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

Author
item YUN, CHANGHONG - University Of Houston
item YIN, TAIJUN - University Of Houston
item SHATZER, KATHERINE - University Of Houston
item Burrin, Douglas - Doug
item CUI, LIWEI - Children'S Nutrition Research Center (CNRC)
item TU, YIFAN - University Of Houston
item HU, MING - University Of Houston

Submitted to: Journal of Chromatography B
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/3/2016
Publication Date: 5/9/2016
Publication URL: http://handle.nal.usda.gov/10113/62878
Citation: Yun, C., Yin, T., Shatzer, K., Burrin, D.G., Cui, L., Tu, Y., Hu, M. 2016. Determination of 7alpha-OH cholesterol by LC-MS/MS: Application in assessing the activity of CYP7A1 in cholestatic minipigs. Journal of Chromatography B. 1025:76-82.

Interpretive Summary: Many preterm infants are fed intravenously using total parenteral nutrition (PN) for several weeks after birth because they have an immature functioning intestine. In some infants fed for long periods on PN of a month or more, the liver becomes diseased due to an accumulation of bile acids. Bile acids are compounds made in the liver from cholesterol that function to digest dietary fats and enable absorption in to the body. The accumulation of bile acids in the liver and bloodstream of PN-fed premature infants is thought to be regulated by the activity of a key liver enzyme, CYP7A1, which first and rate-limiting step of bile acid biosynthesis from cholesterol. The aim of this study was to develop a quicker and more convenient assay to measure the activity of CYP7A1 using a very sensitive mass spectrometry method. This method was developed and used to test whether treatment of neonatal pigs with bile acids would affect the CYP7A1 activity in liver tissue. The results show that the new method had good precision and accuracy. The result also found that the CYP7A1 activity was similar among neonatal piglets fed by PN with or without bile acids.

Technical Abstract: An LC-MS/MS method was developed and validated to determine 7alpha-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 x 4.6mm i.d., 3microm). 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 7alpha-OH cholesterol and D7-cholesterol (I.S.) were 385.1 >= 159.1 and 376.4 >= 266.3, respectively. The linear response range of 7alpha-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.