Phase 1 trial of dichloroacetate (DCA) in adults with recurrent malignant brain tumors.

BACKGROUND: Recurrent malignant brain tumors (RMBTs) carry a poor prognosis. Dichloroacetate (DCA) activates mitochondrial oxidative metabolism and has shown activity against several human cancers. DESIGN: We conducted an open-label study of oral DCA in 15 adults with recurrent WHO grade III - IV gliomas or metastases from a primary cancer outside the central nervous system. The primary objective was detection of a dose limiting toxicity for RMBTs at 4 weeks of treatment, defined as any grade 4 or 5 toxicity, or grade 3 toxicity directly attributable to DCA, based on the National Cancer Institute's Common Toxicity Criteria for Adverse Events, version 4.0. Secondary objectives involved safety, tolerability and hypothesis-generating data on disease status. Dosing was based on haplotype variation in glutathione transferase zeta 1/maleylacetoacetate isomerase (GSTZ1/MAAI), which participates in DCA and tyrosine catabolism. RESULTS: Eight patients completed at least 1 four week cycle. During this time, no dose-limiting toxicities occurred. No patient withdrew because of lack of tolerance to DCA, although 2 subjects experienced grade 0-1 distal parasthesias that led to elective withdrawal and/or dose-adjustment. All subjects completing at least 1 four week cycle remained clinically stable during this time and remained on DCA for an average of 75.5 days (range 26-312). CONCLUSIONS: Chronic, oral DCA is feasible and well-tolerated in patients with recurrent malignant gliomas and other tumors metastatic to the brain using the dose range established for metabolic diseases. The importance of genetic-based dosing is confirmed and should be incorporated into future trials of chronic DCA administration.

Quantitative tests of liver function measure hepatic improvement after sustained virological response: results from the HALT-C trial.

BACKGROUND: The impact of virologic response on hepatic function has not been previously defined. AIM: To determine the relationships of quantitative liver function tests (QLFTs) with virological responses to peginterferon (PEG) +/- ribavirin (RBV) in patients with chronic hepatitis C and to use serial QLFTs to define the spectrum of hepatic improvement after sustained virological response (SVR). METHODS: Participants (n = 232) were enrolled in the Hepatitis C Antiviral Long-term Treatment against Cirrhosis (HALT-C) Trial, had failed prior therapy, had bridging fibrosis or cirrhosis and were retreated with PEG/RBV. All 232 patients had baseline QLFTs; 24 patients with SVR and 68 nonresponders had serial QLFTs. Lidocaine, [24-(13)C]cholate, galactose and (99m)Tc-sulfur colloid were administered intravenously; [2,2,4,2-(2)H]cholate, [1-(13)C]methionine, caffeine and antipyrine were administered orally. Clearances (Cl), breath (13)CO(2), monoethylglycylxylidide (MEGX), perfused hepatic mass (PHM) and liver volume were measured. RESULTS: Rates of SVR were 18-26% in patients with good function by QLFTs, but < or =6% in patients with poor function. Hepatic metabolism, measured by caffeine k(elim) (P = 0.02), antipyrine k(elim) (P = 0.05) and antipyrine Cl (P = 0.02) and the portal circulation, measured by cholate Cl(oral) (P = 0.0002) and cholate shunt (P = 0.0003) and PHM (P = 0.03) improved after SVR. CONCLUSION: Hepatic dysfunction impairs the virological response to PEG/RBV. SVR improves hepatic metabolism, the portal circulation and PHM.

The spectrum of hepatic functional impairment in compensated chronic hepatitis C: results from the Hepatitis C Anti-viral Long-term Treatment against Cirrhosis Trial.

BACKGROUND: The spectrum of functional impairment in patients with compensated chronic hepatitis C is incompletely defined. AIM: To define hepatic impairment by quantitative tests (quantitative liver function tests) and correlate results with disease severity in patients with chronic hepatitis C. METHODS: We studied 285 adult patients with chronic hepatitis C prior to treatment in the Hepatitis C Anti-viral Long-term Treatment against Cirrhosis Trial; 171 had Ishak fibrosis stages 2-4 (fibrosis) and 114 had stage 5 or 6 (cirrhosis). None had had clinical decompensation. A battery of 12 quantitative liver function test assessed the spectrum of hepatic microsomal, mitochondrial and cytosolic functions, and hepatic and portal blood flow. RESULTS: Twenty-six to 63% of patients with fibrosis and 45-89% with cirrhosis had hepatic impairment by quantitative liver function test; patients with cirrhosis had the greatest impairment (P-value ranging from 0.15 to <0.0001). Cholate Cl(oral), cholate shunt and perfused hepatic mass correlated with cirrhosis, stage of fibrosis (r = -0.51, +0.49, -0.51), varices and variceal size (r = -0.39, +0.36, -0.41). PHM < 95 and cholate shunt >35% identified 91% of patients with medium- or large-sized varices. CONCLUSIONS: Hepatic impairment is common in compensated patients with fibrosis or cirrhosis because of chronic hepatitis C. Cholate shunt, and cholate Cl(oral) and perfused hepatic mass, identify patients at risk for cirrhosis or varices.

Evaluation of the Ez-HBT Helicobacter blood test to establish Helicobacter pylori eradication.

BACKGROUND: The urea blood test (Ez-HBT) has been shown to compare favourably with the urea breath test in the diagnosis of active Helicobacter pylori infection. AIM: To examine the performance characteristics of the Ez-HBT Helicobacter blood test in establishing success or failure of therapy in H. pylori-infected adults using the 13C urea breath test as the reference method. METHODS: 13C urea breath test and Ez-HBT Helicobacter blood test were performed 4-6 weeks after completion of treatment in H. pylori positive subjects. Basal urea breath samples were collected; basal Ez-HBT Helicobacter blood test samples were not. Ez-HBT Helicobacter blood test results were reported as positive, negative, or indeterminate. RESULTS: Seventy patients generated 126 measurable sets of urea breath and blood tests. The H. pylori cure rate was 93%. The sensitivity, specificity, and accuracy of the Ez-HBT Helicobacter blood test were 100%, 97%, and 97%, respectively. Six of eight false positive and indeterminate Ez-HBT Helicobacter blood test results could be attributed to incomplete fasting or a 13C enriched diet. After correcting for the non-fasting state, the positive predictive value of the Ez-HBT Helicobacter blood test improved from 56% to 86%. CONCLUSION: The performance characteristics of the Ez-HBT Helicobacter blood test are comparable with that of 13C-urea breath test in establishing H. pylori eradication after therapy. Errors related to incomplete fasting can be mitigated by collection of a basal blood sample.

Phase and size distribution of polycyclic aromatic hydrocarbons in diesel and gasoline vehicle emissions.

Emission measurements were obtained for a variety of military vehicles at Hill Air Force Base (Ogden, UT) in November 2000 as part of a Strategic Environmental Research and Development Program. Aircraft ground support equipment vehicles using gasoline, diesel, and JP8 fuels were tested using chassis dynamometers under predetermined load. The exhaust from the tested vehicle was passed to a dilution tunnel where it was diluted 30-40 times and collected using Micro-Orifice Uniform Deposit Impactor (MOUDI) fitted with aluminum substrates, an XAD-coated annular denuder, and a filter followed by a solid adsorbent. All MOUDI substrates were analyzed for mass and for organic and elemental (EC) carbon by the thermal/optical reflectance method and for polycyclic aromatic hydrocarbons (PAHs) by GC/MS. Black carbon was measured with a photoacoustic instrument. The denuder and filter/solid adsorbent samples were analyzed for semivolatile PAH. Overall, there is more mass and higher EC contribution when the vehicle is run under higher load in comparison with the low load. However, older vehicles generally show more mass and EC emissions than newer vehicles, and there is a shift toward smaller particle sizes for the low load, which is most pronounced for newer vehicles. The particle-associated semivolatile PAHs and nonvolatile four-through six-ring PAHs are present predominantly on the submicron particles collected on MOUDI stages 0.1-0.18, 0.18-0.32, and 0.32-0.56 microm. For the low-load runs, the distribution of PAHs seems to be shifted toward smaller size particles. The gas-particle phase distribution of semivolatile PAHs depends also on the engine loading. For idle, not only are the more volatile two- and three-ring PAHs, from naphthalene to dimethylphenanthrenes, retained on the denuder portion, but also less volatile four-ring PAHs, such as fluoranthene and pyrene, are retained by the denuder at the 80-90% range, which implies that they are present predominantly in the gas phase. In contrast, for engines under high loads, a much larger portion of three- and four-ring PAHs are partitioned to the particle phase.

Phenylalanine kinetics in healthy volunteers and liver cirrhotics: implications for the phenylalanine breath test.

Expired 13CO2 recovery from an oral l-[1-13C]phenylalanine ([13C]Phe) dose has been used to quantify liver function. This parameter, however, does not depend solely on liver function but also on total CO2 production, Phe turnover, and initial tracer distribution. Therefore, we evaluated the impact of these factors on breath test values. Nine ethyl-toxic cirrhotic patients and nine control subjects received intravenously 2 mg/kg of [13C]Phe, and breath and blood samples were collected over 4 h. CO2 production was measured by indirect calorimetry. The exhaled 13CO2 enrichments were analyzed by isotope ratio mass spectrometry and the [13C]Phe and l-[1-13C]tyrosine enrichments by gas chromatography-mass spectrometry. The cumulative 13CO2 recovery was significantly lower in cirrhotic patients (7 vs. 12%; P < 0.01), in part due to lower total CO2 production rates. Phe turnover in cirrhotic patients was significantly lower (33 vs. 44 micro mol. kg(-1). h(-1); P < 0.05). When these extrahepatic factors were considered in the calculation of the Phe oxidation rate, the intergroup differences were even more pronounced (3 vs. 7 micro mol. kg(-1). h(-1)) than those for 13CO2 recovery data. Also, the Phe-to-Tyr conversion rate, another indicator of Phe oxidation, was significantly reduced (0.7 vs. 3.0 micro mol. kg(-1). h(-1)).

Evidence for direct protein kinase-C mediated modulation of N-methyl-D-aspartate receptor current.

Protein kinase-C (PKC) activation differentially affects currents from N-methyl-D-aspartate (NMDA) type glutamate receptors depending upon their subunit composition. Experiments using chimeras initially indicated that the cytoplasmic C-terminal tails of NR2B (responsive to PKC) and NR2C (unresponsive to PKC) subunits contain the amino acid residues responsible for the observed disparity of PKC effects. However, truncation and point mutation experiments have suggested that PKC action on NMDA receptors may be entirely indirect, working via the phosphorylation of associated proteins. Here we suggest that PKC does, in fact, affect NR2B/NR1-011 NMDA currents by direct phosphorylation of the NR2B tail at residues S1303 and S1323. Replacement of either of these residues with Ala severely reduces PKC potentiation. To verify that S1303 and S1323 are sites of direct phosphorylation by PKC, synthetic peptides from the regions surrounding these sites were used as substrates for in vitro assays with purified rat brain PKC. These results indicate that PKC can directly phosphorylate S1303 and S1323 in the NR2B C terminus, leading to enhanced currents through NMDA receptor channels. The direct action of PKC on certain NMDA receptor subtypes may be important in any physiological or pathological process where PKC and NR2B/NR1 receptors interact.

Protein mobility and GABA-induced conformational changes in GABA(A) receptor pore-lining M2 segment.

Protein movements underlying ligand-gated ion channel activation are poorly understood. Here we used disulfide bond trapping to examine the proximity and mobility of cysteines substituted for aligned GABAA receptor alpha1 and beta1 M2 segment channel-lining residues in resting and activated receptors. With or without GABA, disulfide bonds formed at alpha1N275C/beta1E270C (20') and alpha1S272C/beta1H267C (17'), near the extracellular end, suggesting that this end is more mobile and/or flexible than the rest of the segment. Near the middle of M2, at alpha1T261C/beta1T256C (6'), a disulfide bond formed only in the presence of GABA and locked the channels open. Channel activation must involve an asymmetric rotation of two adjacent subunits toward each other. This would move aligned engineered cysteines on different subunits into proximity and allow disulfide bond formation without blocking conduction. Asymmetric rotation of M2 segments is probably a common gating mechanism in other ligand-gated ion channels.

Structure and dynamics of the GABA binding pocket: A narrowing cleft that constricts during activation.

Photo-affinity labeling and mutagenesis studies have identified several amino acids that may contribute to the ligand binding domains of ligand-gated ion channels. These types of studies, however, only generate a one-dimensional, static description of binding site structure. In this study, we used the substituted cysteine accessibility method not only to identify binding pocket residues but also to elicit information about binding site dynamics and structure. Residues surrounding the putative loop C ligand binding domain of the GABA(A) receptor (beta(2)V199 to beta(2)S209) were individually mutated to cysteine, and the mutant subunits were coexpressed with wild-type alpha(1) subunits in Xenopus oocytes. N-biotinylaminoethyl methanethiosulfonate (MTSEA-biotin) reacts with cysteines introduced at positions G203, S204, Y205, P206, R207, and S209. This accessibility pattern is not consistent with either an alpha-helix or beta-strand. Instead, G203-S209 seems to form a water-accessible extended coil, whereas V199-T202 appears to buried in the protein or membrane. Coapplication of either GABA or the competitive antagonist SR-95531 significantly slows MTSEA-biotin modification of cysteines introduced at positions S204, Y205, R207, and S209, demonstrating that these residues line and face into the GABA binding pocket. MTSEA-biotin reaction rates reveal a steep accessibility gradient from G203-S209 and suggests that the binding pocket is a deep narrowing cleft. Pentobarbital activation of the receptor significantly slows MTSEA-biotin modification of cysteines at S204, R207, and S209, suggesting that the binding site may constrict during gating.

Identification of benzodiazepine binding site residues in the gamma2 subunit of the gamma-aminobutyric acid(A) receptor.

gamma-Aminobutyric acid(A) receptor gamma-subunits are important for benzodiazepine (BZD) binding and modulation of the gamma-aminobutyric acid-mediated Cl(-) current. Previously, by using gamma2/alpha1 chimeric subunits, we identified two domains of the gamma2-subunit, Lys-41-Trp-82 and Arg-114-Asp-161, that are, in conjunction, necessary and sufficient for high-affinity BZD binding. In this study, we generated additional gamma2/alpha1 chimeric subunits and gamma2 point mutants to identify specific residues within the gamma2 Lys-41-Trp-82 region that contribute to BZD binding. Mutant gamma2 and gamma2/alpha1 chimeric subunits were expressed with wild-type alpha1 and beta2 subunits in HEK 293 cells, and the binding of several BZDs was measured. We present evidence that the gamma2 region Met-57-Ile-62 is important for flunitrazepam binding and that, in particular, gamma2 Met-57 and gamma2 Tyr-58 are essential determinants for conferring high-affinity binding. Furthermore, we identify an additional residue, gamma2 Ala-79, that not only is important for high-affinity binding by flunitrazepam (a strong positive modulator) but also plays a crucial role in the binding of the imidazobenzodiazepines Ro15-1788 (a zero modulator) and Ro15-4513 (a weak negative modulator) in the BZD binding pocket. Results from site-directed mutagenesis of gamma2 Ala-79 suggest that this residue may be part of a microdomain within the BZD binding site that is important for binding imidazobenzodiazepines. This separation of drug-specific microdomains for competitive BZD ligands lends insight into the structural determinants governing the divergent effects of these compounds.

Protein kinase C potentiation of currents from mouse zeta1/epsilon2 NMDA receptors expressed in Xenopus oocytes depends on f-actin/g-actin cycling.

NMDA currents from Xenopus oocytes expressing recombinant zeta1/epsilon2 NMDA receptors can be potentiated by activation of protein kinase-C (PKC) and also demonstrate time-dependent rundown. In order to determine whether cytoskeletal proteins are involved in either of these phenomena, experiments were performed using the f-actin stabilizer phalloidin, the f-actin de-stabilizer cytochalasin-D, and the microtubule stabilizer taxol. Phalloidin treatment both prevented rundown and inhibited PKC-potentiation of whole-cell currents but did not affect baseline current amplitudes. Treatment with cytochalasin-D also prevented rundown and inhibited PKC-potentiation of whole-cell currents, but baseline currents from cytochalasin treated cells were only 50% as large as those from control cells. Taxol had no effect on either rundown or PKC potentiation of NMDA currents. The results indicate that both spontaneous rundown and PKC potentiation of currents from heterologously expressed zeta1/epsilon2 NMDA receptors depend on dynamic actin polymerization/depolymerization but do not involve changes in microtubules.

Gastric emptying in trauma patients.

BACKGROUND/AIM: The aim of this study was to obtain quantitative data on gastric emptying following trauma. METHODS: In order to assess gastric emptying for early enteral feeding, we evaluated the absorption of an amino acid, L-[1-(13)C]phenylalanine, within 24 h of admission and 7 days later in 14 trauma patients (injury severity score 36 +/- 2). Following nasogastric administration of 100 mg L-[1-(13)C]phenylalanine, the plasma L-[1-(13)C]phenylalanine enrichment at 30 and 60 min and the expired (13)CO(2) for 1 h in the breath were used to measure the degree of gastric emptying. RESULTS: The plasma L-[1-(13)C]phenylalanine enrichment concentration at 30 min was 0.53 +/- 0.23 mmol/l during the first study and 2.46 +/- 0. 62 mmol/l during the second study (p = 0.006, a fivefold increase). The L-[1-(13)C]phenylalanine plasma level in historic controls was 4. 57 +/- 1.48 mmol/l. The percent of the dose oxidized and expired as (13)CO(2) in 1 h was 0.51 +/- 0.17 during the first 24-hour study compared to the second study of 3.37 +/- 0.68 (p = 0.0008) 7 days later (an over sixfold increase). The percent of the dose oxidized in 1 h in 37 normal historic controls was 7.08 +/- 0.33. CONCLUSION: These data indicate delayed gastric emptying with limited recovery in 1 week. We conclude that gastric feeding should not be employed, and the route for early nutritional intervention should be transpyloric for the trauma patient.

Cerebral asymmetries in processing strategies for letter and symbol trigrams.

Several studies have shown that laterally presented consonant-vowel-consonant (CVC) strings produce both superior performance, and a more wholistic processing strategy in the right visual field/left hemisphere (RVF/LHEM), and a more sequential strategy in the inferior left visual field (LVF). To determine whether these strategies are applied to other types of trigrams subjects (n = 30) were asked to identify consonant and symbol trigrams briefly projected unilaterally to the LVF or RVF, or bilaterally (the same trigram in both fields--BVF). A second group of subjects (n = 30) first practiced pronouncing consonant trigrams and then viewed them tachistoscopically. Both tasks yield RVF advantages. Symbols are processed more wholistically in the LVF, more sequentially in the RVF and in an intermediate pattern when presented bilaterally. In contrast, subjects seem to chunk letters as bigrams, and do so equally well in all fields, and visual field differences in strategies emerge for consonants only when they are pronounced. Pronounceability of consonant trigrams, assessed with ratings and vocal reaction times, was predicted by orthographic regularity. Since the RHEM has limited phonetic skills, but it, like the LHEM, is privy to information on orthographic regularity, the error pattern on consonant strings indicates non-phonetic processing, whereas the RVF wholistic strategy for consonant-vowel-consonant strings appears to reflect phonetic processing.

Effect of protein kinase-C activation on the Mg(2+)-sensitivity of cloned NMDA receptors.

The mechanisms responsible for protein kinase-c (PKC) mediated potentiation of NMDA receptors are poorly understood. One hypothesis is that PKC-activation reduces the receptor's characteristic voltage-dependent Mg(2+)-blockade. Experiments performed on Xenopus oocytes expressing cloned NMDA receptors demonstrated that PKC-activation induced no change in the sensitivity of zeta 1/epsilon 3 and zeta 1/epsilon 4 receptors to Mg(2+)-blockade and, even though PKC-activation did induce a small shift in Mg2+ sensitivity for the zeta 1/epsilon 1 and zeta 1/epsilon 2 receptors, the change seen was not large enough to account for an appreciable increase in NMDA receptor activity. Baseline Mg(2+)-sensitivities and levels of PKC-mediated potentiation were also quantified for each of the di-heteromeric NMDA receptors. The order of Mg(2+)-sensitivity is zeta 1/epsilon 1 (most sensitive) > zeta 1/epsilon 2 > zeta 1/epsilon 4 > zeta 1/epsilon 3 (least sensitive). PKC-activation caused a 2-fold increase in zeta 1/epsilon 1 currents, a 4-fold increase in zeta 1/epsilon 2 currents and no change in either zeta 1/epsilon 3 or zeta 1/epsilon 4 currents. These data suggest that PKC-potentiation of the cloned di-heteromeric NMDA receptors does not involve a reduction in Mg(2+)-blockade. The di-heteromeric receptors possess varied properties in regard to PKC-potentiation and Mg(2+)-blockade which have been quantified here.

Determination of the isotope enrichment of one or a mixture of two stable labelled tracers of the same compound using the complete isotopomer distribution of an ion fragment; theory and application to in vivo human tracer studies.

Calculations of flux rates for stable isotope tracer studies are based upon enrichment values of an infused tracer. We propose the determination of enrichment values by gas chromatography/mass spectrometry, which is based on tracer mole fraction and mass spectrometer signals, normalized over the total signal of an ion fragment isotopomer distribution. The method accounts for overlap of the signals of one or two tracers and the tracee, high tracer mole fraction and incomplete labelling of the (infused) tracer. For the single and multiple tracer case a linear relationship between tracer mole fraction (from zero to one) and all normalized mass spectrometer signals is derived. This linearity over the entire range is demonstrated with a single (1-13C)glucose tracer and for mixtures of (1-13C)- and (3,3-2H2)tyrosine tracers. The linearity allows determination of the tracer mole fraction for two tracers, using multiple linear regression. The corresponding calibration can rely on measurements of the pure tracer and tracee compound, without weighing or check for chemical purity. This is compared with a calibration based on tracer/tracee mixtures. Estimates for the tracer mole fraction are slightly better if based on a calibration, using standard mixtures. In all cases the tracer mole fraction can be determined with high precision (coefficient of variation smaller than 5%) and high accuracy. For tyrosine it is demonstrated that the measurement of seven channels rather than three, for the main isotopomers, does not reduce the precision in the prediction of the tracer mole fraction. Equations are also derived to use the tracer mole fraction to estimate the endogenous production of the tracee under study conditions, assuming a steady state of the host metabolism.

Methionine kinetics and balance at the 1985 FAO/WHO/UNU intake requirement in adult men studied with L-[2H3-methyl-1-13C]methionine as a tracer.

The upper range of the requirement for methionine plus cystine in healthy adults was proposed in 1985 by FAO/WHO/UNU to be 13 mg.kg body wt-1.d-1. To explore the validity of this estimate, five healthy, young adult men were given for 7 d a diet based on an L-amino acid mixture supplying 13 mg methionine.kg-1.d-1 (87 mumol.kg-1.d-1) without cystine. Constant intravenous infusions of L-[2H3-methyl-1-13C]methionine were given on days 5 and 7 while subjects were in the fed and postabsorptive states, respectively. Estimates were made of methionine oxidation, and daily methionine balance was derived from the intake-oxidation data. For the five subjects, methionine balances were -0.9, +0.7, +3.5, -3.1, and -3.8 mg kg-1.d-1, or -6, +5, +23, -21, and -26 mumol.kg-1.d-1. These findings lead to the conclusion that the upper range of the requirement for methionine plus cystine probably exceeds 13 mg.kg-1.d-1 in healthy young adults. The implications of this conclusion for establishing an appropriate amount of sulfur amino acids in an amino acid requirement pattern for adults is discussed.

Methionine kinetics in adult men: effects of dietary betaine on L-[2H3-methyl-1-13C]methionine.

The effects of a daily 3-g supplement of betaine on kinetic aspects of L-[2H3-methyl-1-13C]methionine (MET) metabolism in healthy young adult men were explored. Four groups of four subjects each were given a control diet, based on an L-amino acid mixture supplying 29.5 and 21.9 mg.kg-1.d-1 of L-methionine and L-cystine for 4 d before the tracer study, conducted on day 5 during the fed state. Two groups received the control diet and two groups received the betaine supplement. Tracer was given intravenously (iv) or orally. The transmethylation rate of MET (TM), homocysteine remethylation (RM), and oxidation of methionine were estimated from plasma methionine labeling and 13C enrichment of expired air. RM tended to increase (P = 0.14) but the TM and methionine oxidation were significantly (P less than 0.05) higher after betaine supplementation when estimated with the oral tracer. No differences were detected with the intravenous tracer. Methionine concentration in plasma obtained from blood taken from subjects in the fed state was higher (P less than 0.01) with betaine supplementation. These results suggest that excess methyl-group intake may increase the dietary requirement for methionine.

Branched-chain amino acid interactions with reference to amino acid requirements in adult men: valine metabolism at different leucine intakes.

We explored whether the oxidation of valine and by implication the physiological requirement for this amino acid are affected by changes in leucine intake over a physiological range. Six young adult men received, in random order, four L-amino acid-based diets for 5 d supplying either 20 or 10 mg valine.kg body wt-1.d-1, each in combination with 80 or 40 mg leucine.kg-1.d-1. On day 6 subjects were studied with an 8-h continuous intravenous infusion of [1-13C]valine (and [2H3]leucine) to determine valine oxidation in the fasted state (first 3 h) and fed state (last 5 h). Valine oxidation in the fasted state was similar among all diets but was lower (P less than 0.05) in the fed state for the 10 vs 20 mg valine.kg-1.d-1 intake. Leucine intake did not affect valine oxidation. Mean daily valine balance approximated +1.3 mg.kg-1.d-1 for the 20-mg intake and -1.6 mg.kg-1.d-1 for the 10-mg intake. These findings support our previously suggested mean valine requirement estimate of approximately 20 mg.kg-1.d-1.

Branched-chain amino acid interactions with reference to amino acid requirements in adult men: leucine metabolism at different valine and isoleucine intakes.

Recent estimates of the leucine requirement of adult men based on 13C-tracer studies are substantially higher than those proposed by FAO/WHO/UNU (1985). To explore whether leucine oxidation and requirements are affected by the dietary amount of valine. 11 healthy young adult men received, in random order, for 5 d, one of four L-amino acid diets providing 40 or 15 mg leucine.kg-1.d-1 together with variable amounts mg.kg-1.d-1 of valine and isoleucine in the following combinations (Val:Ile): 80:62 and 20:62 (six subjects; phase 1); 20:62 and 20:20 (five subjects, phase 2). On the morning of day 6, a continuous intravenous infusion of L-[1-13C]leucine was given for 7-8 h; the subject was in the fasting state for the initial 2.5 or 3 h and in the fed state for the remainder of the time. Also, [2H3]leucine was added to the diet. Leucine oxidation was similar for all diet groups in the fasted state. During the fed state, leucine oxidation was not affected by the Val:Ile pattern. Thus, changes in the pattern of branched-chain amino acid intake within a physiological range do not affect isotopically derived estimates of the leucine requirement.

Proline metabolism in adult male burned patients and healthy control subjects.

Postabsorptive proline flux, oxidation, and endogenous biosynthesis were determined in five severely burned intensive-care-unit patients (mean age 27 y) and in six healthy, young-adult control subjects. Continuous primed, intravenous, 160-min, dual stable-isotope-tracer infusions of L-[1-13C]proline and L-[methyl-2H3]leucine were used in conjunction with measurement of plasma proline concentration and 24-h urinary hydroxyproline output. Burn patients, compared with normal individuals, demonstrated a doubling in proline and leucine flux (P less than 0.01 for both findings), a threefold enhancement of proline oxidation (P less than 0.05), a trend toward decreased proline synthesis, and a 37% reduction in plasma proline concentrations (P less than 0.05). Further, the injured group, unlike the control group, was in a distinct negative body proline balance, as proline oxidation greatly exceeded endogenous proline biosynthesis (P less than 0.01). These studies indicate that significant proline deficits may evolve during the postabsorptive period in severely burned patients and that an exogenous supply of proline might benefit the nitrogen economy of the traumatized patient.