Acute Consumption of Flavan-3-ol-Enriched Dark Chocolate Affects Human Endogenous Metabolism.

Flavan-3-ols and methylxanthines have potential beneficial effects on human health including reducing cardiovascular risk. We performed a randomized controlled crossover intervention trial to assess the acute effects of consumption of flavan-3-ol-enriched dark chocolate, compared with standard dark chocolate and white chocolate, on the human metabolome. We assessed the metabolome in urine and blood plasma samples collected before and at 2 and 6 h after consumption of chocolates in 42 healthy volunteers using a nontargeted metabolomics approach. Plasma samples were assessed and showed differentiation between time points with no further separation among the three chocolate treatments. Multivariate statistics applied to urine samples could readily separate the postprandial time points and distinguish between the treatments. Most of the markers responsible for the multivariate discrimination between the chocolates were of dietary origin. Interestingly, small but significant level changes were also observed for a subset of endogenous metabolites. 1H NMR revealed that flavan-3-ol-enriched dark chocolate and standard dark chocolate reduced urinary levels of creatinine, lactate, some amino acids, and related degradation products and increased the levels of pyruvate and 4-hydroxyphenylacetate, a phenolic compound of bacterial origin. This study demonstrates that an acute chocolate intervention can significantly affect human metabolism.

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.

1H-nuclear magnetic resonance spectroscopy-based metabolic assessment in a rat model of obesity induced by a high-fat diet.

Obesity, whose prevalence is increasing rapidly worldwide, is recognized as a risk factor for diabetes, cardiovascular disease, liver disease, and renal disease. To investigate metabolic changes in the urine of a rat model of obesity induced by a high-fat diet (HFD), rats were divided into the following four groups based on the diet type and degree of weight gain: normal-diet (ND) low gainers, ND high gainers, HFD low gainers, and HFD high gainers. Biochemical analyses of visceral fat-pad weight, plasma, and liver tissues were performed. The (1)H-nuclear magnetic resonance ((1)H-NMR) spectra of urine were analyzed using multivariate statistical analysis to identify the separation of the groups. It was observed that the metabolic profile of urine obtained by (1)H-NMR-spectroscopy-based metabolomic analysis differed between ND low gainers and ND high gainers even though these animals consumed the same normal diet. Several key metabolites in urine, such as betaine, taurine, acetone/acetoacetate, phenylacetylglycine, pyruvate, lactate, and citrate contributed to the classification of these two groups. The metabolic profile of urine also differed between ND low gainers and HFD high gainers, which consumed the different diet and showed a different weight gain. This study has identified features of urine metabolites in various groups and demonstrated the reliability of an NMR-based metabolomics approach to investigate the effects of the diet and the physical constitution on obesity.

The urinary organic acids profile in single large-scale mitochondrial DNA deletion disorders.

Single large-scale mitochondrial DNA deletions disorders are classified into three main phenotypes with frequent clinical overlap: Pearson marrow-pancreas syndrome (PMS), Kearns-Sayre syndrome (KSS) and chronic progressive external ophtalmoplegia (PEO). So far, only few anecdotal studies have reported on the urinary organic acids profile in this disease class. In this single-center retrospective study, we performed quantitative evaluation of urinary organic acids in a series of 15 pediatric patients, 7 with PMS and 8 with KSS. PMS patients showed an organic acids profile almost constantly altered, whereas KSS patients frequently presented with normal profiles. Lactate, 3-hydroxybutyrate, 3-hydroxyisobutyrate, fumarate, pyruvate, 2-hydroxybutyrate, 2-ethyl-3-hydroxypropionate, and 3-methylglutaconate represented the most frequent metabolites observed in PMS urine. We also found novel metabolites, 3-methylglutarate, tiglylglycine and 2-methyl-2,3-dihydroxybutyrate, so far never reported in this disease. Interestingly, patients with a disease onset as PMS evolving overtime into KSS phenotype, presented persistent and more pronounced alterations of organic acid signature than in patients with a pure KSS phenotype. Our study shows that the quantitative analysis of urinary organic acid profile represents a helpful tool for the diagnosis of PMS and for the differential diagnosis with other inherited diseases causing abnormal organic acidurias.

Metabolomics combined with pattern recognition and bioinformatics analysis methods for the development of pharmacodynamic biomarkers on liver fibrosis.

The major obstacle for the development of targeted therapies is the lack of pharmacodynamic (PD) biomarkers to provide an early readout of biological activities. As the modulation of metabolites may reflect the biological changes occurring in the targets, metabolomics is promising to be an efficient way to explore PD biomarkers. In the present study, a liver fibrosis rat model was established by intraperitoneal injection of CCl4 twice weekly for 6 weeks, the treatment of total aglycone extracts of Scutellaria baicalensis (TAES) was begun 4 weeks after the modeling, and gas chromatography-mass spectrometry (GC-MS) based metabolomics combined with pattern recognition and network analysis were carried out for the research on PD biomarkers of TAES on liver fibrosis. After 2 weeks of treatment, TAES shows positive effects on CCl4-induced liver fibrosis. In the metabolomics study, 63 urinary metabolites contributing to liver fibrosis were identified. Six metabolic pathways significantly enriched in metabolomics data were mapped onto a network to determine global patterns of metabolic alterations in liver fibrosis. By topological analysis, 6 metabolites with high centrality in the metabolic sub-network were selected as potential PD biomarkers. Within 24 h of the final administration, the 6 identified urine metabolic biomarkers with response to time variation of TAES were validated as PD biomarkers. This integrative study presents an attractive strategy to explore PD biomarkers, which may give insight into the actual pharmacological effect of target drugs, and the information from PD biomarkers can be combined with pharmacokinetics to select the optimal dose and a schedule of administration for the drugs.

Fullerene-C60-MWCNT composite film based ultrasensitive electrochemical sensing platform for the trace analysis of pyruvic acid in biological fluids.

We propose development of a novel electrochemical sensor based on fullerene-multi-walled carbon nanotubes composite film for the sensitive determination of the pyruvic acid in biological fluids. The developed sensor was characterized by cyclic voltammetry. The nanocomposite film of C60-MWCNTs on GCE exhibits electrocatalytic activity towards pyruvic acid reduction and also decreases the reduction overpotential. The influence of the optimization parameters such as pH and effect of loading of composite mixture of C60 and MWCNTs on the electrochemical performance of the sensor were evaluated. Various kinetic parameters such as electron transfer number (n=2), proton transfer number (m=2) and charge transfer coefficient (α=0.56) were also calculated. Under optimized conditions, the squarewave reduction peak current was linear over the concentration range of 2.0-55 nM with the detection and quantification limit of 0.1 nM and 0.8 nM respectively. The fabricated sensor was successfully applied to the detection of pyruvic acid in biological samples with good recovery ranging from 97.6% to 103.6%.

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.

Preliminary Study on J-Resolved NMR Method Usability for Toxic Kidney's Injury Assessment.

BACKGROUND: Nowadays, the Nuclear Magnetic Resonance (NMR) techniques are tested for metabolomic urine profile in order to detect early damage of kidney. OBJECTIVES: The purpose of this investigation was the initial assessment of two-dimensional J-resolved NMR urine spectra analysis usability for early kidney injuries detection. The amino acids (AA) and acids profile change after the exposure to nephrotoxic agent (the cisplatin infusion) was examined. MATERIAL AND METHODS: The material was the urine of patients with non-small-cell lung cancer, treated with cisplatin in Pulmonology and Lung Cancers Clinic in Wroclaw. The urine of healthy volunteers was also examined. The identification of metabolites in urine was based on two-dimensional JRES signals in spectra, described in Human Metabolites Database (HMD). The molar concentration of metabolites was calculated from the volume under the signals. The analysis was focused on amino acids and organic acids (lactid acid and pyruvic acid) profiles. RESULTS: Any specific amino acids were identified after cisplatin infusion in comparison to the state before infusion. However, the differences in concentration were observed over 2-fold increase in valine, isoleucine and leucine, over 3-fold in alanine. Also, the concentration of pyruvic and lactic acids increased significantly (p≤0.05, p≤0.01). CONCLUSIONS: There were no specific amino acids identified in response to the infusion of cisplatin; however, some changes in the concentrations of amino acids and other small molecules were found. The analysis of two-dimensional JRES spectra showed an increase of alanine, leucine, isoleucine and valine concentration after the application of cisplatin. It seems that it is worth developing the JRES method based on special computer program.

High-performance liquid chromatographic assay of lactic, pyruvic and acetic acids and lactic acid stereoisomers in calf feces, rumen fluid and urine.

To facilitate clinical investigation of metabolic acidosis, a high-performance liquid chromatographic method was adapted and validated for the chiral separation of D-(-) and L-(+)-lactic acid in calf feces, rumen fluid and urine. A non-chiral method was also adapted and validated for the separation of pyruvic, acetic and DL-(+/-)-lactic acids in calf feces and DL-(+/-)-lactic and pyruvic acids in rumen fluid. Separation and quantification were achieved using a reversed phase sulphonated polystyrenedivinylbenzene analytical column for pyruvic, acetic and racemic lactic acids and by a 3 microm octadecylsilane (ODS) packed analytical column coated with N,N-dioctyl-L-alanine as the chiral selector for the separation of lactic acid enantiomers with Cu(II)-containing eluents by stereoselective ligand exchange chromatography. Endogenous analytes were present in validation samples over a range of concentrations (0.2-14.8 mmol/l). For the stereoselective assay, mean intra-day accuracy ranged from 90.6 to 108.4% and intra-day precision from 0.3 to 13.8%. For the non-stereoselective assay, mean intra-day accuracy ranged from 90.4 to 108.8% and intra-day precision from 1.5 to 11.1%. The limit of quantitation was 1.0 mmol/l for D- and L-lactic acid, 0.06125 mmol/l for pyruvic acid, 1.0 mmol/l for DL-lactic acid and 1 mmol/l for acetic acid. These assays can be used to study the role of the gastrointestinal tract and kidney in metabolic acidosis.

Mitochondrial disorders: a potentially under-recognized etiology of infantile spasms.

Infantile spasms represent an age-dependent response of the immature brain to a wide variety of insults. An unselected group of children with infantile spasms were reviewed to determine etiology; a metabolic work-up was undertaken if the etiology was unclear from history and examination (cryptogenic). Of the 56 infants, 34 had a recognizable etiology (symptomatic), 1 had normal development (idiopathic), and 21 had cryptogenic infantile spasms. Among the latter, results of plasma lactate and pyruvate or urine organic acids were available in 17. In 2 infants (monozygotic twins), mitochondrial DNA testing revealed the relatively common A3243G mitochondrial mutation. In these twins and 11 of the remaining 15, body fluid metabolite testing suggested possible defective energy metabolism. Our twins and previous reports suggest that mitochondrial disorders should be considered in the differential diagnosis of infantile spasms. Among our cases remaining cryptogenic, signs of abnormal energy metabolism were prevalent, suggesting that metabolic derangements may be common causes or secondary consequences of infantile spasms.

D-lactate production and excretion in diarrheic calves.

The origin of D-lactate, the most important acid contributing to metabolic acidosis in the diarrheic calf, is unknown. We hypothesized that because D-lactate is produced only by microbes, gastrointestinal fermentation is the source. The objective of this study was to determine whether D-lactate production occurs in the rumen, colon, or both, and to measure D- and L-lactate concentrations in urine. Fecal, rumen, blood, and urine samples were obtained from 16 diarrheic and 11 healthy calves. Serum electrolyte concentrations were measured in both groups, and blood gas analyses were performed for diarrheic calves. All samples were analyzed for D- and L-lactate by high performance liquid chromatography (HPLC). Diarrheic calves were generally hyperkalemic with high serum anion gap, depressed serum bicarbonate, and low blood pH. L-lactate was markedly higher in rumen contents (22.7 mmol/ L [median]) and feces (8.6 mmol/L) of diarrheic calves than healthy calves (0.5 mmol/L and 5.1 mmol/L, respectively), but not different in serum or urine. Rumen, fecal, serum, and urine D-lactate concentrations were all significantly higher (P < .05) in diarrheic calves (17.0, 25.4, 13.9, and 19.2 mmol/L, respectively) than in healthy calves (0.5, 9.1, 1.4, and 0.5 mmol/L, respectively). Higher D-lactate concentrations in the rumen and feces of diarrheic calves suggests these sites as the source of D-lactate in blood and urine.

Lipid storage myopathy in a cocker spaniel.

A six-year-old male cocker spaniel was presented to the Veterinary Medical Teaching Hospital, University of Florida, with a three-week history of generalised weakness and myalgia. Electrodiagnostic evaluation, cerebrospinal fluid analysis and thoracolumbar myelography were unremarkable. Biopsies from vastus lateralis and triceps muscles revealed numerous large lipid droplets within type 1 fibres and to a lesser degree within type 2 fibres. The resting plasma lactate was mildly increased and there was elevated urinary excretion of lactic, pyruvic and acetoacetic acids, increased urinary excretion of carnitine esters, and increased plasma alanine. This pattern of metabolite excretion is consistent with an, as yet undefined, block in oxidative metabolism.

[Mitochondrial DNA T to G mutation 8993 in Leigh encephalopathy and organic aciduria].

We report a 10-month-old female infant with Leigh encephalopathy caused by a T to G mutation at nucleotide 8993 of mitochondrial DNA. Initial manifestations were diarrhea and pyrexia, followed by disturbance of consciousness. Blood chemistry showed lactic acidosis, and cranial T2 weighted magnetic resonance imaging demonstrated symmetric high-intensity areas in the basal ganglia, consistent with Leigh encephalopathy. Analysis of urinary organic acids revealed a increase of alpha-ketoglutamate. Derivatives of branched chain amino acids, which accumulate in maple syrup disease, were also increased. Lipoamide dehydrogenase (E3) deficiency was initially suspected; however, normal activity of pyruvate dehydrogenase complex excluded the diagnosis. The organic aciduria disappeared after two weeks. The CNS lesions in our case were observed more prominently in the floor of the bilateral frontal lobes than in the globus pallidus and putamen. In this case, mitochondrial DNA mutation may have caused organic aciduria and the atypical imaging findings.

Pantothenic acid deficiency may increase the urinary excretion of 2-oxo acids and nicotinamide catabolites in rats.

Pantothenic acid (PaA) is involved in the metabolism of amino acids as well as fatty acid. We investigated the systemic metabolism of amino acids in PaA-deficient rats. For this purpose, urine samples were collected and 2-oxo acids and L-tryptophan (L-Trp) and its metabolites including nicotinamide were measured. Group 1 was freely fed a conventional chemically-defined complete diet and used as an ad lib-fed control, which group was used for showing reference values. Group 2 was freely fed the complete diet without PaA (PaA-free diet) and used as a PaA-deficient group. Group 3 was fed the complete diet, but the daily food amount was equal to the amount of the PaA-deficient group and used as a pair-fed control group. All rats were orally administered 100 mg of L-Trp/kg body weight at 09:00 on day 34 of the experiment and the following 24-h urine samples were collected. The urinary excretion of the sum of pyruvic acid and oxaloacetic acid was higher in rats fed the PaA-free diets than in the rats fed pair-fed the complete diet. PaA deficiency elicited the increased urinary excretion of anthranilic acid and kynurenic acid, while the urinary excretion of xanthurenic acid decreased. The urinary excretion of L-Trp itself, 3-hydroxyanthranilic acid, and quinolinic acid revealed no differences between the rats fed the PaA-free and pair-fed the complete diets. PaA deficiency elicited the increased excretion of N(1)-methylnicotinamide, N(1)-methyl-2-pyridone-5-carboxamide, and N(1)-methyl-4-pyridone-3-carboxamide. These findings suggest that PaA deficiency disturbs the amino acid catabolism.

Detection of urinary metabolomics before and after Pringle maneuver-induced liver ischemia and reperfusion injury in rats using gas chromatography-mass spectrometry.

BACKGROUND: Metabolomics studies can quantitatively detect the dynamic metabolic response of living systems. OBJECTIVE: To detect urinary metabolomics after hepatic ischemia/reperfusion (I/R) injury induced by the Pringle maneuver using gas chromatography-mass spectrometry (GC-MS). METHODS: Male Sprague-Dawley rats (N = 80) were randomly divided into 4 groups (n = 20/group): sham operation, day 1, day 3, and day 5. Rats in the day 1, day 3, and day 5 groups underwent the Pringle maneuver. Serum alanine transaminase (ALT) and total bilirubin (TBIL) were measured, and hematoxylin and eosin (HE) staining of the liver tissue was performed. GC-MS was used to detect urinary metabolomics. RESULTS: Compared with the sham group, the serum ALT and TBIL levels at day 1 were significantly elevated (P < 0.01) and then decreased and reached close to normal levels at day 5. GC-MS detected 7 metabolites which had similar changes as those of liver tissue revealed by histological examination. Significant differences in lactic acid, pyruvic acid, alanine, serine, and glycerol-3-phosphate were found among the groups (P < 0.001). Principle component analysis showed that 7 metabolites distinguished the day 1 and day 3 groups from the sham group. CONCLUSIONS: Noninvasive urinary metabolomic analysis is a potential means for the early detection and diagnosis of hepatic I/R injury.

Authentication of beef production systems using a metabolomic-based approach.

There is a need for new, non-invasive, rapid and reliable analytical methodologies that can easily be implemented and used for authentication of cattle production systems and the meat derived from them. Easily quantifiable markers could strengthen the current tracing methods for beef authentication. This study investigated the use of a nuclear magnetic resonance-based metabolomic approach as a tool to authenticate beef on the basis of the pre-slaughter production system. Urine and muscle samples were collected from animals fed either pasture outdoor, a barley-based concentrate indoor, silage followed by pasture outdoor or silage followed by pasture outdoor with concentrate over 1 year. A metabolomic analysis was performed on urine (n = 68) and muscle (n = 98) samples collected from animals on the different diets. The results showed that separation according to production system was possible indicating the potential use of this approach in beef authentication. Identification of the major discriminating peaks in urine led to the identification of potential markers of production system including creatinine, glucose, hippurate, pyruvate, phenylalanine, phenylacetylglycine and three unassigned resonances.

Determination of pyruvic acid by using enzymic fluorescence capillary analysis.

A new method (P-LE-FCA) for the determination of pyruvic acid was proposed based on liquid enzyme method (LE) and fluorescence capillary analysis (FCA). The optimum experimental conditions were as follows: the excitation and emission wavelengths were 350 and 460 nm, respectively; the reaction time and temperature were 20 min and 38 degrees C, respectively; the pH of phosphate buffer solution was 7.5; the concentrations of nicotinamide adenine dinucleotide and lactate dehydrogenase were 1.0 mmol L(-1) and 5.0 k UL(-1), respectively. The linear range of this method was 0.2-1.2 mmol L(-1) (Delta F=327.13C-10.018, r=0.9942). Its detection limit was 0.012 mmol L(-1). And its relative standard deviation was 0.86%. Only 18 microL of total reaction solution is enough for the detection. P-LE-FCA has some merits such as lower cost, simple operation procedure and micro determination. It has been used for the determination of pyruvic acid content in human urine samples.

The yield of laboratory investigations in children with infantile autism.

PURPOSE: To evaluate the yield of laboratory investigations in infantile autism. METHODS: We retrieved and evaluated the results of investigative procedures recorded in the medical files of autistic infants in four child developmental centers and two pediatric psychiatric outpatient clinics. RESULTS: One-hundred and thirty-two infants were included in the study of whom 47 (36%) underwent autistic regression at an average age of 20 months. The investigative procedures included electroencephalogram (n = 132), neuroimaging (n = 70), genetic studies to detect Fragile-X (n = 59) and a metabolic workup (n = 53). Except for the molecular diagnosis that revealed Fragile-X syndrome in two children (3%), all other tests were negative. The two infants with the Fragile-X syndrome belonged to the non-regressive group. CONCLUSIONS: The only investigative study that contributed to the diagnosis of autistic infants was the molecular diagnosis detecting Fragile-X. In spite of the high frequency of epilepsy and epileptiform abnormalities in the electroencephalograms of autistic children in general, the contribution of epilepsy, both clinical and subclinical, to the etiology of autism is apparently minimal.

1H NMR pattern recognition and 31P NMR studies with d-Serine in rat urine and kidney, time- and dose-related metabolic effects.

Proton NMR spectroscopy of urine has previously been used to gain insight into the site and mechanism of toxic injury to the kidney. d-Serine injures the rat kidney, causing selective necrosis of the proximal straight tubules. Damage is accompanied by proteinuria, glucosuria, and amino aciduria, the latter preceding the onset of necrosis. This study has employed (1)H NMR spectroscopy of urine and (1)H NMR and (31)P NMR spectroscopy of kidney extracts to examine the nephrotoxic action of d-serine. Urine was collected 0-8 h (all doses) and 8-24, 24-48, 48-72, 72-96, and 96-120 h (500 mg/kg only) postdosing from Alderley Park rats given d-serine (62.5, 125, 250, and 500 mg/kg ip). (1)H NMR spectra were monitored for markers of tubular damage. Additionally, ATP and ADP were quantitated in kidney perchloric acid extracts, prepared after 0.5, 1, 2, 4, and 8 h (500 mg/kg) to assess energy status; serine was also measured in these samples. At 500 mg/kg, glucosuria, amino aciduria, and reduced citrate, alpha-ketoglutarate, and succinate were observed in urine at 0-8 h. Furthermore, serine and pyruvate levels were elevated at this time. After 8-24 h, similar changes were observed; however, they were more severe reflecting the development of the lesion prior to recovery. These perturbations were dose-related, in particular, for serine and pyruvate, with no alterations seen at 62.5 mg/kg. Kidney serine concentration rapidly increased, where it was maximal after 30 min and cleared by 8 h. A decline in ATP, to approximately 60-70% of control, was observed within the kidney at 2-4 h postdosing, when necrosis first becomes evident suggesting that mitochondrial function might be impaired in the early stages of d-serine-induced nephrotoxicity. The use of NMR spectroscopy has given a comprehensive overview of the effects of d-serine in vivo. Information on the excretion of serine and its effect on renal energy metabolism provides insight into the possible mechanism of renal tubule injury.