A genome-wide metabolic QTL analysis in Europeans implicates two loci shaped by recent positive selection

We have performed a metabolite quantitative trait locus (mQTL) study of the (1)H nuclear magnetic resonance spectroscopy ((1)H NMR) metabolome in humans, building on recent targeted knowledge of genetic drivers of metabolic regulation. Urine and plasma samples were collected from two cohorts of individuals of European descent, with one cohort comprised of female twins donating samples longitudinally. Sample metabolite concentrations were quantified by (1)H NMR and tested for association with genome-wide single-nucleotide polymorphisms (SNPs). Four metabolites' concentrations exhibited significant, replicable association with SNP variation (8.6×10(-11)相似文献   

Quantitative analysis of metabolite concentrations in human urine samples using 13C{1H} NMR spectroscopy

Targeted profiling is a library-based method of using mathematically modeled reference spectra for quantification of metabolite concentrations in NMR mixture analysis. Metabolomics studies of biofluids, such as urine, represent a highly complex problem in this area, and for this reason targeted profiling of ¹H NMR spectra can be hampered. A number of the issues relating to ¹H NMR spectroscopy can be overcome using ¹³C{¹H} NMR spectroscopy. In this work, a ¹³C{¹H} NMR database was created using Chenomx NMR Suite, incorporating 120 metabolites. The ¹³C{¹H} NMR database was standardized through the analysis of a series of metabolite solutions containing varying concentrations of 19 distinct metabolites, where the metabolite concentrations were varied across a range of values including biological ranges. Subsequently, the NMR spectra of urine samples were collected using ¹³C{¹H} NMR spectroscopy and profiled using the ¹³C{¹H} NMR library. In total, about 30 metabolites were conclusively identified and quantified in the urine samples using ¹³C{¹H} NMR targeted profiling. The proton decoupling and larger spectral window provided easier identification and more accurate quantification for specific classes of metabolites, such as sugars and amino acids with overlap in the aliphatic region of the ¹H NMR spectrum. We discuss potential application areas in which ¹³C{¹H} NMR targeted profiling may be superior to ¹H NMR targeted profiling.     

Variation of metabolites in normal human urine

Urine is often sampled from patients participating in clinical and metabolomic studies. Biological homeostasis occurs in humans, but little is known about the variability of metabolites found in urine. It is important to define the inter- and intra-individual metabolite variance within a normal population before scientific or clinical conclusions are made regarding different pathophysiologies. This study investigates the variability of selected urine metabolites in a group of 60 healthy men and women over a period of 30 days. To monitor individual variation, 6 women from the normal population were randomly selected and followed for 30 days. To determine the influence of extraneous environmental factors urine was collected from 25 guinea pigs with similar genetics, diet, and living environment. For both studies, 24 metabolites were identified and quantified using high-resolution ¹H nuclear magnetic resonance spectroscopy (NMR). The data demonstrated large inter and intra-individual variation in metabolite concentrations in both normal human and control animal populations. A defined normal baseline is essential before any conclusions may be drawn regarding changes in urine metabolite concentrations.     

A note on normalization of biofluid 1D 1H-NMR data

One-dimensional ¹H nuclear magnetic resonance (1D ¹H-NMR) has been used extensively as a metabolic profiling tool for investigating urine and other biological fluids. Under ideal conditions, ¹H-NMR peak intensities are directly proportional to metabolite concentrations and thus are useful for class prediction and biomarker discovery. However, many biological, experimental and instrumental variables can affect absolute NMR peak intensities. Normalizing or scaling data to minimize the influence of these variables is a critical step in producing robust, reproducible analyses. Traditionally, analyses of biological fluids have relied on the total spectral area [constant sum (CS)] to normalize individual intensities. This approach can introduce considerable inter-sample variance as changes in any individual metabolite will affect the scaling of all of the observed intensities. To reduce normalization-related variance, we have developed a histogram matching (HM) approach adapted from the field of image processing. We validate our approach using mixtures of synthetic compounds that mimic a biological extract and apply the method to an analysis of urine from rats treated with ethionine. We show that HM is a robust method for normalizing ¹H-NMR data and propose it as an alternative to the traditional CS method.     

Modelling the acid/base <Superscript>1</Superscript>H NMR chemical shift limits of metabolites in human urine

Introduction

Despite the use of buffering agents the ¹H NMR spectra of biofluid samples in metabolic profiling investigations typically suffer from extensive peak frequency shifting between spectra. These chemical shift changes are mainly due to differences in pH and divalent metal ion concentrations between the samples. This frequency shifting results in a correspondence problem: it can be hard to register the same peak as belonging to the same molecule across multiple samples. The problem is especially acute for urine, which can have a wide range of ionic concentrations between different samples.

Objectives

To investigate the acid, base and metal ion dependent ¹H NMR chemical shift variations and limits of the main metabolites in a complex biological mixture.

Methods

Urine samples from five different individuals were collected and pooled, and pre-treated with Chelex-100 ion exchange resin. Urine samples were either treated with either HCl or NaOH, or were supplemented with various concentrations of CaCl₂, MgCl₂, NaCl or KCl, and their ¹H NMR spectra were acquired.

Results

Nonlinear fitting was used to derive acid dissociation constants and acid and base chemical shift limits for peaks from 33 identified metabolites. Peak pH titration curves for a further 65 unidentified peaks were also obtained for future reference. Furthermore, the peak variations induced by the main metal ions present in urine, Na⁺, K⁺, Ca²⁺ and Mg²⁺, were also measured.

Conclusion

These data will be a valuable resource for ¹H NMR metabolite profiling experiments and for the development of automated metabolite alignment and identification algorithms for ¹H NMR spectra.

     

Quantitative variability of 342 plasma proteins in a human twin population

The degree and the origins of quantitative variability of most human plasma proteins are largely unknown. Because the twin study design provides a natural opportunity to estimate the relative contribution of heritability and environment to different traits in human population, we applied here the highly accurate and reproducible SWATH mass spectrometry technique to quantify 1,904 peptides defining 342 unique plasma proteins in 232 plasma samples collected longitudinally from pairs of monozygotic and dizygotic twins at intervals of 2–7 years, and proportioned the observed total quantitative variability to its root causes, genes, and environmental and longitudinal factors. The data indicate that different proteins show vastly different patterns of abundance variability among humans and that genetic control and longitudinal variation affect protein levels and biological processes to different degrees. The data further strongly suggest that the plasma concentrations of clinical biomarkers need to be calibrated against genetic and temporal factors. Moreover, we identified 13 cis‐SNPs significantly influencing the level of specific plasma proteins. These results therefore have immediate implications for the effective design of blood‐based biomarker studies.     

Structural and dynamical characterization of tubular HIV‐1 capsid protein assemblies by solid state nuclear magnetic resonance and electron microscopy

The wild‐type HIV‐1 capsid protein (CA) self‐assembles in vitro into tubular structures at high ionic strength. We report solid state nuclear magnetic resonance (NMR) and electron microscopy measurements on these tubular CA assemblies, which are believed to contain a triangular lattice of hexameric CA proteins that is similar or identical to the lattice of capsids in intact HIV‐1. Mass‐per‐length values of CA assemblies determined by dark‐field transmission electron microscopy indicate a variety of structures, ranging from single‐wall tubes to multiwall tubes that approximate solid rods. Two‐dimensional (2D) solid state ¹³C? ¹³C and ¹⁵N? ¹³C NMR spectra of uniformly ¹⁵N,¹³C‐labeled CA assemblies are highly congested, as expected for a 25.6 kDa protein in which nearly the entire amino acid sequence is immobilized. Solid state NMR spectra of partially labeled CA assemblies, expressed in 1,3‐¹³C₂‐glycerol medium, are better resolved, allowing the identification of individual signals with line widths below 1 ppm. Comparison of crosspeak patterns in the experimental 2D spectra with simulated patterns based on solution NMR chemical shifts of the individual N‐terminal (NTD) and C‐terminal (CTD) domains indicates that NTD and CTD retain their individual structures upon self‐assembly of full‐length CA into tubes. 2D ¹H‐¹³C NMR spectra of CA assemblies recorded under solution NMR conditions show relatively few signals, primarily from segments that link the α‐helices of NTD and CTD and from the N‐ and C‐terminal ends. Taken together, the data support the idea that CA assemblies contain a highly ordered 2D protein lattice in which the NTD and CTD structures are retained and largely immobilized.     

Study of metabolite composition of eccrine sweat from healthy male and female human subjects by 1H NMR spectroscopy

The aim of the study was to evaluate metabolite variability in human eccrine sweat using a metabonomics based approach. Eccrine sweat is a dilute electrolyte solution whose primary function is to control body temperature via evaporative cooling. Although the composition of sweat is primarily water, previous studies have shown that a diverse array of organic and inorganic compounds are also present. Human eccrine sweat samples from 30 female and 30 male subjects were analysed using high-resolution ¹H nuclear magnetic resonance (NMR) spectroscopy in conjunction with statistical pattern recognition. High-resolution ¹H NMR spectroscopy produced spectra of the sweat samples that readily identified and quantified many different metabolites. The major metabolite classes found to be present were lactate, amino acids and lipids, with lactate being by far the most dominant metabolite found in all samples. Principal Components Analysis, Principal Components-Discriminant Analysis and Partial Least Squares-Discriminant Analysis of the eccrine sweat samples, revealed no significant differences in metabolite composition and concentration between female and male subjects. Also, the variation between subjects did not appear to be correlated with any other clinical information provided by the subjects. Overall, the spectra data set demonstrates the large physiological variability in terms of number of metabolites present and concentrations between subjects i.e. human eccrine sweat samples exhibit a high degree of inter-individual variability.     

Metabolic profiling of Rhodiola rosea rhizomes by 1H NMR spectroscopy

Introduction – Rhodiola rosea is a broadly used medicinal plant with largely unexplored natural variability in secondary metabolite levels. Objective – The aim of this work was to develop a non‐target procedure for ¹H NMR spectroscopic fingerprinting of rhizome extracts for pattern recognition analysis and identification of secondary metabolites responsible for differences in sample composition. To achieve this, plants from three different geographic areas (Swiss Alps, Finland, and Altai region in Siberia) were investigated. Results – A sample preparation procedure was developed in order to remove polymeric polyphenols as the ¹H NMR analysis of low‐molecular‐weight metabolites was hampered by the presence of tannins. Principal component analysis disclosed tight clustering of samples according to population. PCA models based on the aromatic region of the spectra showed that the first two components reflected changes in the content of salidroside and rosavin, respectively, the rosavin content being negatively correlated to that of rhodiocyanoside A and minor aromatics. Score plots and non‐parametric variance tests demonstrated population‐dependent changes according to harvest time. Data consistency was assessed using score plots and box‐and‐whisker graphs. In addition, a procedure for presenting loadings of PCA models based on bucketed data as high‐resolution plots, which are reminiscent of real ¹H NMR spectra and help to identify latent biomarkers, is presented. Conclusion – This study demonstrated the usefulness of the established procedure for multivariate non‐target ¹H NMR metabolic profiling of Rhodiola rosea. Copyright © 2010 John Wiley & Sons, Ltd.     

Urinary metabolomic phenotyping of nickel induced acute toxicity in rat: an NMR spectroscopy approach

The metabolomic approach has been widely used in toxicology to investigate mechanisms of toxicity. To understand the mammalian system??s response to nickel exposure, we analysed the NiCl₂ induced metabolomic changes in urine of rats using ¹H nuclear magnetic resonance (¹H NMR) spectroscopy together with clinically relevant biochemical parameters. Male Sprague?CDawley rats were administered intraperitoneally with NiCl₂ at doses of 4, 10 and 20?mg/kg body weight. Urine samples were collected at 8, 16, 24, 72, 96 and 120?h post treatment. The metabolomic profile of rat urine showed prominent changes in citrate, dimethylamine, creatinine, choline, trimethylamine oxide (TMAO), phenyl alanine and hippurate at all doses. Principal component analysis of urine ¹H NMR spectra demonstrated the dose and time dependent development of toxicity. The metabolomic time trajectory, based on pattern recognition analysis of ¹H NMR spectra of urine, illustrated clear separation of pre and post treatments (temporal). Only animals treated with a low dose of NiCl₂ returned to normal physiology. The ¹H NMR spectral data correlated well with the clinically relevant nephrotoxic biomarkers. The urinary metabolomic phenotyping for NiCl₂ induced nephrotoxicity was defined according to the predictive ability of the known metabolite biomarkers, creatinine, citrate and TMAO. The current approach demonstrates that metabolomics, one of the most important platform in system biology, may be a promising tool for identifying and characterizing biochemical responses to toxicity.     

Individual differences in migratory behavior shape population genetic structure and microhabitat choice in sympatric blackcaps (Sylvia atricapilla)

In migratory birds, traits such as orientation and distance are known to have a strong genetic background, and they often exhibit considerable within‐population variation. How this variation relates to evolutionary responses to ongoing selection is unknown because the underlying mechanisms that translate environmental changes into population genetic changes are unclear. We show that within‐population genetic structure in southern German blackcaps (Sylvia atricapilla) is related to individual differences in migratory behavior. Our 3‐year study revealed a positive correlation between individual migratory origins, denoted via isotope (δ²H) values, and genetic distances. Genetic diversity and admixture differed not only across a recently established migratory polymorphism with NW‐ and SW‐migrating birds but also across δ²H clusters within the same migratory route. Our results suggest assortment based on individual migratory origins which would facilitate evolutionary responses. We scrutinized arrival times and microhabitat choice as potential mechanisms mediating between individual variation in migratory behavior and assortment. We found significant support that microhabitat choice, rather than timing of arrival, is associated with individual variation in migratory origins. Moreover, examining genetic diversity across the migratory divide, we found migrants following the NW route to be genetically more distinct from each other compared with migrants following the traditional SW route. Our study suggests that migratory behavior shapes population genetic structure in blackcaps not only across the migratory divide but also on an individual level independent of the divide. Thus, within‐population variation in migratory behavior might play an important role in translating environmental change into genetic change.     

Evaluation of melanin‐related metabolites as markers of solar ultraviolet‐B radiation

Ultraviolet‐B (UVB) radiation due sunlight can result in sunburns and/or suntans. Sunburn occurs only several hours after solar UVB radiation, while a suntan requires several days to several weeks to develop. In the present study, we measured serum and urine levels of melanin‐related metabolites, 5‐S‐cysteinyldopa (5‐S‐CD) and 6‐hydroxy‐5‐methoxyindole‐2‐carboxylic acid (6H5MI2C), in nine subjects exposed to normal sunlight over the course of 12 months. We collected samples in the middle of each month and examined the variation of the markers, the correlation between them, and their correlation with solar UVB radiation. Those markers exhibited a seasonal variation with lower values in the winter and higher values in the summer. Levels of 5‐S‐CD and 6H5MI2C in the serum showed 48% and 54% increases in the summer compared with those in the winter, respectively. Comparison of 5‐S‐CD in the serum and urine showed the highest correlation (r² = 0.344), followed by the pair of 5‐S‐CD and 6H5MI2C in the serum. Levels of 5‐S‐CD in the serum showed the highest correlation (r² = 0.729) with the mean solar UVB radiation during the first 10 d of the month, while 6H5MI2C in the serum was highly correlated (r² = 0.483) with solar UVB radiation during the previous month. Levels of 5‐S‐CD and 6H5MI2C in the serum appear to reflect the degrees of skin injury and pigmentation in the skin, respectively.     

Function and solution structure of the Arabidopsis thaliana RALF8 peptide

We report the recombinant preparation from Escherichia coli cells of samples of two closely related, small, secreted cysteine‐rich plant peptides: rapid alkalinization factor 1 (RALF1) and rapid alkalinization factor 8 (RALF8). Purified samples of the native sequence of RALF8 exhibited well‐resolved nuclear magnetic resonance (NMR) spectra and also biological activity through interaction with a plant receptor kinase, cytoplasmic calcium mobilization, and in vivo root growth suppression. By contrast, RALF1 could only be isolated from inclusion bodies as a construct containing an N‐terminal His‐tag; its poorly resolved NMR spectrum was indicative of aggregation. We prepared samples of the RALF8 peptide labeled with ¹⁵N and ¹³C for NMR analysis and obtained near complete ¹H, ¹³C, and ¹⁵N NMR assignments; determined the disulfide pairing of its four cysteine residues; and examined its solution structure. RALF8 is mostly disordered except for the two loops spanned by each of its two disulfide bridges.     

Quantitation of the antitumor agent N-(Phosphonacetyl)-l-aspartic acid in human plasma and urine by gas chromatography—mass spectrometry—selected ion monitoring

N-(Phosphonacetyl)-l-aspartic acid (PALA) is an antitumor agent which is currently under clinical study. A gas chromatography—mass spectrometry—selected ion monitoring assay procedure using [¹³C]PALA as the internal standard has been developed for the quantitation of PALA in biological samples. Standard curves which related ion intensity peak height ratios (m/e 220/221) to PALA concentrations in plasma and urine were described by a non-linear least square analysis with correlation coefficients of R² > 0.995 and > 0.996, respectively. Over concentration ranges for PALA of 1–60 μg/ml of plasma and 1–160 μg/ml of urine the coefficient of variation from the fitted curve was 4–18%. This methodology has been used to quantitate PALA in human plasma samples in a study on the clinical pharmacology of the drug.     

Determination of 8-oxoguanine in human plasma and urine by high-performance liquid chromatography with electrochemical detection

A highly sensitive and selective method for determining 8-oxoguanine in plasma and urine was developed by high-performance liquid chromatography with electrochemical detection. The compound was separated by gradient elution on a C₁₈ reversed-phase column with a mobile phase of acetonitrile and 0.1 M sodium acetate, pH 5.2. 8-Hydroxy-2′-deoxyguanosine was used as internal standard. 8-Oxoguanine was detected electrochemically by setting the potential to +300 mV vs. Pd reference. The sensitivity of the assay was 22 ng/ml with a signal-to-noise ratio of 7:1. The within-day relative standard deviations for 8-oxoguanine quality control samples with concentrations of 3340, 1340 and 84 ng/ml were 3.6, 4.3 and 5.7% for plasma, and 4.1, 4.6 and 6.2% for urine, respectively. The day-to-day relative standard deviations for the same samples were 3.8, 6.8 and 7.1% for plasma, and 3.9, 7.0 and 7.9% for urine, respectively. The method is designed to study the pharmacokinetics and metabolic fate of O⁶-benzylguanine in a phase I clinical trial. Previously, O⁶-benzyl-8-oxoguanine was identified as the primary metabolite of O⁶-benzylguanine in humans. We now demonstrate that 8-oxoguanine is a further metabolite of O⁶-benzylguanine.     

Discrimination and Characterization of Two Mediterranean Species from the Laurencia Complex (Rhodomelacea) Using an NMR‐Based Metabolomic Approach

Generic and specific determination among the Laurencia complex is a challenging task. DNA barcoding combined with phenotypic investigations are mandatory for species differentiation. In this study, two morphologically different members of the Laurencia complex were investigated using untargeted ¹H‐NMR‐based metabolomics. Twenty‐one population samples were collected in order to evaluate both temporal and geographical homogeneity. Data obtained from ¹H‐NMR analysis followed by statistical analysis allowed a clear separation of all the samples into two groups. DNA mitochondrial tests confirmed this pattern and identified the two species as Laurenciella sp. and Laurencia obtusa. In addition, metabolites responsible of this discrimination were investigated directly in crude extracts by ¹³C‐NMR using an in‐house computer‐assisted method. The combination of both untargeted (¹H) and targeted (¹³C) NMR‐based metabolomic approaches proves to be a powerful and complementary approach to discriminate species from the Laurencia complex.     

Urine stability for metabolomic studies: effects of preparation and storage

Metabolomic studies attempt to identify and profile unique metabolic differences among test populations, which may be correlated with a specific biological stress or pathophysiology. Due to the ease of collection and the metabolite-rich nature of urine, it is frequently used as a bio-fluid for human and animal metabolic studies. High-resolution ¹H-NMR is an analytical tool used to qualitatively and quantitatively identify metabolites in urine. Urine samples were collected from healthy male and female subjects and prepared: raw, following centrifugation, filtration, or the addition of the bacteriostatic preservative sodium azide and analyzed by NMR. In addition, these samples were stored at room temperature (22 °C), in a refrigerator (4 °C), or in a deep-freeze (−80 °C). Samples were analyzed by NMR every week for a month and changes in concentrations of 55 easily identifiable metabolites were followed. The degree of change in metabolite concentrations following storage over a 4-week period were influenced by the different methods of sample preparation and storage. Significant changes in urine metabolites are likely due to bacterial contamination of the urine. Our study demonstrates that bacterial contamination of urine in normal individuals significantly alters the metabolic profile of urine over time and proper preparation and storage procedures must be followed to reduce these changes. By identifying appropriate methods of urine preparation and storage investigators will preserve the fidelity of the urine samples in order to better reflect the original metabolic state.     

Single-dose Pharmacokinetics of Nestorone, a potential female-contraceptive

A synthetic progestin Nestorone^® is being developed for female-contraception. This study was conducted to determine the distribution, metabolism, and excretion of tritium-labeled Nestorone (³H Nestorone) in adult female rats. Rats were injected subcutaneously (S.C.) with a single dose of 400 μCi ³H Nestorone/kg BW. Its distribution and concentrations in blood, plasma and other tissues were determined at defined times. The excreta were examined for elimination of ³H Nestorone. Radioactivity in all samples was analyzed by liquid scintillation counter. Metabolite profiling was performed by HPLC and LC/MS analysis of the plasma, urine, and feces samples. Following subcutaneous injection of ³H Nestorone, the mean peak concentrations of radioactivity (C_max) in the blood and plasma were 58.1 and 95.5 ng equiv. ³H Nestorone/g, respectively, at 2-h postdose (T_max). Thereafter, the concentration of drug steadily declined through 96-h postdose with a terminal elimination half-life (t_1/2) of 15.6 h. ³H Nestorone-derived radioactivity was widely distributed in most tissues by 0.5 h and attained a mean maximal concentration by 2-h postdose. Approximately, 81.4% and 7.62% of the administered dose was excreted via feces and urine, respectively. In vivo metabolism of ³H Nestorone resulted into a total of 19 metabolites. Among them, two metabolites viz., 17α-deacetyl-Nestorone (M9) and 4,5-dihydro-17α-deacetyl-Nestorone (M19) were identified by HPLC and LC/MS analysis. Metabolite profiling of plasma samples showed that most of the circulating radioactivity was associated with unchanged parent drug, and M19. The M19 was a major metabolite in the profiled urine and feces samples. Presence of large proportion of drug/drug-related material in feces suggested that the biliary excretion is a main elimination route of ³H Nestorone. The distribution, metabolism, and excretion profiles of ³H Nestorone obtained in this study provide a fairly good insight about its fate in women.     

A sample preparation protocol for H nuclear magnetic resonance studies of water-soluble metabolites in blood and urine

We describe a general protocol for preparing protein-containing biofluids for ¹H nuclear magnetic resonance (NMR) metabolomic studies. In this protocol, untreated samples are diluted in deuterated solvents to precipitate proteins and recover metabolites quantitated relative to standard reference compounds such as 3-trimethylsilylpropionic acid (TSP) and 2,2-dimethyl-2-silapentane-5-sulfonic acid (DSS). The efficacy of this protocol was tested using a bovine serum albumin/metabolite mix and human serum samples. This sample preparation method can be readily applied to any protein-containing biofluid for ¹H NMR studies.     

Optical resolution and enantiomeric purity determination of the analgesic cizolirtine

The optical resolution of (±)‐cizolirtine was accomplished with excellent results (>99% ee) by means of crystallization with (+)‐ or (−)‐di‐p‐toluoyltartaric acid. The optical purity of the samples was controlled by three independent methods: ¹H NMR, capillary electrophoresis (CE) (using β‐cyclodextrins as chiral resolving agents), and HPLC (using a glycoproteic column). The use of a rapid analytical technique like ¹H NMR for estimating the relative amounts of each enantiomer, together with the high sensitivity of CE, afforded a convenient strategy for monitoring the entire process leading to enantiopure compounds. Chirality 11:63–69, 1999. © 1999 Wiley‐Liss, Inc.