High Expression of UGT1A1/1A6 in Monkey Small Intestine: Comparison of Protein Expression Levels of Cytochromes P450, UDP-Glucuronosyltransferases, and Transporters in Small Intestine of Cynomolgus Monkey and Human

Cynomolgus monkeys have been widely used for the prediction of drug absorption in humans. The purpose of this study was to clarify the regional protein expression levels of cytochromes P450 (CYPs), UDP-glucuronosyltransferases (UGTs), and transporters in small intestine of cynomolgus monkey using liquid chromatography–tandem mass spectrometry, and to compare them with the corresponding levels in human. UGT1A1 in jejunum and ileum were >4.57- and >3.11-fold and UGT1A6 in jejunum and ileum were >16.1- and >8.57-fold, respectively, more highly expressed in monkey than in human. Also, jejunal expression of monkey CYP3A8 (homologue of human CYP3A4) was >3.34-fold higher than that of human CYP3A4. Among apical drug efflux transporters, BCRP showed the most abundant expression in monkey and human, and the expression levels of BCRP in monkey and human were >1.74- and >1.25-fold greater than those of P-gp and >2.76- and >4.50-fold greater than those of MRP2, respectively. These findings should be helpful to understand species differences of the functions of CYPs, UGTs, and transporters between monkey and human. The UGT1A1/1A6 data would be especially important because it is difficult to identify isoforms responsible for species differences of intestinal glucuronidation by means of functional studies due to overlapping substrate specificity

Identification of blood biomarkers in glioblastoma by SWATH mass spectrometry and quantitative targeted absolute proteomics

<div><p>Molecular biomarkers in blood are needed to aid the early diagnosis and clinical assessment of glioblastoma (GBM). Here, in order to identify biomarker candidates in plasma of GBM patients, we performed quantitative comparisons of the plasma proteomes of GBM patients (n = 14) and healthy controls (n = 15) using SWATH mass spectrometry analysis. The results were validated by means of quantitative targeted absolute proteomics analysis. As a result, we identified eight biomarker candidates for GBM (leucine-rich alpha-2-glycoprotein (LRG1), complement component C9 (C9), C-reactive protein (CRP), alpha-1-antichymotrypsin (SERPINA3), apolipoprotein B-100 (APOB), gelsolin (GSN), Ig alpha-1 chain C region (IGHA1), and apolipoprotein A-IV (APOA4)). Among them, LRG1, C9, CRP, GSN, IGHA1, and APOA4 gave values of the area under the receiver operating characteristics curve of greater than 0.80. To investigate the relationships between the biomarker candidates and GBM biology, we examined correlations between plasma concentrations of biomarker candidates and clinical presentation (tumor size, progression-free survival time, or overall survival time) in GBM patients. The plasma concentrations of LRG1, CRP, and C9 showed significant positive correlations with tumor size (R² = 0.534, 0.495, and 0.452, respectively).</p></div

Summary of the differentially expressed proteins in plasma validated by QTAP analysis.

<p>Summary of the differentially expressed proteins in plasma validated by QTAP analysis.</p

Box plot showing the plasma levels of each differentially expressed protein in GBM plasma (n = 14) compared with healthy plasma (n = 15).

<p>Each dot represents the protein level of an individual sample. In box plots, the band inside the box represents the median. The bottom and top of the box represent the first and third quartiles. The whiskers reflect the minimum and maximum values that fall within 1.5 times the interquartile range. Any data not included between the whiskers is an outlier. *, p < 0.05; **, p < 0.01; ***, p < 0.001. Cont, Healthy controls; GBM, glioblastoma.</p

Identification of blood biomarkers in glioblastoma by SWATH mass spectrometry and quantitative targeted absolute proteomics - Fig 3

<p><b>Kaplan–Meier curve of progression-free survival time (PFS) (A) and overall survival time (OS) (B) in patients with glioblastoma (GBM) showed prognostic significance of gelsolin (GSN).</b> GBM patients were classified into two categories on the basis of GSN level: low (0–472 fmol/μL plasma) and high (> 472 fmol/μL plasma). Mean GSN plasma level in GBM patients was selected as the cut-off point. (A) PFS interval was determined as the interval between the date of initial operation and the date of patient’s recurrence or determined endpoint (for those no recurrent on August 1, 2015). (B) OS interval was determined as the interval between the date of the initial operation and date of patient’s death or determined end point (for those alive on August 1, 2015).</p

Characteristics of subjects.

<p>Characteristics of subjects.</p

Total number of proteins and peptides in spectral library.

<p>Total number of proteins and peptides in spectral library.</p

Flow diagram of GBM biomarker discovery.

<p>Flow diagram of GBM biomarker discovery.</p

Summary of the differentially expressed proteins identified in plasma analyzed by SWATH-MS analysis.

<p>Summary of the differentially expressed proteins identified in plasma analyzed by SWATH-MS analysis.</p

Quantitative Atlas of Cytochrome P450, UDP-Glucuronosyltransferase, and Transporter Proteins in Jejunum of Morbidly Obese Subjects

Protein expression levels of drug-metabolizing enzymes and transporters in human jejunal tissues excised from morbidly obese subjects during gastric bypass surgery were evaluated using quantitative targeted absolute proteomics. Protein expression levels of 15 cytochrome P450 (CYP) enzymes, 10 UDP-glucuronosyltransferase (UGT) enzymes, and NADPH-P450 reductase (P450R) in microsomal fractions from 28 subjects and 49 transporters in plasma membrane fractions from 24 of the same subjects were determined using liquid chromatography–tandem mass spectrometry. Based on average values, UGT1A1, UGT2B15, UGT2B17, SGLT1, and GLUT2 exhibited high expression levels (over 10 fmol/μg protein), though UGT2B15 expression was detected at a high level in only one subject. CYP2C9, CYP2D6, CYP3A5, UGT1A6, P450R, ABCG2, GLUT5, PEPT1, MCT1, 4F2 cell-surface antigen heavy chain (4F2hc), LAT2, OSTα, and OSTβ showed intermediate levels (1–10 fmol/μg protein), and CYP1A1, CYP1A2, CYP1B1, CYP2C18, CYP2C19, CYP2J2, CYP3A7, CYP4A11, CYP51A1, UGT1A3, UGT1A4, UGT1A8, UGT2B4, ABCC1, ABCC4, ABCC5, ABCC6, ABCG8, TAUT, OATP2A1, OATP2B1, OATP3A1, OATP4A1, OCTN1, CNT2, PCFT, MCT4, GLUT4, and SLC22A18 showed low levels (less than 1 fmol/μg protein). The greatest interindividual difference (364-fold) was detected for UGT2B17. However, differences in expression levels of other quantified UGTs (except UGT2B15 and UGT2B17), CYPs (except CYP1A1 and CYP3A5), and P450R, and all quantified transporters, were within 10-fold. Expression levels of CYP1A2 and GLUT4 were significantly correlated with body-mass index. The levels of 4F2hc showed significant gender differences. Smokers showed increased levels of UGT1A1 and UGT1A3. These findings provide a basis for understanding the changes in molecular mechanisms of jejunal metabolism and transport, as well as their interindividual variability, in morbidly obese patients