The concomitant lower concentrations of vitamins B6, B9 and B12 may cause methylation deficiency in autistic children.

Autism spectrum disorder (ASD) is characterized by severe and persistent difficulties in social communication and social interaction at multiple levels. Recently, metabolic disorders have been associated with most cases of patients with ASD. The aim of this study was to investigate, through a new and more sophisticated mass technique, such as UHPLC-mass spectrometry (Q-exactive analyzer), alteration in metabolisms analyzing ASD children urine samples from children showing simultaneous vitamin B6, B9 and B12 deficiencies. This in order to study how these concurrent deficiencies may influence some phenotypic aspects of autistic disorder. Thus, urinary metabolic patterns specific to ASD were explored at an early age in 60 children with ASD, showing lower three vitamins levels, and 60 corresponding controls (age group 3-8, M: F=42:18). The results showed significant block of cystathionine formation with consequent accumulation of homocysteine. A lower glutathione levels (GSH), with reduction of essential intracellular reducing environment required for normal immune function, detoxification capacity and redox-sensitive enzyme activity. Increased concentration of 5-methyltetrahydrofolate, which leads to a lower availability of methyl group and significant decrease in urinary methionine and S-adenosyl-L-methionine (SAM) concentrations, the major methyl donor. The latter justify the well-known reduction in protein and DNA methylation reported in autistic children. As a final consideration, the concomitant deficiencies of all three B vitamins, recorded in a significant number of autistic children, suggests that intestinal dysbiosis in these patients may be the main cause of a reduction in their absorption, in addition to the genetic mutation of a specific gene.

Metabolomic Profiling of Infants With Recurrent Wheezing After Bronchiolitis.

BACKGROUND: Bronchiolitis is associated with a greater risk of developing recurrent wheezing, but with currently available tools, it is impossible to know which infants with bronchiolitis will develop this condition. This preliminary prospective study aimed to assess whether urine metabolomic analysis can be used to identify children with bronchiolitis who are at risk of developing recurrent wheezing. METHODS: Fifty-two infants <1 year old treated in the emergency department at University Hospital of Padova for acute bronchiolitis were enrolled (77% tested positive for respiratory syncytial virus [RSV]). Follow-up visits were conducted for 2 years after the episode of bronchiolitis. Untargeted metabolomic analyses based on mass spectrometry were performed on urine samples collected from infants with acute bronchiolitis. Data modeling was based on univariate and multivariate data analyses. RESULTS: We distinguished children with and those without postbronchiolitis recurrent wheeze, defined as ≥3 episodes of physician-diagnosed wheezing. Pathway overrepresentation analysis pointed to a major involvement of the citric acid cycle (P < .001) and some amino acids (lysine, cysteine, and methionine; P ≤ .015) in differentiating between these 2 groups of children. CONCLUSION: This is the first study showing that metabolomic profiling of urine specimens from infants with bronchiolitis can be used to identify children at increased risk of developing recurrent wheezing.

Metabolic Response in Rabbit Urine to Occurrence and Relief of Unilateral Ureteral Obstruction.

Ureteral obstruction will lead clinically to hydronephrosis, which may further develop into partial or complete loss of kidney function and even cause permanent histological damage. However, there is little knowledge of metabolic responses during the obstructed process and its recoverability. In this study, a complete unilateral ureteral obstruction (CUUO) model was established in the rabbit, and 1H NMR-based metabolomic analysis of urine was used to reveal the metabolic perturbations in rabbits caused by CUUO and the metabolic recovery after the CUUO was relieved. Univariate and multivariate statistical analyses were used to identify metabolic characteristics. The gradually decreased levels of 3-hydroxykynurenine, 3-methylhistidine, creatinine, guanidoacetate, meta- and para-hydroxyphenylacetate, and phenylacetylglycine and the gradually increased levels of acetate, alanine, citrate, glycine, lactate, and methionine in urine could be regarded as potential biomarkers for the occurrence and severity of ureteral obstruction. And the reduced levels of 3-methylhistidine, creatinine, guanidoacetate, hippurate, meta-hydroxyphenylacetate, and methylguanidine and the elevated levels of 2-aminoisobutyrate, acetylcholine, citrate, lactate, lysine, valine, and α-ketoglutarate in urine compared with the obstructed level could characterize the metabolic recovery of ureteral obstruction. Our results depicted the disturbed biochemical pathways involved in ureteral obstruction and demonstrated the practicability of recovering renal functions for the patients with severe hydronephrosis in clinical practice by removing causes for obstruction.

A male case with CDKL5-associated encephalopathy manifesting transient methylmalonic acidemia.

Mutations in the X-linked gene CDKL5 cause early-onset epileptic encephalopathy and severe developmental delay. Because this disorder predominantly affects females, the full clinical spectrum of male patients remains elusive. We herein report a 16-year-old boy, who suffered from intractable seizures 20 days after birth. Serial electroencephalograms detected recurrent focal epileptiform discharges from age 4 months, which evolved to hypsarrhythmia later in infancy. Mass-spectrometric analyses revealed increase in urinary excretion of methylmalonic acid without perturbed concentrations of propionic acid, homocystein and methionine. Whole-exome sequencing identified a de novo, truncating mutation in CDKL5 (NM_003159.2:c.419dupA, p.Asn140Lysfs*8). Targeted sequencing excluded concomitant mutations in methylmalonic academia-associated genes. No methylmalonic acidemia has been reported in children with CDKL5 disorder. Extensive analyses on organic acid metabolism for males with CDKL5 mutations will gain more insight into their biochemical profiles in infancy.

Simultaneous Determination of Methionine and Homocysteine by on-column derivatization with o-phtaldialdehyde.

A fast and simple HPLC-based assay has been developed for the simultaneous determination of homocysteine (Hcy) and methionine (Met) in plasma and urine samples, utilizing as small volume of sample as 10µL. The assay uses on-column derivatization with o-phthaldialdehyde. The separation of Hcy and Met was achieved in 14min on a reversed phase C-18 column, followed by fluorescence detection (excitation at 348nm and emission at 438nm for Met; excitation at 370nm and emission at 480nm for Hcy). Linearity of the detector response was observed in the range of 2-60 µmol L-1 for Met and 2-40 µmol L-1 for Hcy. The method was successfully applied for Met and Hcy quantification in human and mouse plasma and urine samples from cystathionine ß-synthase deficient and unaffected individuals.

Increased homocysteine levels correlate with the communication deficit in children with autism spectrum disorder.

The clinical significance of high levels of homocysteine in autism spectrum disorder (ASD) is unknown. An experimental study was conducted in order to evaluate the concentration of homocysteine in children with ASD and typically developing children and to analyse any relationships with the severity of core symptoms of ASD and other clinical features (drugs, co-morbidities, gender, age, diet). Core symptoms of autism were evaluated by DSM-IV criteria. Homocysteine, glutathione, methionine, 3-nitrotyrosine were measured in urine. The increase in homocysteine concentration was significantly and directly correlated with the severity of the deficit in communication skills, but was unrelated to deficit in socialisation or repetitive/restricted behaviour. Urinary homocysteine concentration may be a possible biomarker for communication deficits in ASD and a potential diagnostic tool useful to evaluate new treatment options since no treatment for core symptoms of ASD are available.

Discovery and validation of urinary metabotypes for the diagnosis of hepatocellular carcinoma in West Africans.

UNLABELLED: There is no clinically applicable biomarker for surveillance of hepatocellular carcinoma (HCC), because the sensitivity of serum alpha-fetoprotein (AFP) is too low for this purpose. Here, we determined the diagnostic performance of a panel of urinary metabolites of HCC patients from West Africa. Urine samples were collected from Nigerian and Gambian patients recruited on the case-control platform of the Prevention of Liver Fibrosis and Cancer in Africa (PROLIFICA) program. Urinary proton nuclear magnetic resonance ((1) H-NMR) spectroscopy was used to metabolically phenotype 290 subjects: 63 with HCC; 32 with cirrhosis (Cir); 107 with noncirrhotic liver disease (DC); and 88 normal control (NC) healthy volunteers. Urine samples from a further cohort of 463 subjects (141 HCC, 56 Cir, 178 DC, and 88 NC) were analyzed, the results of which validated the initial cohort. The urinary metabotype of patients with HCC was distinct from those with Cir, DC, and NC with areas under the receiver operating characteristic (AUROC) curves of 0.86 (0.78-0.94), 0.93 (0.89-0.97), and 0.89 (0.80-0.98) in the training set and 0.81 (0.73-0.89), 0.96 (0.94-0.99), and 0.90 (0.85-0.96), respectively, in the validation cohort. A urinary metabolite panel, comprising inosine, indole-3-acetate, galactose, and an N-acetylated amino acid (NAA), showed a high sensitivity (86.9% [75.8-94.2]) and specificity (90.3% [74.2-98.0]) in the discrimination of HCC from cirrhosis, a finding that was corroborated in a validation cohort (AUROC: urinary panel = 0.72; AFP = 0.58). Metabolites that were significantly increased in urine of HCC patients, and which correlated with clinical stage of HCC, were NAA, dimethylglycine, 1-methylnicotinamide, methionine, acetylcarnitine, 2-oxoglutarate, choline, and creatine. CONCLUSION: The urinary metabotyping of this West African cohort identified and validated a metabolite panel that diagnostically outperforms serum AFP.

Citrin deficiency in a Romanian child living in Spain highlights the worldwide distribution of this defect and illustrates the value of nutritional therapy.

We report citrin deficiency in a neonatal non-East-Asian patient, the ninth Caucasian reported with this disease. The association of intrahepatic cholestasis, galactosuria, very high alpha-fetoprotein and increased plasma and urine citrulline, tyrosine, methionine and threonine levels suggested citrin deficiency. Identification of a protein-truncating mutation (c.1078C>T; p.Arg360*) in the SLC25A13 gene confirmed the diagnosis. An immediate response to a high-protein, lactose-free, low-carbohydrate formula was observed. Our report illustrates the need for awareness on citrin deficiency in Western countries.

Maternal choline intake modulates maternal and fetal biomarkers of choline metabolism in humans.

BACKGROUND: In 1998 choline Adequate Intakes of 425 and 450 mg/d were established for nonpregnant and pregnant women, respectively. However, to our knowledge, no dose-response studies have been conducted to evaluate the effects of pregnancy or maternal choline intake on biomarkers of choline metabolism. OBJECTIVE: We sought to quantify the effects of pregnancy and maternal choline intake on maternal and fetal indicators of choline metabolism. DESIGN: Healthy pregnant (n = 26; 27 wk gestation) and nonpregnant (n = 21) women were randomly assigned to receive 480 or 930 mg choline/d for 12 wk. Fasting blood samples and placental tissue and umbilical cord venous blood were collected and analyzed for choline and its metabolites. RESULTS: Regardless of the choline intake, pregnant women had higher circulating concentrations of choline (30%; P < 0.001) but lower concentrations of betaine, dimethylglycine, sarcosine, and methionine (13-55%; P < 0.001). Obligatory losses of urinary choline and betaine in pregnant women were ∼2-4 times as high (P ≤ 0.02) as those in nonpregnant women. A higher choline intake yielded higher concentrations of choline, betaine, dimethylglycine, and sarcosine (12-46%; P ≤ 0.08) in both pregnant and nonpregnant women without affecting urinary choline excretion. The higher maternal choline intake also led to a doubling of dimethylglycine in cord plasma (P = 0.002). CONCLUSION: These data suggest that an increment of 25 mg choline/d to meet the demands of pregnancy is insufficient and show that a higher maternal choline intake increases the use of choline as a methyl donor in both maternal and fetal compartments. This trial was registered at clinicaltrials.gov as NCT01127022.

Altered D: -methionine kinetics in rats with renal impairment.

D: -Amino acids are now recognized to be widely present in mammals. In rats, exogenously administered D: -methionine is almost converted into the L: -enantiomer via 2-oxo-4-methylthiobutylic acid as an intermediate. D: -Amino acid oxidase is associated with conversion of D: -methionine into the 2-oxo acid. Since D: -amino acid oxidase is present at the highest activity in the kidney compared to other organ, kidney injury is suggested to cause accumulation of D: -methionine. The purpose of the present study is to assess the role of kidney in the elimination of D: -methionine and metabolic conversion into L: -methionine in rats using a stable isotope methodology. After a bolus i.v. administration of D: -[²H3)]methionine to 5/6-nephrectomized rats, plasma concentrations of D: -[²H3]methionine, L: -[²H3]methionine, and endogenous L: -methionine were determined by a stereoselective GC-MS method. Renal mass reduction slowed down the elimination of D: -[²H3]methionine. The clearance values of conversion of D: -[²H3]methionine into the L: -enantiomer in 5/6-nephrectomized rats were one-sixth of those in sham-operated rats. The elimination behavior of D: -[²H3]methionine observed in rats demonstrated that kidney was the principal organ responsible for chiral inversion of D: -methionine.

Potential loss of methionine following extended storage of newborn screening samples prepared for tandem mass spectrometry analysis.

BACKGROUND: Methionine (Met) is a key metabolite used in the newborn screening of homocystinuria by tandem mass spectrometry (MS/MS). Recently, a loss of ion counts in both Met and its deuterium-labeled internal standard ((2)H(3)-Met) was observed by the CDC's Newborn Screening Quality Assurance Program laboratory. We report on the stability of labeled and unlabeled Met solutions and their storage in two types of 96 well microtiter plates to illustrate the potential loss of Met following storage of samples prior to MS/MS analysis. METHODS: Neat labeled and unlabeled Met standards were prepared and added (25, 50 and 100 microl) to two different types of microtiter plates, dried under nitrogen and stored for up to 168 h. All samples were reconstituted in mobile phase and analyzed as free acids for simplification of the study. RESULTS AND CONCLUSIONS: Met appears to interact significantly with polystyrene microtiter plates and to a much lesser extent with polypropylene microtiter plates. Furthermore, the loss is greatest for lower concentrations of methionine. While this loss of Met signal may be unimportant due to a presumption of equal loss of (2)H(3)-Met, a significant decline in ion signals will cause greater error in the calculation of concentration. These results suggest that polypropylene may be a better choice for Met analysis. Furthermore, storing prepared samples prior to analysis may impact the quality of the MS/MS analysis for Met and potentially other metabolites. Plates used by newborn screening laboratories should be evaluated periodically if the signal intensity for Met is reduced.

Identification of urinary modified nucleosides and ribosylated metabolites in humans via combined ESI-FTICR MS and ESI-IT MS analysis.

The physiological response of the human body to several diseases can be reflected by the metabolite pattern in biological fluids. Cancer, like other diseases accompanied by metabolic disorders, causes characteristic effects on cell turnover rate, activity of modifying enzymes, and RNA/DNA modifications. This results in an altered excretion of modified nucleosides and biochemically related compounds. In the course of our metabolic profiling project, we screened 24-h urine of patients suffering from lung, rectal, or head and neck cancer for previously unknown ribosylated metabolites. Therefore, we developed a sample preparation procedure based on boronate affinity chromatography followed by additional prepurification with preparative TLC. The isolated metabolites were analyzed by ion trap mass spectrometry (IT MS) and Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS). IT MS was applied for LC-auto MS(3) screening runs and MS(n(n=4-6)) syringe pump infusion experiments, yielding characteristic fragmentation patterns. FTICR MS measurements enabled the calculation of corresponding molecular formulae based on accurate mass determination (mass accuracy: 1-5 ppm for external and sub-ppm values for internal calibration). We were able to identify 22 metabolites deriving from cellular RNA metabolism and related metabolic pathways like histidine metabolism, purine biosynthesis, methionine/polyamine cycle, and nicotinate/nicotinamide metabolism. The compounds 1-ribosyl-3-hydroxypyridinium, 1-ribosyl-pyridinium, and 3-ribosyl-1-methyl-l-histidinium as well as a series of ribosylated histamines, conjugated to carboxylic acids at the N(omega)-position were found as novel urinary constituents. The occurrence of the modified nucleosides 2-methylthio-N(6)-(cis-hydroxyisopentenyl)-adenosine, 5-methoxycarbonylmethyl-2-thiouridine, N(6)-methyl-N(6)-threonylcarbamoyladenosine, and 2-methylthio-N(6)-threonylcarbamoyladenosine in human urine is verified for the first time.

Simultaneous quantitation of homocysteine, cysteine and methionine in plasma and urine by liquid chromatography-tandem mass spectrometry.

BACKGROUND: A method utilizing liquid chromatography-electrospray tandem mass spectrometry (LC-MS/MS) has been developed and evaluated for the determination of total homocysteine, cysteine and methionine in plasma and urine. The simultaneous measurement of homocysteine and methionine concentrations may help explain the underlying mechanism responsible for hyperhomo-cysteinaemia. METHODS: Samples were prepared by simple protein precipitation after reduction of disulphides by dithiothreitol. Reduced analyte signal caused by ionization suppression effects, seen with plasma samples, was compensated for with matrix-matched standards, and the use of isotopically labelled internal standards. Recovery for each analyte was better than 94%. RESULTS: Concentrations of plasma homocysteine determined by LC-MS/MS were compared with those obtained by two automated commercially available FDA-approved procedures: (i) high-performance liquid chromatography (HPLC) with pre-column derivatization and fluorescence detection and (ii) by fluorescence polarization immunoassay (FPIA). Agreement with the LC-MS/MS method is given by the Deming regression equations LC-MS/MS = 1.062 HPLC-0.01 and LC-MS/MS = 1.104 FPIA-0.43. CONCLUSION: Low reagent costs together with the relative simplicity of sample preparation make the LC-MS/MS method well suited, not only for research work but also in those laboratories with a tandem mass spectrometer, for the measurement of routine clinical samples.

In vivo metabolism of L-methionine in mice: evidence for stereoselective formation of methionine-d-sulfoxide and quantitation of other major metabolites.

Flavin-containing monooxygenases (FMOs) 1-4 oxidize methionine (Met) to methionine sulfoxide (MetO). FMO3, the primary isoform expressed in adult human liver, has the lowest Km and favors methionine-d-sulfoxide (Met-d-O) formation over methionine-l-sulfoxide. Because female mice, but not males, also express FMO3 in liver, levels of Met and its major metabolites were determined in male or female mice dosed with 400 mg/kg Met i.p. The results show that Met levels in male and female mouse liver or plasma increased significantly at both 15 and 30 min after the Met treatment; Met plasma and liver levels at 30 min were similar to or lower than the corresponding levels at 15 min. Liver and plasma MetO levels increased significantly in both sexes at 30 min, and Met-d-O was the major MetO diastereomer detected. Interestingly, less than 0.1% of the Met dose was excreted in the urine (0-24 h) as Met and Met-d-O. S-Adenosylmethionine (SAM) was the major metabolite detected in liver at 15 min. Liver SAM levels at 30 min were lower than the levels at 15 min, and the plasma SAM levels at both 15 and 30 min were much lower than the corresponding levels in the liver. Increases in liver and/or plasma S-adenosyl-l-homocysteine, 5'-deoxy-5'-(methylthio)adenosine, and N-acetyl-l-methionine were also detected. Taken together, these results suggest that mice extensively and rapidly used the Met dose. Although mice exhibited increases in tissue MetO levels, a major role for FMO3 in Met-d-O formation is not certain since the MetO increases were mostly similar in both males and females.

Effect of L-methionine supplementation on plasma homocysteine and other free amino acids: a placebo-controlled double-blind cross-over study.

OBJECTIVE: The essential amino acid L-methionine is a potential compound in the prophylaxis of recurrent or relapsing urinary tract infection due to acidification of urine. As an intermediate of L-methionine metabolism, homocysteine is formed. The objective was to study the metabolism of L-methionine and homocysteine, and to assess whether there are differences between patients with chronic urinary tract infection and healthy control subjects. DESIGN: A randomized placebo-controlled double-blind intervention study with cross-over design. SETTING: Department of Nutritional Physiology, Institute of Nutrition in cooperation with the Department of Internal Medicine III, Friedrich Schiller University of Jena, Germany. SUBJECTS: Eight female patients with chronic urinary tract infection and 12 healthy women (controls). INTERVENTIONS: After a methionine-loading test, the volunteers received 500 mg L-methionine or a placebo three times daily for 4 weeks. MAIN OUTCOME MEASURES: Serum and urinary concentrations of methionine, homocysteine, cystathionine, cystine, serine, glycine and serum concentrations of vitamin B12, B6 and the state of folate. RESULTS: Homocysteine plasma concentrations increased from 9.4+/-2.7 micromol/l (patients) and 8.9+/-1.8 micromol/l (controls) in the placebo period to 11.2+/-4.1 micromol/l (P=0.031) and 11.0+/-2.3 micromol/l (P=0.000), respectively, during L-methionine supplementation. There were significant increases in serum methionine (53.6+/-22.0 micromol/l; P=0.003; n=20) and cystathionine (0.62+/-0.30 micromol/l; P=0.000; n=20) concentrations compared with the placebo period (33.0+/-12.0 and 0.30+/-0.10 micromol/l; n=20). Simultaneously, renal excretion of methionine and homocysteine was significantly higher during L-methionine intake. CONCLUSIONS: Despite an adequate vitamin status, the supplementation of 1500 mg of L-methionine daily significantly increases homocysteine plasma concentrations by an average of 2.0 micromol/l in patients and in control subjects. An optimal vitamin supplementation, especially with folate, might prevent such an increase.

Elastin defects in the lungs of avian and murine models of homocysteinemia.

Homocysteinemia in animals is associated with disruption of the elastic fiber component of the extracellular matrix, resulting in vascular complications. The authors have utilized both avian and murine models to investigate the effects of homocysteinemia on lung development and repair following injury. Days old chicks were fed a diet containing 2% methionine for 3 weeks. Pregnant mice were given 2% methionine in the diet and feeding continued for up to 6 weeks after birth. The lungs were removed and examined for defects in elastin fiber formation. Methionine levels were elevated 20-fold in the serum from chicks receiving the methionine and 10-fold in pregnant mice. The elastic fibers in the parabronchi and air capillaries of chicks receiving methionine were thin and clearly disrupted. In the 2% methionine neonatal pups, normal lung development was prevented and the alveoli were significantly enlarged. However, after the pups reached 10 days of age the 2% methionine lungs did not differ histologically from the normal controls. Fetal mice reflected the same serum methionine levels as the dams fed the 2% methionine diet, yet after birth the serum levels of the neonates returned to control levels within 3 days. The authors found that the high serum methionine levels of the dams were not transferred to the milk, allowing the pups to reverse the histopathology observed early and then develop normally. The ability of the lung to replace elastin following elastase injury was not different in mice raised on the 2% methionine diet compared to controls. The studies show that continuous exposure of the developing lung to high circulating levels of methionine/homocysteine can result in major disruptions of elastic fibers and lung architecture. However, young mammals such as the mouse are protected from extended lung pathology because toxic levels of methionine are not transferred through the mothers milk.

A novel mutation, P126R, in a Japanese patient with HHH syndrome.

Mitochondrial ornithine transporter deficiency, or HHH syndrome, is a metabolic disorder resulting in various neurologic symptoms, including mental retardation, spastic paraparesis with pyramidal signs, cerebellar ataxia, and episodic disturbance of consciousness or coma caused by hyperammonemia. Several mutations have been reported in the ORNT1 gene encoding mitochondrial ornithine transporter of patients with this disorder. In this article, we report a new patient, a male 15 years of age, who had typical clinical features of HHH syndrome. Because the patient did not have any of the three mutations previously described in other Japanese patients with HHH syndrome, and the only material available from the patient was peripheral leukocytes, we established a genomic polymerase chain reaction method using intronic primers to amplify every exon of the ORNT1 gene, and we directly sequenced the polymerase chain reaction products. Using this method, we documented a novel mutation in this patient, P126R, and demonstrated that HHH syndrome is genetically heterogeneous, even in the Japanese population.

Importance of sulfate, cysteine and methionine as precursors to felinine synthesis by domestic cats (Felis catus).

There is conflicting evidence in the literature on the utilization of cysteine and methionine as precursors to the urinary sulfur-containing amino acid felinine in cats. Three entire domestic short-haired male cats, housed individually in metabolism cages, were injected intraperitoneally with either [35S]-sulfate, [35S]-cysteine, or [35S]-methionine. Daily urine samples were collected quantitatively for up to 9 days after injection. Each cat was injected once with each compound after observing an appropriate interval for [35S] to be depleted between injections. All the urine samples were analysed for felinine content and total radioactivity. Felinine was isolated from each urine sample and analysed for radioactivity. No radioactivity was found in felinine from cats injected with [35S]-sulfate. The mean (+/-S.E.M.) cumulative recovery of radioactivity in the urine of the [35S]-sulfate injected cats was 90.6+/-6.1% after 4 days. The mean (+/-S.E.M.) cumulative incorporation rate of radioactivity into felinine by the cats receiving the [35S]-cysteine and [35S]-methionine were 11.6+/-1.6 and 8.6+/-0.6%, respectively, after 9 days. The mean (+/-S.E.M.) cumulative recoveries of radioactivity in the urine were 58.1+/-3.7 and 36.0+/-8.0%, respectively. Cysteine and methionine, but not sulfate, are precursors to felinine, with cysteine being a more quantitatively important precursor compared to methionine.

Synthesis of glutathione in response to methionine load in control subjects and in patients with cirrhosis.

The fasting plasma level of reduced glutathione (GSH), a methionine-derived tripeptide, is reduced in cirrhosis. There is evidence that a reduced activity of S-adenosyl-L-methionine synthetase limiting the flux of methionine along the transmethylation/transsulfuration pathway may contribute to decrease GSH levels. No studies have analyzed plasma GSH in response to a methionine load. In 6 control subjects and in 10 patients with cirrhosis, plasma sulfur amino acid and plasma and erythrocyte GSH levels were measured in response to a L-methionine load (0.1 g/kg). Blood samples were obtained throughout the day after the oral load. Urine was collected for measurement of sulfur excretion. During the study period, all subjects consumed a standard diet of 1,683 kcal containing 2% protein and virtually no methionine. Plasma methionine increased in both groups to a peak level exceeding 20 times the basal value 90 minutes after the load, and declined thereafter. Methionine clearance, calculated on the descending part of the methionine-time curve, was reduced by 50% in cirrhosis (P = .0001). Fasting GSH was higher in controls (mean +/- SD, 3.9 +/- 1.3 v 1.6 +/- 0.7 micromol/L, P = .0004). In response to a methionine load, it peaked at 10.2 +/- 7.2 and 3.2 +/- 1.3 micromol/L, respectively (P = .009). Thereafter, plasma GSH progressively declined, and after 24 hours, it returned to the fasting preinfusion values in both groups. Plasma cysteine and taurine concentrations, as well as the erythrocyte GSH time course, paralleled plasma GSH levels, with less significant differences between groups. Sulfate excretion was delayed. GSH synthesis is stimulated by a methionine load. The reduced flux of methionine along the transmethylation/transsulfuration pathway reduces GSH synthesis in cirrhosis. Defective methionine metabolism also may be responsible for reduced fasting GSH.

Methionine transamination in patients with homocystinuria due to cystathionine beta-synthase deficiency.

To assess the ability of patients with homocystinuria due to cystathionine beta-synthase (CBS) deficiency to perform the reactions of the methionine transamination pathway, the concentrations of the products of this pathway were measured in plasma and urine. The results clearly demonstrate that CBS-deficient patients develop elevations of these metabolites once a threshold near 350 micromol/L for the concurrent plasma methionine concentration is exceeded. The absence of elevated methionine transamination products previously reported among 16 CBS-deficient B6-responsive patients may now be attributed to the fact that in those patients the plasma methionine concentrations were below this threshold. The observed elevations of transamination products were similar to those observed among patients with isolated hypermethioninemia. Plasma homocyst(e)ine did not exert a consistent effect on transamination metabolites, and betaine appeared to effect transamination chiefly by its tendency to elevate methionine. Even during betaine administration, the transamination pathway does not appear to be a quantitatively major route for the disposal of methionine.