Urinary (1)H-NMR-based metabolic profiling of children with NAFLD undergoing VSL#3 treatment.

BACKGROUND: Nowadays, non-alcoholic fatty liver disease (NAFLD) is one of the most common chronic liver diseases in children. Our recent clinical trial demonstrated that dietary and VSL#3-based interventions may improve fatty liver by ultrasound and body mass index (BMI) after 4 months. OBJECTIVES: As in this short-term trial, as in others, it is impracticable to monitor response to therapy or treatment by liver biopsy, we aimed to identify a panel of potential non-invasive metabolic biomarkers by a urinary metabolic profiling. METHODS: Urine samples from a group of 31 pediatric NAFLD patients, enrolled in a VSL#3 clinical trial, were analyzed by high-resolution proton nuclear magnetic resonance spectroscopy in combination with analysis of variance-Simultaneous Component Analysis model and multivariate data analyses. Urinary metabolic profiles were interpreted in terms of clinical patient feature, treatment and chronology pattern correlations. RESULTS: VSL#3 treatment induced changes in NAFLD urinary metabolic phenotype mainly at level of host amino-acid metabolism (that is, valine, tyrosine, 3-amino-isobutyrate or ß-aminoisobutyric acid (BAIBA)), nucleic acid degradation (pseudouridine), creatinine metabolism (methylguanidine) and secondarily at the level of gut microbial amino-acid metabolism (that is, 2-hydroxyisobutyrate from valine degradation). Furthermore, some of these metabolites correlated with clinical primary and secondary trial end points after VSL#3 treatment: tyrosine and the organic acid U4 positively with alanine aminotransferase (R=0.399, P=0.026) and BMI (R=0.36, P=0.045); BAIBA and tyrosine negatively with active glucagon-like-peptide 1 (R=-0.51, P=0.003; R=-0.41, P=0.021, respectively). CONCLUSIONS: VSL#3 treatment-dependent urinary metabotypes of NAFLD children may be considered as non-invasive effective biomarkers to evaluate the response to treatment.

Determination of gamma-hydroxybutyric acid in biofluids using a one-step procedure with "in-vial" derivatization and headspace-trap gas chromatography-mass spectrometry.

A headspace-trap gas chromatography-mass spectrometry (HS-trap GC-MS) method was developed to determine GHB, a low molecular weight compound and drug of abuse, in various biological fluids. Combining this relatively novel and fully automated headspace technique with "in-vial" methylation of GHB allowed for a straightforward approach. One single method could be used for all biofluids (urine, plasma, serum, whole blood or lyzed blood), requiring only 100µl of sample. Moreover, our approach involves mere addition of all reagents and sample into one vial. Following optimization of headspace conditions and trap settings, validation was performed. Although sample preparation only consists of the addition of salt and derivatization reagents directly to a 100µl-sample in a HS-vial, adequate method sensitivity and selectivity was obtained. Calibration curves ranged from 5 to 150µg/ml GHB for urine, from 2 to 150µg/ml for plasma, and from 3.5 to 200µg/ml for whole blood. Acceptable precision and accuracy (<13% bias and imprecision) were seen for all quality controls (QC's) (LLOQ-level, low, medium, high), including for the supplementary serum- and lyzed blood-based QC's, using calibration curves prepared in plasma or whole blood, respectively. Incurred sample reanalysis demonstrated assay reproducibility, while cross-validation with another GC-MS method demonstrated that our method is a valuable alternative for GHB determination in toxicological samples, with the advantage of requiring only 100µl and minimal hands-on time, as sample preparation is easy and injection automated.

¹H NMR-based metabolic profiling of naproxen-induced toxicity in rats.

The dose-dependent perturbations in urinary metabolite concentrations caused by naproxen toxicity were investigated using ¹H NMR spectroscopy coupled with multivariate statistical analysis. Histopathologic evaluation of naproxen-induced acute gastrointestinal damage in rats demonstrated a significant dose-dependent effect. Furthermore, principal component analysis (PCA) of ¹H NMR from rat urine revealed a dose-dependent metabolic shift between the vehicle-treated control rats and rats treated with low-dose (10 mg/kg body weight), moderate-dose (50 mg/kg), and high-dose (100 mg/kg) naproxen, coinciding with their gastric damage scores after naproxen administration. The resultant metabolic profiles demonstrate that the naproxen-induced gastric damage exhibited energy metabolism perturbations that elevated their urinary levels of citrate, cis-aconitate, creatine, and creatine phosphate. In addition, naproxen administration decreased choline level and increased betaine level, indicating that it depleted the main protective constituent of the gastric mucosa. Moreover, naproxen stimulated the decomposition of tryptophan into kynurenate, which inhibits fibroblast growth factor-1 and delays ulcer healing. These findings demonstrate that ¹H NMR-based urinary metabolic profiling can facilitate noninvasive and rapid diagnosis of drug side effects and is suitable for elucidating possible biological pathways perturbed by drug toxicity.

Clinical applications of urinary organic acids. Part I: Detoxification markers.

Modern instrumentation allows the measurement of organic acids in urine in their physiological concentration ranges. Eight of the compounds that are reported can serve as markers for specific toxicant exposure or detoxification challenges. Xylene exposure causes elevation of 2-methylhippurate, and orotic acid elevation reveals ammonia challenge that exceeds the capacity of the urea cycle. General hepatic detoxification stimulation by natural compounds, drugs, or xenobiotic compounds causes elevated levels of glucaric acid. Abnormalities of alpha-hydroxybutyrate, pyroglutamate, and sulfate can indicate up-regulated glutathione biosynthesis, impaired reformation of glutathione in the gamma-glutamyl cycle, and depleted total body glutathione status, respectively. Patterns of these compounds measured in a simple overnight urine specimen help to identify focal areas of clinical concern and monitor patient responses to detoxification interventions.

Anabolic effect of human growth hormone: management of inherited disorders of catabolic pathways.

The effects of growth hormone treatment and dietary alanine supplementation, individually and in combination, were studied in five patients with organic acidemias. Three patients had propionic acidemia, one had 3-hydroxyisobutyric acidemia, and one had a defect in isoleucine metabolism. Two patients with propionic acidemia had decreased growth hormone secretion in response to provocative stimuli (intravenous L-arginine and oral levodopa or clonidine); the remaining subjects had sufficient growth hormone secretion. Three of four subjects in whom IGF1 was measured showed subnormal concentrations at baseline (including two with normal growth hormone secretory responses). All patients showed an increase in linear growth with growth hormone. In the four patients studied, all had a significant increase in nitrogen retention over baseline with alanine or growth hormone alone, or with the combination of growth hormone and alanine, with a much greater effect of growth hormone. Lean body mass and body fat composition tended to become normal with treatment. Protein tolerance increased, and when the patients' dietary protein intakes were increased between 20 and 60% they maintained positive nitrogen balance, without a significant increase in metabolite excretion. One patient with propionic acidemia expired during the time of the study, following a course of recurrent pancreatitis and an episode of acute basal ganglia infarction. All of the other subjects showed clinical improvement (decreased incidence of ketoacidotic episodes and decreased frequency of hospital admission and school absence) during treatment, and even the patient who expired remained metabolically stable up to and through the terminal event. We conclude that growth hormone may be of value in the management of patients with organic acidemia.

Effect of level of input of different proportions of volatile fatty acids on energy utilization in growing ruminants.

Four steers were maintained wholly by intragastric infusion of volatile fatty acids (VFA) and protein, together with a mineral-vitamin supplement. The infusion was given at three levels of energy, namely 450, 675 and 900 kJ/kg live weight0.75, calculated to supply energy at 1.0, 1.5 or 2.0 times that required for maintenance. The VFA provided 0.837 and the protein 0.163 of the energy infused. The molar proportions of individual VFA were varied so that the infusate contained 0.36-0.91 of acetic acid, 0.56-0.01 of propionic acid and a constant 0.08 of butyric acid. Heat production was measured in respiration chambers. Urine was analysed for N, urea, beta-hydroxybutyrate and VFA. Blood plasma was analysed for beta-hydroxybutyrate, free fatty acids, insulin and glucose. As the proportion of acetic acid was increased, and propionic acid reduced, there was no change in blood or urine metabolites or in heat production until acetic acid exceeded a proportion of about 0.75. At higher proportions beta-hydroxybutyrate increased in plasma and urine, blood glucose and insulin tended to fall and urinary N excretion rose. At a proportion of acetic acid of > 0.80, acetate appeared in the urine and at > 0.86 heat production declined. The effect of level of infusion on the molar proportion at which plasma and urine metabolites changed was less clear. There was a tendency for the increase in beta-hydroxybutyrate to occur at a slightly lower proportion of acetic acid at the highest level of infusion. It is concluded that differences in heat production that are observed between diets are probably not caused by differences in rumen VFA proportions.(ABSTRACT TRUNCATED AT 250 WORDS)

Biotinidase activity in patients with liver disease.

To investigate whether biotinidase deficiency may occur in liver disease, we determined biotinidase activity, biotin levels, and organic acids in patients with liver disease. Serum biotinidase activity in patients with liver disease (2.63 +/- 1.40 nmol/min/ml) was significantly lower than in the control group (5.43 +/- 1.06 nmol/min/ml). Serum biotinidase activity in decompensated liver cirrhosis (LC) and hepatoma was significantly lower than in acute viral hepatitis (AVH), chronic viral hepatitis (CVH), and compensated LC. The mean serum level of biotin in decompensated LC (1.8 +/- 0.6 microgram/ml) and hepatoma (1.7 +/- 0.8 microgram/ml) was significantly lower than in the control group (2.5 +/- 1.0 microgram/ml), and urinary excretion of biotin was increased in patients with liver disease, particularly in decompensated LC. Biotinidase activity correlated positively with serum biotin level and correlated negatively with urinary biotin level. Moreover, in four of five patients with severe liver disease the excretion of propionate, lactate, and 3-hydroxybutyrate decreased after biotin supplementation. The data for patients with severe liver disease so resembled those for late-onset multiple carboxylase deficiency that biotinidase deficiency is likely in patients with severe liver disease.

Increase of urinary ketone body excretion in selenium-deficient rats is a ketone-specific change.

The effects of selenium (Se) deficiency on urinary ketone body excretion in starved rats were examined. Rats were fed a basal diet which was Se-deficient (Se content: 0.011 micrograms/g) or a Se-adequate diet (the basal diet supplemented with 0.1 micrograms Se/g as sodium selenite). On the 11th and 22nd week of the feeding period, Se-deficient status in rats fed the basal diet was verified by the observation that the Se content and glutathione peroxidase activity in their plasma, erythrocytes, and livers were markedly lowered. On the 4th, 6th, 11th, 15th, and 22nd week, the rats were starved for 48 h and the urinary excretion of ketone bodies (acetoacetate (AcAc) and 3-hydroxybutyrate (3-OHBA)), urea, and creatinine were examined. The urinary excretion of AcAc and 3-OHBA during the second 24 h of the 48-h starvation period were markedly higher in the Se-deficient rats than in the Se-adequate rats for all weeks examined, while the urine volume and the excretion of urea and creatinine were similar in the Se-deficient and Se-adequate rats, irrespective of the feeding period and the number of hours of starvation. On the 22nd week, the plasma ketone body levels were also determined and significantly higher plasma 3-OHBA levels were observed in the Se-deficient rats than in the Se-adequate rats 72 h after starvation began. These results indicate that Se deficiency causes an increase of urinary ketone body excretion in starved rats and that the increase is ketone-specific with no changes in major urinary profiles.

Effect of fat emulsions containing medium-chain triglycerides and glucose on ketone body production and excretion.

The parenteral application of fat emulsions containing medium-chain triglycerides results in an increase of ketone bodies as expression of the rapid metabolism of medium-chain triglycerides. Although ketoacidosis could not be watched, it should be of advantage to reduce the ketonemia. This might be reached by application of carbohydrates. The simultaneous infusion of glucose--given to 10 healthy persons--together with a 10% fat emulsion containing 75% medium-chain triglycerides almost completely inhibited the increase of ketone bodies in serum and its urinary excretion. Other prosperous metabolic effects, eg, the rapid clearance of the triglycerides from the serum were not affected by this regimen.

The formation of 2-hydroxybutyric acid in experimental animals.

1. The formation of 2-hydroxybutyric acid (2-HB) has been studied by animal experiments. 2. 2-HB was excreted in the urine together with lactic acid following intravenous administration of huge amounts of 3-hydroxybutyric acid (sodium salt) to a dog. 3. Rats made diabetic by an injection of streptozotocin were found to excrete large quantities of 2HB in the urine (up to 300 mumol/24 h) together with the development of ketoacidosis. 4. The use of 14-C-labelled precursors clearly showed that the amino acids methionine, threonine and homoserine can be converted to 2-HB. 5. 2-Aminobutyric acid is also converted to 2-HB, with 2-oxobutyric acid as an intermediate metabolite. The ratio between the urinary concentrations of 2-HB and 2-oxobutyric acid was increased by the ingestion of ethanol. 6. In normal rats neither a prolonged fasting period nor loading with large doses of methionine, threonine and homoserine resulted in the excretion of 2-HB. 7. The mechanisms behind the formation of 2-HB are discussed, and it is concluded that an increased NADH2/NAD ratio in the cytoplasma is the most important factor.