Plasma metabolites were associated with spatial working memory in major depressive disorder.

ABSTRACT: Major depressive disorder (MDD) is a common disease with both affective and cognitive disorders. Alterations in metabolic systems of MDD patients have been reported, but the underlying mechanisms still remains unclear. We sought to identify abnormal metabolites in MDD by metabolomics and to explore the association between differential metabolites and neurocognitive dysfunction.Plasma samples from 53 MDD patients and 83 sex-, gender-, BMI-matched healthy controls (HCs) were collected. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) system was then used to detect metabolites in those samples. Two different algorithms were applied to identify differential metabolites in 2 groups. Of the 136 participants, 35 MDD patients and 48 HCs had completed spatial working memory test. Spearman rank correlation coefficient was applied to explore the relationship between differential metabolites and working memory in these 2 groups.The top 5 metabolites which were found in sparse partial least squares-discriminant analysis (sPLS-DA) model and random forest (RF) model were the same, and significant difference was found in 3 metabolites between MDD and HCs, namely, gamma-glutamyl leucine, leucine-enkephalin, and valeric acid. In addition, MDD patients had higher scores in spatial working memory (SWM) between errors and total errors than HCs. Valeric acid was positively correlated with working memory in MDD group.Gamma-glutamyl leucine, leucine-enkephalin, and valeric acid were preliminarily proven to be decreased in MDD patients. In addition, MDD patients performed worse in working memory than HCs. Dysfunction in working memory of MDD individuals was associated with valeric acid.

Gut Microbial Product Predicts Cardiovascular Risk in Chronic Kidney Disease Patients.

BACKGROUND: The gut microbiota is altered in patients with chronic kidney disease (CKD), and cardiovascular risk increases with progressive CKD. This study examined the potential link between short chain fatty acids (SCFAs), which are produced by the gut microbiota, and cardiovascular outcomes in patients with CKD. METHODS: SCFAs were measured using a targeted liquid chromatography-mass spectrometry platform in baseline plasma samples from 214 patients with CKD enrolled in the Clinical Phenotyping Resource and Biobank Core; 81 patients with coronary artery disease (CAD) and 133 without CAD were randomly assigned to training and validation subsets. The primary outcome was a history of CAD and the secondary outcome was a composite history of cardiovascular disease (CVD) at enrollment. RESULTS: We found significantly higher levels of the SCFA valerate among patients with CAD as compared with patients without CAD in the training set (p < 0.001). The valerate concentrations were also significantly higher among subjects with composite outcomes of CVD compared to those without CVD (p = 0.006). These results were subsequently replicated in the validation set. Logistic regression analysis revealed a strong independent association between plasma valerate levels and CVD in both training and validation sets. When valerate was added to the base clinical model comprising of diabetes, hypertension, urinary protein-creatinine ratio, and estimated glomerular filtration rate, it increased the c-statistics for predicting CVD from 0.68 to 0.79 (p = 0.02) in the training set, an observation which was confirmed in the validation set. -Conclusion: This study provides evidence for alterations in gut-microbiota-derived SCFAs with advancing CKD, demonstrates the association of higher plasma valerate levels with pre-existing CVD, and reveals areas for future exploration of cardiovascular risk in patients with CKD.

GC-MS Analysis of Short-Chain Fatty Acids in Feces, Cecum Content, and Blood Samples.

Short-chain fatty acids, the end products of fermentation of dietary fibers by the gut microbiota, have been shown to exert multiple effects on mammalian metabolism. For the analysis of short-chain fatty acids, gas chromatography-mass spectrometry is a very powerful and reliable method. Here, we describe a fast, reliable, and reproducible method for the separation and quantification of short-chain fatty acids in mouse feces, cecum content, and blood samples (i.e., plasma or serum) using gas chromatography-mass spectrometry. The short-chain fatty acids analyzed include acetic acid, propionic acid, butyric acid, valeric acid, hexanoic acid, and heptanoic acid.

Dimethylguanidino valeric acid is a marker of liver fat and predicts diabetes.

Unbiased, "nontargeted" metabolite profiling techniques hold considerable promise for biomarker and pathway discovery, in spite of the lack of successful applications to human disease. By integrating nontargeted metabolomics, genetics, and detailed human phenotyping, we identified dimethylguanidino valeric acid (DMGV) as an independent biomarker of CT-defined nonalcoholic fatty liver disease (NAFLD) in the offspring cohort of the Framingham Heart Study (FHS) participants. We verified the relationship between DMGV and early hepatic pathology. Specifically, plasma DMGV levels were correlated with biopsy-proven nonalcoholic steatohepatitis (NASH) in a hospital cohort of individuals undergoing gastric bypass surgery, and DMGV levels fell in parallel with improvements in post-procedure cardiometabolic parameters. Further, baseline DMGV levels independently predicted future diabetes up to 12 years before disease onset in 3 distinct human cohorts. Finally, we provide all metabolite peak data consisting of known and unidentified peaks, genetics, and key metabolic parameters as a publicly available resource for investigations in cardiometabolic diseases.

Metabolic syndrome increases dietary α-tocopherol requirements as assessed using urinary and plasma vitamin E catabolites: a double-blind, crossover clinical trial.

Background: Vitamin E supplementation improves liver histology in patients with nonalcoholic steatohepatitis, which is a manifestation of the metabolic syndrome (MetS). We reported previously that α-tocopherol bioavailability in healthy adults is higher than in those with MetS, thereby suggesting that the latter group has increased requirements.Objective: We hypothesized that α-tocopherol catabolites α-carboxyethyl hydroxychromanol (α-CEHC) and α-carboxymethylbutyl hydroxychromanol (α-CMBHC) are useful biomarkers of α-tocopherol status.Design: Adults (healthy or with MetS; n = 10/group) completed a double-blind, crossover clinical trial with four 72-h interventions during which they co-ingested 15 mg hexadeuterium-labeled RRR-α-tocopherol (d6-α-T) with nonfat, reduced-fat, whole, or soy milk. During each intervention, we measured α-CEHC and α-CMBHC excretions in three 8-h urine collections (0-24 h) and plasma α-tocopherol, α-CEHC, and α-CMBHC concentrations at various times ≤72 h.Results: During the first 24 h, participants with MetS compared with healthy adults excreted 41% less α-CEHC (all values are least-squares means ± SEMs: 0.6 ± 0.1 compared with 1.0 ± 0.1 µmol/g creatinine, respectively; P = 0.002), 63% less hexadeuterium-labeled (d6)-α-CEHC (0.04 ± 0.02 compared with 0.13 ± 0.02 µmol/g creatinine, respectively; P = 0.002), and 58% less d6-α-CMBHC (0.017 ± 0.004 compared with 0.041 ± 0.004 µmol/g creatinine, respectively; P = 0.0009) and had 52% lower plasma d6-α-CEHC areas under the concentration curves [area under the curve from 0 to 24 h (AUC0-24h): 27.7 ± 7.9 compared with 58.4 ± 7.9 nmol/L × h, respectively; P = 0.01]. d6-α-CEHC peaked before d6-α-T in 77 of 80 paired plasma concentration curves. Urinary d6-α-CEHC 24-h concentrations were associated with the plasma AUC0-24 h of d6-α-T (r = 0.53, P = 0.02) and d6-α-CEHC (r = 0.72, P = 0.0003), and with urinary d6-α-CMBHC (r = 0.88, P < 0.0001), and inversely with the plasma inflammation biomarkers C-reactive protein (r = -0.70, P = 0.0006), interleukin-10 (r = -0.59, P = 0.007), and interleukin-6 (r = -0.54, P = 0.01).Conclusion: Urinary α-CEHC and α-CMBHC are useful biomarkers to noninvasively assess α-tocopherol adequacy, especially in populations with MetS-associated hepatic dysfunction that likely impairs α-tocopherol trafficking. This trial was registered at clinicaltrials.gov as NCT01787591.

Genotype and phenotype characterization in a Spanish cohort with isovaleric acidemia.

Isovaleric acidemia (IVA) is a rare disorder of leucine metabolism. We carried out a multicenter study of IVA patients diagnosed by newborn screening (NBS) or symptoms clinics over a period of 28 years in Spain. Evaluated at diagnosis, data included age, detection method, levels of C5 and IVG, enzymatic studies, clinical presentation parameters and genotype in 16 patients. Follow-up data included C5 levels, intellectual quotient and correlation genotype-phenotype. IVA was detected by NBS in 8 patients (prevalence of 1/326 629). Except 1, all the 8 patients identified by NBS were asymptomatic at diagnosis and had isovalerylcarnitine (C5) levels of 1.6-6.4 µM and isovalerylglycine (IVG) levels <1100 mmol per mol creatinine; they remained asymptomatic with a natural protein intake ⩾1.5 g kg-1 per day. Symptomatic patients with chronic intermittent or acute neonatal IVA had C5 levels of 3.9-16.3 µM and IVG levels >3400 mmol per mol creatinine. The percentage of isovalerate incorporation in fibroblasts was 64-80% in patients detected by NBS and 4.9-13% in symptomatic patients. Cognitive function was within normal ranges in all patients but was negatively correlated with IVG at detection (-0.592; P<0.05). The genetic analysis revealed nine novel mutations. The clinical/biochemical phenotype correlated fairly well with the phenotype predicted by the mutations found. In conclusion, although blood C5 levels have traditionally been considered the prognostic marker of choice, urine IVG levels would appear to be a better predictor, as they correlated well with severity of mutations and were associated with a lower incorporation rate of IVA in fibroblasts and a less favorable clinical course.

Quantification of [1-(5-fluoropentyl)-1H-indol-3-yl](naphthalene-1-yl)methanone (AM-2201) and 13 metabolites in human and rat plasma by liquid chromatography-tandem mass spectrometry.

AM-2201 is a popular synthetic cannabinoid first synthesized in 2000. AM-2201 pharmacokinetic and pharmacodynamic data are scarce, requiring further investigation. We developed a sensitive method for quantifying AM-2201 and 13 metabolites in plasma to provide a tool to further metabolic, pharmacokinetic and pharmacodynamic studies. Analysis was performed by liquid chromatography-tandem mass spectrometry. Chromatographic separation was performed by gradient elution on a biphenyl column with 0.1% formic acid in water/0.1% formic acid in acetonitrile:methanol 50:50 (v/v) mobile phase. Sample preparation (75µL) consisted of an enzymatic hydrolysis and a supported liquid extraction. The method was validated with human plasma with a 0.025 or 0.050-50µg/L working range, and cross-validated for rat plasma. Analytical recovery was 88.8-110.1% of target concentration, and intra- (n=30) and inter-day (n=30) imprecision<11.9% coefficient of variation. Method recoveries and matrix effects ranged from 58.4-84.4% and -62.1 to -15.6%, respectively. AM-2201 and metabolites were stable (±20%) at room temperature for 24h, at 4°C for 72h, and after three freeze-thaw cycles, and for 72h in the autosampler after extraction. The method was developed for pharmacodynamic and pharmacokinetic studies with controlled administration in rats but is applicable for pre-clinical and clinical research and forensic investigations. Rat plasma specimen analysis following subcutaneous AM-2201 administration demonstrated the suitability of the method. AM-2201, JWH-018 N-(5-hydroxypentyl), and JWH-018 N-pentanoic acid concentrations were 4.8±1.0, 0.15±0.03, and 0.34±0.07µg/L, respectively, 8h after AM-2201 administration at 0.3mg/kg (n=5).

Phenotypic Variability and Newly Identified Mutations of the IVD Gene in Japanese Patients with Isovaleric Acidemia.

Isovaleric acidemia (IVA) is an autosomal recessive inborn error affecting leucine metabolism. It is caused by a deficiency in isovaleryl-CoA dehydrogenase (IVD), a mitochondrial matrix enzyme that catalyzes the oxidation of isovaleryl-CoA to 3-methylcrotonyl-CoA. IVD is a FAD-containing enzyme, consisting of four identical subunits. Clinical features of IVA include poor feeding, vomiting, lethargy, developmental delay, metabolic acidosis, and a characteristic "sweaty foot" odor. IVA is one of the target disorders for newborn screening by tandem mass spectrometry (MS/MS). The human IVD gene is located on chromosome 15q. To date, over 50 disease-causing mutations have been reported worldwide. In this study, we searched for IVD mutations in five Japanese patients with IVA (neonatal type, two patients; chronic intermittent type, two patients; and mild biochemical type, one patient). The diagnosis of IVA was confirmed by urinary organic acid analysis using gas chromatography and mass spectrometry. All coding exons and the flanking introns in the IVD gene were amplified by PCR and were directly sequenced. We thus identified six hitherto unknown mutations (p.G94D, p.E116K, p.M167T, p.L243P, p.L246P, and c.696+1G>T) and four previously reported (p.R53P, p.R395C, p.Y403C, and p.E411K) pathogenic mutations. All patients were compound heterozygotes, and each mutation was identified in a single patient. Pathogenicity of newly identified mutations was validated using computational programs. Among them, the p.M167T is believed to influence FAD binding, as the position 167 is present in one of the FAD-binding sites. Our results have illustrated the heterogeneous mutation spectrum and clinical presentation of IVA in the Japanese patients.

Carcinogenicity assessment of the pan-caspase inhibitor, emricasan, in Tg.rasH2 mice.

Emricasan, formerly IDN-6556, is a small molecule currently being evaluated in clinical trials to reduce hepatic injury and liver fibrosis. Since emricasan is an irreversible pan-caspase inhibitor that potently inhibits caspase-mediated apoptosis and inflammation, its carcinogenic potential was evaluated in a humanized mouse model. Tg.rasH2 mice received LabDiet formulated with 0, 10, 25, and 75mg/kg/day of emricasan, for 26weeks. At terminal sacrifice, blood was collected for clinical pathology analysis and tissues were collected, processed, and evaluated microscopically. There were no treatment related deaths or overt signs of toxicity for the duration of the study. There was no evidence of a carcinogenic effect in the peripheral blood leukocyte counts. Liver microgranulomas, which are background lesions, were slightly increased, especially in males. Increases in the incidence of the activated germinal centers were seen in the spleens and mesenteric lymph nodes of males and females, and in the mandibular lymph nodes of male mice. Atrophy of ovaries and testicular degeneration were also seen in emricasan treated animals. Although several non-neoplastic lesions were observed, there was no evidence of emricasan-related tumor formation in any tissue. In addition, the non-neoplastic lesions were not considered pre-neoplastic. Thus, emricasan is not considered carcinogenic.

Validation of JWH-018 and its metabolites in blood and urine by UPLC-MS/MS: Monitoring in forensic cases.

The herbal products referred to as 'Spice' have been used as 'legal alternatives' to cannabis worldwide since 2004. The first synthetic cannabinoid JWH-018 was detected in 'Spice' products in 2008, and has been banned by many legal authorities since the beginning of 2009. In order to prove use of JWH cannabinoids (JWHs), specific and robust methods were needed. We have developed a specific and reliable method for the detection and quantification of JWH-018, JWH-018 N-pentanoic acid, and JWH-018 N-(5-hydroxypentyl) in blood and urine using solid-phase extraction followed by UPLC-MS/MS analysis. The method has been validated in terms of linearity (0.1-50ng/mL), selectivity, intra-assay and inter-assay accuracy and precision (CV<15%), recovery (85-98%), limits of detection (LOD) (0.08-0.14ng/mL), and quantification (LOQ) (0.10-0.21ng/mL). Matrix effects, stability, and process efficiency were also assessed. The method has been applied to 868 authentic samples received by the Department of Chemistry (Istanbul) in the Council of Forensic Medicine of the Ministry of Justice.

Fasting serum concentration of short-chain fatty acids in subjects with microscopic colitis and celiac disease: no difference compared with controls, but between genders.

BACKGROUND: Short-chain fatty acids (SCFAs), particularly propionic and butyric acids, have been shown to have many positive health effects. The amount and type of SCFAs formed from dietary fibre by the colonic microbiota depends on the substrate available and is reflected in blood. The total intake and type of dietary fibre in people with gastrointestinal diseases differs considerably from healthy subjects. OBJECTIVE: To compare fasting SCFA concentrations in subjects with microscopic colitis (MC), celiac disease and controls without these diseases. SCFAs were also analysed over 6.5 h in young healthy subjects, who had eaten a fibre-rich breakfast, to identify a possible peak concentration of SCFAs after a meal. METHODS: SCFAs in serum were pre-concentrated using hollow fibre-supported liquid membrane extraction and gas chromatography. RESULTS: The MC group had a higher concentration of valeric acid than the control group (p < 0.01). No significant differences in other SCFA concentrations were seen between groups, but the control group tended to have higher concentration of acetic acid (p = 0.1). Furthermore, males had higher concentrations of SCFAs (with the exception of valeric acid) than females (p < 0.05), which were independent of groups. The peaks for acetic, propionic and butyric acids came approximately 5 h, 6.5 h and 2-3 h, respectively, after breakfast. CONCLUSION: The fasting concentrations of SCFAs were quite similar, although the fibre intake had probably been quite different for a long time. The results might have been different if SCFAs had been recorded over a longer period.

PET imaging-based evaluation of hepatobiliary transport in humans with (15R)-11C-TIC-Me.

UNLABELLED: It is well accepted that drug transporters play a pivotal role in hepatobiliary excretion of anionic drugs, in which drug-drug interactions and genetic polymorphisms are known to cause variations. However, PET probes for in vivo functional characterization of these transporters have not been established yet. We used PET to investigate hepatic uptake and subsequent canalicular efflux of (11)C-labeled (15R)-16-m-tolyl-17,18,19,20-tetranorisocarbacyclin methyl ester [(15R)-(11)C-TIC-Me)] in healthy subjects. METHODS: Serial PET scans of the abdominal region in healthy male subjects were obtained with or without the organic anion-transporting polypeptide (OATP) inhibitor rifampicin after intravenous injection of (15R)-(11)C-TIC-Me as a radiotracer. Venous blood samples and PET images were obtained at frequent intervals up to 30 min after administration of the PET tracer. Dynamic imaging data were evaluated by integration plots of data collected for 2-10 min and for 10-30 min after tracer administration for the determination of tissue uptake clearance and biliary efflux clearance, respectively. RESULTS: After rapid hydrolysis in blood, the acid form-(11)C-labeled (15R)-16-m-tolyl-17,18,19,20-tetranorisocarbacyclin [(15R)-(11)C-TIC]-accumulated in the liver (37% of the dose by 17 min), and the radioactivity was then excreted into the bile (6.2% by 30 min). Rifampicin (600 mg by mouth), a potent OATP inhibitor, significantly reduced the radioactivity excreted into the bile (by 44%) by inhibiting both uptake (by 45%) and subsequent canalicular efflux (by 62%). (15R)-(11)C-TIC is an in vitro substrate of OATP1B1 and OATP1B3, and clinically relevant concentrations of rifampicin inhibited uptake by OATP1B1 and OATP1B3. These results demonstrated that in humans, (15R)-(11)C-TIC-associated radioactivity is excreted into the bile by organic anion transport systems. CONCLUSION: We demonstrated that PET image analysis with (15R)-(11)C-TIC-Me is useful for investigating variations in OATP function in the human hepatobiliary transport system.

Newborn screening for isovaleric acidemia using tandem mass spectrometry: data from 1.6 million newborns.

BACKGROUND: Electrospray ionization-tandem mass spectrometry (ESI-MS/MS) has been used in the Bavarian newborn screening (NBS) program since 1999. The use of ESI-MS/MS has led to the inclusion of isovaleric acidemia (IVA) into NBS. We retrospectively evaluated data on more than 1.6 million newborns screened during 9.5 years. METHODS: Acylcarnitines from whole blood spotted on filter paper were converted to their corresponding butyl esters, and the samples were analyzed by use of ESI-MS/MS with stable isotope labeled internal standards. RESULTS: A total of 24 individuals with IVA were detected by use of a multiparametric threshold criteria panel including isovalerylcarnitine (C5) and the ratios of C5 to octanoyl-, butyryl-, and propionylcarnitine. A cutoff set at the 99.99th percentile for isolated C5 or at the 99th percentile for C5 plus at least 2 ratios resulted in a positive predictive value for IVA screening of 7.0% and an overall recall rate of 0.024%. Adjusted reference ranges for age and birth weight were applied, and the incidence of IVA in the study population was calculated to be 1 in 67,000. Missed cases were not brought to our attention. IVA was also detectable in cord blood and early postnatal blood samples. CONCLUSIONS: IVA can be reliably detected in NBS through acylcarnitine analysis in dried blood spots by using multiparametric threshold criteria. Further improvement (positive predictive value 13.0%, recall rate 0.01%) can be achieved by using more stringent recall criteria. In view of the potentially life-threatening natural course of IVA in early life, presymptomatic diagnosis may thus prevent mortality and morbidity.

Chronic intermittent form of isovaleric acidemia mimicking diabetic ketoacidosis.

Isovaleric acidemia is a rare autosomal recessive inborn error of leucine metabolism. Two phenotypes with either an acute neonatal or a chronic intermittent presentation were described. The acute type is observed more frequently and is more fatal. We report the case of a girl in childhood who presented with hyperglycemia and metabolic acidosis with an increased anion gap; and preliminarily diagnosed as diabetic ketoacidosis, but further investigation revealed chronic intermittent isovaleric academia. This case is of interest because of the rarity of this presentation. The importance of thinking for inborn errors of metabolism in children with metabolic acidosis in late childhood is emphasized.

A rapid HPLC/ESI-MS/MS method for quantitative analysis of isovalerylshikonin in rat plasma.

A new, fast and sensitive high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC/ESI-MS/MS) method was developed and validated for isovalerylshikonin in rat plasma using emodin as internal standard (IS). The analyte was extracted from rat plasma with ethyl acetate, after 10% HCl treatment and protein precipitated by methanol. The compound was separated on an Ultimate XB-C(18) analytical column using a mobile phase of methanol-10 mM ammonium acetate in water-acetonitrile containing 0.05% formic acid (45 : 10 : 45, v/v/v) with isogradient elution. The analyte was detected in negative ion mode using multiple-reaction monitoring. The method was validated and the specificity, linearity, lower limit of quantitation (LLOQ), precision, accuracy, recoveries and stability were determined. LLOQ was 9 ng/mL for isovalerylshikonin. Correlation coefficient (r) value for the linear range of the analyte was greater than 0.99. The intra-day and inter-day precision and accuracy were better than 8.52%. The relative and absolute recovery was above 86% and no matrix effects were observed for isovalerylshikonin. This validated method provides a modern, rapid and robust procedure for the pharmacokinetic study of the two compounds in rats after intravenous administration to rats (n = 4).

Abnormal tricarboxylic acid cycle metabolites in isovaleric acidaemia.

BACKGROUND: Although a number of abnormal diagnostic metabolites have previously been described in the urine of patients with isovaleric acidaemia (IVA), they do not fully explain the clinical symptoms associated with this disease. METHODS: On the basis of our current understanding of the TCA cycle and IVA, we predicted a number of abnormal methylated TCA cycle metabolites, initiated by methylsuccinic acid. We subsequently obtained characteristic gas chromatography-mass spectrometry elution times and mass spectra of the chemically synthesized predicted compounds and screened the urine of 6 IVA patients and 24 age-matched controls. Further proof for our findings was generated from a series of in vitro enzyme reactions using the chemically synthesized standards as substrates to their respective TCA cycle enzymes. RESULTS: Apart from the previously described methylsuccinic and methylfumaric acid, 3-methylmalic acid, (2R,3S)- and (2R,3R)-methylcitric acid and 2-methyl-cis-aconitic acid were detected in the urine of all 6 IVA patients in increased amounts. Additionally, although not directly determined, the in vitro enzyme reaction using of 3-methylmalic acid and malate dehydrogenase, in conjunction with the detection of 2-ketobutyric acid in the urine of all 6 IVA patients, strongly suggests an additional synthesis of 3-methyloxaloacetic acid by the same cycle. CONCLUSION: Not only do these newly identified metabolites serve as additional diagnostic markers to those previously identified in IVA, but due to the structural arrangements of the (2R,3R)-methylcitric acid and 2-methyl-cis-aconitinic acid-derived 2-methylisocitric acid, inhibition of normal TCA cycle metabolism results at citrate synthase and isocitrate dehydrogenase, respectively. SYNOPSIS: Methylsuccinic acid acts as the initiating substrate to a series of abnormal, potentially harmful, methylated tricarboxylic acid cycle metabolites in isovaleric acidaemia.

Stable-isotope dilution measurement of isovalerylglycine by tandem mass spectrometry in newborn screening for isovaleric acidemia.

BACKGROUND: Recent neonatal screening for isovaleric acidemia by tandem mass spectrometry based on dried blood-spot levels of C5-acylcarnitines, including isovalerylcarnitine and its isomer, pivaloylcarnitine, which is derived from pivalate-generating antibiotics, has caused many false-positive results. We have developed a method to overcome this interference. METHODS: The amounts of isovalerylglycine were determined by a stable-isotope dilution electrospray tandem mass spectrometric analysis, using multiple-reaction monitoring with product ions of m/z 132, which were generated predominantly from quasi-molecular ions of isovalerylglycine butylester but apparently not from those of pivaloylglycine butylester. RESULTS: Isovalerylglycine concentrations in dried blood spots of control newborns were 0.17+/-0.03 nmol/ml, and those of patients with isovaleric acidemia ranged from 1.3 to 80.0 nmol/ml. Those of the newborns treated with antibiotics, which caused high C5-acylcarnitine levels (1.9+/-1.7 nmol/ml) in dried blood spots, were 0.22+/-0.05 nmol/ml. CONCLUSIONS: Our data showed that the present method is useful in eliminating the false-positive results due to antibiotics use in newborn screening for isovaleric acidemia.

Different spectrum of mutations of isovaleryl-CoA dehydrogenase (IVD) gene in Korean patients with isovaleric acidemia.

Isovaleric acidemia (IVA) is an autosomal recessive inborn error of the leucine metabolism that is caused by a deficiency of isovaleryl-CoA dehydrogenase (IVD). Recent application of tandem mass spectrometry to newborn screening has allowed a significant expansion of the recognition of individuals with IVD deficiency. Although many patients have been reported worldwide, there are no genetically confirmed patients in Korea. This study characterizes IVD mutations in seven Korean IVA patients from six unrelated families. Bi-directional sequencing analysis identified two novel variations affecting consensus splice sites (c.144+1G>T in intron 1 and c.457-3_2CA>GG in intron 4) and three novel variations altering coding sequences (c.149G>T; Arg21Leu, c.832A>G; Ser249Gly, and c.1135T>G; Phe350Val). Five patients from four families were found to be compound heterozygotes while two unrelated patients were homozygous for the c.457-3_2CA>GG variation. Reverse-transcription polymerase chain reaction confirmed that both intron variations cause aberrant splicing. Furthermore, analysis of cultured lymphocyte extracts of the seven patients showed no detectable enzyme activity and reduced levels of IVD protein (<10.0% of control) in all samples. These results confirm IVD mutations in Korean patients with IVA and reveal that the mutation spectrum is different from previously reported patients.

Amino-acid depletion induced by abnormal amino-acid conjugation and protein restriction in isovaleric acidemia.

BACKGROUND: Previously, we detected 19 'new' amino-acid conjugates in the urine of patients with isovaleric acidemia. There is currently a poor understanding of the relationship between the clinical symptoms and the excreted metabolites occurring in these patients, owing to insufficient metabolite characterization and quantification. Consequently, controversial treatment protocols exist, particularly pertaining to dietary protein restriction. OBJECTIVE: To determine the effect of the previously identified amino-acid conjugates and conventional dietary protein restriction therapy, on the free amino-acid concentrations in isovaleric acidemia patients, to better explain the clinical symptoms and develop more effective therapy. DESIGN: Free amino-acid quantification via liquid chromatography mass spectrometry (LC-MS-MS) was performed on pre- and post-treatment urine or serum samples collected from six isovaleric acidemia patients, previously investigated for the presence of new induced N-isovaleryl and N-acetyl-amino-acid conjugates. RESULTS: Depleted amino-acid concentrations were detected in varying degrees in all six patients and did not recover after conventional treatment. CONCLUSIONS: The 19 potentially toxic metabolites previously identified and the consequent amino-acid depletions detected in this study, may explain many of the clinical symptoms associated with isovaleric acidemia. Furthermore, the occurrence of amino-acid depletions in these patients, steers away from the controversial dietary protein restriction treatment protocols, and towards dietary leucine restriction alone with essential amino-acid supplementation, in combination with glycine and L-carnitine supplementation.