Second-tier test for quantification of alloisoleucine and branched-chain amino acids in dried blood spots to improve newborn screening for maple syrup urine disease (MSUD)

BACKGROUND: Newborn screening for maple syrup urine disease (MSUD) relies on finding increased concentrations of the branched-chain amino acids (BCAAs) leucine, isoleucine, and valine by tandem mass spectrometry (MS/MS). d-Alloisoleucine (allo-Ile) is the only pathognomonic marker of MSUD, but it cannot be identified by existing screening methods because it is not differentiated from isobaric amino acids. Furthermore, newborns receiving total parenteral nutrition often have increased concentrations of BCAAs. To improve the specificity of newborn screening for MSUD and to reduce the number of diet-related false-positive results, we developed a LC-MS/MS method for quantifying allo-Ile. METHODS: Allo-Ile and other BCAAs were extracted from a 3/16-inch dried blood spot punch with methanol/H2O, dried under nitrogen, and reconstituted into mobile phase. Quantitative LC-MS/MS analysis of allo-Ile, its isomers, and isotopically labeled internal standards was achieved within 15 min. To determine a reference interval for BCAAs including allo-Ile, we analyzed 541 dried blood spots. We also measured allo-Ile in blinded samples from 16 MSUD patients and 21 controls and compared results to an HPLC method. RESULTS: Intra- and interassay imprecision (mean CVs) for allo-Ile, leucine, isoleucine, and valine ranged from 1.8% to 7.4%, and recovery ranged from 91% to 129%. All 16 MSUD patients were correctly identified. CONCLUSIONS: The LC-MS/MS method can reliably measure allo-Ile in dried blood spots for the diagnosis of MSUD. Applied to newborn screening as a second-tier test, it will reduce false-positive results, which produce family anxiety and increase follow-up costs. The assay also appears suitable for use in monitoring treatment of MSUD patients.     

Determination of branched-chain amino acids and tyrosine in serum of patients with various hepatic diseases, and its clinical usefulness

We developed an automated enzymatic method for determination of the branched-chain amino acids (BCAAs; valine, isoleucine, leucine) and tyrosine in serum, and applied it to the clinical evaluation of patients with various hepatic diseases. Analytically, the test results were acceptably precise and reproducible, and correlated well with results obtained with an amino acid analyzer. Clinically, we found that a decrease in BCAAs, an increase in tyrosine, and the BCAAs/tyrosine ratio in serum paralleled the severity of hepatic parenchymal damage. We conclude that this enzymatic determination of BCAAs and tyrosine is simple and convenient enough for routine clinical laboratory use, and that the ratio of BCAAs/tyrosine obtained may be a good indicator of the severity of hepatic disorders.     

Combined nutritional support and continuous extracorporeal removal therapy in the severe acute phase of maple syrup urine disease

OBJECTIVE: The authors assessed the efficiency, tolerance and outcome of neonates and children with maple syrup urine disease (MSUD) in acute decompensation managed by endogenous and extracorporeal removal of accumulated MSUD metabolites. DESIGN: Single center cohort study. SETTING: Pediatric and neonatal intensive care unit in a tertiary care hospital. PATIENTS: Between January, 1991, and June, 1999, six neonates and six children in acute decompensation of MSUD were included in the study. Each of them had two of the three following criteria: comatose state, gastrointestinal intolerance, leucine plasma levels over 1700 micromol/l. INTERVENTIONS: Patients were treated by combined nutrition manipulation and continuous venovenous extracorporeal removal therapies (CECRT) including hemofiltration, hemodialysis or hemodiafiltration. A clinical and biological evaluation was performed before, during and following the treatment. RESULTS: Eleven out of the 12 patients survived. One child had two acute episodes at 6.5 and 9 years old. Eight patients recovered a normal cerebral performance category score. In all cases, plasma leucine level decreased according to a logarithmic mode within 11-24 h hemodiafiltration combined with nutritional support whereas, with nutrition alone after stopping CECRT, the decrease in leucine plasma levels was slower, following a linear mode. Eight patients were supplemented with valine and isoleucine for mean plasma values of 177+/-92 and 68+/-66, respectively. CONCLUSION: In severe acute decompensation of MSUD, CECRT combined with nutritional support limit central nervous system damage, by dramatically decreasing branched chain amino and keto acid levels.     

Pharmacokinetic and metabolic interrelationships among branched-chain keto and amino acids in humans

Equimolar loads (0.55 mmol/kg) of alpha-ketoisocaproic acid (KICA) or alpha-keto-beta-methyl-n-valeric acid (KMVA), leucine, or isoleucine were given to healthy human subjects. Branched-chain keto acid (BCKA) and amino acid (BCAA) blood levels increased severalfold within minutes for greater than 3 hours. Time course and magnitude of the increments were different for each substance. Keto acids augmented the concentrations of their corresponding amino acids, and amino acids those of the respective keto acids. Each individual keto or amino acid affected all other constituents of the BCKA-BCAA pool. KICA and leucine greatly diminished KMVA, isoleucine, alpha-ketoisovaleric acid, and valine concentrations in blood. Leucine and isoleucine increased significantly more in response to leucine or isoleucine than to administration of equimolar loads of KICA or KMVA. The data suggest that an inappropriate increase in KICA or leucine in BCKA- or BCAA-containing supplements may stimulate the catabolism of BCKAs and BCAAs. Furthermore, if keto acid-containing supplements are given to increase low leucine, isoleucine, or valine concentrations, the dose must be significantly higher than for amino acid-containing supplements.     

Accumulation of 3-hydroxyisobutyric acid, 2-methyl-3-hydroxybutyric acid and 3-hydroxyisovaleric acid in ketoacidosis.

1. Urine and serum samples from patients with ketoacidosis of varying degree and etiology have been examined by gas chromatography and mass spectrometry. 2. In addition to 3-hydroxyisovaleric acid, relatively high concentrations of two analogous hydroxy acids, 3-hydroxyisobutyric acid and 2-methyl-3-hydroxybutyric acid, were found in the urine. 3. There were highly significant positive correlations between the excreted amounts of the three acids. 4. Experiments on rats with isotope-labelled compounds revealed that the acids were formed by the degradation of leucine, isoleucine and valine. 5. The accumulation of the hydroxy acids during ketoacidosis is probably caused by a similar derangement of the metabolism of all three branched-chain amino acids.     

Influence of branched-chain amino acid infusion on arterial concentrations and brain exchange of amino acids in patients with hepatic cirrhosis

Summary.

• 1 The influence of branched-chain amino acid (BCAA) infusion on arterial concentrations and brain exchange of amino acids was studied in seven patients with hepatic cirrhosis and in six healthy control subjects. Arterial levels and arterial-jugular venous (A-JV) concentration differences for amino acids, glucose, ketone bodies and lactate were measured in the basal state and during a constant rate, 150 min intravenous infusion of a BCAA solution (250 fmiol/min, 70% L-leucine, 20% L-valine and 10% L-isoleucine).
• 2 In the basal state the arterial whole blood concentrations of tyrosine, phenylalanine and methionine were 40–130 % higher in the cirrhotic patients compared to the controls, while the levels of the BCAA were 20–35 % lower. The patients' concentration of aspartic acid was 70% below the corresponding control value.
• 3 During BCAA infusion the arterial leucine concentration rose 4–5 fold while valine increased 60–95% and isoleucine rose 45–50%. The arterial levels of several amino acids decreased progressively in a similar manner in patients and controls. At the end of the 150 min infusion period methionine had decreased 35% (P<001), tyrosine 25% (P<0–001) and phenylalanine 35% (P<0–001) in the patient group.
• 4 Positive A-JV differences in amino acid concentration–indicating net brain uptake–were seen for leucine, isoleucine, valine, serine, tyrosine and lysine in both groups in the basal state. The patients had a greater A-JV difference than the controls for tyrosine (P<0–05) while the uptake of the other amino acids, including the BCAA, was similar in the two groups. The fractional uptake of leucine and valine was significantly increased in the patients.
• 5 During BCAA infusion the A-JV difference for leucine increased 2–3 fold in both patients and controls and the net uptake to the brain of tyrosine and phenylalanine was abolished in the patient group.
• 6 It is concluded that (a) patients with hepatic cirrhosis show a decreased whole blood concentration of aspartate indicating a reduced intracellular concentration of this amino acid, (b) brain uptake of tyrosine and fractional uptake of leucine and valine is augmented in patients with hepatic cirrhosis, demonstrating abnormal brain amino acid uptake in this disorder, and (c) BCAA infusion effectively lowers the arterial concentrations for tyrosine, phenylalanine and methionine in cirrhotic patients as well as in healthy controls and blocks the abnormal brain uptake of tyrosine. These findings provide a biochemical background to the suggested beneficial effect of BCAA infusion in patients with hepatic cirrhosis and portal systemic encephalopathy.

     

E2 transacylase-deficient (type II) maple syrup urine disease. Aberrant splicing of E2 mRNA caused by internal intronic deletions and association with thiamine-responsive phenotype.

Maple syrup urine disease (MSUD) or branched-chain alpha-ketoaciduria is an autosomally inherited disorder in the catabolism of branched-chain amino acids leucine, isoleucine, and valine. The disease is characterized by severe ketoacidosis, mental retardation, and neurological impairments. MSUD can be classified into genetic subtypes according to the genes of the branched-chain alpha-ketoacid dehydrogenase (BCKD) complex which are affected in patients. We describe here four intronic deletions and an intronic nucleotide substitution in the E2 transacylase gene of type II MSUD, in which the E2 subunit of the BCKD complex is deficient. These new E2 mutations comprise an internal 3.2-kb deletion in intron 4 (causing a 17-bp insertion in mRNA), an internal 12-bp (ttaccttgttac) deletion in intron 4 (creating a 10-bp insertion), a 10-bp (catttctaG) deletion in intron 10/ exon 11 junction (leading to a 21-bp deletion), a 2-bp deletion in the exon 5/intron 5 junction (ATgt--> A-t) (resulting in the skipping of exon 5), and a G to A transition at nucleotide -7 of intron 9 (causing a 6-bp insertion). These intronic mutations were initially detected by secondary alterations in the mutant E2 mRNA, as a result of aberrant splicing. The 3.2-kb deletion in intron 4 was determined by the amplification of the entire intron from both a normal subject (11.2 kb) and a homozygous patient (8 kb) by long PCR, followed by subcloning and sequencing of regions flanking the deletion. Similar methods were used to identify and characterize the other intronic alterations. Our results depict heretofore undescribed splicing errors caused by the deletion of internal intronic segments, and provide an approach for detecting this class of novel and rare human mutation. The association of the thiamine-responsive phenotype with a subset of the type II MSUD patients studied is also discussed.     

Determination of alpha-keto acids as silylated oximes in urine and serum by combined gas chromatography-mass spectrometry

Nine α-keto acids in serum and urine were analyzed by gas chromatography as their oxime-trimethylsilylated derivatives, following oxime formation with hydroxylamine in pyridine and silylation with BSA containing 10% TMCS. The oxime-TMS derivatives were identified by both low- and high-resolution mass spectrometry. Recoveries from urine were greater than 90% and from serum greater than 80%.Loads of valine, leucine and isoleucine are shown to result in increased serum concentrations of corresponding α-keto acids in healthy adults. In a child with maple-syrup-urine disease under treatment by dietary restriction of the branchedchain amino acids, administration of leucine results in a large excretion of α-keto isocaproic acid and a less striking excretion of α-keto-β-methylvaleric and α-keto isovaleric acid. The excretion of the branched-chain α-keto acids is reported in two patients with maple-syrup-urine disease before and after institution of dietary treatment.Nine α-keto acids and at least 15 phenolic acids and related compounds can be determined in a single analysis using the method described. The ‘metabolic profile’ thus obtained may be used in screening for abnormalities in the metabolism of amino acids. The gas Chromatographic analysis alone is adequate for screening; when an abnormal quantity of a constituent is found, mass spectrometry is used for confirmation of identity.     

ENU mutagenesis identifies mice with mitochondrial branched-chain aminotransferase deficiency resembling human maple syrup urine disease

Tandem mass spectrometry was applied to detect derangements in the pathways of amino acid and fatty acid metabolism in N-ethyl-N-nitrosourea-treated (ENU-treated) mice. We identified mice with marked elevation of blood branched-chain amino acids (BCAAs), ketoaciduria, and clinical features resembling human maple syrup urine disease (MSUD), a severe genetic metabolic disorder caused by the deficiency of branched-chain alpha-keto acid dehydrogenase (BCKD) complex. However, the BCKD genes and enzyme activity were normal. Sequencing of branched-chain aminotransferase genes (Bcat) showed no mutation in the cytoplasmic isoform (Bcat-1) but revealed a homozygous splice site mutation in the mitochondrial isoform (Bcat-2). The mutation caused a deletion of exon 2, a marked decrease in Bcat-2 mRNA, and a deficiency in both BCAT-2 protein and its enzyme activity. Affected mice responded to a BCAA-restricted diet with amelioration of the clinical symptoms and normalization of the amino acid pattern. We conclude that BCAT-2 deficiency in the mouse can cause a disease that mimics human MSUD. These mice provide an important animal model for study of BCAA metabolism and its toxicity. Metabolomics-guided screening, coupled with ENU mutagenesis, is a powerful approach in uncovering novel enzyme deficiencies and recognizing important pathways of genetic metabolic disorders.     

Abnormal amino acid metabolism in diabetes mellitus]

Abnormal amino acid metabolism is sometimes observed among patients with diabetes mellitus. Of many amino acids, alanine and branched-chain amino acids such as valine, leucine, isoleucine show characteristic changes. In diabetic ketoacidosis, plasma concentration of alanine decreases and that of branched-amino acid increases and the oxidation of branched-amino acids is enhanced. Splanchnic amino acid uptake is generally higher in diabetics and this level is partially restored by exercise. Some glycosylated proteins are used to estimate the condition of diabetes mellitus. Increment of urinary glycosylated amino acid excretion is reported in diabetics. Plasma homocysteine, reactive vascular-injuring amino acid, increases in diabetics with nephropathy. Those abnormal amino acid metabolism would be restored after good glycemic control is obtained.     

The amino acids of the blood in patients with peritonitis: the significance of Fisher's index

Blood plasma amino acid levels in 10 patients with peritonitis are analyzed. Nonsurvivors, in contrast to survivors, had severe amino acid derangements. Aminoacidemia and amino acid imbalance were registered early in the course of the illness. Increased concentrations of aromatic amino acids: phenylalanine and tyrosine, and low concentrations of branched-chain amino acids: valine, leucine, and isoleucine were associated with a decrease of Fisher's index (BCAA/AAA molar ratio) from 3.0 +/- 0.5 (normal) to 2.0 +/- 0.5 which was of high diagnostic and prognostic significance. The nature of such imbalance and its role in protein derangements are discussed.     

Analysis of maple syrup urine disease in cell culture: use of substrates

Branched-chain 2-oxo acid dehydrogenase activity in human skin fibroblasts against L-leucine, L-valine, L-isoleucine and derived 2-oxo acids was compared in incubations with 1 mmol/l of 1-14C-labelled substrate. The results suggested that the amino acids are the more suitable substrates for an estimation of decarboxylation activity in intact cells. In control cell lines (n = 12), 14CO2 release from amino acids was highest for valine and least for leucine. In a representative number of fibroblast strains of patients with different forms of MSUD (n = 11; residual decarboxylation activity 2-60% of the controls), 14CO2 release from the different amino acids was reduced to a similar degree. Additional measurement of 2-oxo[1-14C]acid release suggested that substrate supply to the branched-chain 2-oxo acid dehydrogenase complex was not rate limiting in the cell lines under investigation.     

Effect of Circuit Class Training for Eight Weeks on Changes in Ratios of F-Trp/BCAAs and Depression in People with Poststroke Depression

[Purpose] The purpose of the present study was to investigate the potential effects of circuit class training (CCT) on poststroke depression through changes in branched-chain amino acids (BCAAs) (isoleucine, leucine, and valine) and free-tryptophan (f-Trp). [Subjects] The study subjects were 40 stroke patients with major depressive disorder. The subjects were group-matched into an experimental and a control group according to sex, age, height, and weight. [Methods] The experimental CCT group performed gradual task-oriented CCT (80 min per session). The control group performed stretching exercises and weight bearing exercises (80 min per session). Both groups performed the exercises three times per week for eight weeks (24 sessions). Blood samples were collected immediately before the exercise (9:10 a.m.) and after the exercise (10:30 a.m.), every two weeks for eight weeks. [Results] The f-Trp/BCAAs ratio in the CCT group showed a significant increase compared to the control group over time. [Conclusion] The results show that the CCT may help to improve depression in people with poststroke depression (PSD).Key words: Circuit class training, F-Trp/BCAAs ratios, Poststroke depression     

Significance of L-alloisoleucine in plasma for diagnosis of maple syrup urine disease.

BACKGROUND: The significance of plasma L-alloisoleucine, which is derived from L-isoleucine in vivo, for diagnosis of maple syrup urine disease (MSUD) was examined. METHODS: Branched-chain L-amino acids were measured by automatic amino acid analysis. RESULTS: Alloisoleucine reference values in plasma were established in healthy adults [1.9 +/- 0.6 micromol/L (mean +/- SD); n = 35], children 3-11 years (1.6 +/- 0.4 micromol/L; n = 17), and infants <3 years (1.3 +/- 0.5 micromol/L; n = 37). The effect of dietary isoleucine was assessed in oral loading tests. In controls receiving 38 micromol (n = 6; low dose) and 1527 micromol (n = 3; high dose) of L-isoleucine per kilogram of body weight, peak increases of plasma isoleucine were 78 +/- 24 and 1763 +/- 133 micromol/L, respectively; the peak increase of alloisoleucine, however, was negligible for low-dose (<0.3 micromol/L) and minor for high-dose (5. 5 +/- 2.1 micromol/L) load. In patients with diabetes mellitus, ketotic hypoglycemia, phenylketonuria, and obligate heterozygous parents of MSUD patients, alloisoleucine was not significantly different from healthy subjects. Therefore, a plasma concentration of 5 micromol/L was used as a cutoff value. In patients with classical MSUD (n = 7), alloisoleucine was beyond the cutoff value in 2451 of 2453 unselected samples. In patients with variant MSUD (n = 9), alloisoleucine was >5 micromol/L in all samples taken for establishment of diagnosis and in 94% of the samples taken for treatment control (n = 624). With the other branched-chain amino acids, the frequency of diagnostically significant increases was <45%. CONCLUSIONS: The present findings indicate that plasma L-alloisoleucine above the cutoff value of 5 micromol/L is the most specific and most sensitive diagnostic marker for all forms of MSUD.     

Leucine and protein metabolism after bilateral nephrectomy in rats: the role of hepatic tissue

The aim of this study was to evaluate the effect of acute uremia on changes in leucine and protein metabolism in the whole body and in hepatic tissue. Acute renal insufficiency was induced by bilateral nephrectomy (BNX). Twenty-four hours later, parameters of protein and amino acid metabolism were evaluated in the whole body using primed constant intravenous infusion of L-[1-¹⁴C]leucine, and in isolated perfused liver (IPL) using !-keto[1-¹⁴C]isocaproate. The control group consisted of sham-operated rats. BNX induced a marked decrease in proteolysis, protein synthesis, leucine oxidized fraction and leucine clearance. The decrease in protein synthesis was higher than in proteolysis. A significant drop in protein synthesis was observed in muscle, gut, heart and spleen. The study with IPL in BNX animals showed decreased oxidation of ketoisocaproic acid and higher concentrations of the branched-chain amino acids (BCAA) leucine, isoleucine and valine in perfusion solution. We conclude that the cause of rapid depletion of body proteins after BNX is a greater decrease in protein synthesis than in proteolysis associated with an increase in leucine oxidized fraction. The data obtained in the IPL model indicate that BNX causes metabolic changes that enable resynthesis of BCAA from corresponding branched-chain keto acids in liver.     

A novel NMR-based assay to measure circulating concentrations of branched-chain amino acids: Elevation in subjects with type 2 diabetes mellitus and association with carotid intima media thickness

ObjectivesPlasma branched-chain amino acid (BCAA) levels, measured on nuclear magnetic resonance (NMR) metabolomics research platforms or by mass spectrometry, have been shown to be associated with type 2 diabetes mellitus (T2DM) and cardiovascular disease (CVD). We developed a new test for quantification of BCAA on a clinical NMR analyzer and used this test to determine the clinical correlates of BCAA in 2 independent cohorts.Design and methodsThe performance of the NMR-based BCAA assay was evaluated. A method comparison study was performed with mass spectrometry (LC-MS/MS). Plasma BCAA were measured in the Insulin Resistance Atherosclerosis Study (IRAS, n = 1209; 376 T2DM subjects) and in a Groningen cohort (n = 123; 67 T2DM subjects). In addition, carotid intima media thickness (cIMT) was measured successfully in 119 subjects from the Groningen cohort.ResultsNMR-based BCAA assay results were linear over a range of concentrations. Coefficients of variation for inter- and intra-assay precision ranged from 1.8–6.0, 1.7–5.4, 4.4–9.1, and 8.8–21.3%, for total BCAA, valine, leucine, and isoleucine, respectively. BCAA quantified from the same samples using NMR and LC-MS/MS were highly correlated (R² = 0.97, 0.95 and 0.90 for valine, leucine and isoleucine). In both cohorts total and individual BCAA were elevated in T2DM (P = 0.01 to ≤0.001). Moreover, cIMT was associated with BCAA independent of age, sex, T2DM and metabolic syndrome (MetS) categorization or alternatively of individual MetS components.ConclusionsBCAA levels, measured by NMR in the clinical laboratory, are elevated in T2DM and may be associated with cIMT, a proxy of subclinical atherosclerosis.     

Determination of dipeptides in urine

The urine of a patient suffering from dermatological purpura was examined for dipeptides by combined gas chromatography—mass spectrometry. Several dipeptides were identified and all were of the general formula, R proline, where R is one of the amino acids, glycine, alanine, leucine, isoleucine, valine, phenylalanine, glutamic acid, aspartic acid. The prevalence of proline and the percentage distributions of the other amino acids, R, strongly suggested a collagen abnormality.     

Leucine Oxidation and Protein Turnover in Clofibrate-induced Muscle Protein Degradation in Rats

Treatment of hyperlipidemia with clofibrate may result in development of a muscular syndrome. Our previous investigation (1979. J. Clin. Invest.64: 405.) showed that chronic administration of clofibrate to rats causes myotonia and decreases glucose and fatty acid oxidation and total protein of skeletal muscle. In the present experiments we have investigated amino acid and protein metabolism in these rats. Clofibrate administration decreased the concentration of all three branched-chain amino acids without affecting those of others in muscle. Studies to examine the mechanism of decreases in muscle concentrations of branched-chain amino acids showed the following: (a) Plasma concentration of leucine was decreased, whereas there was no significant change in the concentration of isoleucine and valine. (b) Liver concentrations of all three branched-chain amino acids remained unaltered. (c) The uptake of cycloleucine (a nonmetabolizable analogue of leucine) by both muscle and liver was unaffected. (d) The percentage of a trace amount of injected [1-¹⁴C]leucine expired as ¹⁴CO₂ in 1 h was significantly increased. (e) The capacity of muscle homogenate for α-decarboxylation of leucine was enhanced, whereas that of liver was unaffected. (f) The activity of leucine transaminase was unaffected, whereas that of α-ketoisocaproate dehydrogenase was increased in muscle.     

Branched chain amino acids supplementation modulates TGF‐β1/Smad signaling pathway and interleukins in CCl4‐induced liver fibrosis

The alterations and low levels of circulating branched chain amino acids (BCAAs), leucine, isoleucine, and valine, are associated with liver diseases. The study was designed to evaluate hepatoprotective effect of BCAAs on CCl₄‐induced liver fibrosis and to investigate the molecular mechanisms underlying these effects in rats. In all, 30 male rats were divided into three groups. Control group (n = 10) and CCl₄ group (n = 10), where rats were injected with CCl₄ (1 mL/kg of 0.5 : 1 v/v injected i.p. twice weekly for 12 weeks). In CCl₄ + BCAAs group (n = 10), rats were injected with similar doses of CCl₄ and supplemented with a mixture of 600 mg/kg BCAAs (2 : 1 : 1.2 leucine : isoleucine : valine) by oral gavage, three times/week for 12 weeks. Liver fibrosis was assessed by measuring total bilirubin, total protein, alanine aminotransferase, and aspartate aminotransferase, hydroxyproline content, and serum IL‐6 and IL‐10. Histopathologic studies and α‐smooth muscle actin (α‐SMA) were detected immunohistochemically in liver. Serum insulin level, blood glucose, liver malodialdehyde concentration (MDA), glutathione peroxidase, and superoxide dismutase (SOD) activities were quantified. TGF‐β1, Smad3, and Smad7 gene expressions were estimated by qRT‐PCR. BCAAs suppressed liver fibrosis induced by CCl₄ treatment. BCAAs modulated liver indices and downregulated TGF‐β1, Smad3, and Smad7 expressions in hepatocytes. BCAAs enhanced liver antioxidant enzyme activities (P < 0.001), reduced serum levels of TGF‐β1, IL‐6, and IL‐10 compared to CCL₄ group and ameliorated histopathologic changes in rat liver. BCAAs may have a protective role against liver fibrosis via antioxidant and anti‐inflammatory mechanisms.     

Influence of leucine infusion on intracellular amino acids in humans

A continuous intravenous infusion of L-leucine (300 mumols min-1) was given to 12 healthy females over a 2 1/2 h period. Arterial plasma concentrations of amino acids and the keto acids of the branched-chain amino acids (BCAA) were measured. In six subjects muscle biopsies were taken before and at the end of the infusion for determination of intracellular (i.c.) free amino acid concentrations, and leg exchange of amino acids was measured. During infusion the plasma level of leucine rose sixfold. Approximately 40% of the infused amount was taken up by muscle. Of this, half was accumulated intracellularly, where the free leucine concentration increased from basal 190 +/- 22 to 580 +/- 110 mumols l-1 ICW (intracellular water) at the end of infusion. The concentrations of most other amino acids, above all the other BCAA and the aromatic amino acids, decreased, by 17-48% in the i.c. pool and by 17-79% in plasma. The plasma level of ketoisocaproic acid (KIC), the keto acid of leucine, increased in parallel with that of leucine. The concentration of keto valine, ketoisovaleric acid (KIV), decreased by 75%, whereas the keto acid of isoleucine, ketomethylvaleric acid (KMV), was unchanged. Leg release of alanine decreased significantly, whereas the exchange of other amino acids were unchanged. Taken together, decreased i.c. and plasma concentrations but unchanged leg exchange of tyrosine and phenylalanine suggest i.c. accumulation of protein. It can be calculated that approximately 40% of the leucine taken up by muscle was accumulated in the intracellular free pool, some 20% could have been incorporated into protein and 40% was probably oxidized.