Dataset Information

Determination of 7?-OH cholesterol by LC-MS/MS: Application in assessing the activity of CYP7A1 in cholestatic minipigs.

ABSTRACT: An LC-MS/MS method was developed and validated to determine 7?-OH cholesterol in liver microsome. This method was convenient and fast with high specificity and sensitivity. Briefly, a gradient elution was performed on a Synergi polar-C18 column (50×4.6mm i.d., 3?m). The mobile phase (consisting of 0.1% HCOOH solution and acetonitrile) eluted in gradient at a flow rate of 1ml/min. MS detection was operated on APCI (+) mode; the MRM transitions for 7?-OH cholesterol and D7-cholesterol (I.S.) were 385.1?159.1 and 376.4?266.3, respectively. The linear response range of 7?-OH cholesterol was covered from 1.563 to 100.0ng/ml. All of the validation items meet the requirement of FDA guidance for bioanalytical method validation. This method was applied to enzymatic studies for determination of cholesterol 7alpha-hydroxylation activity catalyzed by CYP7A1 in the cholestatic minipigs liver microsomes.

SUBMITTER: Yun C

PROVIDER: S-EPMC5358015 | biostudies-literature | 2016 Jul

REPOSITORIES: biostudies-literature

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Determination of 7α-OH cholesterol by LC-MS/MS: Application in assessing the activity of CYP7A1 in cholestatic minipigs.

Yun Changhong C Yin Taijun T Shatzer Katherine K Burrin Douglas G DG Cui Liwei L Tu Yifan Y Hu Ming M

Journal of chromatography. B, Analytical technologies in the biomedical and life sciences 20160509

An LC-MS/MS method was developed and validated to determine 7α-OH cholesterol in liver microsome. This method was convenient and fast with high specificity and sensitivity. Briefly, a gradient elution was performed on a Synergi polar-C18 column (50×4.6mm i.d., 3μm). The mobile phase (consisting of 0.1% HCOOH solution and acetonitrile) eluted in gradient at a flow rate of 1ml/min. MS detection was operated on APCI (+) mode; the MRM transitions for 7α-OH cholesterol and D7-cholesterol (I.S.) were ...[more]

PMID: 27218859

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Project description:BackgroundDuring the postnatal feeding period, formula-fed infants have higher cholesterol synthesis rates and lower circulating cholesterol concentrations than their breastfed counterparts. Although this disparity has been attributed to the uniformly low dietary cholesterol content of typical infant formulas, little is known of the underlying mechanisms associated with this altered cholesterol metabolism phenotype.ObjectiveWe aimed to determine the molecular etiology of diet-associated changes in early-life cholesterol metabolism with the use of a postnatal piglet feeding model.MethodsTwo-day-old male and female White-Dutch Landrace piglets were fed either sow milk (Sow group) or dairy-based (Milk group; Similac Advance powder) or soy-based (Soy group; Emfamil Prosobee Lipil powder) infant formulas until day 21. In addition to measuring serum cholesterol concentrations, hepatic and intestinal genes involved in enterohepatic circulation of cholesterol and bile acids were analyzed by real-time reverse-transcriptase polymerase chain reaction and Western blot. Bile acid concentrations were measured by liquid chromatography-mass spectrometry in serum, liver, and feces.ResultsCompared with the Sow group, hepatic cholesterol 7α hydroxylase (CYP7A1) protein expression was 3-fold higher in the Milk group (P < 0.05) and expression was 10-fold higher in the Soy group compared with the Milk group (P < 0.05). Likewise, fecal bile acid concentrations were 3-fold higher in the Soy group compared with the Milk group (P < 0.05). Intestinal mRNA expression of fibroblast factor 19 (Fgf19) was reduced in the Milk and Soy groups, corresponding to 54% and 67% decreases compared with the Sow group. In the Soy group, small heterodimer protein (SHP) protein expression was 30% lower compared with the Sow group (P < 0.05).ConclusionsThese results indicate that formula feeding leads to increased CYP7A1 protein expression and fecal bile acid loss in neonatal piglets, and this outcome is linked to reduced efficacy in inhibiting CYP7A1 expression through FGF19 and SHP transcriptional repression mechanisms.

Project description:BackgroundOxysterols are promising biomarkers of neurodegenerative diseases that are linked with cholesterol and vitamin D metabolism. There is an unmet need for methods capable of sensitive, and simultaneous quantitation of multiple oxysterols, vitamin D and cholesterol pathway biomarkers.MethodsA method for simultaneous determination of 5 major oxysterols, 25-hydroxy vitamin D3 and cholesterol in human plasma was developed. Total oxysterols were prepared by room temperature saponification followed by solid phase extraction from plasma spiked with deuterated internal standards. Oxysterols were resolved by reverse phase HPLC using a methanol/water/0.1% formic acid gradient. Oxysterols and 25-hydroxy vitamin D3 were detected with atmospheric pressure chemical ionization mass spectrometry in positive ion mode; in-series photodiode array detection at 204nm was used for cholesterol. Method validation studies were performed. Oxysterol levels in 220 plasma samples from healthy control subjects, multiple sclerosis and other neurological disorders patients were quantitated.ResultsOur method quantitated 5 oxysterols, cholesterol and 25-hydroxy vitamin D3 from 200 μL plasma in 35 minutes. Recoveries were >85% for all analytes and internal standards. The limits of detection were 3-10 ng/mL for oxysterols and 25-hydroxy vitamin D3 and 1 μg/mL for simultaneous detection of cholesterol. Analytical imprecision was <10 %CV for 24(S)-, 25-, 27-, 7α-hydroxycholesterol (HC) and cholesterol and ≤15 % for 7-keto-cholesterol. Multiple Sclerosis and other neurological disorder patients had lower 27-hydroxycholesterol levels compared to controls whereas 7α-hydroxycholesterol was lower specifically in Multiple Sclerosis.ConclusionThe method is suitable for measuring plasma oxysterols levels in human health and disease. Analysis of human plasma indicates that the oxysterol, bile acid precursors 7α-hydroxycholesterol and 27-hydroxycholesterol are lower in Multiple Sclerosis and may serve as potential biomarkers of disease.

Dataset Information

Determination of 7?-OH cholesterol by LC-MS/MS: Application in assessing the activity of CYP7A1 in cholestatic minipigs.

Publications

Determination of 7α-OH cholesterol by LC-MS/MS: Application in assessing the activity of CYP7A1 in cholestatic minipigs.

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