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The aging of Pb added to soils has not been studied by the isotopic technology because of difficulties in determination of isotopically exchangeable Pb in soil, so that a set of 10 typical agricultural soils in China and a one-year aging experiment with the addition of water-soluble Pb to the soils were carried out. A modified stable isotope dilution technique to determine isotopically exchangeable Pb in soil was developed where 0.2 mM EDTA (ethylenediaminetetraacetic acid) as the extractant. When water-soluble Pb was added to soil, the isotopically exchangeable Pb (Eadd%, the percentage of isotopically exchangeable Pb to total Pb added to soil) initially decreased rapidly and gradually slowly. A semi-mechanistic aging model of Pb added to soils, including precipitation/nucleation (Y1), micropore diffusion (Y2), and organic matter encapsulation processes (Y3) was developed with the root mean square error 8.3% where Y1, Y2, and Y3 accounted for 0.02~26.9%, 1.4~21.8% and 3.8~11.3%, respectively, when the pH 4.0~8.0 and organic matter 2.0~6.0%. Soil pH was a vital factor affecting the aging rate. When the pH increased by 1 unit, the Eadd value decreased by approximately 9%. The model could be used to scale ecotoxicological data of Pb in soil generated in different aging times. [ABSTRACT FROM AUTHOR]

Abstract The aging of Pb added to soils has not been studied by the isotopic technology because of difficulties in determination of isotopically exchangeable Pb in soil, so that a set of 10 typical agricultural soils in China and a one-year aging experiment with the addition of water-soluble Pb to the soils were carried out. A modified stable isotope dilution technique to determine isotopically exchangeable Pb in soil was developed where 0.2 mM EDTA (ethylenediaminetetraacetic acid) as the extractant. When water-soluble Pb was added to soil, the isotopically exchangeable Pb (Eadd%, the percentage of isotopically exchangeable Pb to total Pb added to soil) initially decreased rapidly and gradually slowly. A semi-mechanistic aging model of Pb added to soils, including precipitation/nucleation (Y1), micropore diffusion (Y2), and organic matter encapsulation processes (Y3) was developed with the root mean square error 8.3% where Y1, Y2, and Y3 accounted for 0.02~26.9%, 1.4~21.8% and 3.8~11.3%, respectively, when the pH 4.0~8.0 and organic matter 2.0~6.0%. Soil pH was a vital factor affecting the aging rate. When the pH increased by 1 unit, the Eadd value decreased by approximately 9%. The model could be used to scale ecotoxicological data of Pb in soil generated in different aging times.

Accurate measurement of sulfated steroid metabolite concentrations can not only enable the elucidation of the mechanisms regulating steroid metabolism, but also lead to the diagnosis of various related diseases. The present study describes a simple and sensitive method for the simultaneous determination of four sulfated steroid metabolites in saliva, pregnenolone sulfate (PREGS), dehydroepiandrosterone sulfate (DHEAS), cortisol sulfate (CRTS), and 17β-estradiol-3-sulfate (E2S), by online coupling of in-tube solid-phase microextraction (IT-SPME) and stable isotope dilution liquid chromatography–tandem mass spectrometry (LC–MS/MS). These compounds were extracted and concentrated on Supel-Q PLOT capillary tubes by IT-SPME and separated and detected within 6 min by LC–MS/MS using an InertSustain swift C18 column and negative ion mode multiple reaction monitoring systems. These operations were fully automated by an online program. Calibration curves using their stable isotope-labeled internal standards showed good linearity in the range of 0.01–2 ng mL−1 for PREGS, DHEAS, and CRTS and of 0.05–10 ng mL−1 for E2S. The limits of detection (S/N = 3) of PREGS, DHEAS, CRTS, and E2S were 0.59, 0.30, 0.80, and 3.20 pg mL−1, respectively. Moreover, intraday and interday variations were lower than 11.1% (n = 5). The recoveries of these compounds from saliva samples were in the range of 86.6–112.9%. The developed method is highly sensitive and specific and can easily measure sulfated steroid metabolite concentrations in 50 μL saliva samples. [ABSTRACT FROM AUTHOR]

Friedreich's ataxia (FRDA) is an autosomal recessive disease caused by an intronic guanine-adenine-adenine (GAA) triplet expansion in the frataxin (FXN) gene, which leads to reduced expression of full-length frataxin (1–210) also known as isoform 1. Full-length frataxin has a mitochondrial targeting sequence, which facilitates its translocation into mitochondria where it is processed through cleavage at G41-L42 and K80-S81 by mitochondrial processing (MPP) to release mitochondrial mature frataxin (81–210). Alternative splicing of FXN also leads to expression of N-terminally acetylated extra-mitochondrial frataxin (76–210) named isoform E because it was discovered in erythrocytes. Frataxin isoforms are undetectable in serum or plasma, and originally whole blood could not be used as a biomarker in brief therapeutic trials because it is present in erythrocytes, which have a half-life of 115-days and so frataxin levels would remain unaltered. Therefore, an assay was developed for analyzing frataxin in platelets, which have a half-life of only 10-days. However, our discovery that isoform E is only present in erythrocytes, whereas, mature frataxin is present primarily in short-lived peripheral blood mononuclear cells (PBMCs), granulocytes, and platelets, meant that both proteins could be quantified in whole blood samples. We now report a quantitative assay for frataxin proteoforms in whole blood from healthy controls and FRDA patients. The assay is based on stable isotope dilution coupled with immunoprecipitation (IP) and two-dimensional-nano-ultrahigh performance liquid chromatography/parallel reaction monitoring/high resolution mass spectrometry (2D-nano-UHPLC-PRM/HRMS). The lower limit of quantification was 0.5 ng/mL for each proteoform and the assays had 100% sensitivity and specificity for discriminating between healthy controls (n = 11) and FRDA cases (N = 100 in year-1, N = 22 in year-2,3). The mean levels of mature frataxin in whole blood from healthy controls and homozygous FRDA patients were significantly different (p < 0.0001) at 7.5 ± 1.5 ng/mL and 2.1 ± 1.2 ng/mL, respectively. The mean levels of isoform E in whole blood from healthy controls and homozygous FRDA patients were significantly different (p < 0.0001) at 26.8 ± 4.1 ng/mL and 4.7 ± 3.3 ng/mL, respectively. The mean levels of total frataxin in whole blood from healthy controls and homozygous FRDA patients were significantly different (p < 0.0001) at 34.2 ± 4.3 ng/mL and 6.8 ± 4.0 ng/mL, respectively. The assay will make it possible to rigorously monitor the natural history of the disease and explore the potential role of isoform E in etiology of the disease. It will also facilitate the assessment of therapeutic interventions (including gene therapy approaches) that attempt to increase frataxin protein expression as a treatment for this devastating disease. [ABSTRACT FROM AUTHOR]

Friedreich’s ataxia (FRDA) is an autosomal recessive disease caused by an intronic guanine-adenine-adenine (GAA) triplet expansion in the frataxin (FXN) gene, which leads to reduced expression of full-length frataxin (1–210) also known as isoform 1. Full-length frataxin has a mitochondrial targeting sequence, which facilitates its translocation into mitochondria where it is processed through cleavage at G41-L42 and K80-S81 by mitochondrial processing (MPP) to release mitochondrial mature frataxin (81–210). Alternative splicing of FXN also leads to expression of N-terminally acetylated extra-mitochondrial frataxin (76–210) named isoform E because it was discovered in erythrocytes. Frataxin isoforms are undetectable in serum or plasma, and originally whole blood could not be used as a biomarker in brief therapeutic trials because it is present in erythrocytes, which have a half-life of 115-days and so frataxin levels would remain unaltered. Therefore, an assay was developed for analyzing frataxin in platelets, which have a half-life of only 10-days. However, our discovery that isoform E is only present in erythrocytes, whereas, mature frataxin is present primarily in short-lived peripheral blood mononuclear cells (PBMCs), granulocytes, and platelets, meant that both proteins could be quantified in whole blood samples. We now report a quantitative assay for frataxin proteoforms in whole blood from healthy controls and FRDA patients. The assay is based on stable isotope dilution coupled with immunoprecipitation (IP) and two-dimensional-nano-ultrahigh performance liquid chromatography/parallel reaction monitoring/high resolution mass spectrometry (2D-nano-UHPLC-PRM/HRMS). The lower limit of quantification was 0.5 ng/mL for each proteoform and the assays had 100% sensitivity and specificity for discriminating between healthy controls (n = 11) and FRDA cases (N = 100 in year-1, N = 22 in year-2,3). The mean levels of mature frataxin in whole blood from healthy controls and homozygous FRDA patients were significantly different (p < 0.0001) at 7.5 ± 1.5 ng/mL and 2.1 ± 1.2 ng/mL, respectively. The mean levels of isoform E in whole blood from healthy controls and homozygous FRDA patients were significantly different (p < 0.0001) at 26.8 ± 4.1 ng/mL and 4.7 ± 3.3 ng/mL, respectively. The mean levels of total frataxin in whole blood from healthy controls and homozygous FRDA patients were significantly different (p < 0.0001) at 34.2 ± 4.3 ng/mL and 6.8 ± 4.0 ng/mL, respectively. The assay will make it possible to rigorously monitor the natural history of the disease and explore the potential role of isoform E in etiology of the disease. It will also facilitate the assessment of therapeutic interventions (including gene therapy approaches) that attempt to increase frataxin protein expression as a treatment for this devastating disease.

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. [ABSTRACT FROM AUTHOR]

Accurate measurement of sulfated steroid metabolite concentrations can not only enable the elucidation of the mechanisms regulating steroid metabolism, but also lead to the diagnosis of various related diseases. The present study describes a simple and sensitive method for the simultaneous determination of four sulfated steroid metabolites in saliva, pregnenolone sulfate (PREGS), dehydroepiandrosterone sulfate (DHEAS), cortisol sulfate (CRTS), and 17β-estradiol-3-sulfate (E2S), by online coupling of in-tube solid-phase microextraction (IT-SPME) and stable isotope dilution liquid chromatography–tandem mass spectrometry (LC–MS/MS). These compounds were extracted and concentrated on Supel-Q PLOT capillary tubes by IT-SPME and separated and detected within 6 min by LC–MS/MS using an InertSustain swift C18 column and negative ion mode multiple reaction monitoring systems. These operations were fully automated by an online program. Calibration curves using their stable isotope-labeled internal standards showed good linearity in the range of 0.01–2 ng mL−1 for PREGS, DHEAS, and CRTS and of 0.05–10 ng mL−1 for E2S. The limits of detection (S/N = 3) of PREGS, DHEAS, CRTS, and E2S were 0.59, 0.30, 0.80, and 3.20 pg mL−1, respectively. Moreover, intraday and interday variations were lower than 11.1% (n = 5). The recoveries of these compounds from saliva samples were in the range of 86.6–112.9%. The developed method is highly sensitive and specific and can easily measure sulfated steroid metabolite concentrations in 50 μL saliva samples.

The Lipid Metabolites and Pathway Strategy (LIPID MAPS) Consortium is a nationwide initiative that has taken on the task of employing lipidomics to advance our understanding of lipid metabolism at the molecular and mechanistic level in living organisms. An important step toward this goal is to craft enabling analytical procedures to comprehensively measure all lipid species, to establish the precise structural identity of the lipid molecules analyzed, and to generate accurate quantitative information. The LIPID MAPS Consortium has succeeded in the implementation of a complete infrastructure that now provides tools for analysis of the global lipidome in cultured and primary cells. Here we illustrate the advancement of a gas chromatography mass spectrometry (GC/MS) procedure for the analysis of essential fatty acids in RAW 264.7 cells. Our method allows for the specific identification and quantification of over 30 fatty acids present in cells in their free form in a single analytical GC/MS run. Free fatty acids are selectively extracted in the presence of deuterated internal standards, which permit subsequent estimation of extraction efficiencies and quantification with high accuracy. Mass spectrometer conditions were optimized for single-ion monitoring, which provides an extremely sensitive technology to measure fatty acids from biological samples in trace amounts. These methods will be presented in the context of our broader effort to analyze all fatty acids as well as their metabolites in inflammatory cells.

Experiments were conducted to identify a compound responsible for a spicy, woody, incense-like odor note in oak-aged spirits. The target compound was extracted from oak wood and various oak-aged spirits and analyzed by multidimensional (heart-cut) gas chromatography–mass spectrometry–olfactometry (MD–GC–MS–O), and was unambiguously identified as the sesquiterpene ketone, 5-isopropenyl-3,8-dimethyl-3,4,5,6,7,8-hexadydro-1(2H)-azulenone (rotundone). Quantitation of the trace-level target compound was done by stable isotope dilution analysis (SIDA) in a variety of oak-aged spirits, including bourbon, rye, Tennessee whiskey, scotch, rum, and tequila. The content of rotundone was found to increase as a function of years of barrel aging for 4-, 8-, and 12-year-old bourbons obtained from the same manufacturer, thus confirming its origin to be from oak. In addition, odor-activity values (OAVs) were compared for selected potent odorants, including rotundone, in the same 4-, 8-, and 12-year-old bourbons, which indicated the relative importance of rotundone in the overall flavor of oak-aged spirits.

Glycation is a non-enzymatic post-translational modification of proteins, formed by the reaction of reducing sugars and α-dicarbonyl products of their degradation with amino and guanidino groups of proteins. Resulted early glycation products are readily involved in further transformation, yielding a heterogeneous group of advanced glycation end products (AGEs). Their formation is associated with ageing, metabolic diseases, and thermal processing of foods. Therefore, individual glycation adducts are often considered as the markers of related pathologies and food quality. In this context, their quantification in biological and food matrices is required for diagnostics and establishment of food preparation technologies. For this, exhaustive protein hydrolysis with subsequent amino acid analysis is the strategy of choice. Thereby, multi-step enzymatic digestion procedures ensure good recoveries for the most of AGEs, whereas tandem mass spectrometry (MS/MS) in the multiple reaction monitoring (MRM) mode with stable isotope dilution or standard addition represents "a gold standard" for their quantification. Although the spectrum of quantitatively assessed AGE structures is continuously increases, application of untargeted profiling techniques for identification of new products is desired, especially for in vivo characterization of anti-glycative systems. Thereby, due to a high glycative potential of plant metabolites, more attention needs to be paid on plant-derived AGEs. [ABSTRACT FROM AUTHOR]

Glycation is a non-enzymatic post-translational modification of proteins, formed by the reaction of reducing sugars and α-dicarbonyl products of their degradation with amino and guanidino groups of proteins. Resulted early glycation products are readily involved in further transformation, yielding a heterogeneous group of advanced glycation end products (AGEs). Their formation is associated with ageing, metabolic diseases, and thermal processing of foods. Therefore, individual glycation adducts are often considered as the markers of related pathologies and food quality. In this context, their quantification in biological and food matrices is required for diagnostics and establishment of food preparation technologies. For this, exhaustive protein hydrolysis with subsequent amino acid analysis is the strategy of choice. Thereby, multi-step enzymatic digestion procedures ensure good recoveries for the most of AGEs, whereas tandem mass spectrometry (MS/MS) in the multiple reaction monitoring (MRM) mode with stable isotope dilution or standard addition represents “a gold standard” for their quantification. Although the spectrum of quantitatively assessed AGE structures is continuously increases, application of untargeted profiling techniques for identification of new products is desired, especially for in vivo characterization of anti-glycative systems. Thereby, due to a high glycative potential of plant metabolites, more attention needs to be paid on plant-derived AGEs.

Chemical extractants used to measure labile soil metal must ideally select for and solubilise the labile fraction, with minimal solubilisation of non-labile metal. We assessed four extractants (0.43 M HNO 3 , 0.43 M CH 3 COOH, 0.05 M Na 2 H 2 EDTA and 1 M CaCl 2 ) against these requirements. For soils contaminated by contrasting sources, we compared isotopically exchangeable Ni, Cu, Zn, Cd and Pb (E Value , mg kg −1 ), with the concentrations of metal solubilised by the chemical extractants (M Ext , mg kg −1 ). Crucially, we also determined isotopically exchangeable metal in the soil–extractant systems (E Ext , mg kg −1 ). Thus ‘E Ext – E Value ’ quantifies the concentration of mobilised non-labile metal, while ‘E Ext – M Ext ’ represents adsorbed labile metal in the presence of the extractant. Extraction with CaCl 2 consistently underestimated E Value for Ni, Cu, Zn and Pb, while providing a reasonable estimate of E Value for Cd. In contrast, extraction with HNO 3 both consistently mobilised non-labile metal and overestimated the E Value . Extraction with CH 3 COOH appeared to provide a good estimate of E Value for Cd; however, this was the net outcome of incomplete solubilisation of labile metal, and concurrent mobilisation of non-labile metal by the extractant (M Ext < E Ext > E Value ). The Na 2 H 2 EDTA extractant mobilised some non-labile metal in three of the four soils, but consistently solubilised the entire labile fraction for all soil-metal combinations (M Ext ≈ E Ext ). Comparison of E Value , M Ext and E Ext provides a rigorous means of assessing the underlying action of soil chemical extraction methods and could be used to refine long-standing soil extraction methodologies. [ABSTRACT FROM AUTHOR]

Background: The WHO estimates that 190 million preschool children have vitamin A deficiency (VAD). Serum retinol (SR) concentration is a common indicator of vitamin A (VA) status, but SR is homeostatically controlled and suppressed during inflammation, which may lead to misdiagnosis. Objective: The sensitivity and specificity of SR compared with VA total liver reserves (TLRs) were evaluated for VAD in children from Thailand (n = 37) and Zambia (n = 128). SR was adjusted for inflammation in the Zambian children. Design: Each child was classified as VA-deficient or not based on cutoffs of <0.1 µmol VA/g liver with the use of retinol isotope dilution and <0.7 µmol/L for SR concentrations. Four categories of infection status in the Zambian children were based on elevated C-reactive protein (CRP) and α1-acid glycoprotein (AGP). Sensitivity and specificity were calculated with the use of unadjusted and inflammation marker-adjusted SR cutoffs. Results: VAD was 65% and 0% according to TLRs and SR, respectively, in Thai children and 0% and 17%, respectively, in Zambian children. No true positive VAD cases occurred; thus, sensitivity was 0% and indeterminable, respectively; specificity was 100% and 82.8%, respectively. CRP was elevated in 26.6% of Zambian children, whereas 97.7% had elevated AGP, categorizing them as having no infection (2.3%) or in early (26.6%) or late (58.6%) convalescence. With the use of marker-adjusted SR cutoffs of 0.6 µmol/L for late convalescence and 0.5 µmol/L for early convalescence, the adjusted prevalence of SR deficiency was 2.3%, increasing specificity to 97.3%. Conclusions: No cases of VAD were identified by both TLRs and SR (true positives) in Thai or Zambian children. Specificity of SR to evaluate VAD was high, but additional research is needed to investigate sensitivity. Adjusting SR cutoffs for inflammation improved specificity by reducing false positives. SR as a VAD indicator may depend on infection rates, which should be taken into consideration. These studies were registered at clinicaltrials.gov as NCT01061307 (for Thailand) and NCT01814891 (for Zambia). [ABSTRACT FROM AUTHOR]

We have previously demonstrated that the rat brain contains three unconjugated bile acids, and chenodeoxycholic acid (CDCA) is the most abundantly present in a tight protein binding form. The ratio of CDCA to the other acids in rat brain tissue was significantly higher than the ratio in the peripheral blood, indicating a contribution from either a specific uptake mechanism or a biosynthetic pathway for CDCA in rat brain. In this study, we have demonstrated the existence of an enzymatic activity that converts 3β-hydroxy-5-cholenoic acid into CDCA in rat brain tissue. To distinguish marked compounds from endogenous related compounds, 18O-labeled 3β-hydroxy-5-cholenoic acid, 3β,7α-dihydroxy-5-cholenoic acid, and 7α-hydroxy-3-oxo-4-cholenoic acid were synthesized as substrates for in vitro incubation studies. The results clearly suggest that 3β-hydroxy-5-cholenoic acid was converted to 3β,7α-dihydroxy-5-cholenoic acid by microsomal enzymes. The 7α-hydroxy-3-oxo-4-cholenoic acid was produced from 3β,7α-dihydroxy-5-cholenoic acid by the action of microsomal enzymes, and Δ4-3-oxo acid was converted to CDCA by cytosolic enzymes.These findings indicate the presence of an enzymatic activity that converts 3β-hydroxy-5-cholenoic acid into CDCA in rat brain tissue. Furthermore, this synthetic pathway for CDCA may relate to the function of 24S-hydroxycholesterol, which plays an important role in cholesterol homeostasis in the body.

The determination of albendazole (ABZ) and its metabolites, albendazole sulfoxide, albendazole sulfone, and albendazole 2-aminosulfone in crab tissue is a complex analysis involving the extraction of trace levels of potentially unstable analytes from a solid matrix. Here, we report on an online solid-phase extraction (SPE) for automated trapping of ABZ and its metabolites in Chinese mitten crab, Eriocheir sinensis H. Milne Edwards ( E. sinensis), followed by stable isotope dilution UPLC-MS/MS analysis. The performance of online SPE was fully validated. Good linearity was obtained with correlation coefficients >0.99. In the analytical range, the intra-day and inter-day precision and accuracies (RSD %) were 1.44-4.62 and 3.28-6.93 %, respectively. The mean recoveries were in the range of 78.2-109.3 %. All these demonstrated that the online SPE is an excellent technique for trapping and purification of ABZ and its metabolites in crab tissue samples. Finally, this online SPE-UPLC-MS/MS strategy was successfully applied to analyze the crude extracts from several crab tissue samples. [ABSTRACT FROM AUTHOR]

The bile salt export pump, encoded by ABCB11, is the predominant canalicular transport protein for biliary bile acid secretion. The level of ABCB11 expression in humans is widely variable yet the impact of this variability on human disease is not well defined. We aim to determine the effect of hepatic Abcb11 overexpression on the enterohepatic circulation (EHC) in mice. We used a stable isotope dilution technique in transgenic mice overexpressing hepatic Abcb11 (TTRAbcb11) to determine the pool size, fractional turnover rate (FTR), and synthesis rate of the primary bile acid, cholic acid (CA). The gallbladder was cannulated to determine bile flow, bile acid composition, and the biliary secretion rates of CA, total bile acids, phospholipid, and cholesterol. The combined data allowed for estimation of the CA cycling time and the fraction of CA lost per cycle. Hepatic and intestinal gene and protein expression were determined by qPCR and Western blot. Abcb11 overexpression strongly decreased FTR and synthesis rate of CA. Abcb11 overexpression decreased the fraction of CA that was lost per cycle of the EHC. Hepatic expression of Cyp7a1 was suppressed by nearly 50% and ileal expression of FGF15 was increased more than eightfold in TTR-Abcb11 mice. Despite the increased intestinal reabsorption of bile acids, ileal Asbt expression was suppressed. Hepatic Abcb11 overexpression in mice increases the conservation of bile acids within the enterohepatic circulation. These data provide strong evidence for the existence of feed-forward communication between hepatic expression of a bile acid transport protein and the intestine. [ABSTRACT FROM AUTHOR]

Stable isotope dilution-based quantitative proteomics with intact labeled proteins as internal standards in combination with a bottom-up approach, i.e., with quantification on the peptide level, is an established method. To explore the technical precision of this approach, calmodulin-like protein 3 was prepared in non-labeled (light) and SILAC-type labeled (heavy) form by cell-free synthesis, mixed, digested with trypsin, and analyzed by UPLC-ESI-MS. In total, 16 light/heavy peptide pair ratios were determined. Pair-wise comparison of ratios of 12 peptides selected according to S/ N ratios >50 revealed that the majority exhibited ratios, which were different at a high level of statistical significance ( p < 0.001). HPLC-MALDI-MS ratio data confirmed this observation, thus excluding the ionization method as a source of the observed ratio differences. Variation of the digestion time from 0.25 to 4 h showed that the light/heavy ratios of most peptides decrease with time, indicating a kinetic isotope effect leading to preferred cleavage of light calmodulin-like protein 3. The subset of peptides with statistically identical ratios resulted in an average ratio with a RSD of 1.0 %. The light/heavy ratio calculated on the basis of these peptides probably provides the most accurate molar protein ratio. [ABSTRACT FROM AUTHOR]

Abstract: Multiple reaction monitoring mass spectrometry coupled with stable isotope dilution has become the method of choice for quantifying target proteins in complex biological samples. It quantifies signature peptides based on the sequential detection of peptide precursor ions and their fragments generated upon gas-phase collision. Heavy-isotope (13C and/or 15N) labeled peptides or proteomes are commonly used as internal reference standards for quantitation. However, use of these standards is becoming expensive when large numbers/amounts of reference peptides are needed. The search for low-cost, labeled references for proteomic quantitation such as those with 2H-labels is an object of constant pursuit. In order to take the cost advantage of 2H-labels, this work examines whether or not the known chromatographic separation of 2H-based peptides from the native counterparts affects the peptide quantitation using multiple reaction monitoring mass spectrometry. Experimental results from model peptides and proteome digests indicate that targeted mass spectrometry quantitation of the 2H- and 13C/15N-based peptides are comparable. [Copyright &y& Elsevier]

Plant metabolomics has benefited from a rich array of pre-existing methodological approaches and bioanalytical knowledge for the characterization of the many chemically diverse classes of metabolites. While the field has pushed the implementation of unbiased and generally applicable strategies for metabolite extraction, fractionation and detection, significant challenges in fundamental activities such as compound identification and quantification still exist. This review provides an introduction to metabolomics terminology, methods and resources and discusses analytical limitations. Progress in the application of stable isotope dilution and stable isotopic metabolic labeling to metabolite identification and quantification are discussed. New strategies to address spatial distribution of metabolites via mass spectrometry-based imaging are also reviewed. [ABSTRACT FROM PUBLISHER]

Essential fatty acid (EFA) deficiency in mice has been associated with increased bile production, which is mainly determined by the enterohepatic circulation (EHC) of bile salts. To establish the mechanism underlying the increased bile production, we characterized in detail the EHC of bile salts in EFA-deficient mice using stable isotope technique, without interrupting the normal EHC. Farnesoid X receptor (FXR) has been proposed as an important regulator of bile salt synthesis and homeostasis. In Fxr-/- mice we additionally investigated to what extent alterations in bile production during EFA deficiency were FXR dependent. Furthermore, we tested in differentiating Caco-2 cells the effects of EFA deficiency on expression of FXR-target genes relevant for feedback regulation of bile salt synthesis. EFA deficiency-enhanced bile flow and biliary bile salt secretion were associated with elevated bile salt pool size and synthesis rate (+ 146 and +42%, respectively, P < 0.05), despite increased ileal bile salt reabsorption (+228%, P < 0.05). Cyp7a1 mRNA expression was unaffected in EFA-deficient mice. However, ileal mRNA expression of Fgf15 (inhibitor of bile salt synthesis) was significantly reduced, in agreement with absent inhibition of the hepatic bile salt synthesis. Bile flow and biliary secretion were enhanced to the same extent in EFA-deficient wild-type and Fxr-/- mice, indicating contribution of other factors besides FXR in regulation of EHC during EFA deficiency. In vitro experiments show reduced induction of mRNA expression of relevant genes upon chenodeoxycholic acid and a selective FXR agonist GW4064 stimulation in EFA-deficient Caco-2 cells. In conclusion, our data indicate that EFA deficiency is associated with interrupted negative feedback of bile salt synthesis, possibly because of reduced ileal Fgf15 expression. [ABSTRACT FROM AUTHOR]

Sucrose-phosphate synthase (SPS) has attracted the interest of plant scientists for decades. It is the key enzyme in sucrose metabolism and is under investigation in various plant species, e.g. spinach, tobacco, poplar, resurrection plants, maize, rice, kiwi and Arabidopsis thaliana. In A. thaliana, there are four distinct SPS isoforms. Their expression is thought to depend on environmental conditions and plant tissue. However, these data were derived from mRNA expression levels only. No data on SPS protein identification from crude extracts have been available until now. An antibody approach failed to distinguish the four isoforms. Therefore, we developed a method for SPS quantification and isoform-specific identification in A. thaliana complex protein samples. Samples were separated on SDS-PAGE, digested and directly applied to liquid chromatography/triple-stage quadrupole mass spectrometry (LC/TSQ-MS). In this approach, known as mass Western, samples were analysed in multi-reaction monitoring (MRM) mode, so that all four SPS isoforms could be measured in one experiment. In addition to the relative quantification, stable isotope-labelled internal peptide standards allowed absolute quantification of SPS proteins. Protein extracts from various plant tissues, samples harvested during the day or the night, and cold-stressed plants were analysed. The stress-specific SPS5a isoform showed increased concentrations in cold-stressed leaf material. [ABSTRACT FROM AUTHOR]

Background: The conventional deuterated-retinol-dilution (DRD) technique provides a quantitative estimate of total body stores of vitamin A in humans. The procedure requires equilibration of serum deuterated retinol with nondeuterated retinol after administration of an oral dose of deuterated vitamin A. Equilibration takes ≈3 wk to complete. Objective: Our goal was to develop a predictive mathematical formula for quantitative assessment of total body stores of vitamin A in adults by using a procedure that takes less time to perform because serum isotope equilibration is not required, so that blood drawing can be done 3 d, instead of ≈3 wk, after isotope dosing. Design: Ratios of serum deuterated to nondeuterated retinol (D:H retinol) were determined in Filipino adults (n = 68) 3 and 20 d after an oral dose of 0.015 mmol [2H4]retinyl acetate and in Guatemalan adults (n = 15) 3 and 21 d after a 0.030-mmol dose. D:H retinol values 20 or 21 d after the isotope dose were used in a mathematical formula to obtain quantitative estimates of total body stores of vitamin A that were then correlated with serum D:H retinol values 3 d after the isotope dose. Results: The relation between these variables was nonlinear and was described by the following equation: total body stores of vitamin A (in mmol retinol) = 0.00468 × 1037(isotope dose in mmol) /D:H retinol in serum 3 d after the isotope dose. Conclusion: A 3-d DRD technique could be used for quantitative assessment of total body stores of vitamin A; this technique takes less time than does the conventional DRD technique. [ABSTRACT FROM AUTHOR]

Experiments were conducted to identify a compound responsible for a spicy, woody, incense-like odor note in oak-aged spirits. The target compound was extracted from oak wood and various oak-aged spirits and analyzed by multidimensional (heart-cut) gas chromatography–mass spectrometry–olfactometry (MD–GC–MS–O), and was unambiguously identified as the sesquiterpene ketone, 5-isopropenyl-3,8-dimethyl-3,4,5,6,7,8-hexadydro-1(2H)-azulenone (rotundone). Quantitation of the trace-level target compound was done by stable isotope dilution analysis (SIDA) in a variety of oak-aged spirits, including bourbon, rye, Tennessee whiskey, scotch, rum, and tequila. The content of rotundone was found to increase as a function of years of barrel aging for 4-, 8-, and 12-year-old bourbons obtained from the same manufacturer, thus confirming its origin to be from oak. In addition, odor-activity values (OAVs) were compared for selected potent odorants, including rotundone, in the same 4-, 8-, and 12-year-old bourbons, which indicated the relative importance of rotundone in the overall flavor of oak-aged spirits. [ABSTRACT FROM AUTHOR]