John J. Thaden

Proposed minimum reporting standards for chemical analysis

There is a general consensus that supports the need for standardized reporting of metadata or information describing large-scale metabolomics and other functional genomics data sets. Reporting of standard metadata provides a biological and empirical context for the data, facilitates experimental replication, and enables the re-interrogation and comparison of data by others. Accordingly, the Metabolomics Standards Initiative is building a general consensus concerning the minimum reporting standards for metabolomics experiments of which the Chemical Analysis Working Group (CAWG) is a member of this community effort. This article proposes the minimum reporting standards related to the chemical analysis aspects of metabolomics experiments including: sample preparation, experimental analysis, quality control, metabolite identification, and data pre-processing. These minimum standards currently focus mostly upon mass spectrometry and nuclear magnetic resonance spectroscopy due to the popularity of these techniques in metabolomics. However, additional input concerning other techniques is welcomed and can be provided via the CAWG on-line discussion forum at http://msi-workgroups.sourceforge.net/ or http://Msi-workgroups-feedback@lists.sourceforge.net. Further, community input related to this document can also be provided via this electronic forum.

Remarkable longevity and stress resistance of nematode PI3K-null mutants.

The great majority of lifespan‐augmenting mutations were discovered in the nematode Caenorhabditis elegans. In particular, genetic disruption of insulin‐like signaling extends longevity 1.5‐ to 3‐fold in the nematode, and to lesser degrees in other taxa, including fruit flies and mice. C. elegans strains bearing homozygous nonsense mutations in the age‐1 gene, which encodes the class‐I phosphatidylinositol 3‐kinase catalytic subunit (PI3KCS), produce progeny that were thought to undergo obligatory developmental arrest. We now find that, after prolonged developmental times at 15–20 °C, they mature into extremely long‐lived adults with near‐normal feeding rates and motility. They survive to a median of 145–190 days at 20 °C, with nearly 10‐fold extension of both median and maximum adult lifespan relative to N2DRM, a long‐lived wild‐type stock into which the null mutant was outcrossed. PI3K‐null adults, although a little less thermotolerant, are considerably more resistant to oxidative and electrophilic stresses than worms bearing normal or less long‐lived alleles. Their unprecedented factorial gains in survival, under both normal and toxic environments, are attributed to elimination of residual and maternally contributed PI3KCS or its products, and consequent modification of kinase signaling cascades.

Chromosomal Mapping of Osteopenia-Associated Quantitative Trait Loci Using Closely Related Mouse Strains

Peak bone mineral density (BMD) is a highly heritable trait in humans and is currently the best predictor of skeletal fragility underlying osteoporosis. The SAMP6 mouse strain displays unusually low BMD at maturity, and age‐dependent osteopenia associated with defective osteoblastogenesis. To identify quantitative trait loci (QTLs) influencing bone density, we constructed crosses between SAMP6 and either AKR/J or SAMP6, two related mouse strains of higher peak BMD. Due to common ancestry of these strains, intercross parents differed at only 39–40% of 227 highly‐polymorphic genotyping markers, thus restricting our search to this informative portion of the genome and reducing the number of mice required for QTL significance. Using dual energy X‐ray absorptiometry (DEXA), we measured spinal BMD in F2 cross progeny at 4 months of age, and selectively genotyped those in the highest and lowest quartiles for BMD. Based on linear regression of bone density on genotype, including Composite Interval Mapping to enhance mapping precision while adjusting for effects of distal markers, we identified multiple QTLs significantly affecting spinal BMD; these were mapped to regions of chromosomes 2 (two sites, one confirmed in both crosses), 7, 11, 13 and 16. One of these loci had been previously identified as a significant bone‐density QTL, while 3 substantiate QTLs suggested by a low‐power study of 24 recombinant‐inbred mouse lines. Such recurrent appearance of QTLs, especially in crosses involving distantly‐related strains, implies that polymorphism at these loci may be favored by evolution and might underlie variation in peak bone density among humans.

Assessing the clinical significance of botanical supplementation on human cytochrome P450 3A activity: Comparison of a milk thistle and black cohosh product to rifampin and clarithromycin

Phytochemical‐mediated modulation of cytochrome P450 enzymes (CYPs) may underlie many herb‐drug interactions. This studys purpose was to assess the effects of milk thistle and black cohosh supplementation on CYP3A activity and compare them to a clinically recognized inducer, rifampin, and inhibitor, clarithromycin. Healthy volunteers were randomly assigned to receive a standardized milk thistle (900 mg) or black cohosh (80 mg) supplement for 14 days. Subjects also received rifampin (600 mg) and clarithromycin (1000 mg) for 7 days as positive controls for CYP3A induction and inhibition, respectively. Midazolam was administered orally before and after each supplementation and control period. The effects of milk thistle, black cohosh, rifampin, and clarithromycin on midazolam pharmacokinetics were determined using noncompartmental techniques. Unlike those observed for rifampin and clarithromycin, midazolam pharmacokinetics was unaffected by milk thistle or black cohosh. Milk thistle and black cohosh appear to have no clinically relevant effect on CYP3A activity in vivo.

An iterative block-shifting approach to retention time alignment that preserves the shape and area of gas chromatography-mass spectrometry peaks

BackgroundMetabolomics, petroleum and biodiesel chemistry, biomarker discovery, and other fields which rely on high-resolution profiling of complex chemical mixtures generate datasets which contain millions of detector intensity readings, each uniquely addressed along dimensions of time (e.g., retention time of chemicals on a chromatographic column), a spectral value (e.g., mass-to-charge ratio of ions derived from chemicals), and the analytical run number. They also must rely on data preprocessing techniques. In particular, inter-run variance in the retention time of chemical species poses a significant hurdle that must be cleared before feature extraction, data reduction, and knowledge discovery can ensue. Alignment methods, for calibrating retention reportedly (and in our experience) can misalign matching chemicals, falsely align distinct ones, be unduly sensitive to chosen values of input parameters, and result in distortions of peak shape and area.ResultsWe present an iterative block-shifting approach for retention-time calibration that detects chromatographic features and qualifies them by retention time, spectrum, and the effect of their inclusion on the quality of alignment itself. Mass chromatograms are aligned pairwise to one selected as a reference. In tests using a 45-run GC-MS experiment, block-shifting reduced the absolute deviation of retention by greater than 30-fold. It compared favourably to COW and XCMS with respect to alignment, and was markedly superior in preservation of peak area.ConclusionIterative block-shifting is an attractive method to align GC-MS mass chromatograms that is also generalizable to other two-dimensional techniques such as HPLC-MS.

Mutagenic Effects of 4-Hydroxynonenal Triacetate, a Chemically Protected Form of the Lipid Peroxidation Product 4-Hydroxynonenal, as Assayed in L5178Y/Tk+/– Mouse Lymphoma Cells

The lipid peroxidation product 4-hydroxynon-2-enal (4-HNE) is cytotoxic and genotoxic at superphysiological concentrations. To characterize the mechanism of action of 4-HNE, we assessed genotoxic damage by 4-HNE and by 4-HNE triacetate [4-HNE(Ac)3] using the mouse lymphoma assay that measures the mutant frequency in the Tk gene. As a strong electrophile, 4-HNE reacts readily with nucleophilic centers on cellular components. When added extracellularly, it may react preferentially with proteins in culture medium or on the cell surface and not reach deeper cellular targets such as nuclear DNA. Therefore, 4-HNE(Ac)3, a protected form of 4-HNE that is metabolically converted to 4-HNE in cells (Neely MD, Amarnath V, Weitlauf C, and Montine TJ, Chem Res Toxicol 15:40–47, 2002), was assayed in addition to 4-HNE. When added in serum-containing medium, 4-HNE was not mutagenic in the mouse lymphoma assay up to 38 μM (cytotoxicity = 13%). In contrast, exposure to 4-HNE(Ac)3, which mimics intracellular formation of 4-HNE, resulted in dose-dependent induction of mutations. At 17 μM 4-HNE(Ac)3 (cytotoxicity = 33%), the mutant frequency was 719 × 10–6 (>7-fold higher than the spontaneous mutant frequency). Loss of heterozygosity analysis in the Tk mutants revealed that the majority of mutations induced by 4-HNE(Ac)3 resulted from clastogenic events affecting a large segment of the chromosome. The results indicate that, in the presence of serum that approximates physiological conditions, 4-HNE generated intracellularly but not extracellularly is a strong mutagen via a clastogenic action at concentrations that may occur during oxidative stress.

Development of high-throughput HILIC-MS/MS methodology for plasma citrulline determination in multiple species

Circulating citrulline originates almost exclusively from the small intestinal enterocytes in mammals and therefore is a potential biomarker of disease states affecting enterocyte mass including exposure to ionizing radiation. There is a need for a simple and rapid method for citrulline quantification in plasma. To achieve this goal, a high-throughput separation and tandem mass spectrometric detection strategy has been developed and validated in six different species. HILIC separation was achieved on a 1.7 μm fused-core Diol column using an acidic acetonitrile/water gradient. A surrogate analyte (citrulline stable isotope) was used to determine the lower-limit-of-quantitation, extraction recovery, and matrix ion effects. Mass spectrometric detection was achieved in the multiple reaction-monitoring mode using m/z 176 → 159, 177 → 160, and 181 → 164, for citrulline, citrulline+1, and citrulline+5, respectively. The retention time of citrulline and total chromatographic run time were 1.1 min and 2.5 min, respectively, while effectively eliminating matrix-ion effects and achieving baseline separation from the confounding amino acid arginine. Quantitation was precise (CV <4.3%), accurate (90–110%), and sensitive (lower-limit-of-quantitation; 0.125 μM) without interference from the confounding amino acid, arginine. The throughput of the method was enhanced by incorporation of a 96-well filter plate for final sample cleanup. The method was used to determine plasma citrulline in six different species and in mice treated with escalating doses of radiation. This simple, accurate and high-throughput (up to 200 samples/day) methodology provides the first quantitative assay to meet the growing demand for a rapid and simple citrulline assay with high sensitivity.

A Narrow Quantitative Trait Locus in C. elegans Coordinately Affects Longevity, Thermotolerance, and Resistance to Paraquat

By linkage mapping of quantitative trait loci, we previously identified at least 11 natural genetic variants that significantly modulate Caenorhabditis elegans life-span (LS), many of which would have eluded discovery by knock-down or mutation screens. A region on chromosome IV between markers stP13 and stP35 had striking effects on longevity in three inter-strain crosses (each P < 10−9). In order to define the limits of that interval, we have now constructed two independent lines by marker-based selection during 20 backcross generations, isolating the stP13–stP35 interval from strain Bergerac-BO in a CL2a background. These congenic lines differed significantly from CL2a in LS, assayed in two environments (each P < 0.001). We then screened for exchange of flanking markers to isolate recombinants that partition this region, because fine-mapping the boundaries for overlapping heteroallelic spans can greatly narrow the implicated interval. Recombinants carrying the CL2a allele at stP35 were consistently long-lived compared to those retaining the Bergerac-BO allele (P < 0.001), and more resistant to temperature elevation and paraquat (each ∼1.7-fold, P < 0.0001), but gained little protection from ultraviolet or peroxide stresses. Two rounds of recombinant screening, followed by fine-mapping of break-points and survival testing, narrowed the interval to 0.18 Mb (13.35–13.53 Mb) containing 26 putative genes and six small-nuclear RNAs – a manageable number of targets for functional assessment.

Ammonia, respiration, and longevity in nematodes: insights on metabolic regulation of life span from temporal rescaling.

To better understand metabolic correlates of longevity, we used a graphical technique to compare the adult temporal patterns of several markers of metabolic activity — ammonia elimination, oxygen consumption rate, ATP levels, and (in freeze-permeabilized worms) the rate of NADPH-activated, lucigenin-mediated superoxide formation — as observed by us and others in normal and long-lived mutant Caenorhabditis elegans strains. All of these traits declined with time, and when their logarithms were plotted against time, appeared reasonably linear for most of the duration of the experiments. The profiles for ammonia output conformed well to parallel regression lines; those for the other metabolic parameters varied widely in slope as originally plotted by the authors, but much less so when replotted as logarithms against adjusted time, scaled by the reciprocal of strain longevity. This is consistent with coregulation of life span, respiration rate, ATP levels, lucigenin reactivity, but not ammonia excretion, by a physiological clock distinguishable from chronologic time. Plots, scaled appropriately for equalized slopes, highlighted y-axis intercept differences among strains. On rescaled plots, these constitute deviations from the expectation based on ’strain-specific clock’ differences alone. With one exception, y-intercept effects were observed only for mutants in an insulin-like signaling pathway.

Application of Gas Chromatography-Tandem Mass Spectrometry (GC/MS/MS) for the Analysis of Deuterium Enrichment of Water

Incorporation of deuterium from deuterium oxide ((2) H2 O) into biological components is a commonly used approach in metabolic studies. Determining the dilution of deuterium in the body water (BW) pool can be used to estimate body composition. We describe three sensitive GC/MS/MS methods to measure water enrichment in BW. Samples were reacted with NaOH and U-(13) C3 -acetone in an autosampler vial to promote deuterium exchange with U-(13) C3 -acetone hydrogens. Headspace injections were made of U-(13) C3 -acetone-saturated air onto a 30-m DB-1MS column in electron impact-mode. Subjects ingested 30 ml (2) H2 O, and plasma samples were collected. BW was determined by standard equation. Dual-energy X-ray absorptiometry scans were performed to calculate body mass, body volume and bone mineral content. A four-compartmental model was used to estimate body composition (fat and fat free mass). Full-scan experiments generated an m/z 45 peak and to a lesser extent an m/z 61 peak. Product fragment ions further monitored included 45 and 46 using selected ion monitoring (Method1), the 61 > 45 and 62 > 46 transition using multiple reaction monitoring (MRM; Method2) and the neutral loss, 62 > 45, transition (Method3). MRM methods were optimized for collision energy (CE) and collision-induced dissociation (CID) argon gas pressure with 6 eV CE and 1.5 mTorr CID gas being optimal. Method2 was used for final determination of (2) H2 O enrichment of subjects because of lower natural background. We have developed a sensitive method to determine (2) H2 O enrichment in BW to enable measurement of FM and FFM.