GC-MS analysis of organic acids in rat urine: A protocol of direct ultrasound-assisted derivatization.

The aim of the present study was to develop a novel ultrasound-assisted derivatization method for analysis of urine that can be used for preliminary screening and monitoring of metabolic disorders. Here we describe an ultrasound-assisted derivatization method followed by GC-MS analysis to quantify 26 organic acids in urine. The optimum levels of the variables affecting the yield of derivatization were investigated, including urease doses, derivatization reagents and derivatization conditions (duration time, reaction temperature and sonic power). The method exhibited the best results with 80 µl urease. The optimal reaction conditions were 100 µl BSTFA, 80% ultrasound power, 70°C and 40 min. This method showed satisfactory linearity, good reproducibility and an acceptable limit of detection and accuracy. Therefore, it could potentially be used to as a standard method to enable comparisons between laboratories. Finally, we applied our method to urine samples from pregnant rats administered 2 or 10 mg/kg folic acid supplementation.

Extractive Ratio Analysis NMR Spectroscopy for Metabolite Identification in Complex Biological Mixtures.

The complexity of biological mixtures continues to challenge efforts aimed at unknown metabolite identification in the metabolomics field. To address this challenge, we provide a new method to identify related peaks from individual metabolites in complex NMR spectra. Extractive ratio analysis NMR spectroscopy (E-RANSY) builds on our previously described ratio analysis method [ Wei et al. Anal. Chem. 2011 , 83 , 7616 - 7623 ] and exploits the simplified NMR spectra provided by the extraction of metabolites under varied pH conditions. Under such conditions, metabolites from the same biological specimen are extracted differentially, and the resulting NMR spectra exhibit characteristics favorable for unraveling unknown metabolite peaks using ratio analysis. We demonstrate the utility of the E-RANSY method by extracting carboxylic acid containing metabolites from human urine, one of the highly complex biological mixtures encountered in the metabolomics field. E-RANSY performs better than STOCSY and the original RANSY method and offers new avenues to identify unknown metabolites in complex biological mixtures.

Metabonomics Approach To Comparing the Antistress Effects of Four Panax ginseng Components in Rats.

Different components of Panax ginseng have different properties and medicinal effects. Metabonomics was a prospective approach to analyze the global response of endogenous metabolites to physiological and pathological processes. In this study, an untargeted metabonomics method using GC/TOFMS combined with multivariate statistical techniques was applied to compare entire metabolite differences and the antistress variations among four components of P. ginseng, namely, total ginsenosides (TG), panaxadiol (PD), panaxatriol (PT), and ginseng polysaccharide (PS), in Wistar rats. The results of metabolite analysis showed that numerous urine metabolites involving neurotransmitters, amino acids, organic acids, and gut microbiota metabolites were changed after administration of the four components of P. ginseng, with TG having the least impact on urinary metabolites. The urinary metabolite profiling of these rats exposed to acute combined stress (forced swimming and behavior restriction) demonstrated that the four ginseng components attenuated urine metabolite changes involving gut microbiota metabolites, tricarboxylic acid (TCA) cycle and energy metabolites, and organic acids to different degrees, with TG improving most of the metabolites altered by stress.

Robust and High-Throughput Method for Anionic Metabolite Profiling: Preventing Polyimide Aminolysis and Capillary Breakages under Alkaline Conditions in Capillary Electrophoresis-Mass Spectrometry.

Capillary electrophoresis-mass spectrometry (CE-MS) represents a high efficiency microscale separation platform for untargeted profiling of polar/ionic metabolites that is ideal for volume-restricted biological specimens with minimal sample workup. Despite these advantages, the long-term stability of CE-MS remains a major obstacle hampering its widespread application in metabolomics notably for routine analysis of anionic metabolites under negative ion mode conditions. Herein, we report for the first time that commonly used ammonia containing buffers compatible with electrospray ionization (ESI)-MS can compromise the integrity of fused-silica capillaries via aminolysis of their outer polyimide coating. Unlike organic solvent swelling effects, this chemical process occurs under aqueous conditions that is dependent on ammonia concentration, buffer pH, and exposure time resulting in a higher incidence of capillary fractures and current errors during extended operation. Prevention of polyimide aminolysis is achieved by using weakly alkaline ammonia containing buffers (pH < 9) in order to preserve the tensile strength of the polyimide coated fused-silica capillary. Alternatively, less nucleophilic primary/secondary amines can be used as electrolytes without polyimide degradation, whereas chemically resistant polytetrafluoroethylene coating materials offer higher pH tolerance in ammonia. In this work, multisegment injection (MSI)-CE-MS was used as multiplexed separation platform for high throughput profiling of anionic metabolites when using optimized buffer conditions to prevent polyimide degradation. A diverse range of acidic metabolites in human urine were reliably measured by MSI-CE-MS via serial injection of seven urine samples within a single run, including organic acids, food-specific markers, microbial-derived compounds and over-the-counter drugs as their sulfate and glucuronide conjugates. This approach offers excellent throughput (<5 min/sample) and acceptable intermediate precision (average CV ≈ 16%) with high separation efficiency as reflected analysis of 30 anionic metabolites following 238 repeated sample injections of human urine over 3 days while using a single nonisotope internal standard for data normalization. Careful optimization and rigorous validation of CE-MS protocols are crucial for developing a rapid, low cost, and robust screening platform for metabolomics that is amenable to large-scale clinical and epidemiological studies.

Monitoring the Response of the Human Urinary Metabolome to Brief Maximal Exercise by a Combination of RP-UPLC-MS and (1)H NMR Spectroscopy.

The delineation of exercise biochemistry by utilizing metabolic fingerprinting has become an established strategy. We present a combined RP-UPLC-MS and (1)H NMR strategy, supplemented by photometric assays, to monitor the response of the human urinary metabolome to short maximal exercise. Seventeen male volunteers performed two identical sprint sessions on separate days, consisting of three 80 m maximal runs. Using univariate and multivariate analyses, we followed the fluctuation of 37 metabolites at 1, 1.5, and 2 h postexercise. 2-Hydroxyisovalerate, 2-hydroxybutyrate, 2-oxoisocaproate, 3-methyl-2-oxovalerate, 3-hydroxyisobutyrate, 2-oxoisovalerate, 3-hydroxybutyrate, 2-hydroxyisobutyrate, alanine, pyruvate, and fumarate increased 1 h postexercise and then returned toward baseline. Lactate and acetate were higher than baseline at 1 and 1.5 h. Hypoxanthine and inosine remained above baseline throughout the postexercise period. Urate decreased at 1 h and increased at 1.5 h before returning to baseline. Valine, isoleucine, succinate, citrate, trimethylamine, trimethylamine N-oxide, tyrosine, and formate decreased at 1 h and/or 1.5 h postexercise and then returned to baseline. Creatinine gradually decreased over the sampling period. Glycine, 4-aminohippurate, and hippurate remained below baseline throughout the postexercise period. Our findings show that even one-half minute of maximal exercise elicited major perturbations in human metabolism, several of which persisted for at least 2 h.

Metabolomic analysis reveals distinct profiles in the plasma and urine of rats fed a high-protein diet.

A high-protein, low-carbohydrate diet has been regarded as a dietary intervention for weight loss in the obese population. We integrated metabolomics profiles and correlation-based network analysis to reveal the difference in metabolism under diets with different protein:carbohydrate ratios. Rats were fed a control diet (moderate-protein moderate-carbohydrate: MPMC; 20 % protein, 56 % carbohydrate) or HPLC diet (high-protein low-carbohydrate: 45 % protein, 30 % carbohydrate) for 6 weeks. The fat content was equal for both diets. HPLC feeding induced weight loss and reduced adipose weight and plasma triglyceride. Compared to the MPMC diet, HPLC significantly increased plasma α-tocopherol, pyruvate, 2-oxoisocaproate, and ß-hydroxybutyrate, and reduced linoleate, palmitate, α-glycerophosphate and pyroglutamic acid. The HPLC-associated urinary metabolite profile was signified with an increase in palmitate and stearate and a reduction of citrate, 2-ketoglutarate, malate, and pantothenate. Pathway analysis implicated a significant alteration of the TCA cycle in urine. Biomarker screening demonstrated that individual metabolites, including plasma urea, pyruvate, and urinary citrate, robustly distinguished the HPLC group from the MPMC group. Correlation-based network analysis enabled to demonstrate that the correlation of plasma metabolite was strengthened after the HPLC diet, while the energy-metabolism relatives 2-ketoglutarate and fumarate correlated positively with phenylalanine, methionine, and serine. The correlation network between plasma-urinary metabolites revealed a negative correlation of plasma valine with urinary ß-hydroxybutyrate in MPMC rats. In HPLC rats, plasma 2-oxoisocaproate negatively correlated with urinary pyruvate and glycine. This study using metabolomics analysis revealed the systemic metabolism in response to diet treatment and identified the significantly distinct profiles associated with a HPLC diet.

Human metabolism of Δ3-carene and renal elimination of Δ3-caren-10-carboxylic acid (chaminic acid) after oral administration.

We studied the human in vivo metabolism of Δ(3)-carene (CRN), a natural monoterpene which commonly occurs in the human environment. Four healthy human volunteers were orally exposed to a single dose of 10 mg CRN. Each volunteer gave one urine sample before administration and subsequently collected each urine sample within 24 h after administration. The concentration of the proposed CRN metabolites Δ(3)-caren-10-ol (CRN-10-OH), Δ(3)-caren-10-carboxylic acid (chaminic acid, CRN-10-COOH), and Δ(3)-caren-3,4-diol (CRN-3,4-OH) were determined using a very specific and sensitive GC-MS/MS procedure. Other CRN metabolites were investigated using GC-PCI-MS Q1 scan analyses. CRN-10-COOH was detected in each urine sample with maximum concentration (113.0-1,172.9 µg L(-1)) 2-3 h after administration, whereas CRN-10-OH and CRN-3,4-OH were not detected in any of the samples. The renal excretion kinetics of CRN-10-COOH showed an elimination half-life of about 3 h. The cumulative excretion of CRN-10-COOH within 24 h after exposure correlated with about 2 % of the applied dose. The GC-PCI-MS Q1 scan analysis indicated several additional human CRN metabolites; thereof, six spectra enabled the prediction of the corresponding chemical structure. The results of the study indicate that CRN-10-COOH is a relevant product of the human in vivo metabolism of CRN. The oxidation of its allylic methyl group proceeds until the acidic structure without interruption. Thus, the generation of the alcoholic intermediate appeared to be the rate-determining step of this metabolic route. Nevertheless, the proportion of CRN-10-COOH in the CRN metabolism is low, and other oxidative metabolites are likely. This hypothesis was confirmed by the discovery of additional human CRN metabolites, whose predicted chemical structures fit in with further oxidative products of CRN metabolism.

Spectrum analysis of common inherited metabolic diseases in Chinese patients screened and diagnosed by tandem mass spectrometry.

BACKGROUND: Information concerning inherited metabolic diseases in China is scarce. We investigated the prevalence and age distributions of amino acid, organic acid, and fatty acid oxidation disorders in Chinese patients. METHODS: Blood levels of amino acids and acylcarnitines (tandem mass spectrometry) were measured in 18,303 patients with suspected inherited metabolic diseases. Diagnosis was based on clinical features, blood levels of amino acids or acylcarnitines, urinary organic acid levels (gas chromatography-mass spectrometry), and (in some) gene mutation tests. RESULTS: Inherited metabolic diseases were confirmed in 1,135 patients (739 males, 396 females). Median age was 12 months (1 day to 59 years). There were 28 diseases: 12 amino acid disorders (580 patients, 51.1%), with hyperphenylalaninemia (HPA) being the most common; nine organic acidemias (408 patients, 35.9%), with methylmalonic acidemia (MMA) as the most common; and seven fatty acid oxidation defects (147 patients, 13.0%), with multiple acyl-coenzyme A dehydrogenase deficiency (MADD) being the most common. Onset was mainly at 1-6 months for citrin deficiency, 0-6 months for MMA, and in newborns for ornithine transcarbamylase deficiency (OTCD). HPA was common in patients aged 1-3 years, and MADD was common in patients >18 years. CONCLUSIONS: In China, HPA, citrin deficiency, MMA, and MADD are the most common inherited disorders, particularly in newborns/infants.

Urinary loss of tricarboxylic acid cycle intermediates as revealed by metabolomics studies: an underlying mechanism to reduce lipid accretion by whey protein ingestion?

Whey protein intake is associated with the modulation of energy metabolism and altered body composition both in human subjects and in animals, but the underlying mechanisms are not yet elucidated. We fed obesity-prone C57BL/6J mice high-fat diets with either casein (HF casein) or whey (HF whey) for 6 weeks. At equal energy intake and apparent fat and nitrogen digestibility, mice fed HF whey stored less energy as lipids, evident both as lower white adipose tissue mass and as reduced liver lipids, compared with HF-casein-fed mice. Explorative analyses of 48 h urine, both by (1)H NMR and LC-MS metabolomic platforms, demonstrated higher urinary excretion of tricarboxylic acid (TCA) cycle intermediates citric acid and succinic acid (identified by both platforms), and cis-aconitic acid and isocitric acid (identified by LC-MS platform) in the HF whey, relative to in the HF-casein-fed mice. Targeted LC-MS analyses revealed higher citric acid and cis-aconitic acid concentrations in fed state plasma, but not in liver of HF-whey-fed mice. We propose that enhanced urinary loss of TCA cycle metabolites drain available substrates for anabolic processes, such as lipogenesis, thereby leading to reduced lipid accretion in HF-whey-fed compared to HF-casein-fed mice.

Fully automated reagent peak-free liquid chromatography fluorescence analysis of highly polar carboxylic acids using a column-switching system and fluorous scavenging derivatization.

In this study, we combined a column-switching system with a fluorous scavenging derivatization method to develop a fully automated reagent peak-free LC fluorescence detection protocol for the analysis of highly polar carboxylic acids. In this method, highly polar carboxylic acids were derivatized with fluorescent 1-pyrenemethylamine in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 1-hydroxy-1H-benzotriazole. Residual excess of the unreacted reagent was tagged with 2-(perfluorooctyl)ethyl isocyanate and then removed selectively using a fluorous column-switching system placed in front of an analytical reversed-phase column. The signal of the fluorous-tagged unreacted reagent was completely absent in the resulting chromatograms; therefore, it did not interfere with the quantification of each acid especially those eluted before 20 min. The detection limits (S/N = 3) for the examined acids were in the range from 4.0 to 22 fmol per injection. We have applied this method to comparative analysis of highly polar carboxylic acids in urine samples obtained from diabetes mellitus type-II model mice and their control.

The pharmacokinetics and conversion of the lactone to the carboxylate forms of ginkgolide B in rat plasma.

Ginkgolide B consists of three lactone groups, which may undergo hydrolysis, and lead to the rings opening in aqueous solution with different pHs. From mechanisms of pharmacological activity in vivo, the lactone appears to be the active form of the drug. Pharmacokinetics of lactone form (GB-lac) and the total of the lactone and carboxylate form (GB-tot) of ginkgolide B were investigated after intravenous administration of a dose of 4 mg/kg ginkgolide B. The rate of lactone hydrolysis was also studied in plasma in vitro. After intravenous administration, ginkgolide B in the original form was converted to its carboxylate form under simulated physiological conditions. The AUC0 - ∞ of GB-lac constituted 63.5 ± 17.4% of the AUC0 - ∞ of GB-tot. The ratio of average cumulation of excretion of lactone to carboxylate reached approximately 1 to 1 in urine. From the equilibrium of lactone hydrolysis in rat plasma in vitro, the k obs was - 0.0176 min(- 1) and t 1/2 was 39.38 min. In conclusion, the equilibrium existed between lactone of ginkgolide B and its carboxylate form in vivo at physiological pH, which suggested that more attention should be focused on the original and the ionization forms of ginkgolide B and the conversion of the lactone into carboxylate in vivo.

Elevated plasma succinate in PTEN, SDHB, and SDHD mutation-positive individuals.

PURPOSE: Cowden syndrome results from germline mutations in the gene for phosphatase and tensin homologue deleted on chromosome 10 (PTEN) and from variants in succinate dehydrogenase B and D subunits. We hypothesized that succinate accumulation may be common among individuals with SDH variants/mutations and those with PTEN mutations. METHODS: Urine and blood were collected from individuals meeting full or partial Cowden syndrome diagnostic criteria or those with paraganglioma (PGL) or a known susceptibility paraganglioma-associated gene mutation, and succinate was measured. PTEN, SDHB, SDHC, and SDHD genes were sequenced from genomic DNA. RESULTS: Elevated plasma succinate was observed in 13/21 (62%) individuals with germline PTEN, SDHB, or SDHD mutations as compared with 5/32 (16%) controls (P < 0.001), in 10/15 (67%) individuals with pathogenic PTEN mutations but in <20% of mutation-negative individuals meeting identical criteria, and in individuals with mutations in SDHB (1/1, 100%) and SDHD (2/5, 40%). CONCLUSION: Our data suggest that mutations in PTEN, SDHB, and SDHD reduce catalytic activity of succinate dehydrogenase, resulting in succinate accumulation, and identify a common biochemical alteration in these two patient populations (PTEN and SDHx mutation positive individuals). Plasma organic acid analysis may provide an effective and inexpensive screening method to determine when more expensive gene sequencing of PTEN and SDH genes is warranted.

Bezafibrate can be a new treatment option for mitochondrial fatty acid oxidation disorders: evaluation by in vitro probe acylcarnitine assay.

BACKGROUND: The number of patients with mitochondrial fatty acid oxidation (FAO) disorders is recently becoming larger with the spread of newborn mass screening. Despite the advances in metabolic and molecular characterization of FAO disorders, the therapeutic studies are still limited. It was reported recently that bezafibrate (BEZ), an agonist of peroxisome proliferating activator receptor (PPAR), can restore FAO activity in cells from carnitine palmitoyltransferase-2 (CPT2) and very-long-chain acyl-CoA dehydrogenase (VLCAD) deficiencies as well as clinical symptoms in the adult patients. METHODS: In this study, the therapeutic effect of BEZ was determined by in vitro probe acylcarnitine (IVP) assay using cultured fibroblasts and tandem mass spectrometry on various FAO disorders. The clinical trial of BEZ treatment for a boy with the intermediate form of glutaric acidemia type 2 (GA2) was also performed. RESULTS: The effect of BEZ was proven in cells from various FAO disorders including GA2, deficiencies of VLCAD, medium-chain acyl-CoA dehydrogenase, CPT2, carnitine acylcarnitine translocase and trifunctional protein, by the IVP assay. The aberrantly elevated long- or medium-chain acylcarnitines that are characteristic for each FAO disorder were clearly corrected by the presence of BEZ (0.4 mmol/L) in culture medium. Moreover, daily administration of BEZ in a 2-year-old boy with GA2 dramatically improved his motor and cognitive skills, accompanied by sustained reduction of C4, C8, C10 and C12 acylcarnitines in blood, and normalized the urinary organic acid profile. No major adverse effects have been observed. CONCLUSION: These results indicate that BEZ could be a new treatment option for FAO disorders.

Non-extractable proanthocyanidins from grapes are a source of bioavailable (epi)catechin and derived metabolites in rats.

The non-extractable fraction of many fruit and vegetables contains putatively bioactive polyphenolic compounds that, in most cases, have not been well characterised structurally. Non-extractable proanthocyanidins (NEPA) of a polymeric nature are part of the dietary fibre fraction of food. Using liquid chromatography coupled to a mass spectrometer equipped with an electrospray ionisation chamber and a triple quadrupole mass analyser for tandem analysis (HPLC-ESI-QqQ-MS/MS) techniques, we examine the phenolic metabolites present in urine and faeces from rats 24 h after ingestion of an NEPA-rich fraction. We show that NEPA are partially depolymerised during their transit along the intestinal tract, as evidenced by the presence of (epi)catechin (EC) monomers and dimers in faeces and phase II conjugates of EC in urine. Moreover, NEPA are further metabolised by the intestinal microbiota into smaller metabolites including phenolic acids that are present in urine as both free phenolics and conjugates with glucuronate or sulphate moieties. For the first time, we report evidence that NEPA behave in vivo as a source of phenolics that are released progressively and deliver phenolic species that come into contact with the intestinal walls and are bioavailable for at least 24 h after ingestion.

Neonatal intrahepatic cholestasis caused by citrin deficiency: prevalence and SLC25A13 mutations among Thai infants.

BACKGROUND: The most common causes of cholestatic jaundice are biliary atresia and idiopathic neonatal hepatitis (INH). Specific disorders underlying INH, such as various infectious and metabolic causes, including neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD) especially, in East Asian populations are increasingly being identified. Since most NICCD infants recovered from liver disease by 1 year of age, they often are misdiagnosed with INH, leading to difficulty in determining the true prevalence of NICCD. Mutation(s) of human SLC25A13 gene encoding a mitochondrial aspartate/glutamate carrier isoform 2 (AGC2), can lead to AGC2 deficiency, resulting in NICCD and an adult-onset fatal disease namely citrullinemia type II (CTLN2). To study the prevalence of NICCD and SLC25A13 mutations in Thai infants, and to compare manifestations of NICCD and non-NICCD, infants with idiopathic cholestatic jaundice or INH were enrolled. Clinical and biochemical data were reviewed. Urine organic acid and plasma amino acids profiles were analyzed. PCR-sequencing of all 18 exons of SLC25A13 and gap PCR for the mutations IVS16ins3kb and Ex16+74_IVS17-32del516 were performed. mRNA were analyzed in selected cases with possible splicing error. RESULTS: Five out of 39 (12.8%) unrelated infants enrolled in the study were found to have NICCD, of which three had homozygous 851del4 (GTATdel) and two compound heterozygous 851del4/IVS16ins3kb and 851del4/1638ins23, respectively. Two missense mutations (p.M1? and p.R605Q) of unknown functional significance were identified. At the initial presentation, NICCD patients had higher levels of alkaline phosphatase (ALP) and alpha-fetoprotein (AFP) and lower level of alanine aminotransferase (ALT) than those in non-NICCD patients (p< 0.05). NICCD patients showed higher citrulline level and threonine/serine ratio than non-NICCD infants (p< 0.05). Fatty liver was found in 2 NICCD patients. Jaundice resolved in all NICCD and in 87.5% of non-NICCD infants at the median age of 9.5 and 4.0 months, respectively. CONCLUSION: NICCD should be considered in infants with idiopathic cholestasis. The preliminary estimated prevalence of NICCD was calculated to be 1/48,228 with carrier rate of 1/110 among Thai infants. However, this number may be underestimated and required further analysis with mutation screening in larger control population to establish the true prevalence of NICCD and AGC2 deficiency.

Urinary biomarkers of exposure to glycol ethers and chlorinated solvents during pregnancy: determinants of exposure and comparison with indirect methods of exposure assessment.

OBJECTIVES: To describe urine levels of metabolites of glycol ethers and chlorinated solvents in a sample of pregnant women from the general population, to study their occupational and non-occupational determinants and to compare them with the results of indirect assessment methods of solvent exposure. METHODS: A sample of 451 pregnant women was randomly selected from a general population cohort. At inclusion, the women in this sample completed a self-administered questionnaire about their social and medical characteristics, occupation and exposure to different products at work and in non-occupational activities. Occupational exposure to solvents was assessed from the woman's self-report and from a job-exposure matrix. Eight alkoxycarboxylic acids and trichloroacetic acid and trichloroethanol were measured with chromatography in urine samples collected at inclusion. Associations between metabolite levels and job titles, exposure to products used at work, and solvent exposure were studied. RESULTS: The different glycol ether metabolites were detected in 5.3%-96.4% of the urine samples, trichloroacetic acid in 6.4% and trichloroethanol in 5.5%. Nurses had butoxyacetic acid and phenoxyacetic acid in their urine most often, whereas methoxyethoxyacetic acid was the most frequent among nursing aides. Among cleaners, ethoxyacetic acid and ethoxyethoxyacetic acid were the most frequent. The occupation of hairdresser was associated with urinary excretion of ethoxyacetic acid, ethoxyethoxyacetic acid, butoxyacetic acid and phenoxyacetic acid. Among the women classified as exposed to solvents, the agents identified most often were ethoxyacetic acid, ethoxy-ethoxyacetic acid, butoxyacetic acid, phenoxyacetic acid, trichloroacetic acid and trichloroethanol. Ethoxyethoxyacetic acid was the only metabolite associated with non-occupational exposure. CONCLUSIONS: Metabolites of glycol ethers and chlorinated solvents were present at low levels in the urine of pregnant women. Most metabolites were associated with occupational exposure.

Nutritional and metabolic findings in patients with Prader-Willi syndrome diagnosed in early infancy.

OBJECTIVE: Early treatment (growth hormone and nutritional support) improves development in infants with Prader-Willi syndrome. This study aimed to evaluate the nutritional and metabolic condition of nine patients who were diagnosed and treated in early infancy. METHODS: Nine patients were hospitalized at the age of \xe2\u20ac\xa810 days to 11 months because of severe feeding difficulties, failure to thrive, or developmental delay. The diagnosis of Prader-Willi syndrome was confirmed by fluorescence in situ hybridization or other molecular genetic techniques. Nutritional and metabolic investigations including urinary organic acid analysis, blood amino acid, and acylcarnitine profiles were performed. RESULTS: The diagnosis was made at the mean age of 6.3 months. A deletion of the paternal gene in the 15q11-13 region was detected in all patients. Eight patients had ketosis, seven had malnutrition, five had hyperammonemia, three had liver dysfunction, three had low blood cholesterol level, and two had hypoglycemia. All patients had reduction of serum multiple amino acids and free carnitine. Significant arginine deficiency was found in all patients. Six patients had mildly elevated blood long-chain and very long-chain acylcarnitine. After supplementation with l-arginine, medium-chain fatty acids, l-carnitine, and vitamins, all patients responded with improvement of motor development and nutritional conditions. Four patients were almost caught up on physical and psychomotor development. CONCLUSIONS: Patients with Prader-Willi syndrome are in bad metabolic condition in the early period. Early diagnosis and individual nutritional interventions may improve the nutritional and developmental progress and decrease death rate in infancy.

Metabonomic responses in rat urine following subacute exposure to propoxur.

Metabolic profiling of urine from pesticide-treated rats was investigated by the nuclear magnetic resonance (NMR)-based metabonomic strategy. Twenty-four-hour urine samples of rats were collected after administration with propoxur at doses of 0.85, 1.70, and 8.51 mg/kg, respectively, for 28 consecutive days. Liver tissue was fixed and the histopathological alterations were examined. The results showed that propoxur at high dose induced liver histopathological injury. Metabonomic analysis demonstrated that the levels of creatine and taurine markedly increased together with slight elevation of hippurate, glucose, and amino acids in low- and medium-dose groups. However, concentrations of urinary lactate, acetate, acetone, succinate, citrate, and 2-oxoglutarate increased in high-dose group. All these results suggested that propoxur could inhibit liver function through altering the energy and lipid metabolism. These data also supported the contention that the NMR-based metabonomic approach represents a promising new technology for the development of pesticide toxicity screening and mechanism exploration.

Dispersive liquid-liquid microextraction of 11-nor-Δ9-tetrahydrocannabinol-carboxylic acid applied to urine testing.

Aim: THC-COOH is the major metabolite of Δ9-tetrahydrocannabinol commonly tested in urine to determine cannabis intake. In this study, a method based on dispersive liquid-liquid microextraction was developed for testing THC-COOH in urine. Materials & methods: Hydrolyzed urine specimens were extracted via dispersive liquid-liquid microextraction with acetonitrile (disperser solvent) and chloroform (extraction solvent). Derivatization was performed with N,O-Bis(trimethylsilyl)trifluoroacetamide with 1% trichloro(chloromethyl)silane. Analysis was performed by GC-MS/MS. Results: The method showed acceptable linearity (5-500 ng/ml), imprecision (<10.5%) and bias (<4.9%). Limits of detection and quantitation were 1 and 5 ng/ml, respectively. Twenty-four authentic samples were analyzed, with 22 samples being positive for THC-COOH. Conclusion: The proposed method is more environmentally friendly and provided good sensitivity, selectivity and reproducibility.

Tweetable abstract Green analytical toxicology: Dispersive liquid­liquid microextraction applied to the analysis of THC-COOH in urine by GC­MS/MS.