Fiona Bamforth

HMDB: the Human Metabolome Database

The Human Metabolome Database (HMDB) is currently the most complete and comprehensive curated collection of human metabolite and human metabolism data in the world. It contains records for more than 2180 endogenous metabolites with information gathered from thousands of books, journal articles and electronic databases. In addition to its comprehensive literature-derived data, the HMDB also contains an extensive collection of experimental metabolite concentration data compiled from hundreds of mass spectra (MS) and Nuclear Magnetic resonance (NMR) metabolomic analyses performed on urine, blood and cerebrospinal fluid samples. This is further supplemented with thousands of NMR and MS spectra collected on purified, reference metabolites. Each metabolite entry in the HMDB contains an average of 90 separate data fields including a comprehensive compound description, names and synonyms, structural information, physico-chemical data, reference NMR and MS spectra, biofluid concentrations, disease associations, pathway information, enzyme data, gene sequence data, SNP and mutation data as well as extensive links to images, references and other public databases. Extensive searching, relational querying and data browsing tools are also provided. The HMDB is designed to address the broad needs of biochemists, clinical chemists, physicians, medical geneticists, nutritionists and members of the metabolomics community. The HMDB is available at:

The Human Serum Metabolome

Continuing improvements in analytical technology along with an increased interest in performing comprehensive, quantitative metabolic profiling, is leading to increased interest pressures within the metabolomics community to develop centralized metabolite reference resources for certain clinically important biofluids, such as cerebrospinal fluid, urine and blood. As part of an ongoing effort to systematically characterize the human metabolome through the Human Metabolome Project, we have undertaken the task of characterizing the human serum metabolome. In doing so, we have combined targeted and non-targeted NMR, GC-MS and LC-MS methods with computer-aided literature mining to identify and quantify a comprehensive, if not absolutely complete, set of metabolites commonly detected and quantified (with todays technology) in the human serum metabolome. Our use of multiple metabolomics platforms and technologies allowed us to substantially enhance the level of metabolome coverage while critically assessing the relative strengths and weaknesses of these platforms or technologies. Tables containing the complete set of 4229 confirmed and highly probable human serum compounds, their concentrations, related literature references and links to their known disease associations are freely available at http://www.serummetabolome.ca.

Geographical and ethnic variation of the 677C>T allele of 5,10 methylenetetrahydrofolate reductase ( MTHFR ): findings from over 7000 newborns from 16 areas world wide

Since its biochemical characterisation in 19911 and its genetic identification in 1995,2 677C>T allele (T allele) of the 5,10 methylenetetrahydrofolate reductase ( MTHFR ) gene has been a focus of increasing interest from researchers world wide. The expanding spectrum of common conditions linked with the 677C>T allele now includes certain adverse birth outcomes (including birth defects), pregnancy complications, cancers, adult cardiovascular diseases, and psychiatric disorders.3–8 Although several of these associations remain unconfirmed or controversial,4 their scope is such that it becomes of interest to explore the geographical and ethnic distribution of the allele and associated genotypes.9 Accurate information on such distribution can contribute to studies of gene-disease associations (by providing reference population data) and population genetics (by highlighting geographical and ethnic variations suggestive of evolutionary pressures),10 as well as help to evaluate health impact (by allowing estimates of population attributable fraction). Current population data, however, show gaps and even for some ethnic groups or large geographical areas (for example, China) few data are available.3 Our aim was to supplement the available data by collecting a large and diverse sample of newborns from different geographical areas and ethnic groups, and to examine international variations in the distribution of the 677C>T allele. We present findings relating to more than 7000 newborns from 16 areas around the world. The study was conducted under the auspices of the International Clearinghouse for Birth Defect Monitoring Systems (ICBDMS) and was coordinated through its head office, the International Center on Birth Defects (ICBD). ### Sample selection Participating programmes, in consultation with the coordinating group, identified a population sampling approach that would be simple yet minimise sampling bias with respect to the MTHFR genotype. We made an explicit attempt to sample systematically the newborn population. Details of each programme’s approach are listed below, and further …

The human cerebrospinal fluid metabolome.

With continuing improvements in analytical technology and an increased interest in comprehensive metabolic profiling of biofluids and tissues, there is a growing need to develop comprehensive reference resources for certain clinically important biofluids, such as blood, urine and cerebrospinal fluid (CSF). As part of our effort to systematically characterize the human metabolome we have chosen to characterize CSF as the first biofluid to be intensively scrutinized. In doing so, we combined comprehensive NMR, gas chromatography-mass spectrometry (GC-MS) and liquid chromatography (LC) Fourier transform-mass spectrometry (FTMS) methods with computer-aided literature mining to identify and quantify essentially all of the metabolites that can be commonly detected (with todays technology) in the human CSF metabolome. Tables containing the compounds, concentrations, spectra, protocols and links to disease associations that we have found for the human CSF metabolome are freely available at http://www.csfmetabolome.ca.

Hyperhomocysteinemia and inflammatory bowel disease: prevalence and predictors in a cross-sectional study

OBJECTIVE:Homocysteine is a sulfur-containing amino acid formed during the demethylation of methionine. Vitamin B12 and folate deficiency and therapy with antifolate drugs may predispose patients with inflammatory bowel disease (IBD) to hyperhomocysteinemia. The known associations between hyperhomocysteinemia and smoking, osteoporosis, and thrombosis make it an interesting candidate as a pathogenetic link in IBD. The aim of this study was to identify the prevalence and risk factors of hyperhomocysteinemia in patients with IBD.METHODS:Sixty-five consecutive IBD patients were recruited from a tertiary outpatient gastroenterology practice. Fasting plasma homocysteine levels were measured, along with vitamin B12 and folate. Data regarding medication use, multivitamin use, disease location and severity, and extraintestinal manifestations of IBD were gathered. Homocysteine levels in 138 healthy control subjects were compared with the IBD cohort, and adjustments for age and sex were made using logistic regression. Multivariate analysis was performed to seek predictors of homocysteine levels.RESULTS:The mean age in the IBD cohort was 42 ± 13.4 yr (±SD), and 43% were male. The mean disease duration was 13.8 ± 9.4 yr, and 32% had used steroids within the last 3 months. Immunomodulator therapy had been used in 32%, and 75% had had an intestinal resection. Osteoporosis was present in 33% of patients. Five patients had experienced venous thrombosis or stroke, but only one of these had hyperhomocysteinemia. Of the 10 IBD patients (15.4%) with hyperhomocysteinemia, only two had vitamin B12 deficiency. The homocysteine levels in the IBD cohort cases and controls were 8.7 and 6.6 μmol/L, respectively (p < 0.05). IBD significantly increased the risk of hyperhomocysteinemia (adjusted odds ratio = 5.9 [95% CI: 1.5–24]). Advanced age, male sex, vitamin B12 deficiency or lower vitamin B12 serum levels, and multivitamin therapy were independently associated with higher homocysteine levels in the multivariate analysis (R2= 0.55; p = 0.001).CONCLUSIONS:Hyperhomocysteinemia is significantly more common in patients with IBD compared with healthy controls, and is associated with lower (but not necessarily deficient) vitamin B12 levels.

Qualitative Metabolome Analysis of Human Cerebrospinal Fluid by 13C-/12C-Isotope Dansylation Labeling Combined with Liquid Chromatography Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

Metabolome analysis of human cerebrospinal fluid (CSF) is challenging because of low abundance of metabolites present in a small volume of sample. We describe and apply a sensitive isotope labeling LC-MS technique for qualitative analysis of the CSF metabolome. After a CSF sample is divided into two aliquots, they are labeled by 13C-dansyl and 12C-dansyl chloride, respectively. The differentially labeled aliquots are then mixed and subjected to LC-MS using Fourier-transform ion cyclotron resonance mass spectrometry (FTICR MS). Dansylation offers significant improvement in the performance of chromatography separation and detection sensitivity. Moreover, peaks detected in the mass spectra can be readily analyzed for ion pair recognition and database search based on accurate mass and/or retention time information. It is shown that about 14,000 features can be detected in a 25-min LC-FTICR MS run of a dansyl-labeled CSF sample, from which about 500 metabolites can be profiled. Results from four CSF samples are compared to gauge the detectability of metabolites by this method. About 261 metabolites are commonly detected in replicate runs of four samples. In total, 1132 unique metabolite ion pairs are detected and 347 pairs (31%) matched with at least one metabolite in the Human Metabolome Database. We also report a dansylation library of 220 standard compounds and, using this library, about 85 metabolites can be positively identified. Among them, 21 metabolites have never been reported to be associated with CSF. These results illustrate that the dansylation LC-FTICR MS method can be used to analyze the CSF metabolome in a more comprehensive manner.

Two pairs of male monozygotic twins discordant for Wiedemann-Beckwith syndrome

Wiedemann-Beckwith syndrome (WBS) is a congenital anomaly syndrome which classically consists of exomphalos, macroglossia, and gigantism. The syndrome is also associated with a variety of minor anomalies and affected individuals have an increased risk of developing rare embryonal cell tumors. To date, 15 monozygotic (MZ) twin pairs have been reported of which 13 are discordant for WBS. All except one pair of the discordant WBS twin pairs have been female. We report two pairs of male MZ twins, each discordant for WBS.

Zygosity and placental anatomy in 15 consecutive sets of spontaneously conceived triplets

Most triplets are trizygotic because they result from assisted reproduction. Prognosis is generally good. We analyzed 15 sets of triplets who were conceived spontaneously. Six sets were monozygotic, 7 were dizygotic, and only 2 sets were trizygotic. Considered as 45 twin pairs, 25 pairs (56%) were monozygotic. Twenty percent of these twins died as a result of twin-twin transfusion. Spontaneously conceived triplets have high risks compared with those resulting from assisted reproduction. These risks result from a high proportion of monozygotic embryos, many of whom have monochorionic placentas with vascular anastomoses, causing twin-to-twin transfusion.

X-chromosome inactivation is mostly random in placental tissues of female monozygotic twins and triplets

Patterns of X-chromosome inactivation in chorion, amnion, and cord from 79 pairs of twins were examined. Seven sets of triplets were included in the analysis, both as twin pairs and triplets. Twins were stratified as dizygotic (DZ), monozygotic (MZ), monochorionic, and dichorionic and were selected for birth weight discordance, discordance for congenital anomalies, twin-twin transfusion syndrome, and various patterns of vascular anastomosis. X-inactivation was predominantly symmetric. Chorion was the most likely tissue to show asymmetric X-inactivation and was found most frequently in MZ dichorionic twins. There was no correlation of X-inactivation pattern with the selected clinical criteria. This study does not confirm that asymmetric X-inactivation in embryonic tissues is a common phenomenon in female twins, including monozygotic twins.

Evaluation of the accuracy of self-reported smoking in pregnancy when the biomarker level in an active smoker is uncertain

INTRODUCTION Our main objective was to estimate smoking prevalence as well as sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of self-reported smoking among pregnant women in Edmonton, Canada, at 15-16 weeks of gestation. METHODS We used serum samples to assemble a cohort of pregnant women who underwent an optional second-trimester screening for chromosomal and developmental anomalies. We determined cotinine concentrations for 92 self-reported smokers (11% of the cohort) and for 285 self-reported nonsmoking mothers, using adapted urinary cotinine assay. Self-reports were collected at the time of delivery. In a validation study, serum cotinine was determined for known smokers and nonsmokers and used, within a Bayesian statistical framework, to define the distribution of cutoffs that differentiate true smokers from nonsmokers. This distribution of cutoffs was used to construct multiple two-by-two tables to obtain the distribution of sensitivity, specificity, PPV, NPV, and prevalence. RESULTS Sensitivity was poor (M = 47.4%, SD = 17.3%), but specificity was nearly perfect (M = 94.9%, SD = 1.1%). PPV (M = 66.6%, SD = 11.7%) was smaller than NPV (M = 84.7%, SD = 14.3%). In our sample, the prevalence of true smoking at 15-16 weeks of gestation was described by a skewed distribution with a mean of 21.6% (SD = 13.8%) and a median of 16.6%. DISCUSSION The strength of the present study includes blinding of subjects to the intention to test their sera for a biomarker of smoking. A limitation was the use of a nonrandom sample restricted to pregnancies that resulted in live births. We discuss data collection methods that would elicit more accurate smoking histories from pregnant women.