250 WORDS)

Determination of leucine metabolism and protein turnover in sheep, using gas-liquid chromatography-mass spectrometry.

1. Whole-body protein synthetic rates in non-pregnant ewes were determined by the continuous infusion of L-[15N]- and [1-13C]leucine and measuring the plasma enrichment of leucine, alpha-ketoisocaproate (alpha-KIC) and expired carbon dioxide by gas-liquid chromatography-mass spectrometry. 2. The mean whole-body protein synthesis estimated from plasma leucine flux corrected for oxidation was 5.38 (SE 0.54) g/kg per d. 3. Under the conditions of the present study leucine oxidation was 0.323 (SE 0.067) mmol/kg per d and accounted for 10.71 (SE 2.26) % of plasma [13C]leucine flux. Deamination of leucine was 0.55 (SE 0.035) mmol/kg per d and accounted for approximately 17% of plasma [15N]leucine flux. 4. The rate of alpha-KIC reamination to leucine, calculated by subtracting 13C flux from 15N flux, was 0.228 (SE 0.101) mmol/kg per d. 5. The rate of whole-body protein degradation was 4.49 (SE 0.54) g/kg per d and there was a net protein gain of 0.89 (SE 0.21) g/kg per d.

Direct determination of leucine metabolism and protein breakdown in humans using L-[1-13C, 15N]-leucine and the forearm model.

We have used the forearm model to study protein metabolism in six normal healthy subjects in the fed state using L-[1-13C, 15N]-leucine as the substrate tracer. Deep venous and arterialized venous blood samples from the forearm were collected at 10-min intervals 2.5 h into a primed-continuous infusion of the dilabelled tracer. Arterialized venous blood was obtained using a 'hot-box' technique and forearm blood flow was measured by mercury strain-gauge plethysmography. The concentration and isotope enrichment of leucine and its metabolites, alpha-ketoisocaproic acid and CO2, in deep venous and arterialized venous blood were measured by gas chromatography-mass spectrometry and isotope ratio-mass spectrometry. The rates of leucine deamination and reamination were 388 +/- 24 (mean +/- SEM) and 330 +/- 23 nmol (100 ml)-1 min-1 respectively, whilst protein synthesis and breakdown rates were 127 +/- 11 and 87 +/- 10 nmol (100 ml)-1 min-1 respectively across the forearm in the fed state. We have demonstrated that the use of doubly labelled leucine as tracer and application of the mathematical model developed in this study, permits the comprehensive quantification of leucine kinetics including protein breakdown.

Regulation of valine metabolism in man: a stable isotope study

Valine and leucine kinetics were studied in four young healthy men in the postabsorptive state with a 4-h primed infusion of either l-[1-13C,15N]valine or l-[1-13C,15N]-leucine. For 1 wk before each infusion study each subject consumed a diet that provided an adequate amount of energy and 1.6 kg−1 day−1 of protein. During infusion of tracer, plasma valine or leucine, and expired 13CO2 reached isotopic steady state by 2 h. The valine and leucine carbon fluxes (mean ± SE) were 80.3 ± 1.2 and 86.6 ± 2.0 µmol kg−1h−1, respectively, consistent with the lesser content of valine compared with leucine in body protein. Valine and leucine oxidation rates were 11.8 ± 0.6 and 15.9 ± 1.1 µmol kg−1h−1, respectively. From these values and values for valine and leucine nitrogen flux, the rates of valine and leucine transamination were calculated. Valine and leucine deamination were 84.0 ± 3.5 and 103.0 ± 6.5 µmol kg−1h−1, and values for reamination were 72.2 ± 3.3 and 87.1 ± 7.5 µmol kg−1h−1, respectively. Thus, the patterns of valine and leucine catabolism are similar. However, when the plasma substrate levels are used to estimate transamination rate constants, we estimate that the transamination equilibrium favors leucine transamination over valine by 5-fold.

Response of protein and urea kinetics in burn patients to different levels of protein intake.

The effects of two levels of protein intake on protein metabolism in six severely burned adult patients were studied (means of 70% BSA burned). A crossover experimental design enabled the authors to study each patient at the end of two three-day dietary regimens. All diets were isocaloric and provided approximately 25% more calories than the measured energy expenditure (means = 40.8 Kcal/kg X day). In one regimen, each patient received 2.2 g protein/kg X day, while during the other treatment period they received 1.4 g protein/kg X day. The patients were studied in the fed state and after 10 to 12 hours of fasting. Leucine kinetics were determined by means of the primed-constant infusion of [1--13C]--leucine. The authors were able to distinguish the oxidation of plasma leucine from the oxidation of leucine derived from intracellular protein at the site of the deamination of leucine (predominantly muscle) by simultaneously determining both leucine and alpha-ketoisocaproic acid enrichment. Also, rates of whole-body protein synthesis and catabolism were calculated from the leucine flux and oxidation data. Net protein synthesis was also calculated by means of another stable-isotope technique involving the infusion of [15N2]--urea. Finally, a third means of estimating net protein catabolism based on urinary N-excretion data was used at the same time that the isotopic studies were performed. The 13C leucine-data and the N-excretion data indicated that a balance between protein synthesis and catabolism could be achieved with a protein intake of 1.4 protein/kg X day. When protein intake was increased to 2.2 g protein/kg X day, neither isotopic method indicated a further beneficial effect on net protein synthesis, although the absolute rates of protein synthesis and catabolism were stimulated. The N-excretion data, on the other hand, indicated a significant improvement in net protein synthesis with higher protein intake. Regardless of the level of protein intake, the underlying alterations in protein metabolism that occurred as a response to burn injury persisted.