Identification of bile acid-CoA:amino acid N-acyltransferase as the hepatic N-acyl taurine synthase for polyunsaturated fatty acids

N-acyl taurines (NATs) are bioactive lipids with emerging roles in glucose homeostasis and lipid metabolism. The acyl chains of hepatic and biliary NATs are enriched in polyunsaturated fatty acids (PUFAs). Dietary supplementation with a class of PUFAs, the omega-3 fatty acids, increases their cognate NATs in mice and humans. However, the synthesis pathway of the PUFA-containing NATs remains undiscovered. Here, we report that human livers synthesize NATs and that the acyl-chain preference is similar in murine liver homogenates. In the mouse, we found that hepatic NAT synthase activity localizes to the peroxisome and depends upon an active-site cysteine. Using unbiased metabolomics and proteomics, we identified bile acid-CoA:amino acid N-acyltransferase (BAAT) as the likely hepatic NAT synthase in vitro. Subsequently, we confirmed that BAAT knockout livers lack up to 90% of NAT synthase activity and that biliary PUFA-containing NATs are significantly reduced compared with wildtype. In conclusion, we identified the in vivo PUFA-NAT synthase in the murine liver and expanded the known substrates of the bile acid-conjugating enzyme, BAAT, beyond classic bile acids to the synthesis of a novel class of bioactive lipids.     

The origin of long-chain fatty acids required for de novo ether lipid/plasmalogen synthesis

Peroxisomes are single-membrane bounded organelles that in humans play a dual role in lipid metabolism, including the degradation of very long-chain fatty acids and the synthesis of ether lipids/plasmalogens. The first step in de novo ether lipid synthesis is mediated by the peroxisomal enzyme glyceronephosphate O-acyltransferase, which has a strict substrate specificity reacting only with the long-chain acyl-CoAs. The aim of this study was to determine the origin of these long-chain acyl-CoAs. To this end, we developed a sensitive method for the measurement of de novo ether phospholipid synthesis in cells and, by CRISPR-Cas9 genome editing, generated a series of HeLa cell lines with deficiencies of proteins involved in peroxisomal biogenesis, beta-oxidation, ether lipid synthesis, or metabolite transport. Our results show that the long-chain acyl-CoAs required for the first step of ether lipid synthesis can be imported from the cytosol by the peroxisomal ABCD proteins, in particular ABCD3. Furthermore, we show that these acyl-CoAs can be produced intraperoxisomally by chain shortening of CoA esters of very long-chain fatty acids via beta-oxidation. Our results demonstrate that peroxisomal beta-oxidation and ether lipid synthesis are intimately connected and that the peroxisomal ABC transporters play a crucial role in de novo ether lipid synthesis.     

High Sensitivity and Wide Linearity LC-MS/MS Method for Oxylipin Quantification in Multiple Biological Samples

Oxylipins are important biological regulators that have received extensive research attention. Due to the extremely low concentrations, large concentration variations, and high structural similarity of many oxylipins, the quantitative analysis of oxylipins in biological samples is always a great challenge. Here, we developed a liquid chromatography-tandem mass spectrometry-based method with high sensitivity, wide linearity, and acceptable resolution for quantitative profiling of oxylipins in multiple biological samples. A total of 104 oxylipins, some with a high risk of detection crosstalk, were well separated on a 150 mm column over 20 min. The method showed high sensitivity with lower limits of quantitation for 87 oxylipins, reaching 0.05–0.5 pg. Unexpectedly, we found that the linear range for 16, 18, and 17 oxylipins reached 10,000, 20,000, and 40,000 folds, respectively. Due to the high sensitivity, while reducing sample consumption to below half the volume of previous methods, 74, 78, and 59 low-abundance oxylipins, among which some were difficult to detect like lipoxins and resolvins, were well quantified in the tested mouse plasma, mouse liver, and human plasma samples, respectively. Additionally, we determined that analytes with multifarious concentrations of over a 1,000-fold difference could be well quantified simultaneously due to the wide linearity. In conclusion, most likely due to the instrumental advancement, this method effectively improves the quantitative sensitivity and linear range over existing methods, which will facilitate and advance the study of the physiological and pathophysiological functions of oxylipins.     

Evaluation of intestinal microbiota,short-chain fatty acids,and immunoglobulin a in diversion colitis

It is reported that an increase in aerobic bacteria, a lack of short-chain fatty acids (SCFAs), and immune disorders in the diverted colon are major causes of diversion colitis. However, the precise pathogenesis of this condition remains unclear. The aim of the present study was to examine the microbiota, intestinal SCFAs, and immunoglobulin A (IgA) in the diverted colon. Eight patients underwent operative procedures for colostomies. We assessed the diverted colon using endoscopy and obtained intestinal samples from the diverted colon and oral colon in these patients. We analyzed the microbiota and SCFAs of the intestinal samples. The bacterial communities were investigated using a 16S rRNA gene sequencing method. The microbiota demonstrated a change in the proportion of some species, especially Lactobacillus, which significantly decreased in the diverted colon at the genus level. We also showed that intestinal SCFA values were significantly decreased in the diverted colon. Furthermore, intestinal IgA levels were significantly increased in the diverted colon. This study was the first to show that intestinal SCFAs were significantly decreased and intestinal IgA was significantly increased in the diverted colon. Our data suggest that SCFAs affect the microbiota and may play an immunological role in diversion colitis.     

Tracking the Stability of Clinically Relevant Blood Plasma Proteins with Delta-S-Cys-Albumin—A Dilute-and-Shoot LC/MS-Based Marker of Specimen Exposure to Thawed Conditions

Biomolecular integrity can be compromised when blood plasma/serum (P/S) specimens are improperly handled. Compromised analytes can subsequently produce erroneous results—without any indication of having done so. We recently introduced an LC/MS-based marker of P/S exposure to thawed conditions called ΔS-Cys-Albumin which, aided by an established rate law, quantitatively tracks exposure of P/S to temperatures greater than their freezing point of ?30 °C. The purposes of this study were to (1) evaluate ΔS-Cys-Albumin baseline values in gastrointestinal cancer patients and cancer-free control donors, (2) empirically assess the kinetic profiles of ΔS-Cys-Albumin at 23 °C, 4 °C, and ?20 °C, and (3) empirically link ΔS-Cys-Albumin to the stability of clinically relevant proteins. ΔS-Cys-Albumin was measured at ≥ 9 different time points per exposure temperature in serum and K₂EDTA plasma samples from 24 separate donors in aliquots kept separately at 23 °C, 4 °C, and ?20 °C. Twenty-one clinically relevant plasma proteins were measured at four time points per temperature via a multiplexed immunoassay on the Luminex platform. Protein stability was assessed by mixed effects models. Coordinated shifts in stability between ΔS-Cys-Albumin and the unstable proteins were documented by repeated measures and Pearson correlations. Plasma ΔS-Cys-Albumin dropped from approximately 20% to under 5% within 96 h at 23 °C, 28 days at 4 °C, and 65 days at ?20 °C. On average, 22% of the 21 proteins significantly changed in apparent concentration at each exposure temperature (p < 0.0008 with >10% shift). A linear inverse relationship was found between the percentage of proteins destabilized and ΔS-Cys-Albumin (r = ?0.61; p < 0.0001)—regardless of the specific time/temperature of exposure. ΔS-Cys-Albumin tracks cumulative thawed-state exposure. These results now enable ΔS-Cys-Albumin to approximate the percentage of clinically relevant proteins that have been compromised by incidental plasma exposure to thawed-state conditions.     

Metabolic engineering of soybean seeds for enhanced vitamin E tocochromanol content and effects on oil antioxidant properties in polyunsaturated fatty acid-rich germplasm

Soybean seeds produce oil enriched in oxidatively unstable polyunsaturated fatty acids (PUFAs) and are also a potential biotechnological platform for synthesis of oils with nutritional omega-3 PUFAs. In this study, we engineered soybeans for seed-specific expression of a barley homogentisate geranylgeranyl transferase (HGGT) transgene alone and with a soybean γ-tocopherol methyltransferase (γ-TMT) transgene. Seeds for HGGT-expressing lines had 8- to 10-fold increases in total vitamin E tocochromanols, principally as tocotrienols, with little effect on seed oil or protein concentrations. Tocochromanols were primarily in δ- and γ-forms, which were shifted largely to α- and β-tocochromanols with γ-TMT co-expression. We tested whether oxidative stability of conventional or PUFA-enhanced soybean oil could be improved by metabolic engineering for increased vitamin E antioxidants. Selected lines were crossed with a stearidonic acid (SDA, 18:4^Δ6,9,12,15)-producing line, resulting in progeny with oil enriched in SDA and α- or γ-linoleic acid (ALA, 18:3^Δ9,12,15 or GLA, 18:3^Δ6,9,12), from transgene segregation. Oil extracted from HGGT-expressing lines had ≥6-fold increase in free radical scavenging activity compared to controls. However, the oxidative stability index of oil from vitamin E-enhanced lines was ~15% lower than that of oil from non-engineered seeds and nearly the same or modestly increased in oil from the GLA, ALA and SDA backgrounds relative to controls. These findings show that soybean is an effective platform for producing high levels of free-radical scavenging vitamin E antioxidants, but this trait may have negative effects on oxidative stability of conventional oil or only modest improvement of the oxidative stability of PUFA-enhanced oil.     

Delta-6 desaturase (Fads2) deficiency alters triacylglycerol/fatty acid cycling in murine white adipose tissue

The Δ-6 desaturase (D6D) enzyme is not only critical for the synthesis of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) from α-linolenic acid (ALA), but recent evidence suggests that it also plays a role in adipocyte lipid metabolism and body weight; however, the mechanisms remain largely unexplored. The goal of this study was to investigate if a D6D deficiency would inhibit triacylglycerol storage and alter lipolytic and lipogenic pathways in mouse white adipose tissue (WAT) depots due to a disruption in EPA and DHA production. Male C57BL/6J D6D knockout (KO) and wild-type (WT) mice were fed either a 7% w/w lard or flax (ALA rich) diet for 21 weeks. Energy expenditure, physical activity, and substrate utilization were measured with metabolic caging. Inguinal and epididymal WAT depots were analyzed for changes in tissue weight, fatty acid composition, adipocyte size, and markers of lipogenesis, lipolysis, and insulin signaling. KO mice had lower body weight, higher serum nonesterified fatty acids, smaller WAT depots, and reduced adipocyte size compared to WT mice without altered food intake, energy expenditure, or physical activity, regardless of the diet. Markers of lipogenesis and lipolysis were more highly expressed in KO mice compared to WT mice in both depots, regardless of the diet. These changes were concomitant with lower basal insulin signaling in WAT. Collectively, a D6D deficiency alters triacylglycerol/fatty acid cycling in WAT by promoting lipolysis and reducing fatty acid re-esterification, which may be partially attributed to a reduction in WAT insulin signaling.     

Isomeric lipid signatures reveal compartmentalized fatty acid metabolism in cancer

The cellular energy and biomass demands of cancer drive a complex dynamic between uptake of extracellular FAs and their de novo synthesis. Given that oxidation of de novo synthesized FAs for energy would result in net-energy loss, there is an implication that FAs from these two sources must have distinct metabolic fates; however, hitherto, all FAs have been considered part of a common pool. To probe potential metabolic partitioning of cellular FAs, cancer cells were supplemented with stable isotope-labeled FAs. Structural analysis of the resulting glycerophospholipids revealed that labeled FAs from uptake were largely incorporated to canonical (sn-) positions on the glycerol backbone. Surprisingly, labeled FA uptake also disrupted canonical isomer patterns of the unlabeled lipidome and induced repartitioning of n-3 and n-6 PUFAs into glycerophospholipid classes. These structural changes support the existence of differences in the metabolic fates of FAs derived from uptake or de novo sources and demonstrate unique signaling and remodeling behaviors usually hidden from conventional lipidomics.     

A high-throughput platform for the rapid screening of vitamin D status by direct infusion-MS/MS

Vitamin D is an important fat-soluble prohormone with pleiotropic effects on human health, such as immunomodulation of the innate and adaptive immune system. There is an unmet clinical need for a rapid screening platform for 25-hydroxyvitamin D (25OH-D) determination without chromatographic separation that offers better precision and accuracy than immunoassays. Here, we introduce a high-throughput method for assessing vitamin D status from blood specimens based on direct infusion-MS/MS (DI-MS/MS) following click derivatization using 2-nitrosopyridine. We developed an optimized liquid-phase extraction protocol to minimize ion suppression when directly infusing serum or plasma extracts via a capillary electrophoresis system for quantitative determination of 25OH-D. Acceptable reproducibility (mean coefficient of variation = 10.9%, n = 412), recovery (mean = 102% at 15, 30, and 45 nmol/l), and linearity (R² > 0.998) were achieved for 25OH-D with lower detection limits (limit of detection ～1.2 nmol/l, S/N ～ 3), greater throughput (～3 min/sample), and less bias than a commercial chemiluminescence immunoassay prone to batch effects. There was mutual agreement in 25OH-D concentrations from reference blood samples measured by DI-MS/MS as compared with LC-MS/MS (mean bias = 7.8%, n = 18). We also demonstrate that this method could reduce immunoassay misclassification of vitamin D deficiency in a cohort of critically ill children (n = 30). In conclusion, DI-MS/MS offers a viable alternative to LC-MS/MS for assessment of vitamin D status in support of large-scale studies in nutritional epidemiology as well as clinical trials to rapidly screen individual patients who may benefit from vitamin D supplementation.     

Geranylgeranoic acid,a bioactive and endogenous fatty acid in mammals: a review

Geranylgeranoic acid (GGA) was first reported in 1983 as one of the mevalonic acid metabolites, but its biological significance was not studied for a long time. Our research on the antitumor effects of retinoids led us to GGA, one of the acyclic retinoids that induce cell death in human hepatoma-derived cell lines. We were able to demonstrate the presence of endogenous GGA in various tissues of male rats, including the liver, testis, and cerebrum, by LC-MS/MS. Furthermore, the biosynthesis of GGA from mevalonic acid in mammals including humans was confirmed by isotopomer spectral analysis using ¹³C-labeled mevalonolactone and cultured hepatoma cells, and the involvement of hepatic monoamine oxidase B in the biosynthesis of GGA was also demonstrated. The biological activity of GGA was analyzed from the retinoid (differentiation induction) and nonretinoid (cell death induction) aspects, and in particular, the nonretinoid mechanism by which GGA induces cell death in hepatoma cells was found to involve pyroptosis via ER stress responses initiated by TLR4 signaling. In addition to these effects of GGA, we also describe the in vivo effects of GGA on reproduction. In this review, based mainly on our published papers, we have shown that hepatic monoamine oxidase B is involved in the biosynthesis of GGA and that GGA induces cell death in human hepatoma-derived cell lines by noncanonical pyroptosis, one of the mechanisms of sterile inflammatory cell death.     

Effects of dietary β-glucan and rice fermented on growth performance,fatty acids,and Newcastle disease immune response in turkey broilers

Poultry production has been developing in Vietnam with challenges of disease. Thus, feed additive should be investigated not only growth but also health enhancement. Here, we aimed to determine the effects of Saccharomyces cerevisiae-fermented rice (FR) and β-glucan on turkey’s growth performance, carcass characteristics, immune and fatty acid (FA) profiles. A total of 180 turkey chicks aged 1–56 days were randomly assigned to five sextuplicate groups and the birds had ad libitum feed and water access throughout the experiment. The five treatment groups were given the same diet with different proportions of FR and β-glucan. Broilers supplemented with 4% β-glucan and 4% FR presented the highest and second-highest growth performance, respectively. The 4% β-glucan and 4% FR treatments resulted in the highest carcass characteristic values without significantly affecting the breast or thigh meat pH or cooking loss. The 4% β-glucan and 4% FR treatments maximally increased the Newcastle disease (ND) antibody titers at 28, 42 and 56 days, respectively as well as thymus organ index. The foregoing treatments did not significantly affect the blood profiles relative to the control. However, the 4% FR treatment lowered the blood cholesterol levels (p > 0.05). The total FA profiles did not significantly differ among treatments. Nevertheless, both the β-glucan and FR treatments increased the MUFA levels compared to that of the control (p > 0.05). Hence, the dietary administration of 4% β-glucan and FR to turkey broilers could effectively improve their growth performance and immunity.     

Differential involvement of caspase-6 in amyloid-β-induced fragmentation of lamin A and B

Amyloid-β (Aβ), a peptide implicated in Alzheimer's disease, was shown to cause specific fragmentation of lamin proteins, which was mediated by an unidentified protease named nuclear scaffold protease (NSP) independently of caspase-6. Because caspase-6 is responsible for the fragmentation process in many other damage-induced apoptosis, here we further investigated possible involvement of caspase-6 in Aβ-induced lamin fragmentation under various conditions. We found that lamin A fragment generated by NSP (named fragment b) disappeared in cells incubated with Aβ42 for prolonged periods and this product was preserved by a caspase-6 inhibitor. Furthermore, caspase-6 could remove fragment b in nuclei isolated from Aβ42-treated cells (ANU). Lamin B in ANU was fragmented by caspase-6 only after treatment with an alkaline phosphatase. The caspase-mediated fragmentation of lamin B was also achieved with nuclei isolated from cells incubated with Aβ42 plus a Cdk5 inhibitor. The results indicate that Aβ42 induces NSP-mediated fragmentation of lamin A and the following removal process of fragment b by caspase-6 and an Aβ-induced phosphorylation prevents the fragmentation of lamin B by caspase-6. The pathway leading to lamin protein fragmentation in this investigation appears to be specific for Aβ and thus the data will provide novel insights into the toxicity of the peptide.     

Thyroid Immune-Related Adverse Events in Patients with Cancer Treated with anti-PD1/anti-CTLA4 Immune Checkpoint Inhibitor Combination: Clinical Course and Outcomes

ObjectiveThyroid immune-related adverse events (irAEs) have been reported to have prognostic significance among patients with cancer treated with anti-programmed cell death-1 (PD1) and anti-programmed death-ligand 1 monotherapies. We evaluated the clinical course and predictors of thyroid irAEs in relation to outcomes of patients with advanced cancer treated with combination anti-PD1/anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA4).MethodsWe conducted a regional study and identified patients with advanced cancer who received ≥1 cycle of combination anti-PD1/anti-CTLA4 between 2015 and 2019 in Hong Kong. Thyroid function tests (TFTs) were monitored every 3 weeks. Thyroid irAE was defined by ≥2 abnormal TFTs after initiation of combination anti-PD1/anti-CTLA4 in the absence of other causes.ResultsOne hundred and three patients were included (median age: 59 years; 71.8% men). About 45% had prior anti-PD1 exposure. Upon median follow-up of 6.8 months, 17 patients (16.5%) developed thyroid irAEs, where 6 initially presented with thyrotoxicosis (overt, n = 4; subclinical, n = 2) and 11 with hypothyroidism (overt, n = 2; subclinical, n = 9). Eventually, 10 patients (58.8%) required continuous thyroxine replacement. Systemic steroid was not required in all cases. Prior anti-PD1 exposure (odds ratio, 3.67; 95% CI, 1.19–11.4; P = .024) independently predicted thyroid irAEs. Multivariable Cox regression analysis revealed that occurrence of thyroid irAEs was independently associated with better overall survival (adjusted hazard ratio, 0.34; 95% CI, 0.17–0.71; P = .004).ConclusionThyroid irAEs are common in routine clinical practice among patients with advanced cancer treated with anti-PD1/anti-CTLA4 combination and might have potential prognostic significance. Regular TFT monitoring is advised for timely treatment of thyroid irAEs to prevent potential morbidities.     

Cold-induced phase separation for the simple and reliable extraction of sex hormones for subsequent LC-MS/MS analysis

Sex hormones, including androgens, estrogens, and progestogens, are important biomarkers for various diseases. Quantification of sex hormones is typically conducted by LC-MS/MS. At present, most methods require liquid-liquid extraction or solid phase extraction for sample preparation. However, these pretreatments are prone to compromise LC-MS/MS throughput. To improve on the current standard practices, we investigated cold-induced phase separation for sex hormone extraction. After protein precipitation with acetonitrile and adjusting the solution constitution with water, samples were stored at ?30°C for 10 min to generate two distinct phases: an acetonitrile-rich layer on top of a water-rich layer. During this process, the hydrophobic sex hormones spontaneously separate into the upper layer. This simple and reliable cold-induced phase separation-based LC-MS/MS methodology was used here to simultaneously detect estrone, estradiol, estriol, testosterone, androstenedione, dehydroepiandrosterone, progesterone, and 17-hydroxyprogesterone in serum. Validation of this method indicated satisfactory performance, including acceptable linearity, accuracy, precision, and tractability. Compared with the mainstream liquid-liquid extraction-based method, this new method exhibits significant progress in throughput, which shortens the time cost of sample preparation from 90 to 40 min. We propose that this method can be an excellent alternative for sex hormone analysis in routine clinical laboratories.     

Insulin dysregulation drives mitochondrial cholesterol metabolite accumulation: initiating hepatic toxicity in nonalcoholic fatty liver disease

CYP7B1 catalyzes mitochondria-derived cholesterol metabolites such as (25R)26-hydroxycholesterol (26HC) and 3β-hydroxy-5-cholesten-(25R)26-oic acid (3βHCA) and facilitates their conversion to bile acids. Disruption of 26HC/3βHCA metabolism in the absence of CYP7B1 leads to neonatal liver failure. Disrupted 26HC/3βHCA metabolism with reduced hepatic CYP7B1 expression is also found in nonalcoholic steatohepatitis (NASH). The current study aimed to understand the regulatory mechanism of mitochondrial cholesterol metabolites and their contribution to onset of NASH. We used Cyp7b1^−/− mice fed a normal diet (ND), Western diet (WD), or high-cholesterol diet (HCD). Serum and liver cholesterol metabolites as well as hepatic gene expressions were comprehensively analyzed. Interestingly, 26HC/3βHCA levels were maintained at basal levels in ND-fed Cyp7b1^−/− mice livers by the reduced cholesterol transport to mitochondria, and the upregulated glucuronidation and sulfation. However, WD-fed Cyp7b1^−/− mice developed insulin resistance (IR) with subsequent 26HC/3βHCA accumulation due to overwhelmed glucuronidation/sulfation with facilitated mitochondrial cholesterol transport. Meanwhile, Cyp7b1^−/− mice fed an HCD did not develop IR or subsequent evidence of liver toxicity. HCD-fed mice livers revealed marked cholesterol accumulation but no 26HC/3βHCA accumulation. The results suggest 26HC/3βHCA-induced cytotoxicity occurs when increased cholesterol transport into mitochondria is coupled to decreased 26HC/3βHCA metabolism driven with IR. Supportive evidence for cholesterol metabolite-driven hepatotoxicity is provided in a diet-induced nonalcoholic fatty liver mouse model and by human specimen analyses. This study uncovers an insulin-mediated regulatory pathway that drives the formation and accumulation of toxic cholesterol metabolites within the hepatocyte mitochondria, mechanistically connecting IR to cholesterol metabolite-induced hepatocyte toxicity which drives nonalcoholic fatty liver disease.     

Sensitive UHPLC-MS/MS quantification method for 4β- and 4α-hydroxycholesterol in plasma for accurate CYP3A phenotyping

4β-Hydroxycholesterol (4β-OHC) is formed by Cytochrome P450 (CYP)3A and has drawn attention as an endogenous phenotyping probe for CYP3A activity. However, 4β-OHC is also increased by cholesterol autooxidation occurring in vitro due to dysregulated storage and in vivo by oxidative stress or inflammation, independent of CYP3A activity. 4α-hydroxycholesterol (4α-OHC), a stereoisomer of 4β-OHC, is also formed via autooxidation of cholesterol, not by CYP3A, and thus may have clinical potential in reflecting the state of cholesterol autooxidation. In this study, we establish a sensitive method for simultaneous quantification of 4β-OHC and 4α-OHC in human plasma using ultra-high performance liquid chromatography coupled to tandem mass spectrometry. Plasma samples were prepared by saponification, two-step liquid-liquid extraction, and derivatization using picolinic acid. Intense [M+H]+ signals for 4β-OHC and 4α-OHC di-picolinyl esters were monitored using electrospray ionization. The assay fulfilled the requirements of the US Food and Drug Administration guidance for bioanalytical method validation, with a lower limit of quantification of 0.5 ng/ml for both 4β-OHC and 4α-OHC. Apparent recovery rates from human plasma ranged from 88.2% to 101.5% for 4β-OHC, and 91.8% to 114.9% for 4α-OHC. Additionally, matrix effects varied between 86.2% and 117.6% for 4β-OHC and between 89.5% and 116.9% for 4α-OHC. Plasma 4β-OHC and 4α-OHC concentrations in healthy volunteers, stage 3–5 chronic kidney disease (CKD) patients, and stage 5D CKD patients as measured by the validated assay were within the calibration ranges in all samples. We propose this novel quantification method may contribute to accurate evaluation of in vivo CYP3A activity.     

2-fluoro-1-methylpyridinium p-toluene sulfonate: a new LC-MS/MS derivatization reagent for vitamin D metabolites

Vitamin D analysis by MS faces several analytical challenges, including inefficient ionization, nonspecific fragmentation, interferences from epimers, isomers, and isobars, as well as very low concentration levels. In this study, we used 2-fluoro-1-methylpyridinium (FMP) p-toluene sulfonate for derivatization of vitamin D3 metabolites to increase detection sensitivity and allow for full chromatographic separation of vitamin D isomers and epimers. UHPLC-MS/MS was used for measurement of five vitamin D3 metabolites in human serum. Compared with Amplifex and 4-phenyl-1,2,4-triazolin-3,5-dion, the FMP p-toluene sulfonate reaction required less time to be performed. The method was optimized and validated to ensure accuracy, precision, and reliability. In-house and commercial quality control samples were used to assure the quality of the results for 25-hydroxyvitamin D3. The method showed very good linearity and intraday and interday accuracy and precision; coefficients of determination (r²) ranged between 0.9977 and 0.9992, relative recovery from 95 to 111%, and coefficient of variation from 0.9 to 11.3. Stability tests showed that the extracted derivatized serum samples were stable for 24 h after storage at −20°C; 24,25-dihydroxyvitamin D3 and 1,25-dihydroxyvitamin D3-FMP derivatives were stable for 1 week at −80°C. The method was applied to samples of healthy individuals for quantitative determination of vitamin D3, the two epimers of 25-hydroxyvitamin D3 and 24,25-dihydroxyvitamin D3.     

MS2Rescore: Data-Driven Rescoring Dramatically Boosts Immunopeptide Identification Rates

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