Performance Evaluation of TaqMan SARS-CoV-2, Flu A/B, RSV RT-PCR Multiplex Assay for the Detection of Respiratory Viruses.

Purpose: To detect and differentiate co-infection with influenza and respiratory syncytial virus during the COVID pandemic, a rapid method that can detect multiple pathogens in a single test is a significant diagnostic advance to analyze the outcomes and clinical implications of co-infection. Therefore, we validated and evaluated the performance characteristics of TaqMan SARS-CoV-2, Flu A/B, RSV RT-PCR multiplex assay for the detection of SARS-CoV-2, Flu A/B, and RSV using nasopharyngeal and saliva samples. Materials and Methods: The method validation was performed by using culture fluids of Influenza A virus (H3N2) (A/Wisconsin/67/2005), Influenza B virus (B/Virginia/ATCC4/2009), RSV A2 cpts-248, SARS-CoV-2 (USA-WA1/2020) and quantitative RNA controls of Influenza A virus (H1N1) strain A/PR/8/34 (VR-95DQ), RSV A2 (VR-1540DQ) and SARS-CoV-2 (MN908947.3 Wuhan-Hu-1) from ATCC and Zeptometrix, NY, USA. A total of 110 nasopharyngeal specimens and 70 saliva samples were used for the SARS-CoV-2 detection, and a total of 70 nasopharyngeal specimens were used for Influenza and RSV detection. Total RNA was extracted from all the samples and multiplex PCR was performed using TaqMan SARS-CoV-2, Flu A/B, RSV RT-PCR multiplex assay. The assay was used for SARS-CoV-2 variant (B.1.1.7_601443, B.1.617.1_1662307, P.1_792683, B.1.351_678597, B.1.1.529/BA.1). Results: Validation controls showed accurate and precise results. The correlation study found the accuracy of 96.38 to 100% (95% CI) in nasopharyngeal and 94.87 to 100% (95% CI) in saliva for SARS-CoV-2 and 91.1 to 100% (95% CI) for both Influenza A/B and RSV. The diagnostic efficiency of this assay was not affected by SARS-CoV-2 variant, including Omicron. Conclusion: The TaqMan SARS-CoV-2, Flu A/B, RSV RT-PCR multiplex assay is a rapid method to detect and differentiate SAR-CoV-2, Flu A and B, and RSV in nasopharyngeal and saliva samples. It has a significant role in the diagnosis and management of respiratory illnesses and the clinical implications of co-infection.

Oxidative Stress and Lipid Dysregulation in Lipid Droplets: A Connection to Chronic Kidney Disease Revealed in Human Kidney Cells.

Chronic kidney disease (CKD), which is defined as a condition causing the gradual loss of kidney function, shows renal lipid droplet (LD) accumulation that is associated with oxidative damage. There is a possibility that an LD abnormality in quality plays a role in CKD development. This study aimed to explore the chemical composition of LDs that are induced in human kidney cells during exposure to free fatty acids as an LD source and oxidized lipoproteins as oxidative stress. The LDs were aspirated directly from cells using nanotips, followed by in-tip microextraction, and the LD lipidomic profiling was conducted using nanoelectrospray mass spectrometry. As a result, the free fatty acids increased the LD lipid content and, at the same time, changed their composition significantly. The oxidized lipoproteins caused distorted proportions of intact lipids, such as triacylglycerols (TG), phosphatidylcholines (PC), phosphatidylethanolamines (PE), and cholesteryl esters (CE). Notably, the oxidized lipids, including the hydroperoxides of TG, PC, and PE, exhibited significant elevations in dose-dependent manners. Furthermore, the dysregulation of intact lipids was paralleled with the accumulation of lipid hydroperoxides. The present study has revealed that the oxidation of lipids and the dysregulation of the lipid metabolism coexisted in LDs in the kidney cells, which has provided a potential new target for diagnosis and new insights into CKD.

SARS-CoV-2 Variants Detection Using TaqMan SARS-CoV-2 Mutation Panel Molecular Genotyping Assays.

PURPOSE: For rapid detection and tracking of SARS-CoV-2, a simple screening method alternative to laborious and expensive sequencing is highly desirable. Here, we evaluated performance characteristics of TaqMan SARS-CoV-2 mutation panel genotyping molecular assay for detection of most common reported SARS-CoV-2 variants using specific RT-PCR assays targeting single nucleotide polymorphisms (SNP). PATIENTS AND METHODS: A total of 150 SARS-CoV-2 positive samples from March to July were included for this study. In addition, five controls comprised of synthetic RNA B.1.1.7_601443, B.1.351_678597, P.1_792683, B.1.617.1_1662307 and MN908947.3-Wuhan-hu-1 from Twist bioscience and B.1.1.7 (England/204820464/2020) and B.1.351 (South Africa/KRISP-K005325/2020) from Zeptometrix, NY, USA were used for validation. Total RNA from specimens was extracted using Omega Bio-Tek Mag-Bind Viral RNA Xpress Extraction Kit and tested for known SARS-CoV2 variants using ThermoFisher TaqMan SARS-CoV-2 mutation panel molecular assay on the QuantStudio 12K Flex. Nine representative samples have been compared with sequencing. Data were analyzed by genotype calling using QuantStudio™ design and analysis software v 2.5 with the genotyping analysis module. RESULTS: All validation controls were tested in triplicate and repeated in singlet on three different days and all reported variants were matched as expected. Out of 150 SARS-CoV-2 positive specimens, 69 (46%) were B.1.617.2, 49 (32.7%) were B.1.1.7, P.1 and P.2 were 4 (2.7%) each and B.1.351 and B.1.427/B.1429 were 2 (1.3%) each. Three (2%) were B.1.526, and 17 (11.3%) have a mutation in D614G. Genotyping results from the present study showing B.1.617.2, B.1.1.7, and B.1.526 variants and their mutation genes were concordant with sequencing results. CONCLUSION: Our study indicates that TaqMan SARS-CoV-2 mutation panel molecular genotyping assays detect and differentiate all published common variants B.1.617.2 (Delta), B.1.1.7 (Alpha), B.1.526 (Iota), B.1.351 (Beta), P.1 (Gamma), P.2 (Zeta), B.1.617.1 (Kappa) and B.1.427/B.1.429 (Epsilon) that can be used for surveillance and epidemic control and prevention.

Comparison of dimension reduction methods on fatty acids food source study.

Serum fatty acids (FAs) exist in the four lipid fractions of triglycerides (TGs), phospholipids (PLs), cholesteryl esters (CEs) and free fatty acids (FFAs). Total fatty acids (TFAs) indicate the sum of FAs in them. In this study, four statistical analysis methods, which are independent component analysis (ICA), factor analysis, common principal component analysis (CPCA) and principal component analysis (PCA), were conducted to uncover food sources of FAs among the four lipid fractions (CE, FFA, and TG + PL). Among the methods, ICA provided the most suggestive results. To distinguish the animal fat intake from endogenous fatty acids, FFA variables in ICA and factor analysis were studied. ICA provided more distinct suggestions of FA food sources (endogenous, plant oil intake, animal fat intake, and fish oil intake) than factor analysis. Moreover, ICA was discovered as a new approach to distinguish animal FAs from endogenous FAs, which will have an impact on epidemiological studies. In addition, the correlation coefficients between a published dataset of food FA compositions and the loading values obtained in the present ICA study suggested specific foods as serum FA sources. In conclusion, we found that ICA is a useful tool to uncover food sources of serum FAs.

HPLC with spectrophotometric or mass spectrometric detection for quantifying very-long chain fatty acids in human plasma and its association with cardiac risk factors.

BACKGROUND: We developed and compared two liquid chromatography methods, one with UV/Visible spectrophotometric detection (HPLC) and the other with mass spectrometric detection (LC-MS), for quantifying very-long chain fatty acids (VLCFA) in human plasma. Association of VLCFA with various cardiovascular risk factors were evaluated. METHOD: Fasting blood samples were collected from 541 human volunteers (242 men and 299 women; mean age ±SD, 58.9 ± 12.4 years), including 429 and 112 individuals with and without hypertriglyceridemia, respectively. Esterified VLCFA were saponified and derivatized with 2-nitrophenylhydrazine. Separation of VLCFA species was achieved with C4 Mightysil column (HPLC) and Ascentis Express Phenyl-Hexyl column (LC-MS) followed by spectrophotometric and selected-reaction monitoring mode of mass spectrometric detection, respectively. RESULTS: The HPLC assay of VLCFA was precise with intra-assay imprecision of 2.5% to 6.9% and inter-assay imprecision of 3.2% to 9.5%. Moreover, there was an excellent correlation (r > 0.96) between HPLC and LC-MS methods. The 95 percentile reference intervals (RI; upper limit) of VLCFA were determined to be 41.3 µmol/L in healthy volunteers. Plasma VLCFA were significantly correlated with triglycerides (Spearman's ρ = 0.306, P < 0.001) and total cholesterol (Spearman's ρ = 0.251, P < 0.001). All species of VLCFA were significantly elevated in hypertriglyceridaemic individuals compared with control. CONCLUSION: We established LC-based assays of VLCFA with either spectrophotometry or mass spectrometry as a detection system. Hypertriglyceridaemia is significantly associated with elevated concentration of each species of VLCFA.

Evaluation of Analytical Performance of Seven Rapid Antigen Detection Kits for Detection of SARS-CoV-2 Virus.

BACKGROUND: Early diagnosis of the novel coronavirus disease of 2019 (COVID-19) in asymptomatic and symptomatic patients is crucial to identify infectious individuals and to help prevent the spread of the virus in the community. Several assays have been developed and are in use in today's clinical practice. These assays vary in their analytical and clinical performance. For an accurate diagnosis, medical professionals must become more familiar with the test's utility to select the most appropriate test. This study aims to evaluate the analytical performance of rapid antigen tests used for the detection of SARS-CoV-2 viral antigen compared to RT-PCR SARS-CoV-2 molecular assay. METHODS: Oropharyngeal swab specimens from five COVID-19 patients were tested by seven rapid antigen tests developed by different IVD companies. RT-PCR to detect specific RNA fragments of SARS-CoV-2 was used as a confirmatory test. The cycle threshold (Ct) value, which often reflects viral load, in these specimens ranged from 15 to 35. For the analytical evaluation, extraction fluid of each antigen kit was spiked with attenuated ATCC virus at different concentrations ranging from 4.6x104/mL to 7.5x105/mL and tested with antigen testing kits. RESULTS: Out of five confirmed positive SARS-CoV-2 specimens by RT-PCR, only one sample showed a positive result by one of the seven evaluated antigen testing kits. The positive result was observed in the specimen with a Ct value of 15. All other evaluated rapid tests were negative for all five positive specimens. This was further confirmed with the spiking study using ATCC attenuated virus, where extraction fluid of each rapid test was spiked with concentrations ranging from 4.6x104/mL to 7.5x105/mL. None of these spiked specimens showed positive results, indicating very low sensitivity of these antigen kits. CONCLUSION: This comparison study shows that rapid antigen tests are less sensitive than RT-PCR tests and are not reliable tests for testing asymptomatic patients, who often carry low viral load. Analytical performance of rapid antigen tests should be thoroughly evaluated before implementing it at clinical decision level.

A phase II, open-label clinical trial on the combination therapy with medium-chain triglycerides and ghrelin in patients with chronic obstructive pulmonary disease.

The aim of this study was to investigate the effect of activated ghrelin with dietary octanoic acids or medium-chain triglyceride (MCT) administration to underweight patient with chronic obstructive pulmonary disease (COPD). Eleven severe and very severe COPD patients received a 5-day treatment with edible MCT. Sequentially, 10 patients received a 3-week combination treatment with MCT and intravenous acyl ghrelin. Five-day MCT treatment increased endogenous acyl ghrelin (p = 0.0049), but the total ghrelin level was unchanged. MCT-ghrelin combination treatment improved the peak oxygen uptake (p = 0.0120) during whole treatment course. This effect was attributed to the resultant improvements in cardiac function by O2 pulse, and to the difference between inspired and expired oxygen concentration rather than minute ventilation. Addition of dietary MCT to ghrelin treatment improved the aerobic capacity of underweight COPD patients, likely by mechanisms of increased O2 delivery through improvements in primary cardiocirculatory and muscular crosstalk.

Development of a simultaneous quantitation for short-, medium-, long-, and very long-chain fatty acids in human plasma by 2-nitrophenylhydrazine-derivatization and liquid chromatography-tandem mass spectrometry.

Fatty acids (FA) have been important in clinical diagnosis for long, which makes the increasing need for a fast, reliable, and economic approach to determine FA of short-, medium-, long-, and very long-chain by widely available equipment and with high-throughput capacity. In the present work, 2­nitrophenylhydrazine derivatization coupling with LC-MS/MS detection was utilized to simultaneously quantitate 18 FAs ranging from C4 to C26 in human plasma. The sample preparation protocol was optimized and extracting with diethyl ether­potassium phosphate buffer twice was found as the highest efficiency along with economic feasibility. Under the optimized conditions, all the FA showed excellent linearity (R2 > 0.999 for each), sufficient sensitivity (LOD 0.2-330 fmol and LOQ 2.3-660 fmol for all), favorable accuracy (recovery ranged from 98.1 ±â€¯3.6% to 104.9 ±â€¯5.5% with coefficient of variation no >8.6% for all), and negligible matrix effect. In the clinical application on 30 healthy subjects, compared with the previous HPLC-UV method, the developed method showed high reliability, as well as reduced time and reagent costs. The established method showed the potential to apply to not only diagnostic practice, but also nutritional and epidemiological studies.

Triglyceride deposit cardiomyovasculopathy: a rare cardiovascular disorder.

Triglyceride deposit cardiomyovasculopathy (TGCV) is a phenotype primarily reported in patients carrying genetic mutations in PNPLA2 encoding adipose triglyceride lipase (ATGL) which releases long chain fatty acid (LCFA) as a major energy source by the intracellular TG hydrolysis. These patients suffered from intractable heart failure requiring cardiac transplantation. Moreover, we identified TGCV patients without PNPLA2 mutations based on pathological and clinical studies. We provided the diagnostic criteria, in which TGCV with and without PNPLA2 mutations were designated as primary TGCV (P-TGCV) and idiopathic TGCV (I-TGCV), respectively. We hereby report clinical profiles of TGCV patients. Between 2014 and 2018, 7 P-TGCV and 18 I-TGCV Japanese patients have been registered in the International Registry. Patients with I-TGCV, of which etiologies and causes are not known yet, suffered from adult-onset severe heart disease, including heart failure and coronary artery disease, associated with a marked reduction in ATGL activity and myocardial washout rate of LCFA tracer, as similar to those with P-TGCV. The present first registry-based study showed that TGCV is an intractable, at least at the moment, and heterogeneous cardiovascular disorder.

Determination of total, free and esterified short-chain fatty acid in human serum by liquid chromatography-mass spectrometry.

BACKGROUND: Short-chain fatty acids are primarily absorbed through the portal vein during lipid digestion, which is utilized as the energy source, as well as prevent type 2 diabetes and some cancers. However, reports on the determination of these short-chain fatty acids in human serum are limited. METHODS: Blood samples from human subjects ( n = 547, male/female = 246/301, age 58.85 ± 12.57) were collected. Saponification was applied to obtain total fatty acid. After derivatization by 2-nitrophenylhydrazine, fatty acid 4:0 and fatty acid 6:0 were measured by liquid chromatography-mass spectrometry. RESULTS: The developed method exhibited good linearity (R2 = 0.9996 for both). All the coefficients of variation of reproducibility and accuracy for fatty acid 4:0 and fatty acid 6:0 ranged 3.0%-6.1%, with the average recoveries of 87.8%-102.4% and 92.2%-98.2%, respectively. In all the samples, the concentration of fatty acid 4:0 (162.4 ± 76.4 µmol/L) was significantly higher than fatty acid 6:0 (2.0 ± 2.5 µmol/L, P < 0.001). Furthermore, the esterified form was predominant in both fatty acid 4:0 and fatty acid 6:0 (98.2% and 82.4% of total fatty acids, respectively). Besides, short-chain fatty acids showed no significant differences with regard to sex or age differences. CONCLUSION: This developed liquid chromatography-mass spectrometry method is convenient and reliable, which might be useful for monitoring the variations of short-chain fatty acids in blood.

Lipidomic Profiling on Oxidized Phospholipids in Type 2 Diabetes Mellitus Model Zebrafish.

Type 2 diabetes mellitus is a serious metabolic disorder in the world. Oxidative stress, as a key role on the pathogenesis of diabetes, also results in the oxidation of phospholipids. However, studies on phospholipid oxidation in diabetes, especially directly focusing on oxidized and degraded phospholipid species, are quite limited. In this study, phospholipid profiles of diabetic zebrafish plasma were characterized by LC-HRMS and MS/MS, and the total amounts of each lipid class were compared. Furthermore, the key molecular species as biomarkers in distinguishing control and diabetic samples were investigated by orthogonal partial least squares discriminant analysis. Among the identified 114 phospholipid species in total, there were 11 hydroperoxides, 7 aldehydes, and 19 lysophospholipids found significantly elevated along with the increasing blood glucose, which were known as oxidation or degradation products. Furthermore, lysophosphatidylcholine 20:5 and lysophosphatidylcholine 22:6 were assessed as potential biomarkers in diabetic zebrafish. The current work would not only help to gain further insights into diabetes, but also contribute to find new clinical parameters for the screening of the promising antioxidant agents for its therapies.

Microwave-assisted Derivatization of Fatty Acids for Its Measurement in Milk Using High-Performance Liquid Chromatography.

Fatty acid (FA) profiling of milk has important applications in human health and nutrition. Conventional methods for the saponification and derivatization of FA are time-consuming and laborious. We aimed to develop a simple, rapid, and economical method for the determination of FA in milk. We applied a beneficial approach of microwave-assisted saponification (MAS) of milk fats and microwave-assisted derivatization (MAD) of FA to its hydrazides, integrated with HPLC-based analysis. The optimal conditions for MAS and MAD were determined. Microwave irradiation significantly reduced the sample preparation time from 80 min in the conventional method to less than 3 min. We used three internal standards for the measurement of short-, medium- and long-chain FA. The proposed method showed satisfactory analytical sensitivity, recovery and reproducibility. There was a significant correlation in the milk FA concentrations between the proposed and conventional methods. Being quick, economic, and convenient, the proposed method for the milk FA measurement can be substitute for the convention method.

In vitro biofilm formation by Staphylococcus aureus isolated from wounds of hospital-admitted patients and their association with antimicrobial resistance.

INTRODUCTION: Staphylococcus aureus including methicillin-resistant S. aureus (MRSA) has the propensity to form biofilms, and causes significant mortality and morbidity in the patients with wounds. Our aim was to study the in vitro biofilm-forming ability of S. aureus isolated from wounds of hospitalized patients and their association with antimicrobial resistance. MATERIALS AND METHODS: Forty-three clinical isolates of S. aureus were obtained from 150 pus samples using standard microbiological techniques. Biofilm formation in these isolates was detected by tissue culture plate (TCP) method and tube adherence method (TM). Antimicrobial susceptibility test was performed using the modified Kirby-Bauer disk diffusion method as per Clinical and Laboratory Standards Institute guidelines. MRSA was detected using the cefoxitin disk test. RESULTS: Biofilm formation was observed in 30 (69.8%) and 28 (65.1%) isolates of S. aureus via TCP method and TM, respectively. Biofilm-producing S. aureus exhibited a higher incidence of antimicrobial resistance when compared with the biofilm nonproducers (P<0.05). Importantly, 86.7% of biofilm-producing S. aureus were multidrug resistant (MDR), whereas all the biofilm nonproducers were non-MDR (P<0.05). Large proportions (43.3%) of biofilm producers were identified as MRSA; however, none of the biofilm nonproducers were found to be MRSA (P<0.05). CONCLUSION: Both the in vitro methods showed that S. aureus isolated from wound infection of hospitalized patients have high degree of biofilm-forming ability. Biofilm-producing strains have very high tendency to exhibit antimicrobial resistance, multidrug resistance and methicillin resistance. Regular surveillance of biofilm formation by S. aureus and their antimicrobial resistance profile may lead to the early treatment of the wound infection.

Change in Plasma Total, Esterified and Non-esterified Capric Acid Concentrations during a Short-term Oral Administration of Synthetic Tricaprin in Dogs.

We studied change in the plasma total, esterified and non-esterified capric acid (FA10:0) and its effect on longer fatty acid concentrations during the short-term oral administration of synthetic tricaprin in dogs. We administered 150 and 1500 mg tricaprin/kg body weight per day orally to dogs for 7 consecutive days. Blood samples were collected at 0, 0.5, 1, 2, 4, 8, and 24 h on the 1st and 7th days for measuring the total-, esterified- and non-esterified-FA10:0. The total-FA10:0 concentration increased in a dose-dependent manner, reaching a peak at 1 h on the 1st day and at 2 to 4 h on the 7th day; it then mostly disappeared within 24 h. The mean esterified FA10:0 concentration was found be 75.5 and 60.3% of total-FA10:0 in dogs fed 150 and 1500 mg of tricaprin/kg body weight, respectively. The plasma level of FA10:0 depends on the duration and dose of tricaprin administration, but are rapidly cleared from circulation within several hours.

Absolute quantification of cholesteryl esters using liquid chromatography-tandem mass spectrometry uncovers novel diagnostic potential of urinary sediment.

BACKGROUND: Urine has been utilized as a source of biomarkers in renal disease. However, urinary lipids have not attracted much attention so far. Here we studied urinary cholesteryl ester (CE) and its relevance in renal disease. METHODS: Quantitative analysis of CE molecular species in serum, urinary supernatant, and urinary sediment from patients with renal disease (N=64) and non-renal disease (N=23) was carried out using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and deuterated CEs as internal standards. RESULTS: Validation study showed good precision and accuracy of LC-MS/MS. Many CE species were detected in the urinary sediment and supernatant in the renal disease group, whereas only a few CE species were detected in the other group. In the renal disease group, the sum of the concentrations of all CE species showed a significant correlation between the sediment and the supernatant from urinary samples (r=0.876, p<0.001); however, the composition of CEs was significantly different between them. Further, the composition of CEs of the supernatant was similar to that of the serum. CONCLUSIONS: Our LC-MS/MS analysis uncovered a distinct CE profile in urinary sediment from patients with renal disease, suggesting a possible contribution of CEs in urothelial cells to the development of renal disease.

A fatty acid profiling method using liquid chromatography-high resolution mass spectrometry for improvement of assisted reproductive technology.

BACKGROUND: Liquid chromatography-high resolution mass spectrometry (LC-HRMS) can be useful to improve in vitro fertilization (IVF). This study aims to find out an association between embryonic growth and embryonic uptake of free fatty acid (FFA) from culture media by using LC-HRMS. METHODS: Embryos (n=55) from 15 couples undergoing IVF were studied. An embryo was cultivated for up to 6 days in a 20 µl-medium drop under mineral oil, and classified by a morphological grading system into the good-growth group (n=32; good quality blastocysts) and the poor-growth group (n=23; non-blastocysts). The control study was set up without embryo. Extracted ion chromatogram of FFAs was collected in negative-ion mode for each medium sample obtained after use. RESULTS: The percent change from control to sample in mass area for docosahexaenoic acid showed a decrease in the good-growth group than that in the poor-growth group (p<0.05). Decrease in %change of docosahexaenoic acid might indicate proper embryonic growth. Similar but insignificant change was observed for other essential FFAs, but not for non-essential FFAs. CONCLUSION: The proposed metabolomic approach using LC-HRMS might be a powerful tool for non-invasive evaluation of embryonic growth.

Development of homogeneous assay for simultaneous measurement of apoE-deficient, apoE-containing, and total HDL-cholesterol.

BACKGROUND: Pathophysiological role for high-density lipoprotein (HDL) subclasses remains to be elucidated. Homogeneous assay for simultaneous measurements of apoE-deficient HDL-cholesterol (HDL-C), apoE-containing HDL-C, and total HDL-C is desired, because apoE plays important roles in lipid metabolism. METHODS: The proposed assay consists of a primary reaction to remove non-HDL-C, a secondary reaction to measure apoE-deficient HDL-C, and a tertiary reaction to measure apoE-containing HDL-C. The assay is completed within 10 min. For control study, 13% polyethylene glycol precipitation assay and phosphotungstate-dextran sulfate-magnesium precipitation assay were carried out. RESULTS: Good correlations between the control assays and the proposed assay was obtained in serum samples from patients without liver disease (n=33): r=0.987, 0.957, and 0.991 for apoE-deficient, apoE-containing, and total HDL-C, respectively. ApoE-containing HDL-C by the proposed method in healthy individuals (n=12) and patients with hyper-HDL-cholesterolemia (n=5) were 0.11±0.03 and 0.26±0.05 mmol/l (4.1±1.3 and 10.1±2.0 mg/dl), respectively. ApoE-containing HDL-C increased rapidly at >2.59 mmol/l (100 mg/dl) of total HDL-C, suggesting a unique regulating mechanism of apoE-containing HDL-C. CONCLUSIONS: The established homogeneous assay might be useful for clinical and epidemiological studies on apoE-deficient and apoE-containing HDL subclasses.

Synthesis of (2ß,3α,6-²H3cholesteryl linoleate and cholesteryl oleate as internal standards for mass spectrometry.

The accurate analysis of trace component in complex biological matrices requires the use of reliable standards. For liquid chromatography/mass spectrometry analysis, the stable isotope-labeled derivatives of the analyte molecules are the most appropriate internal standards. We report here the synthesis of (2ß,3α,6-(2)H3)cholesteryl linoleate and oleate containing three non-exchangeable deuterium in the steroid ring. The principal reactions used were: (1) trans diaxial opening of 2α,3α-epoxy-6-oxo-5α-cholestane with LiAlD4 and subsequent oxidation of the resulting (2ß,6α-(2)H2)-3α,6ß-diol with Jones' reagent, followed by reduction of the resulting (2ß-(2)H)-3,6-dione with NaBD4 leading to the (2ß,3α,6α-(2)H3)-3ß,6ß-dihydroxy-5α-cholestane, (2) selective protection of the 3ß-hydroxy group as the tert-butyldimethylsilyl ether, (3) dehydration of the 6ß-hydroxy group with POCl3 and removal of tert-butyldimethylsilyloxy groups with 5M HCl in acetone, and (4) esterification of the resultant (2ß,3α,6-(2)H3)cholesterol with linoleic and oleic acids using 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide. The isotopic purity was found to be satisfactory by mass spectrometry, and nuclear magnetic resonance properties of the new compounds were tabulated. The labeled compounds can be used as internal standards in liquid chromatography/mass spectrometry assays for clinical and biochemical studies.

Mass Spectrometric Quantification of Amphipathic, Polyphenolic Antioxidant of the Pacific Oyster (Crassostrea gigas).

A novel amphipathic phenolic compound, 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA), that can be isolated from the Pacific oyster (Crassostrea gigas) has been found to protect human hepatocytes against oxidative stress. This study aims to establish a method for the measurement of DHMBA for industrial application. Liquid chromatography-tandem mass spectrometry using deuterated DHMBA as an internal standard and a polar end-capped ODS (Hypersil GOLD aQ) as the solid phase was validated. The limit of detection was 0.04 pmol (S/N = 5), and the limit of quantitation was 0.1 pmol (S/N = 10). The calibration curve was linear throughout the range of 0.1 - 16 pmol (r(2) = 0.9995). This method successfully quantified DHMBA in oysters from 11 sea areas in Japan. The results showed that the yield of DHMBA was variable from 9.8 to 58.8 µg g(-1) whole oyster meat wet weight but not affected by the seawater temperature. The proposed LC-MS/MS method is useful in quantitative studies for DHMBA and potentially for other amphipathic substances.