Deuterium-labeled Internal Standards Research Articles

Enhanced determination of abscisic acid (ABA) and abscisic acid glucose ester (ABA-GE) in Cistus albidus plants by liquid chromatography–mass spectrometry in tandem mode

An improved, quick and simple method for the extraction and quantification of the phytohormones (+)-abscisic acid (ABA) and its major glucose conjugate, abscisic acid glucose ester (ABA-GE) in plant samples is described. The method includes the addition of deuterium-labeled internal standards to the leaves at the beginning of the extraction for quantification, a simple extraction/centrifugation process and the injection into the liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS-MS) system in multiple reaction monitoring mode (MRM). Quality parameters of the method (detection limits, repeatability, reproducibility and linearity) have been studied. The objective of this work is to show the applicability of this method for quantifying the endogenous content of both ABA and ABA-GE in Cistus albidus plants that have been grown during an annual cycle under Mediterranean field conditions. Leaf samples from winter plants have low levels of ABA which increase in spring and summer showing two peaks that corresponded to April and August. These increases are coincident with the high temperature and solar radiation and the low RWC and RH registered along the year. On the other hand, the endogenous levels of ABA-GE increase until maximum values in July just before the ABA content reaches its highest concentration, decreasing in August and during autumn and winter. Our results suggest that the method is useful for quantifying both compounds in this plant material and represents the advantage of a short-time sample preparation with a high accuracy and viability.

Determination of opiates and cocaine in urine by high pH mobile phase reversed phase UPLC–MS/MS

A fast and selective ultra performance liquid chromatography-tandem mass spectrometry (UPLC–MS/MS) method for the determination of opiates (morphine, codeine, 6-monoacetylmorphine (6-MAM), pholcodine, oxycodone, ethylmorphine), cocaine and benzoylecgonine in urine has been developed and validated. Sample preparation was performed by solid phase extraction (SPE) on a mixed mode cation exchange (MCX) cartridge. For optimized chromatographic performance with repeatable retention times, narrow and symmetrical peaks, and focusing of all analytes at the column inlet at gradient start, a basic mobile phase consisting of 5 mM ammonium bicarbonate, pH 10.2, and methanol (MeOH) was chosen. Positive electrospray ionization (ESI +) MS/MS detection was performed with a minimum of two multiple reaction monitoring (MRM) transitions for each analyte. Deuterium labelled-internal standards were used for six of the analytes. Between-assay retention time repeatabilities ( n = 10 series, 225 injections in total) had relative standard deviation (RSD) values within 0.1–0.6%. Limit of detection (LOD) and limit of quantification (LOQ) values were in the range 0.003–0.05 μM (0.001–0.02 μg/mL) and 0.01–0.16 μM (0.003–0.06 μg/mL), respectively. The RSD values of the between-assay repeatabilities of concentrations were ≤10% at five concentration levels for all analytes, except for pholcodine. Specificity was investigated by determination of the retention times of 96 drugs and internal standards in total. Co-eluting compounds were in all cases separated by the MS/MS detection. No or only minor matrix effects were observed. Total run time, including injection and equilibration time was 5.7 min. The method has been routinely used at the Norwegian Institute of Public Health (NIPH) since August 2007 for qualitative detection of opiates, cocaine and benzoylecgonine in more than 2000 urine samples with two replicates of each sample.

Confirmatory method for the determination of resorcylic acid lactones in urine sample using immunoaffinity cleanup and liquid chromatography–tandem mass spectrometry

The presence of zeranol (α-zearalanol) in urine samples due to natural contamination or illegal treatment is under debate within the European Union. The simultaneous determination of zeranol, its epimer taleranol (β-zearalanol), zearalanone and the structurally related mycotoxin zearalenone with the corresponding α- and β-zearalenol metabolites appears to be critical in deciding whether an illegal use has occurred. The aim of this study is to develop and validate a simple analytical procedure applicable to bovine and swine urine samples for the determination of all six resorcylic acid lactones. After an enzymatic deconjugation, the urine was subjected to a one-step cleanup on a commercially available immunoaffinity chromatography cartridge. The analytes were detected by liquid chromatography–negative-ion electrospray tandem mass spectrometry using deuterium-labelled internal standards. The method was validated as a quantitative confirmatory method according to European Commission Decision 2002/657/EC. The evaluated parameters were: linearity, specificity, precision (repeatability and intra-laboratory reproducibility), recovery, decision limit, detection capability and ruggedness. The decision limits (CCα) obtained, were between 0.56 and 0.68 μg L −1; recovery above 66% for all the analytes. Repeatability was between 1.4 and 5.3% and within-laboratory reproducibility between 1.9 and 16.1% for the six resorcylic acid lactones.

Direct Injection Mass Spectrometric Confirmation of Multiple Drugs in Overdose Cases from Postmortem Blood Using Electrospray Ionization-Tandem Mass Spectrometry and MS3

Five cases of confirmed multiple-drug overdose were previously screened and quantified by the West Virginia Office of the Chief Medical Examiner; 26 different drugs and metabolites were identified and quantified in blood at > or = 10 ng/mL. In this study, whole blood from those five case samples was analyzed by a direct injection multi-stage mass spectrometric (MSn) method to confirm the identity of 26 analytes at or above 10 ng/mL using 16 different deuterium-labeled internal standards. Samples were spiked with internal standards, precipitated with acetonitrile, and centrifuged. Samples were further diluted with either 0.1% formic acid or 0.1% ammonium hydroxide in methanol prior to injection into an electrospray ionization ion trap mass spectrometer (MS). Ions were monitored as MS-MS or MS3 product ions. In all cases, analysis by MS-MS confirmed the presence of the drugs and metabolites when the internal standards were detected. Detection of characteristic MS3 ions was used for further confirmation of the presence of parent drugs in all but three instances. Total analysis time was less than 1 h. Although only useful for qualitative or confirmatory purposes, this direct injection MSn method provides a simple and rapid confirmation of multiple drugs that have been previously identified and quantified by gas chromatographic-MS or liquid chromatographic-MS analytical methods.

Screening method for petroleum-derived aromatic hydrocarbons in wine

A novel screening method was developed using gas chromatography-mass spectrometry (GC-MS) combined with headspace-solid phase microextraction (SPME) for the detection of various aromatic hydrocarbons in wine. Petroleum-derived products such as toluene, styrene, alkylbenzenes and alkylnaphthalenes were targeted for screening. They were detected by monitoring their characteristic ions (selected ion monitoring) and subsequently quantified using selected deuterium-labelled internal standards. The individual compounds were detected at levels as low as 1 μg/L in red or white wine. The method developed was rapid, simple, solvent-free and sensitive. Application of the method to a tainted red wine confirmed the presence and allowed determination of the level of taint hydrocarbons and agreed with the sensory evaluation (kerosene-like).

Rapid measurement of estrogens and their metabolites in human serum by liquid chromatography-tandem mass spectrometry without derivatization

Objectives The steroids estradiol (E2), estrone (E1), and estriol (E3) are the major estrogens. E1/E2 and their metabolite 16-hydroxyestrone (16-OHE1, known to be carcinogenic) could be involved in the development of many cancers including human breast cancer. The aim of the current study was to develop a rapid and simple high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) assay to simultaneously measure E1, E2, E3 and 16-OHE1 in human serum without the need for solid phase extraction or derivatization. Methods An API-5000 triple-quadrupole mass spectrometer coupled with electrospray ionization (ESI) source and Shimadzu HPLC system was used employing isotope dilution with deuterium-labeled internal standard (IS) for each analyte. Quantitation by multiple reaction monitoring (MRM) analysis was performed in negative ion mode. Results The limits of detection were 1.0 pg/mL for E1 and 16-OHE1 and 2.0 pg/mL for E2 and E3. Within-day CVs were < 6.5% for all analytes tested and between-day CVs ranged from 4.5% to 9.5%. Recovery ranged from 88% to 108%. Conclusion This method allows for the simultaneous measurement of four estrogens in human serum within 8 min. It can be routinely employed in a clinical environment and is attractive because of its simplicity in sample processing, micro sample requirement, and high throughput.

Association between Tortilla Consumption and Human Urinary Fumonisin B1 Levels in a Mexican Population

Fumonisins are mycotoxins produced by Fusarium spp. and commonly contaminate maize and maize products worldwide. Fumonisins are rodent carcinogens and have been associated with human esophageal cancer. However, the lack of a valid exposure biomarker has hindered both the assessment of human exposure and the evaluation of disease risk. A sensitive liquid chromatography-mass spectrometry method to measure urinary fumonisin B1 (FB1) following extraction on Oasis MAX cartridges was established and applied to urine samples from women in a cohort recruited in Morelos County, Mexico. Urinary FB1 was compared with dietary information on tortilla consumption. FB1 recovery in spiked samples averaged 94% as judged by deuterium-labeled FB1 internal standard. Urinary FB1 was determined in 75 samples from women selected based on low, medium, or high consumption of maize-based tortillas. The geometric mean (95% confidence interval) of urinary FB1 was 35.0 (18.8-65.2), 63.1 (36.8-108.2), and 147.4 (87.6-248.0) pg/mL and the frequency of samples above the detection limit (set at 20 pg FB1/mL urine) was 45%, 80%, and 96% for the low, medium, and high groups, respectively. Women with high intake had a 3-fold higher average FB1 levels compared with the "low intake" group (F = 7.3; P = 0.0015). Urinary FB1 was correlated with maize intake (P(trend) = 0.001); the correlation remained significant after adjusting for age, education, and place of residence. This study suggests that measurement of urinary FB1 is sufficiently sensitive for fumonisin exposure assessment in human populations and could be a valuable tool in investigating the associated health effects of exposure.

Driving under the influence of cannabis: a 10‐year study of age and gender differences in the concentrations of tetrahydrocannabinol in blood

Delta(9)-Tetrahydrocannabinol (THC) is the major psychoactive constituent of cannabis and its various preparations. Increasing use of cannabis for recreational purposes has created a problem for road-traffic safety. This paper compares age, gender and the concentrations of THC in blood of individuals apprehended for driving under the influence of drugs (DUID) in Sweden, where a zero-tolerance law operates. Specimens of blood or urine were subjected to a broad screening analysis by enzyme immunoassay methods. THC positives were verified by analysis of blood by gas chromatography-mass spectrometry (GC-MS) with a deuterium-labelled internal standard (d(3)-THC). All toxicology results were entered into a database (TOXBASE) along with the age and gender of apprehended drivers. Over a 10-year period (1995-2004), between 18% and 30% of all DUID suspects had measurable amounts of THC in their blood (> 0.3 ng/ml) either alone or together with other drugs. The mean age [+/- standard deviation (SD)] of cannabis users was 33 +/- 9.4 years (range 15-66 years), with a strong predominance of men (94%, P < 0.001). The frequency distribution of THC concentrations (n = 8794) was skewed markedly to the right with mean, median and highest values of 2.1 ng/ml, 1.0 ng/ml and 67 ng/ml, respectively. The THC concentration was less than 1.0 ng/ml in 43% of cases and below 2.0 ng/ml in 61% of cases. The age of offenders was not correlated with the concentration of THC in blood (r = -0.027, P > 0.05). THC concentrations in blood were higher when this was the only psychoactive substance present (n = 1276); mean 3.6 ng/ml, median 2.0 ng/ml compared with multi-drug users; mean 1.8 ng/ml, median 1.0 ng/ml (P < 0.001). In cases with THC as the only drug present the concentration was less than 1.0 ng/ml in 26% and below 2.0 ng/ml in 41% of cases. The high prevalence of men, the average age and the concentrations of THC in blood were similar in users of illicit drugs (non-traffic cases). The concentration of THC in blood at the time of driving is probably a great deal higher than at the time of sampling (30-90 minutes later). The notion of enacting science-based concentration limits of THC in blood (e.g. 3-5 ng/ml), as discussed in some quarters, would result in many individuals evading prosecution. Zero-tolerance or limit of quantitation laws are a much more pragmatic way to enforce DUID legislation.

Assay of the Set of All Sudan Azodye (I, II, III, IV, and Para-Red) Contaminating Agents by Liquid Chromatography−Tandem Mass Spectrometry and Isotope Dilution Methodology

A high-throughput mass spectrometric method is presented for the simultaneous detection of Sudan I, II, III, IV, and Para-Red azodyes in foodstuff. The method is based on the use of deuterium-labeled internal standards and atmospheric pressure chemical ionization (APCI) in a triple-quadrupole instrument. The gas-phase breakdown pattern of each labeled and unlabeled analogue displays the naphthoic moiety as a common fragment. The search for the parents of this common species (parent ion scans) allows, by flow injection and in a single run, the evaluation of the presence of each polluting species spiked in typical foodstuffs. A detailed assay of each azodye was performed by LC-APCI and isotope dilution method, through the multiple reaction monitoring approach, using deuterium-labeled internal standards. Sudan dyes can be quantified above the threshold of 10 ppb except for Sudan Para-Red, for which the limit of quantification was 20 ppb, likely due to the different ionization efficiency.

Simultaneous Determination of Salsolinol Enantiomers and Dopamine in Human Plasma and Cerebrospinal Fluid by Chemical Derivatization Coupled to Chiral Liquid Chromatography/Electrospray Ionization-Tandem Mass Spectrometry

A sensitive, specific, and robust method to simultaneously determine enantiomeric salsolinol (1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline, SAL), a potential biomarker implicated in alcohol-related neurotoxicity in a stereoselective manner, and its precursor dopamine (DA) has been developed using simple chemical derivatization and chiral separation coupled with electrospray ionization-tandem mass spectrometry (ESI-MS/MS). SAL enantiomers and DA were converted to stable pentafluorobenzyl (PFB) derivatives directly from aqueous media. Bulky PFB groups introduced into the SAL structure enabled baseline separation of SAL stereoisomers on a chiral column without cumbersome chiral derivatization to unstable SAL diastereomers. Subsequent analysis by ESI-MS/MS with multiple reaction monitoring (MRM) in the presence of deuterium-labeled internal standards allowed specific detection of both derivatives with a wide dynamic range (SAL, 0.5-5000 pg; DA, 0.02-20 ng). The limit of quantitation assayed in the plasma matrix was below 10 pg for each SAL enantiomer and 100 pg for DA. Both coefficient of variance and error for inter- and intraday measurements in the blank plasma were less than 10% for SAL and DA in the concentration range of 10-4000 pg/mL and 0.1-8 ng/mL, respectively. This strategy enabled routine and specific determination of both SAL enantiomers and DA from 0.5 mL of human plasma and cerebrospinal fluid, which has not been possible using existing methodologies.

Direct-Injection Mass Spectrometric Method for the Rapid Identification of Fentanyl and Norfentanyl in Postmortem Urine of Six Drug-Overdose Cases

A rapid mass spectrometric method was developed for the identification of fentanyl and its major hepatic metabolite norfentanyl in postmortem urine of six drug-overdose victims involving fentanyl use. To reduce matrix effects or ion suppression, sample preparation consisted of centrifugation and solid-phase extraction. Deuterium-labeled internal standards ((2)H(5)-fentanyl and (2)H(5)-norfentanyl) were used to compensate for instrument variation in signal, analyte recovery during sample preparation, and ion suppression. Structural information for fentanyl and norfentanyl were collected using mass spectrometry (MS) with electrospray ionization (ESI) operated in the positive ion mode. Fentanyl (m/z 337) was found in each of the six overdose cases by the appearance of the MS-MS daughter ion on both an ion trap and a triple-quadrupole MS resulting from the fragmentation pathway of fentanyl (m/z 337 --> 188). Norfentanyl was detected in all six cases by the appearance of the MH(+) ion, m/z 233, with a single-quadrupole MS and confirmed in an ion trap MS. Ion suppression, as determined by the comparison of ion intensities from spiked samples in water with postmortem urine from the cases, ranged from 18% to 98% in three ESI sources. The use of stable isotope-labeled internal standards obviates sample preparation because ratios of analyte/internal standard remain constant in the presence of extensive matrix effects. This MS method provided sufficient sensitivity and selectivity for the rapid identification of fentanyl and norfentanyl in urine at levels >/= 10 ng/mL without prior analyte resolution by chromatography and with a total analysis time of less than 1 h.

Application of solid-phase extraction coupled with freezing-lipid filtration clean-up for the determination of endocrine-disrupting phenols in fish

An analytical method has been developed for the determination of endocrine-disrupting phenols (eight alkylphenols and bisphenol A) in fish samples. The extraction of nine phenols from fish samples was carried out by ultrasonification. After the extraction, high levels of lipids were removed by freezing-lipid filtration instead of the traditional methods of column chromatography or saponification. During freezing-lipid filtration, about 90% of the lipids were eliminated without any significant loss of phenolic compounds. For further purification, hydrophilic–lipophilic balanced copolymer (HLB) sorbent with a poly(divinylbenzene-co- N-vinylpyrrolidone) phase and Florisil-solid-phase extraction (SPE) cartridges were used to eliminate the remaining interferences. Silyl-derivatization, with N, N′-methyl-( tert-butyldimethylsilyl) trifluoroacetamide (MTBSTFA), was applied to enhance the sensitivity of detection of phenolic compounds. Quantification was performed by gas chromatography/mass spectrometry (GC/MS)-selected ion monitoring (SIM) mode, using deuterium-labeled internal standards. Spiking experiments were carried out to determine the recovery, precision and detection limit of the method. The overall recoveries ranged between 70 and 120%, with relative standard deviations of 3–17% for the entire procedure. The detection limits of the method for the nine phenols ranged from 0.02 to 0.41 ng g −1. The method provided simultaneous screening and accurate confirmation of each phenol when applied to biological samples.

Detection of the abundance of diacylglycerol and triacylglycerol molecular species in cells using neutral loss mass spectrometry

Triacylglycerols (TAGs) are neutral lipids present in all mammalian cells as energy reserves, and diacylglycerols (DAGs) are present as intermediates in phospholipid biosynthesis and as signaling molecules. The molecular species of TAGs and DAGs present in mammalian cells are quite complex, and previous investigations revealed multiple isobaric species having molecular weights at virtually every even mass between 600 and 900 Da, making it difficult to assess changes of individual molecular species after cell activation. A method has been developed, using tandem MS and neutral loss scanning, to quantitatively analyze changes in those glyceryl ester molecular species containing identical fatty acyl groups. This was carried out by neutral loss scanning of 18 common fatty acyl groups where the neutral loss corresponded to the free carboxylic acid plus NH 3. Deuterium-labeled internal standards were used to normalize the signal for each nominal [M + NH 4] + ion undergoing this neutral loss reaction. This method was applied in studies of TAGs in RAW 264.7 cells treated with the toll-like receptor 4 ligand Kdo 2–lipid A. A 50:1-TAG containing 18:1 was found to increase significantly over a 24-h time course after Kdo 2–lipid A exposure, whereas an isobaric 50:1-TAG containing 16:1 did not change relative to controls.

Effect of residues remaining in the injection liner of a gas chromatograph on the quantification of polycyclic aromatic hydrocarbons by isotope dilution mass spectrometry using deuterium-labeled internal standards

We have investigated the differences in response ratios of native polycyclic aromatic hydrocarbons (native PAHs)/ 13C-labeled PAHs ( 13C-PAHs) and of native PAHs/deuterium-labeled PAHs (PAHs- d) in a calibration solution containing at trace concentrations (<1 μg mL −1) by gas chromatography–mass spectrometry. Only the ratios of native PAHs/PAHs- d decreased significantly ( p < 0.05) accompanied with increase of residue amounts originated from immediately before injected solution. Since the analytical results using PAHs- d as internal standards were significantly lower than those using 13C-PAHs whatever solutions were injected immediately before ( p < 0.05, 1.9–13.2%), 13C-PAHs gave more accurate results.

Kdo2-Lipid A of Escherichia coli, a defined endotoxin that activates macrophages via TLR-4

The LIPID MAPS Consortium (www.lipidmaps.org) is developing comprehensive procedures for identifying all lipids of the macrophage, following activation by endotoxin. The goal is to quantify temporal and spatial changes in lipids that occur with cellular metabolism and to develop bioinformatic approaches that establish dynamic lipid networks. To achieve these aims, an endotoxin of the highest possible analytical specification is crucial. We now report a large-scale preparation of 3-deoxy-D-manno-octulosonic acid (Kdo)(2)-Lipid A, a nearly homogeneous Re lipopolysaccharide (LPS) sub-structure with endotoxin activity equal to LPS. Kdo(2)-Lipid A was extracted from 2 kg cell paste of a heptose-deficient Escherichia coli mutant. It was purified by chromatography on silica, DEAE-cellulose, and C18 reverse-phase resin. Structure and purity were evaluated by electrospray ionization/mass spectrometry, liquid chromatography/mass spectrometry and (1)H-NMR. Its bioactivity was compared with LPS in RAW 264.7 cells and bone marrow macrophages from wild-type and toll-like receptor 4 (TLR-4)-deficient mice. Cytokine and eicosanoid production, in conjunction with gene expression profiling, were employed as readouts. Kdo(2)-Lipid A is comparable to LPS by these criteria. Its activity is reduced by >10(3) in cells from TLR-4-deficient mice. The purity of Kdo(2)-Lipid A should facilitate structural analysis of complexes with receptors like TLR-4/MD2.

Effect of Arachidonic Acid Reacylation on Leukotriene Biosynthesis in Human Neutrophils Stimulated with Granulocyte-macrophage Colony-stimulating Factor and Formyl-methionyl-leucyl-phenylalanine

Priming of human neutrophils with granulocyte-macrophage colony-stimulating factor (GM-CSF) followed by treatment with formyl-methionyl-leucyl-phenylalanine (fMLP) stimulates cells in a physiologically relevant manner with modest 5-lipoxygenase activation and formation of leukotrienes. However, pretreatment of neutrophils with thimerosal, an organomercury thiosalicylic acid derivative, led to a dramatic increase (>50-fold) in the production of leukotriene B(4) and 5-hydroxyeicosatetraenoic acid, significantly higher than that observed after stimulation with calcium ionophore A23187. Little or no effect was observed with thimerosal alone or in combination with either GM-CSF or fMLP. Elevation of [Ca(2+)](i) induced by thimerosal in neutrophils stimulated with GM-CSF/fMLP was similar but more sustained compared with samples where thimerosal was absent. However, [Ca(2+)](i) was significantly lower compared with calcium ionophore-treated cells, suggesting that a sustained calcium rise was necessary but not sufficient to explain the effects of this compound on the GM-CSF/fMLP-stimulated neutrophil. Thimerosal was found to directly inhibit neutrophil lysophospholipid:acyl-CoA acyltransferase activity at the doses that stimulate leukotriene production, and analysis of lysates from neutrophil preparations stimulated in the presence of thimerosal showed a marked increase in free arachidonic acid, supporting the inhibition of the reincorporation of this fatty acid into the membrane phospholipids as a mechanism of action for this compound. The dramatic increase in production of leukotrienes by neutrophils when a physiological stimulus such as GM-CSF/fMLP is employed in the presence of thimerosal suggests a critical regulatory role of arachidonate reacylation that limits leukotriene biosynthesis in concert with 5-lipoxygenase and cytosolic phospholipase A(2)alpha activation.

Changes of Xanthoxin Levels during Fruit Development in Sweet Cherries

Xanthoxin (XAN) in the pulp and seeds of sweet cherry fruit (Prunus avium L.) was identified and quantified by using deuterium-labeled internal standard. The high concentration of total XAN in the pulp was observed in the early stage of fruit development which subsequently declined and remained at a low level until the initial stage of fruit maturation but increased toward harvest. Total XAN concentrations in seeds were high in the immature seed and decreased significantly with seed maturation. Total XAN levels in the immature seeds were approximately 10-times higher than the levels in fruit pulp. This study is the first elucidation of XAN in a non-climacteric fruit.

Regulation of bile acid biosynthesis by hepatocyte nuclear factor 4α

Hepatocyte nuclear factor 4alpha (HNF4alpha) regulates many genes that are preferentially expressed in liver. Mice lacking hepatic expression of HNF4alpha (HNF4alphaDeltaL) exhibited markedly increased levels of serum bile acids (BAs) compared with HNF4alpha-floxed (HNF4alphaF/F) mice. The expression of genes involved in the hydroxylation and side chain beta-oxidation of cholesterol, including oxysterol 7alpha-hydroxylase, sterol 12alpha-hydroxylase (CYP8B1), and sterol carrier protein x, was markedly decreased in HNF4alphaDeltaL mice. Cholesterol 7alpha-hydroxylase mRNA and protein were diminished only during the dark cycle in HNF4alphaDeltaL mice, whereas expression in the light cycle was not different between HNF4alphaDeltaL and HNF4alphaF/F mice. Because CYP8B1 expression was reduced in HNF4alphaDeltaL mice, it was studied in more detail. In agreement with the mRNA levels, CYP8B1 enzyme activity was absent in HNF4alphaDeltaL mice. An HNF4alpha binding site was found in the mouse Cyp8b1 promoter that was able to direct HNF4alpha-dependent transcription. Surprisingly, cholic acid-derived BAs, produced as a result of CYP8B1 activity, were still observed in the serum and gallbladder of these mice. These studies reveal that HNF4alpha plays a central role in BA homeostasis by regulation of genes involved in BA biosynthesis, including hydroxylation and side chain beta-oxidation of cholesterol in vivo.

Effects of Drought Stress on Abscisic Acid and Jasmonate Metabolism in Citrus

Drought stress was applied to ‘Shiranui’ trees [ (Citrus unshiu Marc. × Citrus unshiu Osbeck) × Citrus reticulata Blanco], and xanthoxin, abscisic acid (ABA), phaseic acid (PA), dihydrophaseic acid (DPA), epi-dihydrophaseic acid (epi-DPA), and jasmonates were monitored in fruit and leaf. They were quantified using the respective deuterium-labeled internal standards. Xanthoxin, ABA, and DPA concentrations in the pulp of the drought-stressed trees were found to be higher than those of the well-watered trees throughout development. Xanthoxin concentrations, however, were lower than those of ABA. These results may imply a rapid turnover from xanthoxin to ABA. In addition, ABA concentrations in the leaf were higher in drought-stressed trees than the well-watered trees, although PA and DPA levels between the drought-stressed and well-watered trees were not clear. DPA was found to be the primary metabolite in the pulp, skin, and leaf. Both jasmonic acid (JA) and ABA concentrations in the pulp, skin, and leaf showed high values in the drought-stressed trees. However, JA levels in the pulp decreased as the harvest approached. Sucrose concentrations in the pulp of the drought-stressed trees were higher than those of the well-watered trees. ABA and jasmonate applications induced stomatal closure in leaf The effect of ABA on stomatal closure was stronger than that of jasmonate. We discuss herein the relationship between ABA, jasmonates, and drought stress.

Relation between bloodand urine-amphetamine concentrations in impaired drivers as influenced by urinary pH and creatinine

Amphetamine undergoes extensive renal excretion and significant amounts are present in urine as the unchanged parent drug. This prompted us to investigate whether a quantitative relationship existed between blood and urine concentrations of amphetamine in the body fluids of drug-impaired drivers apprehended in Sweden, where this stimulant is the major drug of abuse. The relationship between blood and urine concentrations of amphetamine was determined by multivariate analysis with urinary pH and creatinine as predictor variables. Amphetamine was determined in blood and urine by gas chromatography-mass spectrometry with deuterium-labelled internal standards. The concentration of amphetamine in urine was about 200 times greater than the concentration in blood; the mean and median urine/blood ratios were 214 and 160, respectively, with large individual variations. The Pearson correlation coefficient between urine (y) and blood (x) amphetamine was r = 0.53, n = 48, which was statistically highly significant (P < 0.001), although the residual standard deviation (SD) was large (+/- 181 mg/L). The correlation coefficient increased (r = 0.60) when the concentration of amphetamine in urine was normalized for dilution by dividing with the creatinine content. When urinary pH and creatinine were both included as predictor variables, the correlation coefficient was even higher (r = 0.69), now explaining 48% (r2 = 0.48) of the variation in urine-amphetamine concentration. However, the partial regression coefficient for creatinine (53 +/- 28.7) was not statistically significant (t = 1.85, P > 0.05), whereas the corresponding regression coefficient for pH was highly significant and had a negative sign (-102 +/- 32.6, t= -3.12, P < 0.005). Other factors could impact on the urine-blood amphetamine relationship, such as route of administration, pattern of voiding and time elapsed after use of the drug.