Fraction Of Liver Research Articles

Glucose transporter expression and regulation following a fast in the ruby-throated hummingbird, Archilochus colubris.

Hummingbirds, subsisting almost exclusively on nectar sugar, face extreme challenges to blood sugar regulation. The capacity for transmembrane sugar transport is mediated by the activity of facilitative glucose transporters (GLUTs) and their localisation to the plasma membrane (PM). In this study, we determined the relative protein abundance of GLUT1, GLUT2, GLUT3 and GLUT5 via immunoblot using custom-designed antibodies in whole-tissue homogenates and PM fractions of flight muscle, heart and liver of ruby-throated hummingbirds (Archilochus colubris). The GLUTs examined were detected in nearly all tissues tested. Hepatic GLUT1 was minimally present in whole-tissue homogenates and absent win PM fractions. GLUT5 was expressed in flight muscles at levels comparable to those of the liver, consistent with the hypothesised uniquely high fructose uptake and oxidation capacity of hummingbird flight muscles. To assess GLUT regulation, we fed ruby-throated hummingbirds 1 mol l-1 sucrose ad libitum for 24 h followed by either 1 h of fasting or continued feeding until sampling. We measured relative GLUT abundance and concentration of circulating sugars. Blood fructose concentration in fasted hummingbirds declined (∼5 mmol l-1 to ∼0.18 mmol l-1), while fructose-transporting GLUT2 and GLUT5 abundance did not change in PM fractions. Blood glucose concentrations remained elevated in fed and fasted hummingbirds (∼30 mmol l-1), while glucose-transporting GLUT1 and GLUT3 in flight muscle and liver PM fractions, respectively, declined in fasted birds. Our results suggest that glucose uptake capacity is dynamically reduced in response to fasting, allowing for maintenance of elevated blood glucose levels, while fructose uptake capacity remains constitutively elevated promoting depletion of blood total fructose within the first hour of a fast.

Drug-gut microbiota metabolic interactions: the case of UniPR1331, selective antagonist of the Eph-ephrin system, in mice

The interest on the role of gut microbiota in the biotransformation of drugs and xenobiotics has grown over the last decades and a deeper understanding of the mutual interactions is expected to help future improvements in the fields of drug development, toxicological risk assessment and precision medicine. In this paper, a microbiome drug metabolism case is presented, involving a lipophilic small molecule, N-(3β-hydroxy-Δ5-cholen-24-oyl)-l-tryptophan, UniPR1331, active as antagonist of the Eph–ephrin system and effective in vivo in a murine orthotopic model of glioblastoma multiforme (GBM). Following the administration of a single 30 mg/kg dose (p.o.) to mice, maximal plasma levels were reached 30 min after dosing and rapidly declined thereafter.To explain the observed in vivo behaviour, in vitro phase I and II metabolism assays were conducted employing mouse and human liver subcellular fractions and profiling main metabolites by means of tandem (HPLC-ESI-MS/MS) and high resolution mass spectrometry (HPLC-ESI-HR-MS).In the presence of in vitro mouse liver fractions, UniPR1331 showed a low phase I metabolic clearance, despite the identification of a 3-oxo and several hydroxylated metabolites. Conversely, after oral administration of UniPR1331 to mice, a novel isobaric metabolite was detected that (i) was subjected, as parent UniPR1331, to enterohepatic circulation (ii) had not been previously identified in vitro in mouse liver microsomes and (iii) was not observed forming after intraperitoneal (i.p.) administration of UniPR1331. An in vitro faecal fermentation assay produced the same chemical entity supporting a major role of gut microbiota in the in vivo clearance of UniPR1331.

The influence of carboxylesterase 1 polymorphism and cannabidiol on the hepatic metabolism of heroin

Heroin (diamorphine) is a highly addictive opioid drug synthesized from morphine. The use of heroin and incidence of heroin associated overdose death has increased sharply in the US. Heroin is primarily metabolized via deacetylation (hydrolysis) forming the active metabolites 6-monoacetylmorphine (6-MAM) and morphine. A diminution in heroin hydrolysis is likely to cause higher drug effects and toxicities. In this study, we sought to determine the contribution of the major hepatic hydrolase carboxylesterase 1 (CES1) to heroin metabolism in the liver as well as the potential influence of one of its known genetic variants, G143E (rs71647871). Furthermore, given the potential therapeutic application of cannabidiol (CBD) for heroin addiction and the frequent co-abuse of cannabis and heroin, we also assessed the effects of CBD on heroin metabolism. In vitro systems containing human liver, wild-type CES1, and G143E CES1 S9 fractions were utilized in the assessment. The contribution of CES1 to the hydrolysis of heroin to 6-MAM was determined as 3.66%, and CES1 was unable to further catalyze 6-MAM under our assay conditions. The G143E variant showed a 3.2-fold lower intrinsic clearance of heroin as compared to the WT. CBD inhibited heroin and 6-MAM hydrolysis in a reversible manner, with IC50s of 14.7 and 12.1 μM, respectively. Our study results suggested only minor involvement of CES1 in heroin hydrolysis in the liver. Therefore, the G143E variant is unlikely to cause significant impact despite a much lower hydrolytic activity. CBD exhibited potent in vitro inhibition toward both heroin and 6-MAM hydrolysis, which may be of potential clinical relevance.

A compartmental approach to isosteviol's disposition in Sprague-Dawley rats.

Isosteviol has been reported to reverse hypertrophy and related inflammatory responses in in vitro models representative of cardiac muscle cells. The disposition of isosteviol is, however, characterized by secondary peaks and long plasma residence time despite reports of a relatively short half-life in liver fractions. The present study describes a compartmental approach to modelling the secondary peaks characteristic of isosteviol's concentration-time data in Sprague-Dawley rats. Oral (4mg/kg) and intravenous (4mg/kg) doses of isosteviol were administered to male and female Sprague-Dawley rats. Plasma samples collected between 0 and 72h, and total bile secreted in 24h, were analysed for isosteviol content with LC-MS/MS techniques. The disposition of isosteviol was, thereafter, described with a structural model that accounted for the sampling, liver and biliary secretion compartments, with a gap-time characterizing the accumulation and subsequent emptying of isosteviol for re-absorption. The half-life of isosteviol following oral dosing was about 103% greater in female rats than in the male, and the model-derived area under the concentration-time curve (AUC) in 72h was about 756% greater in female animals than in males. Following the administration of intravenous doses of isosteviol, half-life and AUC in 24h were about 332% and 595%, respectively, higher in female rats than in males. Isosteviol equivalent secreted into bile over 24h accounted for about 94% of orally administered dose in male rats, and about 59% of oral dose in females. These findings show a differential systemic removal of isosteviol in Sprague-Dawley rats, likely explainable by gender-related differences in the glucuronidation-capacity of isosteviol.

In vitro metabolism of triclosan studied by liquid chromatography-high-resolution tandem mass spectrometry.

Triclosan (TCS) is an antibacterial and antifungal compound found in many hygiene products, including toothpaste, soap, and detergents. However, this molecule can act as an endocrine disruptor and can induce harmful effects on human health and the environment. In this study, triclosan was biotransformed in vitro using human and rat liver fractions, to evaluate oxidative metabolism, the formation of reactive metabolites via the detection of GSH adducts, as well as glucuronide and sulfate conjugates using liquid chromatography coupled to high-resolution tandem mass spectrometry (LC-HRMS/MS). A deuterated analog of triclosan was also employed for better structural elucidation of specific metabolic sites. Several GSH adducts were found, either via oxidative metabolism of triclosan or its cleavage product, 2,4-dichlorophenol. We also detected glucuronide and sulfated conjugates of triclosan and its cleaved product. This study was aimed at understanding the routes of detoxification of this xenobiotic, as well as investigating any potential pathways related to additional toxicity via reactive metabolite formation. Graphical abstract.

Cytochrome P450 1A2 Is Incapable of Oxidizing Bilirubin Under Physiological Conditions.

Background: Bilirubin (BR) is metabolized mainly by uridine diphosphate (UDP)-glucuronosyltransferase 1A1 (UGT1A1) through glucuronidation in the liver. Some studies have shown that several subtypes of cytochrome P450 (CYP) enzymes, including CYP1A2, are upregulated by inducers and proposed to be alternative BR degradation enzymes. However, no information is available on the BR degradation ability of CYP in normal rats without manipulation by CYP inducers. Methods: Quantitative real-time polymerase chain reaction (QRT-PCR), western blot, immunofluorescence, and confocal microscopy were used to find expression of CYP1A2 in the brain and the liver. BR metabolites in microsomal fractions during development were examined by high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (LC-MS/MS). Results: In the present study, we observed that CYP1A2 mRNA levels increased at postnatal days (P)14 and P30 with respect to the level at P7 both in liver and brain, this increment was especially pronounced in the brain at P14. The expression of CYP1A2 in the brainstem (BS) was higher than that in the cerebellum (CLL) and cortex (COR). Meanwhile, the CYP1A2 protein level was significantly higher in the COR than in the brainstem and CLL at P14. The levels of BR and its metabolites (m/z values 301, 315, 333 and biliverdin) were statistically unaltered by incubation with liver and brain microsomal fractions. Conclusion: Our results indicated that the region-specific expression of CYP1A2 increased during development, but CYP family enzymes were physiologically incapable of metabolizing BR. The ability of CYPs to oxidize BR may be triggered by CYP inducers.

2230 Benign Intermittent Intestinal Alkaline Phosphatase Elevation

INTRODUCTION: Liver function tests (LFTs) are commonly checked during routine clinical encounters without specific signs or symptoms. An elevated alkaline phosphatase (AP) level is followed by investigative algorithms to identify the etiology. Total AP comprises liver, bone and intestinal fractions. Elevated intestinal alkaline phosphatase (IAP) can occur as a rare benign condition or due to intestinal pathologies. Unlike previously published reports of benign persistent IAP elevation, our patient had intermittent elevation of IAP. CASE DESCRIPTION/METHODS: A 48-year-old obese female was seen in liver clinic for evaluation of elevated AP for the last two years. She had undergone work-up for elevated AP on several occasions but the exact etiology was not found. She did not report any prior history of jaundice, pruritis, nausea, vomiting, diarrhea, abdominal pain, arthralgias or arthritis. She denied any family history of elevated IAP. She was found to have elevated total AP on 6 separate occasions over the last 18 months ranging from 186 to 239 U/l (normal < 117). Her fasting isozyme fractionation was checked 4 times. She had elevated total AP and normal liver/bone fractions on all occasions but twice had elevated IAP of 62 and 93 U/l (normal < 15 U/l), 26-33% of total AP (normal < 14%). Her other LFTs, TFT, PTH, Ca, CBC, BMP, GGT, viral hepatitis serologies, AMA and Vit D were unremarkable. Abdominal imaging revealed a fatty liver while a bone scan was normal. Our patient remained asymptomatic and did not require any treatment. DISCUSSION: Elevated AP can be physiological during rapid bone growth and pregnancy or pathological due to hepatobiliary disease or abnormal bone metabolism. Non-hepatic causes of elevated AP include heart failure, renal failure, hyperthyroidism, hyperparathyroidism and certain malignancies. IAP normally accounts for up to 14% of total AP. IAP can be elevated in conditions such as intestinal perforation, intestinal ischemia, extensive erosions/ulceration in GI tract, liver cirrhosis, familial benign elevation of IAP or post-prandial rise after a fatty meal particularly in patients with blood group B or O. Our patient did not have any of the above-listed conditions. Although she was blood group B, the blood draws were done after an overnight fast. While investigating elevated AP clinicians should be aware of pathological and benign causes of IAP elevation including benign intermittent IAP elevation which may require repeated testing of AP isozymes.

2286 Cholestatic Hepatitis as a Paraneoplastic Syndrome of Endometrial Carcinoma

INTRODUCTION: Cholestatic hepatitis is a rare manifestation of both solid organ and hematologic malignancies. It has been reported to occur with ovarian cancer, but not other female genitourinary tract malignancies to our knowledge. We report a case of simultaneous cholestatic hepatitis and acute sensorimotor polyneuropathy as paraneoplastic syndromes of endometrial adenocarcinoma. CASE DESCRIPTION/METHODS: A 66-year-old woman with past medical history of newly diagnosed stage IIIC endometrial adenocarcinoma and cholecystectomy presented with ascending bilateral lower extremity weakness and numbness for 14 days. Physical exam revealed bilateral foot drop and distal lower extremity numbness. Patient did not have jaundice or scleral icterus. Laboratory assessment showed ALT 172 U/L (normal < 61 U/L), AST 149 U/L (normal < 40U/L), ALP 243 U/L (normal < 126 U/L), and GGT 461 U/L (normal < 85 U/L). Lumbar puncture showed CSF albuminocytologic disassociation. Further neurologic workup was consistent with an acute sensorimotor polyneuropathy, which was attributed to a paraneoplastic process of her cancer. During investigation of her polyneuropathy, transaminases and ALP continued to rise to peak values of ALT 245 U/L, AST 202 U/L, and ALP 659 U/L so workup of cholestatic hepatitis was pursued. Total bilirubin and INR remained normal. Fractionated ALP showed a majority liver fraction. Infectious, autoimmune, pharmacologic, malignant, metabolic, and drug-induced causes of cholestatic hepatitis were screened for and ruled out. Liver ultrasound with doppler was negative for acute biliary or liver pathology. CT of the chest, abdomen, and pelvis with IV contrast showed a normal liver. Additional workup for cholestatic hepatitis, including liver biopsy, was not pursued as ascending paralysis progressed to paraplegia and patient opted for hospice care. Patient was diagnosed with paraneoplastic, acute cholestatic hepatitis from endometrial adenocarcinoma. DISCUSSION: To our knowledge, this is the first case report of cholestatic hepatitis as a paraneoplastic syndrome of endometrial adenocarcinoma. Our patient’s liver injury may have occurred due to paraneoplastic proinflammatory state or tumor antigen-antibody cross reactivity with liver cell antigens, or a combination of both. Gastroenterologists should include paraneoplastic processes of cancer in the differential diagnosis for liver injury, especially when workup of alternative causes is unrevealing.

Relationship between abdominal fat stores and liver fat, pancreatic fat, and metabolic comorbidities in a pediatric population with non-alcoholic fatty liver disease.

To define the relationship between compartmental abdominal fat stores, liver and pancreatic fat fractions, and type 2 diabetes mellitus (T2DM) in children with non-alcoholic fatty liver disease (NAFLD). This was a retrospective study of patients with NAFLD who underwent abdominal MRI between August 2015 and July 2017. Using an axial multi-echo Dixon-based sequence, liver fat fraction (LFF) and pancreatic fat fraction (PFF) were measured. The fat image was used to quantify abdominal fat depots (thickness, cross-sectional area) at the L2 vertebral level. Multivariable models with stepwise selection were created for prediction of LFF, PFF, and T2DM status based upon variables of clinical interest. 86 patients (70% male, 25% Hispanic, 58% Caucasian, 11% African American) with a mean age of 14.2 ± 3.2years were included. 19 (22%) patients were pre-diabetic or diabetic. Only ethnicity was a predictor of LFF (P = 0.0023) with Hispanic ethnicity associated with the highest LFF. Depending on the model, either total abdominal fat area (P = 0.0003) or patient weight (P = 0.008) were the only predictors of PFF. No patient variable predicted T2DM status. In our population, there was an association between ethnicity and LFF, with the highest LFF in Hispanics. The presence or severity of hepatic steatosis could not be predicted based on patient size or the distribution of abdominal fat in our cohort. Neither LFF nor PFF were predictive of T2DM.

Tentative identification of the metabolites of (1-(cyclohexylmethyl)-1H-indol-3-yl)-(2,2,3,3-tetramethylcyclopropyl)methanone, and the product of its thermal degradation, by in vitro and in vivo methods.

Synthetic cannabinoids (SCs), mimicking the psychoactive effects of cannabis, consist of a vast array of structurally diverse compounds. A novel compound belonging to the SC family, (1-(cyclohexylmethyl)-1H-indol-3-yl)-(2,2,3,3-tetramethylcyclopropyl)methanone (named TMCP-CHM in this article) contains a cyclopropane ring that isomerizes during the smoking process, resulting in a ring-opened thermal degradant with a terminal double bond in its structure. Metabolites of TMCP-CHM were tentatively identified in vitro (after incubation of the parent substance with S9 pooled human liver fraction) and in vivo (rat experimental model) studies by accurate-mass liquid chromatography-tandem mass spectrometry (LC-MS/MS). For the identification of the degradant metabolites, and to study biotransformation of parent substance in the human, urine and hair samples from patients, who had ingested the compound and were subsequently admitted to hospital with drug intoxications, were analyzed. Products of mono-, di-, trihydroxylation, carboxylation, and carboxylation combined with hydroxylation of TMCP-CHM and its degradant were detected in human urine. Metabolism of the degradant included addition of water to the terminal double bond followed by dehydration and formation of a cyclic metabolite. Degradant metabolites prevailed in comparison with metabolites of the parent substance in each metabolite group examined, except carboxylation. N-Dealkylated metabolites found in human urine originated only from the degradant. Most of the hydroxy metabolites were detected in human urine in both the free form and as glucuronides. The detection of monohydroxylated (M1.1-M1.3, M/A1.10) and carboxylated/hydroxylated (M4.2, M/A4.3) metabolites of TMCP-CHM and the hydrated form of the monohydroxylated metabolite of the degradant was found to be convenient for routine analysis.

Intrahepatic fluorine-18-fluorodeoxyglucose kinetics measured by least squares nonlinear computer modelling and Gjedde-Patlak-Rutland graphical analysis.

We aimed to use simple physiological equations to show similarities and differences in blood fluorine-18-fluorodeoxyglucose (F-FDG) clearance in the liver (Ki) and distribution volume (V0) in the liver, respectively, generated from nonlinear least squares computer modelling and Gjedde-Patlak-Rutland graphical analysis of dynamic F-FDG PET. We show theoretically that when, as is usually the case, vascular fraction (fraction of liver occupied by blood, Vb) is included as a parameter in modelling but ignored in graphical analysis, the ratio of Ki values, respectively, generated by graphical anlaysis (Ki) and by modelling (Ki) is equal to 1-Vb. This theoretical prediction was then confirmed from dynamic PET data acquired in a clinical population of patients undergoing routine F-FDG PET/computed tomography and from a review of the literature in which it can be seen that Ki/Ki ranges from 0.47 to 0.98. When Vb is not included as a parameter in modelling, Ki is theoretically equal to Ki and to V0·k3, and V0 is equal to V0. There are several attractions to normalising Ki to V0 with respect to liver. Thus, first, there is no need to correct imaging for photon attenuation. Second, it makes no difference whether uptake constant is expressed as blood or plasma clearance. Third, it circumvents the effects of hepatic fat, which, because it accumulates negligible F-FDG, physically dilutes the F-FDG signal and reduces both uptake constant and distribution volume.

Serum alkaline phosphatase and γ-glutamyl transferase in acute pyelonephritis.

BackgroundElevated serum alkaline phosphatase (AP) and γ-glutamyl transferase (γ-GT) are commonly observed in patients with acute pyelonephritis. The goal of this study was to examine the clinical significance of elevated serum AP and γ-GT levels and to explore the mechanisms underlying these changes.MethodsWe examined serum AP and γ-GT levels in 438 patients with acute pyelonephritis. Urine AP/creatinine (Cr), urine γ-GT/Cr, fractional excretion of AP, and fractional excretion of γ-GT (FEγ-GT) were evaluated in patients with elevated and normal serum levels. AP isoenzymes were also examined.ResultsWe identified 77 patients (17.6%) with elevated serum AP and 134 patients (30.6%) with elevated serum γ-GT. Among them, both enzymes were elevated in 64 patients (14.6%). Older age, longer hospital stay, elevated baseline serum Cr, and complicated pyelonephritis were associated with increases in serum AP and γ-GT. Multivariate analysis showed that high serum AP levels were significantly correlated with renal impairment (odds ratio, 2.13; 95% confidence interval, 1.08–4.19; P = 0.029). FEγ-GT was significantly lower in patients with elevated serum enzyme levels. The liver fraction for AP isoenzyme profile did not increase in patients with elevated serum AP.ConclusionOur results demonstrated that elevated serum AP and γ-GT levels are associated with complicated pyelonephritis and renal impairment. Lower FEγ-GT levels in patients with elevated serum enzymes may be the result of decreased urinary excretion of these enzymes.

Tissue-Specific Sample Dilution: An Important Parameter to Optimise Prior to Untargeted LC-MS Metabolomics.

When developing a sample preparation protocol for LC–MS untargeted metabolomics of a new sample matrix unfamiliar to the laboratory, selection of a suitable injection concentration is rarely described. Here we developed a simple workflow to address this issue prior to untargeted LC–MS metabolomics using pig adipose tissue and liver tissue. Bi-phasic extraction was performed to enable simultaneous optimisation of parameters for analysis of both lipids and polar extracts. A series of diluted pooled samples were analysed by LC–MS and used to evaluate signal linearity. Suitable injected concentrations were determined based on both the number of reproducible features and linear features. With our laboratory settings, the optimum concentrations of tissue mass to reconstitution solvent of liver and adipose tissue lipid fractions were found to be 125 mg/mL and 7.81 mg/mL respectively, producing 2811 (ESI+) and 4326 (ESI−) linear features from liver, 698 (ESI+) and 498 (ESI−) linear features from adipose tissue. For analysis of the polar fraction of both tissues, 250 mg/mL was suitable, producing 403 (ESI+) and 235 (ESI−) linear features from liver, 114 (ESI+) and 108 (ESI−) linear features from adipose tissue. Incorrect reconstitution volumes resulted in either severe overloading or poor linearity in our lipid data, while too dilute polar fractions resulted in a low number of reproducible features (<50) compared to hundreds of reproducible features from the optimum concentration used. Our study highlights on multiple matrices and multiple extract and chromatography types, the critical importance of determining a suitable injected concentration prior to untargeted LC–MS metabolomics, with the described workflow applicable to any matrix and LC–MS system.

Berberine alleviates hyperglycemia by targeting hepatic glucokinase in diabetic db/db mice

Berberine (BBR) is a widely used anti-diabetic agent, and liver glucokinase (GK) has been reported to be involved. However, the mechanisms of BBR in regulating GK are still unknown. Here, we found that BBR upregulated GK immunofluorescence expression in AML12 cells cultured in high glucose and increased glycogen content simultaneously. BBR improved hyperglycemia in db/db mice, and increased liver glucose-6-phosphate/glucose-1-phosphate (G-6-P/G-1-P) was found by analyzing metabolites (serum, liver, and feces) based on gas chromatography-mass spectrometry (GC-MS) metabolomics. Pharmacokinetics-pharmacodynamics (PK-PD) assessment revealed enriched BBR distribution in the liver, and liver G-6-P had the same trend as the concentration-time curve of BBR. G-6-P is solely catalyzed by GK, and GK activity and expression showed a positive correlation with liver BBR levels. In db/db mice, BBR also upregulated GK in liver fractions (cytoplasm and nucleus) and liver glycogen content. GK functionally worked by dissociating from GK regulatory protein (GKRP), and although GKRP expression was not affected, we found a decreased ratio of GK binding with GKRP in BBR treated db/db mice. In conclusion, our study suggests the dissociation of GK from GKRP as the potential mechanism for liver GK increase upon BBR treatment, which contributes to the anti-diabetic effect of BBR.

Hemisynthesis of Anthocyanin Phase II Metabolites by Porcine Liver Enzymes.

The aim of this work was to obtain phase II metabolites of cyanidin-3- O-glucoside and its aglycone using porcine liver enzymes. For this purpose, anthocyanins extracted from blackberry concentrate and containing mostly cyanidin-3- O-glucoside were incubated with the S9, microsomal, and cytosolic fractions of porcine liver. The reactions were targeted to the direction of the respective phase II transformation by the addition of activated cofactors. LC-MS n and LC-IMS-QTOF-MS analyses showed that one methylated, three glucuronidated and three sulfated metabolites of cyanidin-3- O-glucoside were generated. The aglycone, cyanidin, was sulfated and glucuronidated by the liver enzymes. In addition, both were glucuronidated and methylated simultaneously. The detected compounds and the generated data like exact masses, mass spectra, and CCS values may serve as a basis in the search for metabolites formed in vivo. As their effects are largely unexplored, the described synthesis may contribute to a better understanding of the metabolism of anthocyanins.

In Vitro Metabolism by Aldehyde Oxidase Leads to Poor Pharmacokinetic Profile in Rats for c-Met Inhibitor MET401.

MET401 is a potent and selective c-Met inhibitor with a novel triazolopyrimidine scaffold. The aim of this study was to determine the pharmacokinetic profile of MET401 in preclinical species, and to identify the metabolic soft spot and enzyme involved, in order to help medicinal chemists to modify the compound to improve the pharmacokinetic profile. A metabolite identification study was performed in different liver fractions from various species. Chemical inhibition with selective cytochrome P450 (CYP) and molybdenum hydroxylase inhibitors was carried out to identify the enzyme involved. The deuterium substitution strategy was adopted to reduce metabolism. Pharmacokinetic studies were performed in rats to confirm the effect. Although M-2 is a minor metabolite in liver microsomal incubations, it became the predominant metabolite in incubations with liver S9, cytosol, hepatocytes and rat pharmacokinetic study. M-2 was synthesized enzymatically and the structure was identified as a mono-oxidation on the triazolopyrimidine moiety. The M-2 formation was ascribed to aldehyde oxidase (AO)-mediated metabolism based on the following evidence-M-2 production was NADPH independent, pan-CYP inhibitor 1-aminobenzotriazole and xanthine oxidase inhibitor allopurinol did not inhibit M-2 formation, and AO inhibitors menadione and raloxifene inhibited M-2 formation. The deuterated analog MET763 demonstrated an improved pharmacokinetic profile with lower clearance, longer terminal half-life and double oral exposure compared with MET401 in rats. These results indicate that the main metabolic pathway of MET401 is AO-mediated metabolism, which leads to poor in vivo pharmacokinetic profiles in rodents. The deuterium substitution strategy could be used to reduce AO-mediated metabolism liability.

Biochemical and morphological changes in mouse liver induced by mistletoe toxins

Natural compounds are often characterized by high biological activity and sometimes toxicity. This also applies to compounds contained in the herb mistletoe.The objective of this study was to investigate short-term effects (up to 48 h) of mistletoe toxins on mouse hepatocytes. Standardized mistletoe extract Iscador P was given to female mice as a single injection (0.1 mg/kg b.w., 1 mg/kg b.w., or 2 mg/kg b.w). Activities of lysosomal hydrolases: acid phosphatase, cathepsins D and L, N-acetyl-β-D-hexosaminidase, β-D-glucuronidase, β-D-glucosidase and cytosolic proteases: arginine and leucine aminopeptidases were analyzed in the liver fractions 24 and 48 h after the injection. The morphology of hepatocytes was examined by light and transmission electron microscopy.Iscador P caused a decrease in the activity of all lysosomal hydrolases (except cathepsins) in the lysosomal pellet, and an increase in the activity of both aminopeptidases and β-D-glucuronidase in the cytosol. However, despite membranotropic properties of the viscotoxins, we did not find a significant labilising effect on the lysosomal membranes. Only β-D-glucuronidase activity was relocated to the supernatant of lysosomal fraction. Microscopic examinations revealed that hepatocyte mitochondria were enlarged and increased in number, whereas the surface of the rough endoplasmic reticulum was decreased significantly.

A simple GC–MS method for the determination of diphenylamine, tolylfluanid propargite and phosalone in liver fractions

In this study, an accurate and robust gas chromatography/mass spectrometry method was developed for quantitative analysis of diphenylamine, tolylfluanid, propargite and phosalone in liver fractions. Different injector parameters were optimized by an experimental design technique (central composite design). An optimal combination of injector temperature (°C), splitless time (min) and overpressure (kPa) values enabled to maximize the chromatographic responses. Sample preparation was based on protein precipitation using trichloroacetic acid followed by liquid-liquid extraction (LLE) of the pesticides with hexane. All compounds and endrin as internal standard were quantified without interference in selected ion monitoring mode. The calibration curves for diphenylamine, tolylfluanid, propargite and phosalone compounds were linear over the concentration range of 0.1 to 25 μM with determination coefficients (R2) higher than 0.999. A lower limit of quantification of 0.1 μM was obtained for all analytes, i.e. 422.5, 868.0, 876.2 and 919.5 μg/kg of liver fraction (hepatocytes) for diphenylamine, tolylfluanid, propargite and phosalone, respectively. All compounds showed extraction recoveries higher than 93%, with a maximum RSD of 3.4%. Intra- and inter-day accuracies varied from 88.4 to 102.9% and, imprecision varied from 1.1 to 6.7%. Stability tests demonstrated that all pesticides were stable in liver extracts during instrumental analysis (20 °C in the autosampler tray for 72 h) following three successive freeze-thaw cycles and, at −20 °C for up to 12 months. This simple and efficient analytical procedure is thus suitable for metabolism studies or for assessing mammals liver contamination.

04:12 PM Abstract No. 333 How does number of microspheres affect absorbed-dose uniformity in Y90 radioembolization? An in vivo 3D evaluation in a porcine model

To characterize the effect of the number of Y90 microspheres on the microscopic dosimetry and absorbed-dose uniformity in a porcine model. Four healthy pigs received lobar infusions of Y90 glass microspheres at 4, 8, 12 and 16 days post-calibration. The administration dates resulted in the specific activity per microsphere of approximately 855, 302, 106, and 38 Bq at 4, 8, 12 and 16 days, respectively. CBCT was used to evaluate the perfused tissue volume at the time of delivery and the activity was selected to deliver a consistent absorbed dose of 50-60 Gy. After a 1 month survival, livers were explanted, fixed in formalin and gross pathologic evaluation performed. The embolized lobes were scanned on a Bruker MicroCT with a 9 um3 resolution. A blob-detection algorithm was used to identify all microspheres and a complete microdosimetric evaluation was performed. Twelve and 16-day pigs demonstrated focally distributed areas of necrosis on gross exam at organ harvest. Average lobar absorbed-doses were 50.9, 48.7, 53.5 and 50.0 Gy in the 4, 8, 12 and 16 day pigs, as calculated based on MicroCT identification of individual spheres. 3D analysis of microsphere deposition patterns showed average number of microspheres per cluster was 2.3, 3.4, 5.8 and 8.6 in 4, 8, 12 and 16 day pigs, with 4%, 7%, 11% and 14% of clusters in each pig containing >10 spheres, respectively. The percentage of liver tissue receiving more than 40Gy was 32%, 47%, 52% and 58% (days 4, 8, 12, 16), suggesting that microsphere number increases the fraction of normal tissue receiving a higher absorbed dose (p<.01). An analysis of absorbed dose to individual lobules shows higher maximum lobular dose (3501 Gy) in the 4 day pig compared to the 12 day pig (766 Gy, p<.01), but with lower median lobular dose in the 4 day pig (23 Gy) vs the 12 day pig (40 Gy, p<.01). While average organ doses were comparable, earlier treatments (i.e., 4 days post calibration) resulted in a greater fraction of liver receiving lower absorbed-doses (<40 Gy) compared to treatments at 12 or 16 days post calibration. These data suggest that treatments closer to calibration (higher specific activity/sphere) may be less hepatotoxic.

In vitro metabolic stability and biotransformation of isosteviol in human and rat liver fractions

Isosteviol is a lead compound whose cardioprotective property has been partly explained by its regulation of ion channels and interference with signalling pathways in the metabolism of some fatty acids. This study determined the metabolic stability of isosteviol in human liver microsomes and H9c2 cell line, and the identity of its metabolites in human and rat liver fractions. Isosteviol was largely unmetabolized in H9c2 cells and in NADPH-only supplemented human liver fractions, suggesting a very limited contribution of phase I biotransformation to its hepatic clearance. The in vitro half-life of isosteviol in UDPGA-only supplemented medium was observed to be 24.9 min with an estimated intrinsic clearance of 0.349 mL/min/kg in man. Analysis by LC-MS/MS and Q-tof showed that isosteviol is mainly metabolised to its acyl-β-D-glucuronide in humans and rats. Mono-hydroxy-isosteviol and dihydroisosteviol were also identified. Rat liver fraction, however, generated dihydroxy-isosteviol in addition to two mono-hydroxy derivatives. Further studies confirmed that dihydroisosteviol is subsequently biotransformed to its acyl-β-D-glucuronide in man and rat. These findings suggest that future studies of the efficacy and toxicity of isosteviol might have to consider xenobiotics that alter the glucuronidation pathways significantly in man.