A 70-year-old Woman with Asymptomatic Ferroportin Disease.

A 59-year-old Japanese woman presented with hyperferritinemia. We decided against iron removal treatment because there were no symptoms or signs of iron-induced organ damage. A follow-up study revealed a gradual increase in transferrin saturation. The patient underwent a second examination at 66 years old. A liver biopsy showed substantial iron deposits in hepatocytes and Kupffer cells but no inflammation or fibrosis. Serum hepcidin-25 levels were highly parallel with hyperferritinemia. A genetic analysis revealed a G80S mutation in SLC40A1. These features are compatible with those of ferroportin disease. The patient remained asymptomatic at 70 years old, suggesting that the iron-loading condition may have been benign.

Treatment with hepatocyte transplantation in a novel mouse model of persistent liver failure.

Background and Aim: Cell-based transplantation therapy using hepatocytes, hepatic stem cells, hepatocyte-like cells induced from stem cells, etc. is thought to be an attractive alternative to liver transplantation, and have been studied to date. For its clinical application, however, it is extremely important to develop a model that reproduces the pathological conditions with indication for treatment and enables the study for the ideal treatment strategy. Methods: The transgenic mice which express the thymidine kinase (TK) gene of human herpes simplex virus (HSV) in their hepatocytes with normal immunity has been developed (designated as HSVtk). After ganciclovir (GCV) administration which injure TK-expressing hepatocytes, the primary hepatocytes (PHs) isolated from green fluorescent protein (GFP) transgenic mouse (GFP-tg) were transplanted to HSVtk intrasplenically, and replacement index (RI) with transplanted PHs in the liver, liver histology, and mRNA expressions in the liver were analyzed up to 8 weeks after transplantation. Results: HSVtk without PH transplantation after GCV administration developed persistent liver failure with degenerated hepatocytes, persistent elevation of ALT and hepatic p16 mRNA levels, suggesting the existence of cellular senescence in the base of the disease. When autologous GFP-PHs were transplanted to HSVtk, the transplanted cells were successfully engrafted in the liver. Eight weeks after transplantation, serum ALT levels and liver histology were almost normalized, while RIs varied from 19.8 to 73.8%. Since the hepatic p16 mRNA levels were decreased significantly in these mice, the senescence of hepatocytes associated with liver injury was thought to be resolved. On the other hand, allogenic GFP-PHs transplanted to HSVtk were eliminated as early as 1 week after transplantation. In these mice, hepatic p16 mRNA levels were significantly increased at 8 weeks after transplantation, suggesting the aggravation of hepatocyte senescence. FK506 administration to HSVtk protected the transplanted hepatocytes with allogenic background from rejection at 2 weeks after transplantation, but the condition of mice and the senescent status in the liver seemed worsened. Conclusions: The mouse model with HSVtk/GCV system was useful for studying the mechanism of liver regeneration and the immune rejection responses in the hepatocyte transplantation treatment. It may also be utilized to develop the effective remedies to avoid immune rejection.

Short-term high-fat and high-carbohydrate diets increase susceptibility to liver injury by inducing hepatic procoagulant and proinflammatory conditions with different balances.

OBJECTIVES: High-fat diet (HFD) and high-carbohydrate diet (HCD) are strongly linked to nonalcoholic fatty liver disease, a hepatic manifestation of metabolic syndrome. The mechanism of pathologic progression from nonalcoholic fatty liver disease to nonalcoholic steatohepatitis, which is a more severe form associated with inflammation and fibrosis, remains poorly understood. Thus, the aim of this study was to investigate and compare the inflammatory and coagulative state of the liver in short-term HFD- or HCD-fed mice with acute liver injury induced by concanavalin A (Con A). METHODS: Histopathologic evaluation, real-time polymerase chain reaction, and immunohistochemical evaluation were performed on the liver of mice fed HFDs and HCDs for 4 d before and after Con A administration. RESULTS: The liver of the HFD-fed mice had larger fibrinogen/fibrin depositions than those fed the HCD. HCD induced the expression of the proinflammatory cytokine tumor necrosis factor-α in the liver. Moreover, the expression of proinflammatory cytokines and chemokines was further enhanced after Con A stimulation in HCD (e.g., interleukin-1α, interleukin-6 at 1 h), with a strong tendency for inflammatory cell infiltration also found (24 h). CONCLUSIONS: Short-term HCD and HFD increased susceptibility to liver injury. HCD tended to induce more intense inflammation, whereas HFD tended to induce more intense hypercoagulation, suggesting that HCD and HFD may have different mechanisms of pathologic progression to nonalcoholic steatohepatitis.

Association of self-reported moderate vegetable juice intake with small decline in kidney function in a five-year prospective study.

OBJECTIVES: Although consumption of vegetable and 100% fruit juices are an acceptable alternative for vegetable and fruit intake, information about their actual effects on kidney function is sparse. The aim of this study was to determine the association between the consumption of vegetable and fruit juices and changes in kidney function in a Japanese population over a 5-y period. METHODS: In this prospective study, we analyzed 2755 Japanese (742 men and 2013 women) individuals who participated in both the baseline and follow-up surveys in the Daiko study (a study within the Japan Multi-Institutional Collaborative Cohort study). Estimated glomerular filtration rate (eGFR) was calculated by age, sex, and serum creatinine level. For each beverage, we categorized all participants into four groups-rare (rarely consumed), low (≤2 cups/wk), moderate (3-4 cups/wk), or frequent (≥5 cups/wk) consumers of the beverage-based on a food frequency questionnaire. RESULTS: The mean baseline and follow-up eGFR (SD) were 82.4 (14.6) and 72.2 (12.6), respectively. In fully adjusted regression analyses, moderate consumption of vegetable juice was associated a lower decline in eGFR compared with the rare consumption group (ß = -1.30; P = 0.01). Moreover, stratified analyses revealed that this significant association remained in those who were young, female, non-obese, normotensive, smoked cigarettes, consumed alcohol, or exercised. However, no significant association was found in analyses for fruit juices. CONCLUSIONS: This 5-y prospective study suggested an association between self-reported moderate consumption of vegetable juice and changes (possibly smaller decline) in kidney function in a relatively healthy Japanese population.

RECiQ: A Rapid and Easy Method for Determining Cyanide Intoxication by Cyanide and 2-Aminothiazoline-4-carboxylic Acid Quantification in the Human Blood Using Probe Electrospray Ionization Tandem Mass Spectrometry.

In this study, we developed a rapid and easy method to determine cyanide (CN) intoxication by quantification of CN and 2-aminothiazoline-4-carboxylic acid (ATCA), which is a new and reliable indicator of CN exposure, in the human blood using probe electrospray ionization tandem mass spectrometry (PESI/MS/MS) named RECiQ. For CN, we applied the previously reported one-pot derivatization method using 2,3-naphthalenedialdehyde and taurine, which can directly derivatize CN in the blood. The analytical conditions of the CN derivatization were optimized as a 10 min reaction time at room temperature. In contrast, ATCA could be directly detected in the blood by PESI/MS/MS. We developed quantitative methods for the derivatized CN and ATCA using an internal standard method and validated them using quality control samples, demonstrating that the linearities of each calibration curve were greater than 0.995, and intra- and interday precisions and accuracies were 5.1-15 and 1.1-14%, respectively. Moreover, the lower limit of detections for CN and ATCA were 42 and 43 ng/mL, respectively. Finally, we applied RECiQ to three postmortem blood specimens obtained from victims of fire incidents, which resulted in the successful quantification of CN and ATCA in all samples. As PESI/MS/MS can be completed within 0.5 min, and the sample volume requirement of RECiQ is only 2 µL of blood, these methods are useful not only for the rapid determination of CN exposure but also for the estimation of the CN intoxication levels during an autopsy.

A preliminary study of rapid-fire high-throughput metabolite analysis using nano-flow injection/Q-TOFMS.

In this study, we demonstrated nano-flow injection analysis (nano-FIA) with quadrupole time-of-flight mass spectrometry (Q-TOFMS) for 17 highly polar intermediates produced during glycolysis, the tricarboxylic acid (TCA) cycle, and the pentose phosphate pathway (PPP). We optimized the analytical conditions for nano-flow injection/Q-TOFMS, and set the flow rate and ion source temperature to 1000 nL/min and 150 °C, respectively. Under optimal conditions, a single run was finished within 3 min, and the RSD value of 50 sequential injections was 4.2%. The method also showed quantitativity of four stable-isotope-labeled compounds (r2 > 0.99), demonstrating its robustness, high repeatability, and specificity. In addition, we compared three sample-preparation methods for rodent blood samples and found that protein precipitation with threefold methanol was the most effective. Finally, we applied the method to plasma samples from the serotonin syndrome (SS) model and control rats, the results of which were evaluated by principal component analysis (PCA). The two groups showed clearly separated PCA score plots, suggesting that the method could successfully catch the differences in metabolic profiles between SS and control rats. The results obtained from our new method were further validated by using the established gas chromatography/tandem mass spectrometry method, which demonstrated that there were good correlations between the two methods (R = 0.902 and 0.958 for lactic acid and malic acid, respectively, each at p < 0.001), thus proving the validity of our method. The method described here enables high-throughput analysis of metabolites and will be of use for the rapid analysis of metabolic profiles. Graphical abstract.

Short-term high-fat diet intake leads to exacerbation of concanavalin A-induced liver injury through the induction of procoagulation state.

Obesity and high-fat diet (HFD) are known to cause proinflammatory and procoagulation states and suggested to become a risk of developing thromboembolic diseases. Non-alcoholic fatty liver disease (NAFLD) is usually associated with obesity and HFD, and a part of NAFLD is known to progress to nonalcoholic steatohepatitis (NASH), the pathogenesis of which has not been fully elucidated. In the current study, we examined the influence of short-term HFD on hepatic expression of the molecules related to inflammation, coagulation, metabolism, and cellular stresses from the perspective that HFD itself can be a risk for the development to NASH. In the analysis in short-term (4 days to 14 days) HFD-fed mice, we found out that HFD increased hepatic expression of IFN-γ, TNF-α, IL-10, monocyte chemotactic protein-1 (MCP-1), tissue factor (TF), plasminogen activator inhibitor-1 (PAI-1) mRNAs, and fibrin/fibrinogen deposition in the liver tissues. And it was suggested that metabolic alterations and endoplasmic reticulum (ER) stresses induced by the HFD intake were associated with this proinflammatory and procoagulation states. When we administered concanavalin A (Con A) to these HFD-fed mice, the extent of liver injury was dramatically exacerbated in HFD-fed mice. Heparin treatment to Con A-administered, HFD-fed mice (for 4 days) profoundly ameliorated the extent of liver injury. These suggest that even short-term of HFD intake induces proinflammatory and procoagulation states in the liver and thereby increases the susceptibility of the liver to circulating inflammatory stimuli. We think that it may explain a part of NASH pathogenesis.

Synthesis of the Antimalarial Peptide Aldehyde, a Precursor of Kozupeptin A, Utilizing a Designed Hydrophobic Anchor Molecule.

This Letter describes an efficient method of synthesizing highly bioactive peptide aldehydes without any concern about epimerization by liquid-phase peptide synthesis through the use of newly designed hydrophobic anchor molecules. Peptide elongation reactions effectively proceeded in less polar solvents, and direct crystallization by the addition of polar solvents enabled easy purification. This method also represents a new concept for the efficient synthesis of peptide derivatives. The development of new antimalarial drug candidates will be accelerated using this methodology.

Kozupeptins, Antimalarial Agents Produced by Paracamarosporium Species: Isolation, Structural Elucidation, Total Synthesis, and Bioactivity.

Kozupeptins A and B, novel antimalarial lipopeptides, were isolated from the culture broths of Paracamarosporium sp. FKI-7019. They exhibited potent antimalarial activity against chloroquine-sensitive and -resistant Plasmodium falciparum strains in vitro. The structural elucidation was accomplished by a combination of spectroscopic analyses and chemical approaches including a total synthesis of kozupeptin A. Synthetic kozupeptin A demonstrated a therapeutic effect in vivo, and an intermediate exhibited much higher antimalarial activity than kozupeptin A.

In utero exposure to di(2-ethylhexyl)phthalate suppresses blood glucose and leptin levels in the offspring of wild-type mice.

Exposure of pregnant mice to di(2-ethylhexyl)phthalate (DEHP) induces maternal lipid malnutrition and decreases the number of live fetuses/pups. In this study, we aimed to clarify the relationship between maternal lipid malnutrition and the nutritional status of the neonatal, lactational, and adult offspring, as well as the role of peroxisome proliferator-activated receptor α (PPARα) in these relationships. Sv/129 wild-type (mPPARA), Ppara-null, and PPARα-humanized (hPPARA) mice were fed diets containing 0, 0.01, 0.05, or 0.1% DEHP in utero and/or during the lactational stage. The male offspring were killed on postnatal day 2 or 21, or after 11 weeks. Exposure to either 0.05% or 0.1% DEHP during both the in utero and lactational periods decreased serum glucose concentrations in 2-day-old mPPARA offspring. These dosages also decreased both serum and plasma leptin levels in both 2- and 21-day-old mPPARA offspring. In contrast, exposure to DEHP only during the lactational period did not decrease leptin levels, suggesting the importance of in utero exposure to DEHP. Exposure to 0.05% DEHP during the in utero and lactational periods also increased food consumption after weaning in both mPPARA and hPPARA mice; this was not observed in Ppara-null offspring. In conclusion, in utero exposure to DEHP induces neonatal serum glucose malnutrition via PPARα. DEHP also decreases serum and plasma leptin concentrations in offspring during the neonatal and weaning periods, in association with PPARα, which presumably results in increased of food consumption after weaning.

Metabolome analysis of the serotonin syndrome rat model: Abnormal muscular contraction is related to metabolic alterations and hyper-thermogenesis.

AIMS: Serotonin syndrome (SS) is an adverse outcome of selective serotonin reuptake inhibitors, though its mechanism is not understood and there is no specific clinical biomarker. In this article, metabolic profiles of the SS model rats and causes of metabolome disruption were investigated. MAIN METHODS: Gas chromatography-tandem mass spectrometry (GC/MS/MS)-based metabolomics, clinical biomarker measurements and qRT-PCR analysis for UCP-3 in skeletal muscles were performed. KEY FINDINGS: Metabolome analysis demonstrated that 55, 22, 49 and 41 of those were significantly altered in plasma, liver, gastrocnemius muscle, and trapezius, respectively. In particular, lactic acid significantly accumulated in the gastrocnemius muscle of the model, while the branched chain amino acids were not consumed in the trapezius, suggesting site differences in abnormal muscular contractions in the model. This result was supported by UCP-3 expression analysis. Alteration of the urea cycle was observed in the liver of the model, attributed mainly to catabolism of proteins and/or amino acids from excess skeletal muscle activity, which was supported by plasma BUN: BUN levels in the model were significantly higher than those in the control. In contrast, almost all metabolites including amino acids and TCA-cycle intermediates significantly increased in plasma of the model, suggesting these were not consumed in some parts of the muscle due to acceleration of anaerobic respiration. SIGNIFICANCE: Metabolic profiling revealed that abnormal muscular contractions occurred in specific skeletal muscles and enhanced energy production by up-regulation of anaerobic respiration, followed by excess expression of UCP-3, which contributes to the hyper-thermogenesis observed in the SS model.

In Vivo Real-Time Monitoring System Using Probe Electrospray Ionization/Tandem Mass Spectrometry for Metabolites in Mouse Brain.

Recent improvements in ambient ionization techniques combined with mass spectrometry has enabled to achieve real-time monitoring of analytes of interest, even for biogenic molecules in living animals. Here, we demonstrate a newly developed system for in vivo real-time monitoring of metabolites in a living mouse brain. It consists of a semiautomated manipulation system and a unique probe electrospray ionization unit, which uses an extremely thin solid needle (tip dia.: 700 nm) for direct sampling and ionization, coupled to a conventional tandem mass spectrometer. The system successfully monitored 8 cerebrum metabolites related to central energy metabolism in an isoflurane-anesthetized mouse in real time with a 20 s interval. Moreover, our system succeeded in capturing dynamics of energy metabolism in a mouse administered with cannabinoid type-1 receptor agonist, which is known to disrupt cerebrum energy metabolism. The present system now opens the door to the next stage of cutting-edge technique in achieving in vivo real-time monitoring.

Bile acid detoxifying enzymes limit susceptibility to liver fibrosis in female SHRSP5/Dmcr rats fed with a high-fat-cholesterol diet.

During middle age, women are less susceptible to nonalcoholic steatohepatitis (NASH) than men. Thus, we investigated the underlying molecular mechanisms behind these sexual differences using an established rat model of NASH. Mature female and male stroke-prone spontaneously hypertensive 5/Dmcr rats were fed control or high-fat-cholesterol (HFC) diets for 2, 8, and 14 weeks. Although HFC-induced hepatic fibrosis was markedly less severe in females than in males, only minor gender differences were observed in expression levels of cytochrome P450 enzymes (CYP)7A1, CYP8B1 CYP27A1, and CYP7B1, and multidrug resistance-associated protein 3, and bile salt export pump, which are involved in fibrosis-related bile acid (BA) kinetics. However, the BA detoxification-related enzymes UDP-glucuronosyltransferase (UGT) and sulfotransferase (SULT) 2A1, and the nuclear receptors constitutive androstane receptor (CAR) and pregnane X receptor (PXR), were strongly suppressed in HFC-fed males, and were only slightly changed in HFC-diet fed females. Expression levels of the farnesoid X receptor and its small heterodimer partner were similarly regulated in a gender-dependent fashion following HFC feeding. Hence, the pronounced female resistance to HFC-induced liver damage likely reflects sustained expression of the nuclear receptors CAR and PXR and the BA detoxification enzymes UGT and SULT.

Fatal intoxication by 5F-ADB and diphenidine: Detection, quantification, and investigation of their main metabolic pathways in humans by LC/MS/MS and LC/Q-TOFMS.

Despite the implementation of a new blanket scheduling system in 2013, new psychoactive substance (NPS) abuse remains a serious social concern in Japan. We present a fatal intoxication case involving 5F-ADB (methyl 2-[1-(5-fluoropentyl)-1H-indazole-3-carboxamido]-3,3-dimethylbutanoate) and diphenidine. Postmortem blood screening by liquid chromatography/quadrupole time-of-flight mass spectrometry (LC/Q-TOFMS) in the information-dependent acquisition mode only detected diphenidine. Further urinary screening using an in-house database containing NPS and metabolites detected not only diphenidine but also possible 5F-ADB metabolites; subsequent targeted screening by LC/tandem mass spectrometry (LC/MS/MS) allowed for the detection of a very low level of unchanged 5F-ADB in postmortem heart blood. Quantification by standard addition resulted in the postmortem blood concentrations being 0.19 ± 0.04 ng/mL for 5F-ADB and 12 ± 2.6 ng/mL for diphenidine. Investigation of the urinary metabolites revealed pathways involving ester hydrolysis (M1) and oxidative defluorination (M2), and further oxidation to the carboxylic acid (M3) for 5F-ADB. Mono- and di-hydroxylated diphenidine metabolites were also found. The present case demonstrates the importance of urinary metabolite screening for drugs with low blood concentration. Synthetic cannabinoids (SCs) fluorinated at the terminal N-alkyl position are known to show higher cannabinoid receptor affinity relative to their non-fluorinated analogues; 5F-ADB is no exception with high CB1 receptor activity and much greater potency than Δ9 -THC and other earlier SCs, thus we suspect its acute toxicity to be high compared to other structurally related SC analogues. The low blood concentration of 5F-ADB may be attributed to enzymatic and/or non-enzymatic degradation, and further investigation into these possibilities is underway.

Intact metabolite profiling of mouse brain by probe electrospray ionization/triple quadrupole tandem mass spectrometry (PESI/MS/MS) and its potential use for local distribution analysis of the brain.

Probe electrospray ionization (PESI), which is an ambient ionization technique, enables us to analyze intact endogenous metabolites without sample preparation. In this study, we applied the newly developed method of PESI coupled to tandem mass spectrometry (PESI/MS/MS) to analyze metabolites in mouse brain, where its lipid composition often interfere with MS-based metabolome analysis. As a result, PESI/MS/MS directly detected 25 metabolites in a mouse frontal cortex, and clearly discriminated the metabolic profiles of mice model with energy metabolism disruption from control mice. PESI/MS/MS also allowed us to perform local distribution analysis of the hippocampus as well as the frontal cortex in each mouse (n = 5), discriminating their subtle metabolic differences. These results showed high potential of PESI/MS/MS for direct metabolome profiling of mouse brain.

Immunomodulatory Effects of Adipose Tissue-Derived Stem Cells on Concanavalin A-Induced Acute Liver Injury in Mice.

Cell therapy with adipose tissue-derived stem cells (ASCs) is expected to be a candidate for the treatment of fulminant hepatic failure (FHF), which is caused by excessive immune responses. In order to evaluate the therapeutic effects of ASCs on FHF, the in vitro and in vivo immunomodulatory effects of ASCs were examined in detail in the mouse model. The in vitro effects of ASCs were examined by assessing their influence on the proliferation of lymphomononuclear cells (LMCs) stimulated with three kinds of mitogens: phorbol 12-myristate 13-acetate (PMA) plus ionomycin, concanavalin A (ConA), and lipopolysaccharide (LPS). The proliferation of LMCs was efficiently suppressed in a dose-dependent manner by ASCs in the cases of PMA plus ionomycin stimulation and ConA stimulation, but not in the case of LPS stimulation. The in vivo effects of transplanted ASCs were examined in the murine FHF model induced by ConA administration. The ALT levels and histological inflammatory changes in the ConA-administered mice were apparently relieved by the transplantation of ASCs. The analysis of mRNA expression patterns in the livers indicated that the expressions of the cytokines such as Il-6, Il-10, Ifn-γ, and Tnf-α, and the cell surface markers such as Cd3γ, Cd4, Cd8α, Cd11b, and Cd11c were downregulated in the ASC-transplanted mice. The immunomodulatory and therapeutic effects of ASCs were confirmed in the mouse model both in vitro and in vivo. These suggest that the cell therapy with ASCs is beneficial for the treatment of FHF.

Development of a mass spectrometric hydroxyl-position determination method for the hydroxyindole metabolites of JWH-018 by GC-MS/MS.

One of the many issues of designer drugs of abuse like synthetic cannabinoids (SCs) such as JWH-018 is that details on their metabolism has yet to be fully elucidated and that multiple metabolites exist. The presence of isomeric compounds poses further challenges in their identification. Our group has previously shown the effectiveness of gas chromatography-electron ionization-tandem mass spectrometry (GC-EI-MS/MS) in the mass spectrometric differentiation of the positional isomers of the naphthoylindole-type SC JWH-081, and speculated that the same approach could be used for the metabolite isomers. Using JWH-018 as a model SC, the aim of this study was to differentiate the positional isomers of its hydroxyindole metabolites by GC-MS/MS. Standard compounds of JWH-018 and its hydroxyindole metabolite positional isomers were first analyzed by GC-EI-MS in full scan mode, which was only able to differentiate the 4-hydroxyindole isomer. Further GC-MS/MS analysis was performed by selecting m/z 302 as the precursor ion. All four isomers produced characteristic product ions that enabled the differentiation between them. Using these ions, MRM analysis was performed on the urine of JWH-018 administered mice and determined the hydroxyl positions to be at the 6-position on the indole ring. GC-EI-MS/MS allowed for the regioisomeric differentiation of the hydroxyindole metabolite isomers of JWH-018. Furthermore, analysis of the fragmentation patterns suggests that the present method has high potential to be extended to hydroxyindole metabolites of other naphthoylindole type SCs in identifying the position of the hydroxyl group on the indole ring. Copyright © 2016 John Wiley & Sons, Ltd.

Importance of detoxifying enzymes in differentiating fibrotic development between SHRSP5/Dmcr and SHRSP rats.

OBJECTIVES: High-fat and -cholesterol diet (HFC) induced fibrotic steatohepatitis in stroke-prone spontaneously hypertensive rat (SHRSP) 5/Dmcr, the fifth substrain from SHRSP, by dysregulating bile acid (BA) kinetics. This study aimed to clarify the histopathological and BA kinetic differences in HFC-induced fibrosis between SHRSP5/Dmcr and SHRSP. METHODS: Ten-week-old male SHRSP5/Dmcr and SHRSP were randomly allocated to groups and fed with either control diet or HFC for 2 and 8 weeks. The liver histopathology, biochemical features, and molecular signaling involved in BA kinetics were measured. RESULTS: HFC caused more severe hepatocyte ballooning, macrovesicular steatosis and fibrosis in SHRSP5/Dmcr than in SHRSP. It was noted that fibrosis was disproportionately formed in retroperitoneal side of both strains. As for BA kinetics, HFC greatly increased the level of Cyp7a1 and Cyp7b1 to the same degree in both strains at 8 weeks, while multidrug resistance-associated protein 3 was greater in SHRSP5/Dmcr than SHRSP. The diet decreased the level of bile salt export pump by the same degree in both strains, while constitutive androstane receptor, pregnane X receptor, and UDP-glucuronosyltransferase activity more prominent in SHRSP5/Dmcr than SHRSP at 8 weeks. In the fibrosis-related genes, only expression of collagen, type I, alpha 1 mRNA was greater in SHRSP5/Dmcr than SHRSP. CONCLUSIONS: The greater progression of fibrosis in SHRSP5/Dmcr induced by HFC may be due to greater suppression of UDP-glucuronosyltransferase activity detoxifying toxicants, such as hydrophobic BAs.

Intact Endogenous Metabolite Analysis of Mice Liver by Probe Electrospray Ionization/Triple Quadrupole Tandem Mass Spectrometry and Its Preliminary Application to in Vivo Real-Time Analysis.

Probe electrospray ionization (PESI) is a recently developed ionization technique that enables the direct detection of endogenous compounds like metabolites without sample preparation. In this study, we have demonstrated the first combination use of PESI with triple quadrupole tandem mass spectrometry (MS/MS), which was then applied to intact endogenous metabolite analysis of mice liver, achieving detection of 26 metabolites including amino acids, organic acids, and sugars. To investigate its practicality, metabolic profiles of control and CCl4-induced acute hepatic injury mouse model were measured by the developed method. Results showed clear separation of the two groups in score plots of principal component analysis and identified taurine as the primary contributor to group separation. The results were further validated by the established gas chromatography/MS/MS method, demonstrating the present method's usefulness. In addition, we preliminarily applied the method to real-time analysis of an intact liver of a living mouse. We successfully achieved monitoring of the real-time changes of two tricarboxylic acid cycle intermediates, α-ketoglutaric acid and fumaric acid, in the liver immediately after pyruvic acid injection via a cannulated tube to the portal vein. The present method achieved an intact analysis of metabolites in liver without sample preparation, and it also demonstrates future possibility to establish in vivo real-time metabolome analysis of living animals by PESI/MS/MS.