Diet-resistant obesity is characterized by a distinct plasma proteomic signature and impaired muscle fiber metabolism.

BACKGROUND/OBJECTIVES: Inter-individual variability in weight loss during obesity treatment is complex and poorly understood. Here we use whole body and tissue approaches to investigate fuel oxidation characteristics in skeletal muscle fibers, cells and distinct circulating protein biomarkers before and after a high fat meal (HFM) challenge in those who lost the most (obese diet-sensitive; ODS) vs the least (obese diet-resistant; ODR) amount of weight in a highly controlled weight management program. SUBJECTS/METHODS: In 20 weight stable-matched ODS and ODR women who previously completed a standardized clinical weight loss program, we analyzed whole-body energetics and metabolic parameters in vastus lateralis biopsies and plasma samples that were obtained in the fasting state and 6 h after a defined HFM, equivalent to 35% of total daily energy requirements. RESULTS: At baseline (fasting) and post-HFM, muscle fatty acid oxidation and maximal oxidative phosphorylation were significantly greater in ODS vs ODR, as was reactive oxygen species emission. Plasma proteomics of 1130 proteins pre and 1, 2, 5 and 6 h after the HFM demonstrated distinct group and interaction differences. Group differences identified S-formyl glutathione hydratase, heat shock 70 kDA protein 1A/B (HSP72), and eukaryotic translation initiation factor 5 (eIF5) to be higher in ODS vs ODR. Group-time differences included aryl hydrocarbon interacting protein (AIP), peptidylpropyl isomerase D (PPID) and tyrosine protein-kinase Fgr, which increased in ODR vs ODS over time. HSP72 levels correlated with muscle oxidation and citrate synthase activity. These proteins circulate in exosomes; exosomes isolated from ODS plasma increased resting, leak and maximal respiration rates in C2C12 myotubes by 58%, 21% and 51%, respectively, vs those isolated from ODR plasma. CONCLUSIONS: Findings demonstrate distinct muscle metabolism and plasma proteomics in fasting and post-HFM states corresponding in diet-sensitive vs diet-resistant obese women.

Biochemical and Hematologic Manifestations of Gastric Intrinsic Factor (GIF) Deficiency: A Treatable Cause of B12 Deficiency in the Old Order Mennonite Population of Southwestern Ontario.

Intrinsic factor deficiency (OMIM #261000, IFD) is a rare inherited disorder of vitamin B12 metabolism due to mutations in the gastric intrinsic factor (GIF) gene.We report three individuals from an Old Order Mennonite community who presented with B12 deficiency. Two cases are siblings born to consanguineous parents and the third case is not known to be closely related. The older male sib presented at 4 years with gastrointestinal symptoms, listlessness, and pallor. He had pancytopenia with megaloblastic anemia. Serum B12 was 61 (198-615 pmol/L). Methylmalonic aciduria was present. C3 was elevated on acylcarnitine profile. Homocysteine was high at 16.7 (5.0-12.0 umol/L). His asymptomatic female sibling was also found to have B12 deficiency. Genetic testing for methylmalonic aciduria (MMAA), transcobalamin deficiency (TCN2), and Imerslund-Gräsbeck syndrome (AMN) showed no mutation in both siblings. The third patient, a 34-year-old woman, had presented in infancy with a diagnosis of pernicious anemia. Mutation analysis of GIF revealed compound heterozygosity for a c.79+1G>A substitution and a c.973delG deletion in all three individuals. Oral or parenteral vitamin B12 has led to complete recovery of clinical parameters and vitamin B12 levels. Newborn screening samples on the siblings revealed normal methylcitrate, C3, and C3/C2 ratios thus indicating no disruption of propionic or methylmalonic acid metabolism.A high index of suspicion should be maintained if children present with megaloblastic anemia since GIF deficiency is a treatable disorder and newborn screening may not be able to detect this condition.

Clinical, biochemical and molecular characterization of peroxisomal diseases in Arabs.

Peroxisomes are single membrane-bound cellular organelles that carry out critical metabolic reactions perturbation of which leads to an array of clinical phenotypes known as peroxisomal disorders (PD). In this study, the largest of its kind in the Middle East, we sought to comprehensively characterize these rare disorders at the clinical, biochemical and molecular levels. Over a 2-year period, we have enrolled 17 patients representing 16 Arab families. Zellweger-spectrum phenotype was observed in 12 patients and the remaining 5 had the rhizomelic chondrodysplasia punctata phenotype. We show that homozygosity mapping is a cost-effective strategy that enabled the identification of the underlying genetic defect in 100% of the cases. The pathogenic nature of the mutations identified was confirmed by immunofluorescence and complementation assays. We confirm the genetic heterogeneity of PD in our population, expand the pool of pathogenic alleles and draw some phenotype/genotype correlations.

Zellweger syndrome caused by PEX13 deficiency: report of two novel mutations.

Peroxisomal biogenesis disorders represent a group of genetically heterogeneous conditions that have in common failure of proper peroxisomal assembly. Clinically, they are characterized by a spectrum of dysmorphia, neurological, liver, and other organ involvement. To date, mutations in 13 PEX genes encoding peroxins have been identified in patients with peroxisomal biogenesis disorders. Mutations in PEX13, which encodes peroxisomal membrane protein PEX13, are among the least common causes of peroxisomal biogenesis disorders with only three mutations reported so far. Here, we report on two infants whose clinical and biochemical profile was consistent with classical Zellweger syndrome and whose complementation analysis assigned them both to group H of peroxisomal biogenesis disorders. We show that they harbor two novel mutations in PEX13. One patient had a genomic rearrangement resulting in a 147 kb deletion that spans the whole of PEX13, while the other had an out-of-frame deletion of 14 bp. This represents the first report of a PEX13 deletion and suggests that further work is needed to examine the frequency of PEX13 mutations among Arab patients with peroxisomal biogenesis disorders.

LC-MS/MS determination of dibasic amino acids for the diagnosis of cystinuria. Application in a family affected by a novel splice-acceptor site mutation in the SLC7A9 gene.

Cystinuria is an autosomal recessive disorder caused by defective transport of cystine and the dibasic amino acids ornithine, lysine and arginine across cell membranes. Poor solubility of cystine in urine leads to kidney stones and associated symptoms and complications. Mutations of genes SLC3A1 and SLC7A9 encoding for amino acid transport systems are responsible for different types of cystinuria. In this study we describe a new LC-MS/MS assay for these amino acids in urine. Moreover, we report a novel splice-acceptor site mutation in the SLC7A9 gene that we believe is the cause of the phenotype observed in four siblings from a first-cousin marriage. Into the wells of a 96-well microtitre plate, 10 microl of urine was mixed with 90 microl of a solution containing [(2)H4]cystine, [(2)H2]ornithine, [(13)C,(2)H4]arginine and [(2)H5]glutamine that was used as an internal standard for lysine. Chromatographic separation was achieved isocratically and detection was in the selected-reaction monitoring mode. The injection-to-injection time was 8 min. Calibration curves were linear up to 1000 micromol/L. Intra-day (n = 10) and inter-day (n = 6) variations (750 and 10 micromol/L) were less than 11.4%. Urine samples from healthy individuals (n = 135) were analysed and age-matched reference ranges were generated. The method was applied retrospectively and prospectively to analyse samples (n = 13) from nine cystinuria patients. The mutation reported here was not found in 100 controls with similar ethnicity to the studied family and is believed to have consequences for the transcribed mature RNA and protein structure and function.

Quantification of N-acetylaspartic acid in urine by LC-MS/MS for the diagnosis of Canavan disease.

Canavan disease is an autosomal recessive leukodystrophy characterized by excessive excretion of N-acetylaspartic acid (NAA) in urine. The disease is caused by deficiency of aspartoacylase, the enzyme responsible for the hydrolysis of NAA into acetate and l-aspartate. Patients, who are often asymptomatic in their early months, show a wide spectrum of clinical presentation thereafter that includes macrocephaly, poor head control, seizures, abnormal muscle tone, optic atrophy, significant developmental delay and death. In this work, we describe a simple liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of NAA in urine. The internal standard d3-NAA was added to untreated urine and the mixture was injected into the LC-MS/MS system operated in the negative ion mode. Detection was achieved in multiple reaction monitoring (MRM) mode by monitoring m/z 174 --> 88, 174 --> 130 and 174 --> 58 for NAA and 177 --> 89 for the internal standard. Separation was carried out on a C8 column (2.1 x 150 mm) using a mixture of acetonitrile and water (1:1 v/v) containing 0.05% formic acid at a flow rate of 0.25 ml/min. NAA was eluted at 1.6 min and the run time was approximately 2 min. Using spiked urine, the assay was linear up to 2 mmol/L with limit of quantification at 1 micromol/L (S/N = 12). NAA in patients' urine (n = 17) ranged between 366 and 21,235 mmol/mol creatinine compared to controls of <39 mmol/mol creatinine (n = 159). This LC-MS/MS method for NAA as described involved no extraction and no derivatization, showed no interference, and gave excellent recovery with low variability and short analytical time.

Derivatization reagents in liquid chromatography/electrospray ionization tandem mass spectrometry for biomedical analysis.

Liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESIMS/MS) is one of the most prominent analytical techniques owing to its inherent selectivity and sensitivity. In LC/ESI-MS/MS, chemical derivatization is frequently used to enhance the MS/MS detectability. The derivatization improves the separation and ionization efficiency. Moreover, the generated derivatives give particular product ions by CID (collision induced dissociation), which allow for the sensitive detection. In this review, we present an overview of the derivatization reagents which have been applied to LC/ESI-MS/MS, focusing on the applications involving small molecules in biomatrices.

Drugs of abuse in a non-conventional sample; detection of methamphetamine and its main metabolite, amphetamine in abusers' clothes by HPLC with UV and fluorescence detection.

In this paper, we report the detection of methamphetamine and its major metabolite, amphetamine, in garments belong to known-abusers. These compounds were extracted from the textile using a mixture of chloroform:propan-2-ol (3:1, v/v), derivatized with 4-(4,5-diphenyl-1H-imidazol-2-yl) benzoyl chloride and separated using a reversed-phase high-performance liquid chromatography. The derivatives were detected by measuring either fluorescence at 440 nm or absorbance at 330 nm. By using 1-methyl-3-phenyl propylamine as an internal standard, calibration curves of spiked textile samples were linear over a wide range with correlation coefficients of 0.997 or better. Detection limits at a signal-to-noise ratio of 3 were less than or equal to 37.3 and 0.4 pg on column for the high-performance liquid chromatography-ultraviolet and -fluorescence detection methods, respectively. Intra- and inter-day variations at high and low concentrations (n > or = 3) were < or =12.7%. The developed methods were successfully applied to the determination of methamphetamine and amphetamine in clothes samples belong to abusers.

Hyphenated chromatographic methods for biomaterials.

The improvement in hyphenated analytical techniques has significantly widened their applications to the analysis of biomaterials. In this article, we discuss recent advances in applications of hyphenated chromatographic techniques including capillary electrophoresis to the analyses of biological samples. As tools of separation, gas chromatography, high-performance liquid chromatography and capillary electrophoresis are considered with special emphasis on applications utilizing the hyphenation of these methods to mass spectrometry. Moreover, applications using other detection methods such as Fourier transform infrared spectroscopy hyphenated to gas chromatography and photodiode array detector combined with high-performance liquid chromatography or capillary electrophoresis are also discussed. Owing to their high sensitivity, luminescence-based detection systems such as laser-induced fluorescence and chemiluminescence are also included in this review.

Quantification of methamphetamine, amphetamine and enantiomers by semi-micro column HPLC with fluorescence detection; applications on abusers' single hair analyses.

Achiral and chiral semi-micro column high-performance liquid chromatographic methods with fluorescence detection to determine methamphetamine and amphetamine in human hair are described. These compounds were extracted into 5% trifluoroacetic acid (TFA) in methanol, derivatized with 4-(4,5-diphenyl-1H-imidazol-2-yl)-benzoyl chloride and separated either on a 250 x 1.5 mm i.d. octadecyl-silane (ODS) or a 150 x 2 mm i.d. OD-RH column. Linear calibration curves extending over a wide range of concentration that covers the practical samples were obtained for amphetamine, methamphetamine and their enantiomers (r = 0.999). Resolution values for amphetamine and methamphetamine enantiomers were 3.4 and 1.1, respectively. Intra- and inter-day variations of both the methods were not larger than 8.9% expressed as relative standard deviations (n >/= 5). The limits of detection at a signal-to-noise ratio of 3 obtained by both the methods were in the range of 1.0-4.7 fmol/5 microL injection with the achiral method being more sensitive. Abusers' hair samples were analyzed by the two methods and only the S(+)-enantiomers were found in eight Japanese abusers' hair samples. The achiral method was used to study the concentrations of these compounds in single black and white hair strands of abusers.

Achiral and chiral quantification of methamphetamine and amphetamine in human urine by semi-micro column high-performance liquid chromatography and fluorescence detection.

In this paper, miniaturized achiral and chiral high-performance liquid chromatographic procedures for the determination of methamphetamine and amphetamine in human urine are described. After a simple pretreatment of human urine (i.e., 10 microL of urine or diluted urine were acidified and dried-up under N2 at room temperature) and fluorescence derivatization with 4-(4,5-diphenyl-1H-imidazol-2-yl)-benzoyl chloride under mild conditions (pH 9.0, 10 min at room temperature), the derivatives were isocratically separated on a semi-micro ODS column with Tris-HCl buffer (0.1 M, pH 7.0): acetonitrile (45 + 55 v/v) at a flow rate of 0.2 mL/min or their enantiomers were separated on a semi-micro OD-RH column with sodium hexafluorophosphate (0.3 M aq.): acetonitrile (44 + 56 v/v) at a flow rate of 0.1 mL/min as the mobile phase. Wide-ranged calibration curves were obtained with detection limits for the achiral and chiral analyses in the atto and femtomol levels, respectively, per injected volume. Satisfactory within- and between-day reproducibility data were obtained with both the methods with the highest relative standard deviation being 9.6%. The methods were applied to the determination of methamphetamine and amphetamine in human urine samples and the concentrations determined by the two methods were well correlated (r = 0.994).