SLC13A3 variants cause acute reversible leukoencephalopathy and α-ketoglutarate accumulation.

OBJECTIVE: SLC13A3 encodes the plasma membrane Na+ /dicarboxylate cotransporter 3, which imports inside the cell 4 to 6 carbon dicarboxylates as well as N-acetylaspartate (NAA). SLC13A3 is mainly expressed in kidney, in astrocytes, and in the choroid plexus. We describe two unrelated patients presenting with acute, reversible (and recurrent in one) neurological deterioration during a febrile illness. Both patients exhibited a reversible leukoencephalopathy and a urinary excretion of α-ketoglutarate (αKG) that was markedly increased and persisted over time. In one patient, increased concentrations of cerebrospinal fluid NAA and dicarboxylates (including αKG) were observed. Extensive workup was unsuccessful, and a genetic cause was suspected. METHODS: Whole exome sequencing (WES) was performed. Our teams were connected through GeneMatcher. RESULTS: WES analysis revealed variants in SLC13A3. A homozygous missense mutation (p.Ala254Asp) was found in the first patient. The second patient was heterozygous for another missense mutation (p.Gly548Ser) and an intronic mutation affecting splicing as demonstrated by reverse transcriptase polymerase chain reaction performed in muscle tissue (c.1016 + 3A > G). Mutations and segregation were confirmed by Sanger sequencing. Functional studies performed on HEK293T cells transiently transfected with wild-type and mutant SLC13A3 indicated that the missense mutations caused a marked reduction in the capacity to transport αKG, succinate, and NAA. INTERPRETATION: SLC13A3 deficiency causes acute and reversible leukoencephalopathy with marked accumulation of αKG. Urine organic acids (especially αKG and NAA) and SLC13A3 mutations should be screened in patients presenting with unexplained reversible leukoencephalopathy, for which SLC13A3 deficiency is a novel differential diagnosis. ANN NEUROL 2019;85:385-395.

Metabolite diagnosis of primary hyperoxaluria type 3.

BACKGROUND: Primary hyperoxaluria type 3 (PH3) is a recently described cause of childhood renal calculi. It results from mutations in the HOGA1 gene and most cases have been diagnosed after clinical ascertainment, exclusion of other genetic hyperoxalurias and mutation testing. Metabolite testing has not been widely applied but holds promise for the rapid screening and diagnosis of patients who are not specifically suspected to have PH3. CASE-DIAGNOSIS/TREATMENT: Two cases presented with renal calculi. Urine metabolite testing by tandem mass spectrometry was performed as part of the routine diagnostic work-up for this condition. Both had significantly increased levels of the PH3 urine marker 4-hydroxyglutamate and related metabolites. The diagnosis of PH3 was confirmed by the finding of bi-allelic damaging HOGA1 mutations. CONCLUSIONS: Urine screening by tandem mass spectrometry is a rapid, high-throughput test that can detect PH3 cases that may otherwise not be diagnosed.

High-performance liquid chromatographic method for profiling 2-oxo acids in urine and its application in evaluating vitamin status in rats.

B-group vitamins are involved in the catabolism of 2-oxo acids. To identify the functional biomarkers of B-group vitamins, we developed a high-performance liquid chromatographic method for profiling 2-oxo acids in urine and applied this method to urine samples from rats deficient in vitamins B1 and B6 and pantothenic acid. 2-Oxo acids were reacted with 1,2-diamino-4,5-methylenebenzene to produce fluorescent derivatives, which were then separated using a TSKgel ODS-80Ts column with 30 mmol/L of KH2PO4 (pH 3.0):acetonitrile (7:3) at a flow rate of 1.0 mL/min. Vitamin B1 deficiency increased urinary levels of all 2-oxo acids, while vitamin B6 deficiency only increased levels of sum of 2-oxaloacetic acid and pyruvic acid, and pantothenic acid deficiency only increased levels of 2-oxoisovaleric acid. Profiles of 2-oxo acids in urine samples might be a non-invasive way of clarifying the functional biomarker of B-group vitamins.

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.

The role of previously unmeasured organic acids in the pathogenesis of severe malaria.

INTRODUCTION: Severe falciparum malaria is commonly complicated by metabolic acidosis. Together with lactic acid (LA), other previously unmeasured acids have been implicated in the pathogenesis of falciparum malaria. METHODS: In this prospective study, we characterised organic acids in adults with severe falciparum malaria in India and Bangladesh. Liquid chromatography-mass spectrometry was used to measure organic acids in plasma and urine. Patients were followed until recovery or death. RESULTS: Patients with severe malaria (n=138), uncomplicated malaria (n=102), sepsis (n=32) and febrile encephalopathy (n=35) were included. Strong ion gap (mean ± SD) was elevated in severe malaria (8.2 mEq/L ± 4.5) and severe sepsis (8.6 mEq/L ± 7.7) compared with uncomplicated malaria (6.0 mEq/L ± 5.1) and encephalopathy (6.6 mEq/L ± 4.7). Compared with uncomplicated malaria, severe malaria was characterised by elevated plasma LA, hydroxyphenyllactic acid (HPLA), α-hydroxybutyric acid and ß-hydroxybutyric acid (all P<0.05). In urine, concentrations of methylmalonic, ethylmalonic and α-ketoglutaric acids were also elevated. Multivariate logistic regression showed that plasma HPLA was a strong independent predictor of death (odds ratio [OR] 3.5, 95 % confidence interval [CI] 1.6-7.5, P=0.001), comparable to LA (OR 3.5, 95 % CI 1.5-7.8, P=0.003) (combined area under the receiver operating characteristic curve 0.81). CONCLUSIONS: Newly identified acids, in addition to LA, are elevated in patients with severe malaria and are highly predictive of fatal outcome. Further characterisation of their sources and metabolic pathways is now needed.

α-Ketoglutaramate: an overlooked metabolite of glutamine and a biomarker for hepatic encephalopathy and inborn errors of the urea cycle.

Glutamine metabolism is generally regarded as proceeding via glutaminase-catalyzed hydrolysis to glutamate and ammonia, followed by conversion of glutamate to α-ketoglutarate catalyzed by glutamate dehydrogenase or by a glutamate-linked aminotransferase (transaminase). However, another pathway exists for the conversion of glutamine to α-ketoglutarate that is often overlooked, but is widely distributed in nature. This pathway, referred to as the glutaminase II pathway, consists of a glutamine transaminase coupled to ω-amidase. Transamination of glutamine results in formation of the corresponding α-keto acid, namely, α-ketoglutaramate (KGM). KGM is hydrolyzed by ω-amidase to α-ketoglutarate and ammonia. The net glutaminase II reaction is: L - Glutamine + α - keto acid + H2O → α - ketoglutarate + L - amino acid + ammonia. In this mini-review the biochemical importance of the glutaminase II pathway is summarized, with emphasis on the key component KGM. Forty years ago it was noted that the concentration of KGM is increased in the cerebrospinal fluid (CSF) of patients with hepatic encephalopathy (HE) and that the level of KGM in the CSF correlates well with the degree of encephalopathy. In more recent work, we have shown that KGM is markedly elevated in the urine of patients with inborn errors of the urea cycle. It is suggested that KGM may be a useful biomarker for many hyperammonemic diseases including hepatic encephalopathy, inborn errors of the urea cycle, citrin deficiency and lysinuric protein intolerance.

An investigation of the antidepressant action of xiaoyaosan in rats using ultra performance liquid chromatography-mass spectrometry combined with metabonomics.

A rapid, highly sensitive, and selective method was applied in a non-invasive way to investigate the antidepressant action of Xiaoyaosan (XYS) using ultra performance liquid chromatography-mass spectrometry (UPLC-MS) and chemometrics. Many significantly altered metabolites were used to explain the mechanism. Venlafaxine HCl and fluoxetine HCl were used as chemical positive control drugs with a relatively clear mechanism of action to evaluate the efficiency and to predict the mechanism of action of XYS. Urine obtained from rats subjected to chronic unpredictable mild stress (CUMS) was analyzed by UPLC-MS. Distinct changes in the pattern of metabolites in the rat urine after CUMS production and drug intervention were observed using partial least squares-discriminant analysis. The results of behavioral tests and multivariate analysis showed that CUMS was successfully reproduced, and a moderate-dose XYS produced significant therapeutic effects in the rodent model, equivalent to those of the positive control drugs, venlafaxine HCl and fluoxetine HCl. Metabolites with significant changes induced by CUMS were identified, and 17 biomarker candidates for stress and drug intervention were identified. The therapeutic effect of XYS on depression may involve regulation of the dysfunctions of energy metabolism, amino acid metabolism, and gut microflora changes. Metabonomic methods are valuable tools for measuring efficacy and mechanisms of action in the study of traditional Chinese medicines.

[Urine metabonomic study of intervention effects of Morinda officinalis how. on 'kidney-yang deficiency syndrome'].

To investigate the intervention effects of Morinda officinalis How. on 'Kidney-yang deficiency syndrome' induced by hydrocortisone in rats, the metabolic profiles of rat urine were characterized using proton nuclear magnetic resonance and principal component analysis (PCA) was applied to study the trajectory of urinary metabolic phenotype of rats with 'Kidney-yang deficiency syndrome' under administration of M. officinalis at different time points. Meanwhile, the intervention effects of M. officinalis on urinary metabolic potential biomarkers associated with 'Kidney-yang deficiency syndrome' were also discussed. The experimental results showed that in accordance to the increased time of administration, an obvious tendency was observed that clustering of the treatment group moved gradually closed to that of the control group. Eight potential biomarkers including citrate, succinate, alpha-ketoglutarate, lactate, betaine, sarcosine, alanine and taurine were definitely up- or down-regulated. In conclusion, the effectiveness of M. oficinalis on 'Kidney-yang deficiency syndrome' is proved using the established metabonomic method and the regulated metabolic pathways involve energy metabolism, transmethylation and transportation of amine. Meanwhile, the administration of M. officinalis can alleviate the kidney impairment induced by 'Kidney-yang deficiency syndrome'.

Development of a non-targeted metabolomics method to investigate urine in a rat model of polycystic kidney disease.

AIM: The purpose of this research was to use metabolomics to investigate the cystic phenotype in the Lewis polycystic kidney rat. METHODS: Spot urine samples were collected from four male Lewis control and five male Lewis polycystic kidney rats aged 5 weeks, before kidney function was significantly impaired. Metabolites were extracted from urine and analysed using gas chromatography-mass spectrometry. Principal component analysis was used to determine key metabolites contributing to the variance observed between sample groups. RESULTS: With the development of a metabolomics method to analyse Lewis and Lewis polycystic kidney rat urine, 2-ketoglutaric acid, allantoin, uric acid and hippuric acid were identified as potential biomarkers of cystic disease in the rat model. CONCLUSION: The findings of this study demonstrate the potential of metabolomics to further investigate kidney disease.

Mitochondrial involvement and erythronic acid as a novel biomarker in transaldolase deficiency.

BACKGROUND: Sedoheptulose, arabitol, ribitol, and erythritol have been identified as key diagnostic metabolites in TALDO deficiency. METHOD: Urine from 6 TALDO-deficient patients and TALDO-deficient knock-out mice were analyzed using ¹H-NMR spectroscopy and GC-mass spectrometry. RESULTS: Our data confirm the known metabolic characteristics in TALDO-deficient patients. The ß-furanose form was the major sedoheptulose anomer in TALDO-deficient patients. Erythronic acid was identified as a major abnormal metabolite in all patients and in knock-out TALDO mice implicating an as yet unknown biochemical pathway in this disease. A putative sequence of enzymatic reactions leading to the formation of erythronic acid is presented. The urinary concentration of the citric acid cycle intermediates 2-oxoglutaric acid and fumaric acid was increased in the majority of TALDO-deficient patients but not in the knock-out mice. CONCLUSION: Erythronic acid is a novel and major hallmark in TALDO deficiency. The pathway leading to its production may play a role in healthy humans as well. In TALDO-deficient patients, there is an increased flux through this pathway. The finding of increased citric acid cycle intermediates hints toward a disturbed mitochondrial metabolism in TALDO deficiency.

[Study on influence of glucoside Tripterygium total tablets on metabolism in rats by NMR metabonomic technique].

OBJECTIVE: To investigate the toxic effects of Glucoside Tripterygium total on rats with nuclear magnetic resonance (NMR)-based metabonomic method. METHOD: The influence of intragastric administration of Glucoside Tripterygium total suspension at two different doses on endogenetic metabolites in normal rat urine was determined with bio-NMR method then analyzed by pattern recognition technique and partial least-squares discriminant analysis (PLS-DA). Histopathological analysis was carried out. RESULT: Escalations of concentrations of urinary taurine, TMAO and glucose as well as reductions of concentrations of urinary citrate and 2-oxoglutarate were found by analysis of the 1H-NMR spectra, which was coincident with the result of histopathological analysis. The result of pathological examination indicated that pathologic change was not observed in nephridial tissue, but there were obvious changes in hepatic tissue. CONCLUSION: The urinary metabomic spectra were closely associated with the hepatic toxicity, which manifested the mitochondrial dysfunctions, the abnormal energy metabolism in TCA cycle as well as the abnormal glucose metabolism.

Toxicometabolomics of urinary biomarkers for human gastric cancer in a mouse model.

Toxicometabolomics of urinary biomarkers for human gastric cancer in a mouse model was investigated using (1)H-nuclear magnetic resonance (NMR) spectroscopy. A human gastric adenocarcinoma cell line (1 × 10(7) cells/ml) was grafted onto the skin of the back of intact male BALB/c-nu/nu mice. After the xenografted tumors developed, urine was collected and analyzed for endogenous metabolites. Global profiling combined with principal components analysis (PCA), partial least squares-discriminant analysis (PLS-DA), and orthogonal projections to latent squares-discriminant analysis (OPLS-DA) showed distinct separation of clusters between control and tumor-bearing mice. Targeted profiling revealed significant changes in trimethylamine oxide (TMAO), 3-indoxylsulfate, hippurate, and citrate levels in mice carrying human gastric cancer cells compared to normal mice. The levels of TMAO (0.41-fold) and hippurate (0.26-fold) in tumor-bearing mice were significantly decreased, whereas the levels of 3-indoxylsulfate (3.39-fold), 2-oxoglutarate (2.32-fold), and citrate (1.9-fold) were significantly increased in urine samples of tumor-bearing mice. Data suggest that TMAO, hippurate, 3-indoxylsulfate, 2-oxoglutarate, and citrate may serve as useful urinary biomarkers for gastric tumorigenesis in a mouse model.

Eleven novel mutations and clinical characteristics in seven Chinese patients with thiamine metabolism dysfunction syndrome.

Thiamine metabolism dysfunction syndrome (THMD) comprises a group of clinically and genetically heterogeneous encephalopathies with autosomal recessive inheritance. Four genes, SLC19A3, SLC25A19, SLC19A2, and TPK1, are associated with this disorder. This study aimed to explore the clinical, biochemical and molecular characteristics of seven Chinese patients with THMD. Targeted next-generation sequencing of mitochondrial DNA and nuclear DNA was used to identify the causative mutations. The patients presented with subacute encephalopathy between the ages of 1-27 months. Brain magnetic resonance imaging (MRI) revealed abnormalities in the basal ganglia, indicating Leigh syndrome. Urine α-ketoglutarate in five patients was elevated. In four patients, five novel mutations (c.1276_1278delTAC, c.265A > C, c.197T > C, c.850T > C, whole gene deletion) were found in SLC19A3, which is associated with THMD2. In two patients, four novel mutations (c.194C > T, c.454C > A, c.481G > A, and c.550G > C) were identified in SLC25A19, supporting a diagnosis of THMD4. In one patient, two novel mutations (c.395T > C and c.614-1G > A) were detected in TPK1, which is indicative of THMD5. The patients received thiamine, biotin, and symptomatic therapy, upon which six patients demonstrated clinical improvement. Our findings expanded the phenotypic and genotypic spectrum of THMD, with eleven novel mutations identified in seven Chinese patients. Early diagnosis and treatment have a significant impact on prognosis.

The absorption, tissue distribution and excretion of enteraly administered alpha-ketoglutarate in rats.

The absorption, tissue distribution and excretion of enteral alpha-ketoglutarate (AKG) was studied in four experiments. Six male Sprague Dawley rats were used to investigate the excretion of AKG in urine and faeces. Thirty rats, randomly assigned to five groups, were used to investigate the distribution of AKG in body tissues. They were gavaged with AKG enriched with 3 muCi/kg BW of (14)C uniformly marked AKG. Fourteen male Sprague Dawley rats were used to study the absorption of AKG (duodenum vs. ileum). Intestinal recovery of NaAKG vs. CaAKG was investigated in 36 rats. There was no significant excretion of non-metabolized AKG in the urine and faeces. There was no significant difference in the systemic levels of AKG when comparing the proximal to distal small intestine infusion. Up to 50%, 30% and 20% of gastrically delivered AKG was recovered in the stomach, 0.5, 1 and 2 h after gavage; the jejunal recovery achieved a maximum of 3%, 30 min after gavage, and was not detectable 2 h later. There was a relatively high distribution of (14)C-AKG in the tissues (e.g. liver, brain, bones, skin, muscles), 3 h after gavage, up to 70% of the administered dose. In conclusion, the high rate of retention of the carbon from AKG allows the postulation that there is a non-energetic mode of metabolism of intragastrically administered AKG. After conversion to final metabolites, AKG penetrates into all tissues and organs of rats, including the bone tissue. Intestinal absorption of AKG does not depend on the type of AKG salt administered.

[Metabonomics profile of urine from rats administrated with different treatment period of isoniazid].

OBJECTIVE: To study the effect of different treatment period, of isoniazid (INH) on the metabonomic profile of rat urine and its relationship with traditional toxicity evaluation of blood biochemical indicators and histopathology and to explore the feasibility of metabonomics in the application of drug toxicity. METHODS: Sixty male Wistar rats were orally administrated with 0, 50, 100, 200, and 400 mg x kg(-1) INH for 3, 7, and 14 days, respectively. Rat urine was then collected and its 1H nuclear magnetic resonance (NMR) spectra were acquired. All animals underwent traditional toxicity evaluation. RESULTS: Hepatotoxicity was revealed by traditional toxicity evaluation in rats treated with higher dosage and longer treatment of INH. Time-response relationship existed during the treatment. Time-dependent metabonomics changes conformed with the results of traditional toxicity evaluation. The urine metabonomics showed a trajectory bias from those of the controls or pre-administration, and such bias exaggerated along with the prolongation of treatment, indicating a severer toxic injury. Along with the increase of the concentrations of urinary taurine and glucose and the decrease of the concentrations of urinary citrate and 2-oxoglutarate, the 1H NMR spectra of urine in rats treated with INH also changed. CONCLUSIONS: The metabonomics technique can distinguish the onset and development of toxicity, which helps track and identify biomarkers. The hepatic toxicity induced by INH is related to the injury of mitochondrial function, reduction of energy metabolism in tricarboxylic acid cycle, and perturbations in the metabolism of glucose and lipid. The effect of INH on the rat urine metabonomic profile is related with INH toxicology. Therefore, metabonomics can be recognized as an ideal technique to explore and evaluate the drug toxicities.

A Metabolic Study on the Biochemical Effects of Chiral Illegal Drugs in Rats Using 1H-NMR Spectroscopy.

Considering the pharmacological effects of chiral drugs, enantiopure drugs may differ from their racemic mixture formulation in efficacy, potency, or adverse effects. Levomethorphan (LVM) and Dextromethorphan (DXM) act on the central nervous system and exhibit different pharmacological features. LVM, the l-stereoisomer of methorphan, shows many similarities to opiates such as heroin, morphine and codeine, including the potential for addiction, while the d-stereoisomer, DXM, does not have the same opioid effect. In the present study, NMR-based metabolomics were performed on the urine of rats treated with these stereoisomers, and showed significant differences in metabolic profiles. In urine within 24 h after treatment of these samples, levels of citrate, 2-oxoglutarate, creatine, and dimethylglycine were higher in LVM-treated rats than in DXM-treated rats. While urinary levels of hippurate and creatinine gradually increased over 72 h in DXM-treated rats, these metabolites were decreased in the urine by 48-72 h after treatment with LVM. The levels of these changed metabolites may provide the first evidence for different cellular responses to the metabolism of stereoisomers.

Changes in urinary metabolomic profile during relapsing renal vasculitis.

Current biomarkers of renal disease in systemic vasculitis lack predictive value and are insensitive to early damage. To identify novel biomarkers of renal vasculitis flare, we analysed the longitudinal urinary metabolomic profile of a rat model of anti-neutrophil cytoplasmic antibody (ANCA) vasculitis. Wistar-Kyoto (WKY) rats were immunised with human myeloperoxidase (MPO). Urine was obtained at regular intervals for 181 days, after which relapse was induced by re-challenge with MPO. Urinary metabolites were assessed in an unbiased fashion using nuclear magnetic resonance (NMR) spectroscopy, and analysed using partial least squares discriminant analysis (PLS-DA) and partial least squares regression (PLS-R). At 56 days post-immunisation, we found that rats with vasculitis had a significantly different urinary metabolite profile than control animals; the observed PLS-DA clusters dissipated between 56 and 181 days, and re-emerged with relapse. The metabolites most altered in rats with active or relapsing vasculitis were trimethylamine N-oxide (TMAO), citrate and 2-oxoglutarate. Myo-inositol was also moderately predictive. The key urine metabolites identified in rats were confirmed in a large cohort of patients using liquid chromatography-mass spectrometry (LC-MS). Hypocitraturia and elevated urinary myo-inositol remained associated with active disease, with the urine myo-inositol:citrate ratio being tightly correlated with active renal vasculitis.

Investigations into biochemical changes of genetic hypertensive rats using 1H nuclear magnetic resonance-based metabonomics.

Genetically hypertensive rats provide a simple and accessible model for studying essential hypertension, which is a polygenic, heterogenous and multifactorial disease. Their genetic and metabolic features are of great interest because they may provide insight into the pathophysiological processes underlying essential hypertension. We have investigated the genetic influence on metabolic balance and metabolite excretion patterns in stroke-prone spontaneously hypertensive rats (SHRSP) with established hypertension using 1H NMR-based metabonomics. Urinary metabolite profiles for SHRSP and their age-matched normotensive controls, Wistar Kyoto rats, were acquired using 1H NMR spectroscopy. Principal components analysis was applied to these complex NMR data to facilitate differentiation and determine metabolic differences between urine samples collected from the hypertensive and normotensive rats. Consequently, it was possible to distinguish urine samples between the two strains in the principal components scores plot. The loadings plot showed that taurine, creatine and some unidentified metabolites resonating at around delta 2.48, 3.10 and 3.58 predominantly contributed to the separation. In SHRSP, the urinary levels of taurine and creatine were found to be higher and the intensities of the unknown signals much lower than those in the Wistar Kyoto rats. Although the pathophysiological significance of these components remains to be elucidated, this study suggests that 1H NMR-based metabonomics is a promising approach to provide new information on metabolic changes related to the pathophysiological processes of the genetically hypertensive rats.

Compound identification in gas chromatography/mass spectrometry-based metabolomics by blind source separation.

Metabolomics GC-MS samples involve high complexity data that must be effectively resolved to produce chemically meaningful results. Multivariate curve resolution-alternating least squares (MCR-ALS) is the most frequently reported technique for that purpose. More recently, independent component analysis (ICA) has been reported as an alternative to MCR. Those algorithms attempt to infer a model describing the observed data and, therefore, the least squares regression used in MCR assumes that the data is a linear combination of that model. However, due to the high complexity of real data, the construction of a model to describe optimally the observed data is a critical step and these algorithms should prevent the influence from outlier data. This study proves independent component regression (ICR) as an alternative for GC-MS compound identification. Both ICR and MCR though require least squares regression to correctly resolve the mixtures. In this paper, a novel orthogonal signal deconvolution (OSD) approach is introduced, which uses principal component analysis to determine the compound spectra. The study includes a compound identification comparison between the results by ICA-OSD, MCR-OSD, ICR and MCR-ALS using pure standards and human serum samples. Results shows that ICR may be used as an alternative to multivariate curve methods, as ICR efficiency is comparable to MCR-ALS. Also, the study demonstrates that the proposed OSD approach achieves greater spectral resolution accuracy than the traditional least squares approach when compounds elute under undue interference of biological matrices.

Determination of α-ketoglutaric and pyruvic acids in urine as potential biomarkers for diabetic II and liver cancer.

BACKGROUND: A simple and sensitive hollow fiber-liquid phase microextraction with in situ derivatization method was developed for the determination of α-ketoglutaric (α-KG) and pyruvic acids (PA) in small-volume urine samples. 2,4,6-trichloro phenyl hydrazine was used as derivatization agent. RESULTS: Under the optimum extraction conditions, enrichment factors of 742 and 400 for α-KG and PA, respectively, were achieved. Calibration curves were linear over the range 1 to 1000 ng/ml (r(2) ≥ 0.998). Detection and quantitation limits were 0.03 and 0.02, and 0.10 and 0.05 ng/ml for α-KG and PA, respectively. CONCLUSION: The concentrations in diabetic II and liver cancer samples were significantly lower than those from healthy people, showing their potential as biomarkers for these diseases.