5 research outputs found
Identification of a New Reactive Metabolite of Pyrrolizidine Alkaloid Retrorsine: (3<i>H</i>‑Pyrrolizin-7-yl)methanol
Pyrrolizidine alkaloids (PAs) such
as retrorsine are common food
contaminants that are known to be bioactivated by cytochrome P450
enzymes to putative hepatotoxic, genotoxic, and carcinogenic metabolites
known as dehydropyrrolizidine alkaloids (DHPs). We compared how both
electrochemical (EC) and human liver microsomal (HLM) oxidation of
retrorsine could produce short-lived intermediate metabolites; we
also characterized a toxicologically important metabolite, (3<i>H</i>-pyrrolizin-7-yl)Âmethanol. The EC cell was coupled online
or offline to a liquid chromatograph/mass spectrometer (LC/MS), whereas
the HLM oxidation was performed in 100 mM potassium phosphate (pH
7.4) in the presence of NADPH at 37 °C. The EC cell oxidation
of retrorsine produced 12 metabolites, including dehydroretrorsine
(<i>m</i>/<i>z</i> 350, [M + H<sup>+</sup>]),
which was degraded to a new reactive metabolite at <i>m</i>/<i>z</i> 136 ([M + H<sup>+</sup>]). The molecular structure
of this small metabolite was determined using high-resolution mass
spectrometry and NMR spectroscopy followed by chemical synthesis.
In addition, we also identified another minor but reactive metabolite
at <i>m</i>/<i>z</i> 136, an isomer of (3<i>H</i>-pyrrolizin-7-yl)Âmethanol. Both (3<i>H</i>-pyrrolizin-7-yl)Âmethanol
and its minor isomer were also observed after HLM oxidation of retrorsine
and other hepatotoxic PAs such as lasiocarpine and senkirkin. In the
presence of reduced glutathione (GSH), each isomer formed identical
GSH conjugates at <i>m</i>/<i>z</i> 441 and <i>m</i>/<i>z</i> 730 in the negative ESI-MS. Because
(3<i>H</i>-pyrrolizine-7-yl)Âmethanol) and its minor isomer
subsequently reacted with GSH, it is concluded that (3<i>H</i>-pyrrolizin-7-yl)Âmethanol may be a common toxic metabolite arising
from PAs
CMPF concentrations before and after the intervention (0wk and 12 wk) in the Healthy Diet, WGED and Control groups.
<p>Changes between the groups were tested using ANOVA and Bonferroni’s post-hoc tests.</p
Absolute changes in glucose parameters according to quartiles<sup>1</sup> based on absolute changes in CMPF (n = 106).
<p><sup>1</sup> Average changes in quartiles: quartile 1: -7.6 μmol/l; quartile 2: -2.2 μmol/l; quartile 3: 0.89 μmol/l; quartile 4: 7.7 μmol/l</p><p><sup>2</sup> Area under the curve in 2-hour oral glucose tolerance test</p><p><sup>3</sup> Homeostasis model of insulin resistance, HOMA IR = (fasting glucose mmol/l x fasting insulin mU/l) / 22.5</p><p><sup>4</sup> Insulinogenic index, IGI = (insulin 30 min—insulin 0 min, pmol/l) / (glucose 30 min – glucose 0 min, mmol/l)</p><p><sup>5</sup> Quantitative insulin sensitivity check index, Quicky = 1 / (lg10(insulin 0 min, mU/l) + lg10(glucose 0 min, mg/dl))</p><p><sup>6</sup> IGI x Quicky</p><p>Absolute changes in glucose parameters according to quartiles<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124379#t002fn001" target="_blank"><sup>1</sup></a> based on absolute changes in CMPF (n = 106).</p
Baseline correlations and associations between absolute changes in CMPF and glucose parameters adjusted for effect of intervention group (n = 106).
<p><sup>1</sup> Area under the curve in 2-hour oral glucose tolerance test</p><p><sup>2</sup> Homeostasis model of insulin resistance, HOMA IR = (fasting glucose mmol/l x fasting insulin mU/l) / 22.5</p><p><sup>3</sup> Insulinogenic index, IGI = (insulin 30 min—insulin 0 min, pmol/l) / (glucose 30 min – glucose 0 min, mmol/l)</p><p><sup>4</sup> Quantitative insulin sensitivity check index, Quicky = 1 / (lg10(insulin 0 min, mU/l) + lg10(glucose 0 min, mg/dl))</p><p><sup>5</sup> DI = IGI x Quicky</p><p>Baseline correlations and associations between absolute changes in CMPF and glucose parameters adjusted for effect of intervention group (n = 106).</p
Inner Blood–Retinal Barrier Dominantly Expresses Breast Cancer Resistance Protein: Comparative Quantitative Targeted Absolute Proteomics Study of CNS Barriers in Pig
The purpose of this study was to
determine absolute protein expression
levels of transporters at the porcine inner blood–retinal barrier
(BRB) and to compare the transporter protein expression quantitatively
among the inner BRB, outer BRB, blood–brain barrier (BBB),
and blood–cerebrospinal fluid barrier (BCSFB). Crude membrane
fractions of isolated retinal capillaries (inner BRB) and isolated
retinal pigment epithelium (RPE, outer BRB) were prepared from porcine
eyeballs, while plasma membrane fractions were prepared from isolated
porcine brain capillaries (BBB) and isolated choroid plexus (BCSFB).
Protein expression levels of 32 molecules, including 16 ATP-binding-cassette
(ABC) transporters and 13 solute-carrier (SLC) transporters, were
measured using a quantitative targeted absolute proteomic technique.
At the inner BRB, five molecules were detected: breast cancer resistance
protein (BCRP, <i>ABCG2</i>; 22.8 fmol/μg protein),
multidrug resistance protein 1 (MDR1, <i>ABCB1</i>; 8.70
fmol/μg protein), monocarboxylate transporter 1 (MCT1, <i>SLC16A1</i>; 4.83 fmol/μg protein), glucose transporter
1 (GLUT1, <i>SLC2A1</i>; 168 fmol/μg protein), and
sodium–potassium adenosine triphosphatase (Na<sup>+</sup>/K<sup>+</sup>-ATPase; 53.7 fmol/μg protein). Other proteins were
under the limits of quantification. Expression of MCT1 was at least
17.6-, 11.0-, and 19.2-fold greater than those of MCT2, 3, and 4,
respectively. The transporter protein expression at the inner BRB
was most highly correlated with that at the BBB (<i>R</i><sup>2</sup> = 0.8906), followed by outer BRB (<i>R</i><sup>2</sup> = 0.7988) and BCSFB (<i>R</i><sup>2</sup> =
0.4730). Sodium-dependent multivitamin transporter (SMVT, <i>SLC5A6</i>) and multidrug resistance-associated protein 1 (MRP1, <i>ABCC1</i>) were expressed at the outer BRB (0.378 and 1.03 fmol/μg
protein, respectively) but were under the limit of quantification
at the inner BRB. These findings may be helpful for understanding
differential barrier function