Stable Isotope Dilution Method Research Articles

Improved Quantification of Geosmin and 2-Methylisoborneol in Farmed Fish Using Stable Isotope Dilution Gas Chromatography-Mass Spectrometry.

A requisite to improving the taste and odor attributes of farmed fish is the availability of accurate and practical analytical methods to quantify 2-methylisoborneol (MIB) and geosmin (GSM). Solid-phase microextraction (SPME) enables reliable measurement of nanogram per liter quantities of MIB and GSM in water. In contrast, direct headspace (HS)-SPME of biological matrices with variable proximate compositions can increase bias and uncertainty in off-flavor determinations. Analytical recovery plays a crucial role in the accurate determination of MIB and GSM in fish, and this study investigates strategies to maximize and account for this recovery factor. MIB and GSM values in off-flavor catfish and trout were measured using direct HS-SPME and distillation as sample preparation techniques. Trout samples prepared by distillation yielded 10-fold higher GSM recoveries than those from direct HS-SPME (31% versus 3%). A stable isotope dilution method (SIDM) was implemented by routinely spiking samples with known quantities of deuterium-labeled MIB and GSM, allowing for the correction of sample-to-sample recovery deviations. SIDM-determined GSM values generated recoveries of 106 and 95% for direct HS-SPME and distilled trout, respectively. Aspects of the strategies and techniques presented can be incorporated into existing analytical methods to improve the accuracy and sample throughput. Particularly, routine inclusion of SIDM in the evaluation of MIB and GSM can facilitate identification of reliable practices to control off-flavors in aquaculture.

Comparison of Bioelectrical Impedance Indices for Skeletal Muscle Mass and Intracellular Water Measurements of Physically Active Young Men and Athletes

BackgroundBioelectrical impedance analysis (BIA) is a minimally invasive, safe, easy, and quick technology used to determine body composition. ObjectivesWe compared the relationship among impedance indices obtained using single-frequency BIA, multi-frequency BIA, bioelectrical impedance spectroscopy (BIS), and skeletal muscle mass (SMM) of physically active young men and athletes using the creatine (methyl-d3) dilution method. We also compared the SMM and intracellular water (ICW) of athletes and active young men measured using a reference stable isotope dilution and BIS method, respectively. MethodsWe analyzed data from 28 men (mean age, 20 ± 2 y) who exercised regularly. Single-frequency BIA at 5 kHz and 50 kHz (R5 and R50), multi-frequency BIA (R250–5), and BIS (RICW) methods of determining the SMM were compared. The deuterium and sodium bromide dilution methods of obtaining the total body water, ICW, and extracellular water measurements were also used, and the results were compared to those acquired using bioimpedance methods. ResultsThe correlation coefficients between SMM and L2/R5, L2/R50, L2/R250–5, and L2/RICW were 0.738, 0.762, 0.790, and 0.790, respectively (P < 0.01). The correlation coefficients between ICW and L2/R5, L2/R50, L2/R250–5, and L2/RICW were 0.660, 0.687, 0.758, and 0.730, respectively (P < 0.001). However, the correlation coefficients of L2/R50, L2/R250–5, and L2/RICW for SMM and ICW were not significantly different. ConclusionsOur findings suggest that single-frequency BIA at L2/R50, multi-frequency BIA, and BIS are valid for assessing the SMM of athletes and active young men. Additionally, we confirmed that the SMM and ICW were correlated with single-frequency BIA, multi-frequency BIA, and BIS. Bioimpedance technologies may be dependable and practical means for assessing SMM and hydration compartment status of active young adult males; however, cross-validation is needed.

Estimation of skeletal muscle mass in 4-year-old children using the D3-creatine dilution method

BackgroundGiven limited experience in applying the creatine-(methyl-D3) (D3Cr) dilution method to measure skeletal muscle mass (SMM) in young children, the feasibility of deployment in a fielding setting and performance of the method was assessed in a cohort of 4-year-old children in Dhaka, Bangladesh.MethodsFollowing D3Cr oral dose (10 mg) administration, single fasting urine samples were collected at 2–4 days (n = 100). Twenty-four-hour post-dose collections and serial spot urine samples on days 2, 3 and 4 were obtained in a subset of participants (n = 10). Urinary creatine, creatinine, D3Cr and D3-creatinine enrichment were analyzed by liquid chromatography–tandem mass spectrometry. Appendicular lean mass (ALM) was measured by dual-energy x-ray absorptiometry and grip strength was measured by a hand-held dynamometer.ResultsSMM was measured successfully in 91% of participants, and there were no adverse events. Mean ± SD SMM was greater than ALM (4.5 ± 0.4 and 3.2 ± 0.6 kg, respectively). Precision of SMM was low (intraclass correlation = 0.20; 95% CI: 0.02, 0.75; n = 10). Grip strength was not associated with SMM in multivariable analysis (0.004 kg per 100 g of SMM; 95% CI: −0.031, 0.038; n = 91).ConclusionsThe D3Cr dilution method was feasible in a community setting. However, high within-child variability in SMM estimates suggests the need for further optimization of this approach.ImpactThe D3-creatine (D3Cr) stable isotope dilution method was considered a feasible method for the estimation of skeletal muscle mass (SMM) in young children in a community setting and was well accepted among participants.SMM was weakly associated with both dual-energy x-ray absorptiometry-derived values of appendicular lean mass and grip strength.High within-child variability in estimated values of SMM suggests that further optimization of the D3Cr stable isotope dilution method is required prior to implementation in community research settings.

Metabolic dysfunction in obesity is related to impaired suppression of fatty acid release from adipose tissue by insulin.

The aims of this study were: 1) to assess relationships among insulin-mediated glucose uptake with standard clinical outcomes and deep-phenotyping measures (including fatty acid [FA] rate of appearance [FA Ra] into the systemic circulation); and 2) to examine the contribution of adipocyte size, fibrosis, and proteomic profile to FA Ra regulation. A total of 66 adults with obesity (BMI=34 [SD 3] kg/m2 ) were assessed for insulin sensitivity (hyperinsulinemic-euglycemic clamp), and stable isotope dilution methods quantified glucose, FA, and glycerol kinetics in vivo. Abdominal subcutaneous adipose tissue (aSAT) and skeletal muscle biopsies were collected, and magnetic resonance imaging quantified liver and visceral fat content. Insulin-mediated FA Ra suppression associated with insulin-mediated glucose uptake (r=0.51; p < 0.01) and negatively correlated with liver (r=-0.36; p < 0.01) and visceral fat (r=-0.42; p < 0.01). aSAT proteomics from subcohorts of participants with low FA Ra suppression (n=8) versus high FA Ra suppression (n=8) demonstrated greater extracellular matrix collagen protein in low versus high FA Ra suppression. Skeletal muscle lipidomics (n=18) revealed inverse correlations of FA Ra suppression with acyl-chain length of acylcarnitine (r=-0.42; p=0.02) and triacylglycerol (r=-0.51; p < 0.01), in addition to insulin-mediated glucose uptake (acylcarnitine: r=-0.49; p < 0.01, triacylglycerol: r=-0.40; p < 0.01). Insulin's ability to suppress FA release from aSAT in obesity is related to enhanced insulin-mediated glucose uptake and metabolic health in peripheral tissues.

Quantitative Determination of 2-Oxo-Imidazole-Containing Dipeptides by High-Performance Liquid Chromatography/Tandem Mass Spectrometry.

2-Oxo-imidazole-containing dipeptides (2-oxo-IDPs), novel imidazole-containing dipeptide (IDP) derivatives, exhibit a much higher antioxidant capacity than that of IDPs. However, quantitative methods have only been developed for IDPs, and methods for the quantitative analysis of 2-oxo-IDPs are needed. In this study, we developed methods for the quantitative analysis of 2-oxo-IDPs by high-performance liquid chromatography with online electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS) coupled with a stable isotope dilution method. First, we prepared stable isotope-labeled IDP and 2-oxo-IDP standards for MS analyses. Next, using these standards, we established highly sensitive, selective, and absolute quantitative analysis methods for five IDPs and five 2-oxo-IDPs by HPLC-ESI-MS/MS, achieving a limit of detection in the fmol range. Finally, we applied the method to various types of meat, such as beef, pork, chicken, and whale meat, demonstrating the detection of both IDPs and 2-oxo-IDPs. Furthermore, we provide the first evidence for the endogenous production of 2-oxo-balenine in meats. The methods developed in this study enable the detection of trace levels of 2-oxo-IDPs in biological samples and could be helpful for understanding the biological relevance of 2-oxo-IDPs.

Corticotropin-stimulated steroid profiles to predict shock development and mortality in sepsis: From the HYPRESS study

RationaleSteroid profiles in combination with a corticotropin stimulation test provide information about steroidogenesis and its functional reserves in critically ill patients.ObjectivesWe investigated whether steroid profiles before and after corticotropin stimulation can predict the risk of in-hospital death in sepsis.MethodsAn exploratory data analysis of a double blind, randomized trial in sepsis (HYPRESS [HYdrocortisone for PRevention of Septic Shock]) was performed. The trial included adult patients with sepsis who were not in shock and were randomly assigned to placebo or hydrocortisone treatment. Corticotropin tests were performed in patients prior to randomization and in healthy subjects. Cortisol and precursors of glucocorticoids (17-OH-progesterone, 11-desoxycortisol) and mineralocorticoids (11-desoxycorticosterone, corticosterone) were analyzed using the multi-analyte stable isotope dilution method (LC–MS/MS). Measurement results from healthy subjects were used to determine reference ranges, and those from placebo patients to predict in-hospital mortality.Measurements and main resultsCorticotropin tests from 180 patients and 20 volunteers were included. Compared to healthy subjects, patients with sepsis had elevated levels of 11-desoxycorticosterone and 11-desoxycortisol, consistent with activation of both glucocorticoid and mineralocorticoid pathways. After stimulation with corticotropin, the cortisol response was subnormal in 12% and the corticosterone response in 50% of sepsis patients. In placebo patients (n = 90), a corticotropin-stimulated cortisol-to-corticosterone ratio > 32.2 predicted in-hospital mortality (AUC 0.8 CI 0.70–0.88; sensitivity 83%; and specificity 78%). This ratio also predicted risk of shock development and 90-day mortality.ConclusionsIn this exploratory analysis, we found that in sepsis mineralocorticoid steroidogenesis was more frequently impaired than glucocorticoid steroidogenesis. The corticotropin-stimulated cortisol-to-corticosterone ratio predicts the risk of in-hospital death.Trial registration Clinical trial registered with www.clinicaltrials.gov Identifier: NCT00670254. Registered 1 May 2008, https://clinicaltrials.gov/ct2/show/NCT00670254.

Comparing amniotic fluid mass spectrometry assays and amniocyte gene analyses for the prenatal diagnosis of methylmalonic aciduria

BackgroundMethylmalonic aciduria (MMA), a rare inherited disorder, is the most common organic aciduria in China, and prenatal diagnosis has contributed to its prevention. However, the prenatal diagnosis of MMA using cultured amniocytes or chorionic villi to detect gene mutations is exclusively applicable to families with a definite genetic diagnosis. To evaluate the reliability of mass spectrometry assays for the prenatal diagnosis of MMA, we conducted a retrospective study of our 10 years’ experience.Materials and methodsThis retrospective compare study reviewed the medical records for maternal and fetuses data for 287 mothers with a family history of MMA from June 2010 to December 2020. Methylmalonate and propionylcarnitine in cell-free amniotic fluid were measured using a stable isotope dilution method (GC/MS) and MS/MS-based method (LC/MS/MS). Total homocysteine (tHcy) was measured by fluorescence polarization immunoassay. Depending on the presence of disease-causing gene mutations in probands, gene studies on amniocytes from 222 pregnant women were performed.ResultsFor 222 fetuses of the families with definite genetic diagnosis, gene analyses were performed using cultured amniocytes. 52 fetuses were affected by MMA, whereas 170 were “unaffected”. For GC/MS and LC/MS/MS, the specificity was 96.5% and 95.9%, sensitivity was 71.2% and 84.6%, respectively. The positive and negative predictive values were 86.0% and 91.6% and 86.3% and 95.3%, respectively. Propionylcarnitine/butyrylcarnitine ratio showed the highest accuracy and could thus serve as a sensitive indicator to identify those at a risk for MMA. When GC/MS and LC/MS/MS were performed in parallel, the specificity was 92.5% and sensitivity was 95.6%. When evaluating tHcy, the positive and negative predictive values were 95.0% and 96.1%, respectively. In 65 fetuses without family genetic diagnosis, 11 were finally confirmed to have MMA and 54 were “unaffected” by amniotic fluid biochemical assays. The 54 children showed normal urine organic acids and healthy development after birth.ConclusionsAmniotic fluid biochemical assays using GC/MS and LC/MS/MS in parallel increased the accuracy of prenatal diagnosis of MMA. Propionylcarnitine is a more reliable marker than methylmalonic acid in amniotic fluid. Further, tHcy is recommended for the prenatal diagnosis of combined MMA and homocysteinemia.

Quantitation of γ-Aminobutyric Acid in Cerebrospinal Fluid Using Liquid Chromatography-Electrospray-Tandem Mass Spectrometry.

We describe a simple stable isotope dilution method for accurate and precise measurement of γ-aminobutyric acid (GABA), a major inhibitory neurotransmitter in human cerebrospinal fluid (CSF) as a clinical diagnostic test. Determination of CSF GABA has clinical utility in diagnosing inborn errors of GABA metabolism, specifically for deficiencies of GABA-transaminase and succinic semialdehyde dehydrogenase. Quantitation of CSF GABA is performed utilizing high-performance liquid chromatography coupled with electrospray positive ionization tandem mass spectrometry (HPLC-ESI-MS/MS). Analysis of free and total GABA requires two individual sample preparations and mass spectrometry analyses. Free GABA in CSF is determined by a 1:2 dilution with internal standard (GABA-D2) and injected directly onto the HPLC-ESI-MS/MS system. Quantitation of total GABA in CSF requires additional sample preparation in order to hydrolyze all the conjugated GABA in the sample to free GABA. Complete hydrolysis is performed incubating sample at >100°C in acidic conditions (hydrochloric acid) for 4h. The sample is then further diluted 1:10 with a 90% acetonitrile/0.1% formic acid solution and injected into the HPLC-ESI-MS/MS system. Each assay is quantified using a five-point standard curve and is linear from 6 to 1000nM and 0.63 to 80μM for free and total GABA, respectively.

Quantification of Plasma S-adenosylmethionine and S-adenosylhomocysteine Using Liquid Chromatography-Electrospray-Tandem Mass Spectrometry.

We describe a simple stable isotope dilution method for accurate determination of S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) in plasma as a clinical diagnostic test. Determination of SAM/SAH in plasma (20μL) was performed by high-performance liquid chromatography coupled with electrospray positive ionization tandem mass spectrometry (HPLC-ESI-MS/MS). Calibrators (SAM and SAH) and internal standards (2H3-SAM and 2H4-SAH) were included in each analytical run for calibration. Sample preparation involved combining 20μL sample with 180μL of internal standard solution consisting of heavy-isotope-labeled internal standards in mobile phase A and filtering by ultracentrifugation through a 10 kd MW cutoff membrane. Sample filtrate (3μL) was injected by a Shimadzu Nexera LC System interfaced with a 5500 QTRAP® (Sciex). Chromatographic separation was achieved on a 250mm×2.0mm EZ-faast column from Phenomenex. Samples were eluted at a flow rate of 0.20mL/min with a binary gradient with a total run time of 10min. The source operated in positive ion mode at an ion spray voltage of +5000V. SAM and SAH resolved by a gradient to 100% methanol with retention times of 5.8 and 5.5min, respectively. HPLC chromatographic conditions did not produce complete separation of SAM and SAH, but they were completely discerned by their different fragmentation pattern in the mass spectrometer working in the MS-MS mode. The observed m/z values of the fragment ions were m/z 399→250 for SAM, m/z 385→136 for SAH, m/z 402→250 for 2H3-SAM, and m/z 203→46. The calibration curve was linear over the range of 12.5-5000nmol/L for SAM and SAH.

Quantitation of Lactate in Cerebrospinal Fluid Using Liquid Chromatography-Electrospray-Tandem Mass Spectrometry.

We describe a simple stable isotope dilution method for accurate and precise measurement of cerebrospinal fluid (CSF) lactate as a clinical diagnostic test. Lactate is produced from cellular metabolism, primarily in muscle cells, and provides a source of energy especially during instances of low oxygen levels. Measurement of lactate in CSF provides diagnostic information regarding the body's oxidative metabolism including diagnosis of lactate acidosis, aiding in the diagnosis of blood-brain barrier glucose transporter defect and differentiation between bacterial and viral meningitis. Determination of lactate in CSF (20μL) was performed utilizing high-performance liquid chromatography coupled with electrospray positive ionization tandem mass spectrometry (HPLC-ESI-MS/MS). Lactate in CSF is determined by a 1:10 dilution with internal standard (sodium lacate-d3) and injected directly onto the HPLC-ESI-MS/MS system. Each assay is quantified using a six-point standard curve (0.625-20mM) and has an analytical measurement range of 0.3-20mM.

Quantitation of 5-Methyltetrahydrofolate in Cerebrospinal Fluid Using Liquid Chromatography-Electrospray-Tandem Mass Spectrometry.

We describe a simple stable isotope dilution method for accurate and precise measurement of cerebrospinal fluid (CSF) 5-methyltetrahydrofolate (5-MTHF) as a clinical diagnostic test. 5-MTHF is the main biologically active form of folate and is involved in the regulation of homocysteine and numerous methylation reactions, including synthesis of neurotransmitters, lipids, DNA, and RNA. Measurement of 5-MTHF in CSF provides diagnostic information regarding disorders affecting folate metabolism within the central nervous system, in particular inborn errors of folate metabolism and cerebral folate deficiency. Determination of 5-MTHF in CSF (50μL) was performed utilizing high-performance liquid chromatography coupled with electrospray positive ionization tandem mass spectrometry (HPLC-ESI-MS/MS). 5-MTHF in CSF is determined by a 1:2 dilution with internal standard (5-MTHF-13C5) and injected directly onto the HPLC-ESI-MS/MS system. Each assay is quantified using a five-point standard curve (25-400nM) and has an analytical measurement range of 3-1000nM.

DNA-Protein Cross-Links Formed by Reacting Lysine with Apurinic/Apyrimidinic Sites in DNA and Human Cells: Quantitative Analysis by Liquid Chromatography-Tandem Mass Spectrometry Coupled with Stable Isotope Dilution.

Accumulating evidence suggests that DNA lesion-induced DNA-protein cross-links (DPCs) interrupt normal DNA metabolic processes, such as transcription, replication, and repair, resulting in profound biological consequences, including the development of many human diseases, such as cancers. Although apurinic/apyrimidinic (AP) sites are among the most predominant DNA lesions and are in close proximity to the histone proteins that they wrap around in the nucleosome, knowledge of the chemical structure or biological consequences of their associated DPCs is limited in part due to a lack of sensitive and selective analytical methods. We developed liquid chromatography-tandem mass spectrometry coupled with a stable isotope dilution method for rigorous quantitation of DPCs formed by reacting a DNA AP site with a lysine residue. In combination with chemical derivatization with fluorenylmethoxycarbonyl chloride to form a hydrophobic conjugate, the developed LC-MS/MS method allows sensitive detection of AP site-Lys cross-links down to sub-1 adduct per 106 nt. After validation using a synthetic AP site-lysine-cross-linked peptide and an oligodeoxyribonucleotide, the method was used to determine the concentration of AP site-lysine cross-links in hot acid-treated DNA and in human cells exposed to methyl methanesulfonate.

Quantitative metabolomics for dynamic metabolic engineering using stable isotope labeled internal standards mixture (SILIS)

The production of chemicals and fuels from renewable resources using engineered microbes is an attractive alternative for current fossil-dependent industries. Metabolic engineering has contributed to pathway engineering for the production of chemicals and fuels by various microorganisms. Recently, dynamic metabolic engineering harnessing synthetic biological tools has become a next-generation strategy in this field. The dynamic regulation of metabolic flux during fermentation optimizes metabolic states according to each fermentation stage such as cell growth phase and compound production phase. However, it is necessary to repeat the evaluation and redesign of the dynamic regulation system to achieve the practical use of engineered microbes. In this study, we performed quantitative metabolome analysis to investigate the effects of dynamic metabolic flux regulation on engineered Escherichia coli for γ-amino butyrate (GABA) fermentation. We prepared a stable isotope-labeled internal standard mixture (SILIS) for the stable isotope dilution method (SIDM), a mass spectrometry-based quantitative metabolome analysis method. We found multiple candidate bottlenecks for GABA production. Some metabolic reactions in the GABA production pathway should be engineered for further improvement in the direct GABA fermentation with dynamic metabolic engineering strategy.

Probing the Hidden Role of Mitochondrial DNA Damage and Dysfunction in the Etiology of Aristolochic Acid Nephropathy.

Aristolochic acid nephropathy (AAN) is a unique type of progressive renal interstitial fibrotic disease caused by prolonged exposure to aristolochic acids (AAs) through AA-containing herbal medicines or AA-tainted food. Despite decades of research and affecting millions of people around the world, the pathophysiology of AAN remains incompletely understood. In this study, we tested the potential causative role of mitochondrial dysfunction in AAN development. Our findings revealed AA exposure induces an exposure concentration and duration dependent lowering of adenosine triphosphate in both cultured human kidney and liver cells, highlighting an AA exposure effect on mitochondrial energy production in the kidney and liver, which both are highly metabolically active and energy-demanding organs. Analysis with liquid chromatography-tandem mass spectrometry coupled with stable isotope dilution method detected high levels of mutagenic 8-oxo-2'-deoxyguanosine and 7-(deoxyadenosine-N6-yl)-aristolactam adduct on mitochondrial DNA isolated from AA-treated cells, unmasking a potentially important causative, but previously unknown role of mitochondrial DNA mutation in the pathophysiology of AAN development.

Quantitation of γ-Glutamylcysteine-Formaldehyde Conjugate in Formaldehyde- and Oxidative Stress-Exposed Cells by Liquid Chromatography-Tandem Mass Spectrometry.

Humans are constantly exposed to formaldehyde (FA) of both exogenous and endogenous sources, and FA exposure is associated with the development of many human diseases, including cancers. Marker molecules that can provide information on exposure history and amounts will assist disease risk assessment and early interventions. To develop marker signatures of FA exposure, we explored in this study the conjugation reaction of FA with γ-glutamylcysteine (GGC), one of the precursors to glutathione biosynthesis, under physiologically relevant conditions. The results showed that the reaction produced a stable metabolite of FA, (S)-1-((((R)-2-amino-2-carboxyethyl)thio)methyl)-5-oxopyrrolidine-2-carboxylic acid (COCA). Using liquid chromatography-tandem mass spectrometry coupled to a stable isotope-dilution method, we then quantitated for the first time the formation of this novel metabolite in FA- and Fe2+-EDTA-exposed human cells. The results revealed the exposure time- and concentration-dependent formation of COCA in FA- or Fe2+-EDTA-exposed cells, suggesting that COCA may serve as a biomarker of FA and oxidative stress exposure. Furthermore, the study sheds light on a previously unknown protective role of GGC against FA and oxidative stress.

Distribution and quantitative analysis of homoanserine and its 2-oxo derivative in mouse tissues

Imidazole-containing dipeptides (IDPs), such as carnosine and anserine, are endogenously produced and have been shown to function as antioxidants. Recently, we have characterized the endogenous production of 2-oxo-histidine-containing dipeptides (2-oxo-IDPs), such as 2-oxo-carnosine, 2-oxo-anserine, and 2-oxo-homocarnosine in mouse tissues, including brain, and demonstrated that 2-oxo-IDPs exhibit higher antioxidant activities than the corresponding IDPs. In this study, we established a highly sensitive, specific, and quantitative method for the detection of the IDP homoanserine and its oxidized derivative 2-oxo-homoanserine via high-performance liquid chromatography tandem mass spectrometry coupled with a stable-isotope dilution method, and quantitatively analyzed its tissue distribution and age-related intra-brain distribution in C57BL/6J mice. The quantitative analysis revealed that homoanserine exists abundantly not only in the mouse brain but also in other tissues, such as the muscle and lungs. Further, we successfully detected the endogenous production of 2-oxo-homoanserine in the mouse brain. The mass spectrometric analysis revealed that homoanserine predominantly exists in the cerebrum and cerebellum and the concentrations in 10-week-old mice were approximately 50-fold higher than those in 1-week-old mice. Accordingly, this is the first study that reports the spatial and temporal expression patterns of homoanserine and its 2-oxo derivative in C57BL/6J mice.

1222-P: Adipose Tissue Antilipolytic Insulin Sensitivity Protects against Whole-Body Insulin Resistance and Lowers Acylcarnitine Accumulation in Adults with Obesity

The aims of this study were; 1) to assess clinical and subclinical factors associated with insulin-stimulated glucose uptake and 2) compare skeletal muscle lipid profile (untargeted lipidomics) in a sub-cohort of insulin sensitive vs. insulin resistant subjects. 66 obese adults (F=46 [BMI=34±3kg/m2], M=20 [BMI=33±2.9 kg/m2]) with homogeneous body composition completed a 2h hyperinsulinemic-euglycemic clamp, and stable isotope dilution methods to assess glucose rate of disappearance (Glucose Rd: index of insulin sensitivity), fatty acid rate of appearance (FA Ra), and hepatic glucose production (Glucose Ra) under basal and hyperinsulinemic conditions. We also performed an array of metabolic measurements (e.g., hepatic and visceral fat accumulation [via MRI], fat oxidation, lipid profile, circulating cytokines). Covariate analysis (LASSO) identified insulin-mediated suppression of FA Ra, basal glucose Ra, and fat oxidation as the cluster of measurements best predicting Glucose Rd (r=0.76, p&amp;lt;0.01). Insulin-mediated suppression of FA Ra was also the strongest independent predictor of Glucose Rd (r=0.51; p&amp;lt;0.01). As a result of greater adipose tissue anti-lipolytic response to insulin, lower FA delivery to skeletal muscle may improve insulin-mediated glucose uptake via lower muscle lipid accumulation. Importantly, we found the accumulation of several long-chain acylcarnitine species (measure of incomplete β-oxidation: C18:0,18:1,20:1,20:3) to be lower (p&amp;lt;0.05) in muscle samples from an insulin sensitive sub-cohort (Glucose Rd &amp;gt; 8.5 µmol/(min•FFM•[Insulin]); n=10) compared with an insulin resistant sub-cohort (&amp;lt; 7.2 µmol/(min•FFM•[Insulin]); n=8). In conclusion, our findings indicate that greater adipose tissue anti-lipolytic response to insulin is an important predictor of insulin-mediated glucose uptake - and acylcarnitine accumulation in skeletal muscle may mediate this effect. Disclosure M. W. Schleh: None. B. J. Ryan: None. C. Ahn: None. A. Ludzki: None. P. Varshney: None. J. B. Gillen: None. J. F. Horowitz: None. Funding National Institutes of Health (R01DK077966, P30DK089503, T32DK007245, F32DK117522); Canadian Institutes of Health Research (DFS146190, 338735)

A stable isotope dilution method for a highly accurate analysis of karrikins

BackgroundKarrikins (KARs) are recently described group of plant growth regulators with stimulatory effects on seed germination, seedling growth and crop productivity. So far, an analytical method for the simultaneous targeted profiling of KARs in plant tissues has not been reported.ResultsWe present a sensitive method for the determination of two highly biologically active karrikins (KAR1 and KAR2) in minute amounts of plant material (< 20 mg fresh weight). The developed protocol combines the optimized extraction and efficient single-step sample purification with ultra-high performance liquid chromatography-tandem mass spectrometry. Newly synthesized deuterium labelled KAR1 was employed as an internal standard for the validation of KAR quantification using a stable isotope dilution method. The application of the matrix-matched calibration series in combination with the internal standard method yields a high level of accuracy and precision in triplicate, on average bias 3.3% and 2.9% RSD, respectively. The applicability of this analytical approach was confirmed by the successful analysis of karrikins in Arabidopsis seedlings grown on media supplemented with different concentrations of KAR1 and KAR2 (0.1, 1.0 and 10.0 µmol/l).ConclusionsOur results demonstrate the usage of methodology for routine analyses and for monitoring KARs in complex biological matrices. The proposed method will lead to better understanding of the roles of KARs in plant growth and development.

Quantitative analysis of seven plant hormones in Lotus japonicus using standard addition method.

Plant hormones have been identified to be versatile signaling molecules essential for plant growth, development, and stress response. Their content levels vary depending on the species, and they also change in response to any external stimuli. Thus, simultaneous quantification of multiple plant hormones is required to understand plant physiology. Sensitive and quantitative analysis using liquid chromatography-linked mass spectrometry (LC-MS/MS) has been used in detecting plant hormones; however, quantification without stable isotopes is yet to be established. In this study, we quantified seven representative plant hormones of Lotus japonicus, which is a model legume for standard addition method. Accurate masses for monoisotopic ions of seven phytohormones were determined for high-resolution mass spectrometry (HR-MS). Selected ion monitoring (SIM) mode based on accurate masses was used in detecting phytohormones in the roots, stems, and leaves. Evaluation of matrix effects showed ion suppression ranging from 10.2% to 87.3%. Both stable isotope dilution and standard addition methods were able to detect plant hormones in the roots, stems, and leaves, with no significant differences in using both approaches and thus a standard addition method can be used to quantify phytohormones in L. japonicus. The method will be effective, especially when stable isotopes are not available to correct for matrix effects.

LC-MS/MS Coupled with a Stable-Isotope Dilution Method for the Quantitation of Thioproline-Glycine: A Novel Metabolite in Formaldehyde- and Oxidative Stress-Exposed Cells.

Formaldehyde (FA) is a human carcinogen that is ubiquitous in the ambient environment and also generated endogenously in oxidatively stressed cells. There is accumulated evidence that FA is an etiological agent of leukemia development in humans. To develop a biomarker for FA exposure, we have, in this study, developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) coupled with stable isotope-dilution method to explore the reactivity of FA with glutathione (GSH) in physiologically relevant conditions. Interestingly, analysis of the reaction mixture by LC-MS/MS revealed exposure concentration- and duration-dependent formation of thioproline-glycine (SPro-Gly) produced by reaction of FA with cysteinyl-glycine (Cys-Gly) as a novel metabolite. Furthermore, dose-dependent formation of the thioproline adduct was observed in human cells separately exposed to FA and Fe2+-EDTA, a hydroxyl radical source. To the best of our knowledge, this is the first study reporting a thiazolidine carboxylic acid formed by reaction of FA and Cys-Gly is a major metabolite of FA. The results suggest a variety of GSH-derived thiazolidine metabolites may serve as potential biomarkers for FA and oxidative stress exposure, and the developed LC-MS/MS method provides a means for accurate determination of SPro-Gly as a dosimeter of oxidative stress and formaldehyde exposure.