title: Targeted CYP2E1 quantification and its correlation to currently acceptable clinical biochemical indices.
abstract: BACKGROUND
  The Cytochrome P450 enzymes are commonly known for their major role in metabolism. Besides its metabolic role, CYP2E1 gene expression has been associated with the onset of diabetic nephropathy. CYP2E1 protein elevation has also been reported to be responsible for the production of reactive oxygen species. The aims of this study were (i) to optimize and validate a targeted proteomic approach for quantitating CYP2E1 and validating it as a suitable clinical test, (ii) to investigate the concurrency between ESI-LCMS-MS quantitated circulating CYP2E1 and gold standard indices in the context of outpatient point-of-care clinical settings involving various groups of diabetic patients and (iii) to investigate the concurrency profile of circulating CYP2E1 protein, CYP2E1 gene expression and reactive oxygen species (ROS). This is a cross sectional study involving three groups of subjects (n = 166): control, pre-diabetes, and diabetes. We optimized a targeted proteomic approach for absolute quantification of CYP2E1. "YPEIEEK" and "GTVVVPTLYDNQEFPDPEK" were the representative peptides of CYP2E1 for our analytical method. Deuterated forms of "YPEIEEK" and "GTVVVPTLYDNQEFPDPEK" were used as internal standards. Lymphocytes were isolated from whole blood, microsomes were prepared, followed by in-solution digestion for production of tryptic peptides. Amounts of "YPEIEEK" and "GTVVVPTLYDNQEFPDPEK" from patients' samples were calculated from a calibration curve.
  
  
  RESULTS
  "YPEIEEK" is a unique and reliable representative peptide for CYP2E1 quantification. "GTVVVPTLYDNQEFPDPEK" showed poor reproducibility and sensitivity. Incremental amounts of CYP2E1 protein in the peripheral circulation clearly showed concurrency with CYP2E1 gene expression and ROS levels in our study population. Elevations of CYP2E1 were observed even when gold standard clinical indicator for glycemic control (HbA1c) was within normal reference limits. Quantitated amounts of CYP2E1 protein in the pre-diabetes and diabetes groups showed significant difference relative to control group (p < 0.001). No significant differences were observed between the medians of pre-diabetes and diabetes groups (p = 0.870).
  
  
  CONCLUSIONS
  CYP2E1 protein in peripheral blood can be reliably quantitated by the validated targeted proteomic approach method. Quantifiable amounts of CYP2E1 preceded abnormal HbA1C levels which indicates quantitation of CYP2E1 could be useful as an additional tool for early indication of diabetic risks and it complications.
authors.affiliation: Jeffrey Cheah School of Medicine and Health Sciences, Monash University Malaysia, Pesiaran Lagoon Selatan, 46150 Petaling Jaya, Selangor Malaysia.
authors.forename: Christina Gertrude
authors.initials: CG
authors.lastname: Yap
authors.affiliation: Jeffrey Cheah School of Medicine and Health Sciences, Monash University Malaysia, Pesiaran Lagoon Selatan, 46150 Petaling Jaya, Selangor Malaysia.
authors.forename: Anuar
authors.initials: A
authors.lastname: Zaini
authors.affiliation: Jeffrey Cheah School of Medicine and Health Sciences, Monash University Malaysia, Pesiaran Lagoon Selatan, 46150 Petaling Jaya, Selangor Malaysia.
authors.forename: Iekhsan
authors.initials: I
authors.lastname: Othman
keywords: Absolute quantitation; CYP2E1; Diabetes progression; Early risks predictor; LCMS/MS/MS; ROS