Sphingosine kinase 1 is involved in triglyceride breakdown by maintaining lysosomal integrity in brown adipocytes.

Sphingosine 1-phosphate (S1P) has been implicated in brown adipose tissue (BAT) formation and energy consumption; however, the mechanistic role of sphingolipids, including S1P, in BAT remains unclear. Here, we showed that, in mice, BAT activation by cold exposure upregulated mRNA and protein expression of the S1P-synthesizing enzyme sphingosine kinase 1 (SphK1) and S1P production in BAT. Treatment of wild-type brown adipocytes with exogenous S1P or S1P receptor subtype-selective agonists stimulated triglyceride (TG) breakdown only marginally, compared with noradrenaline. However, genetic deletion of Sphk1 resulted in hypothermia and diminished body weight loss upon cold exposure, suggesting that SphK1 is involved in thermogenesis through mechanisms different from receptor-mediated, extracellular action of S1P. In BAT of wild-type mice, SphK1 was localized largely in the lysosomes of brown adipocytes. In the brown adipocytes of Sphk1-/- mice, the number of lysosomes was reduced and lysosomal function, including proteolytic activity, acid esterase activity, and motility, was impaired. Concordantly, nuclear translocation of transcription factor EB, a master transcriptional regulator of lysosome biogenesis, was reduced, leading to decreased mRNA expression of the lysosome-related genes in Sphk1-/- BAT. Moreover, BAT of Sphk1-/- mice showed greater TG accumulation with dominant larger lipid droplets in brown adipocytes. Inhibition of lysosomes with chloroquine resulted in a less extent of triglyceride accumulation in Sphk1-/- brown adipocytes compared with wild-type brown adipocytes, suggesting a reduced lysosome-mediated TG breakdown in Sphk1-/- mice. Our results indicate a novel role of SphK1 in lysosomal integrity, which is required for TG breakdown and thermogenesis in BAT.

Desynchronization between Food Intake and Light Stimulations Induces Uterine Clock Quiescence in Female Mice.

BACKGROUND: Dysmenorrhea is associated with breakfast skipping in young women, suggesting that fasting in the early active phase disrupts uterine functions. OBJECTIVES: To investigate the possible involvement of the uterine clock system in fasting-induced uterine dysfunction, we examined core clock gene expressions in the uterus using a 28-h interval-fed mouse model. METHODS: Young female mice (8 wk of age) were divided into 3 groups: group I (ad libitum feeding), group II (time-restricted feeding, initial 4 h of the active period every day), and group III (time-restricted feeding for 8 h with a 28-h cycle). Groups II and III have the same fasting interval of 20 h. After analyzing feeding and wheel running behaviors during 2 wk of dietary restriction, mice were sacrificed at 4-h intervals, and the expression profiles of clock genes in the uterus and liver were examined by qPCR. RESULTS: The mice in group I took food mainly during the dark phase and those in group II during the initial 4 h of the dark phase, whereas those in group III delayed feeding time by 4 h per cycle. In all groups, spontaneous wheel running was observed during the dark phase. There was no difference in the quantity of feeding and the amount of running exercise among the 3 groups during the second week. The mRNA expressions of peripheral clock genes, Bmal1, Clock, Per1, Per2, Cry1, Nr1d1, and Dbp and a clock-controlled gene, Fabp1, in the uterus showed rhythmic oscillations with normal sequential expression cascade in groups I and II, whereas their expressions decreased and circadian cycles disappeared in group III. In contrast, liver core clock genes in group III showed clear circadian cycles. CONCLUSIONS: Fluctuations in the timing of the first food intake impair the uterine clock oscillator system to reduce clock gene expressions and abolish their circadian rhythms.

Imeglimin profoundly affects the circadian clock in mouse embryonic fibroblasts.

OBJECTIVE: Imeglimin is a novel antidiabetic drug structurally related to metformin. Metformin has been shown to modulate the circadian clock in rat fibroblasts. Accordingly, in the present study, we aimed to determine whether imeglimin can impact the circadian oscillator in mouse embryonic fibroblasts (MEFs). METHODS: MEFs carrying a Bmal1-Emerald luciferase (Bmal1-ELuc) reporter were exposed to imeglimin (0.1 or 1 mM), metformin (0.1 or 1 mM), a nicotinamide phosphoribosyltransferase inhibitor FK866, and/or vehicle. Subsequently, Bmal1-ELuc expression and clock gene mRNA expression levels were measured at 10-min intervals for 55 h and 4-h intervals for 32 h, respectively. RESULTS: Imeglimin significantly prolonged the period (from 26.3 to 30.0 h at 0.1 mM) and dose-dependently increased the amplitude (9.6-fold at 1 mM) of the Bmal1-ELuc expression rhythm; however, metformin exhibited minimal effects on these parameters. Moreover, imeglimin notably impacted the rhythmic mRNA expression of clock genes (Bmal1, Per1, and Cry1). The concurrent addition of FK866 partly inhibited the effects of imeglimin on both Bmal1-ELuc expression and clock gene mRNA expression. CONCLUSION: Collectively, these results reveal that imeglimin profoundly affects the circadian clock in MEFs. Further studies are needed to evaluate whether imeglimin treatment could exert similar effects in vivo.

Impact of genetic polymorphisms on the pharmacokinetics and pharmacodynamics of lenvatinib in patients with hepatocellular carcinoma.

This prospective study examined the impact of genetic polymorphisms on the pharmacokinetics and clinical efficacy and safety of lenvatinib, a substrate of ATP-binding transporters, in a cohort of 48 Japanese patients with hepatocellular carcinoma (HCC). Pharmacokinetic studies were performed at the start of lenvatinib therapy (day 1) and on day 15. The coefficients of variation in AUC0-24h of lenvatinib on days 1 and 15 were 44.0% and 52.4%, respectively. Although the ABCB1 3435C > A, 1236C > T, and 2677G>T/A polymorphisms did not influence pharmacokinetic parameters, the AUC0-24h values on days 1 and 15 of the ABCG2 C/A or A/A group were approximately 1.1-fold and 1.4-fold that in the ABCG2 C/C group (P = 0.164 and 0.024). There were no significant differences in AUC0-24h on days 1 and 15 between the responders (complete or partial response) and non-responders (stable or progressive disease). The AUC0-24h on day 15 in those developing anorexia of any grade was significantly higher than that without such development (P = 0.017). In multivariate analysis, ABCG2 421C > A C/A or A/A was significantly associated with the development of anorexia (odds ratio 9.009, P = 0.009). ABCG2 421C > A polymorphism could affect exposure to lenvatinib and the development of anorexia.

Effects of ABCB1 and ABCG2 polymorphisms on the pharmacokinetics of abemaciclib.

PURPOSE: Adverse events after the use of the CDK4/6 inhibitor abemaciclib are dose-dependent. However, its pharmacokinetics varies among individuals. Abemaciclib is reportedly transported by P-glycoprotein and breast cancer resistance protein. Therefore, we evaluated whether ABCB1 and ABCG2 polymorphisms are pharmacokinetic predictive factors of abemaciclib. METHODS: A total of 45 patients with breast cancer taking abemaciclib (150 mg twice per day) for 2 weeks were evaluated to determine the associations among abemaciclib concentration; adverse events; and ABCB1 1236 T > C, 2677G > T/A, 3435C > T, and ABCG2 421C > A gene polymorphisms. RESULTS: The trough concentration of abemaciclib was significantly higher in the group with grade 2 or greater neutropenia and thrombocytopenia than in those with grades 0 or 1. For ABCB1 2677G > T/A polymorphisms, the concentration of abemaciclib tended to be higher in the homozygous group (TT + AT) than in the wild-type + heterozygous group (GG + GA + GT) (median [range], 222.8 [80.5-295.8] ng/mL vs. 113.5 [23.6-355.2] ng/mL, P = 0.09), Moreover, the ABCB1 2677G > T/A homozygous group had a higher tendency of abemaciclib withdrawal or dose reduction within 4 weeks than the wild-type + heterozygous group (odds ratio, 4.22; 95% confidence interval, 0.86-20.7; P = 0.08). No significant association was observed among abemaciclib concentration; adverse reactions; and ABCB1 1236 T > C, 3435C > T, and ABCG2 421C > A polymorphisms. CONCLUSION: ABCB1 2677G > T/A polymorphism might be a predictor of the pharmacokinetics and tolerability of abemaciclib.

Chronic Treatment with Metformin Has No Disrupting Effect on the Hepatic Circadian Clock in Mice.

Background and Objectives: The antidiabetic agent metformin is known to activate AMP-activated protein kinase (AMPK) in various tissues. Because AMPK can modulate intracellular circadian clocks through regulating the stability of clock components, a single dose of metformin has been reported to affect circadian clocks in the peripheral tissues. In this study, therefore, we investigated whether chronic treatment with metformin causes the impairment of circadian clocks, especially if given at an inappropriate time. Materials and Methods: Non-diabetic C57BL/6J mice were allowed access to food only during 4 h at the beginning of the dark period, and repeatedly i.p. injected with a nearly maximum non-toxic dose of metformin, once daily either at 4 h after the beginning of the dark period or at the beginning of the light period. Diabetic ob/ob mice were given free access to food and treated with metformin in drinking water. Results: Under the controlled feeding regimen, 8-day treatment with metformin did not alter the mRNA expression rhythms of clock genes in both liver and adipose tissue of C57BL/6J mice, regardless of dosing time. In addition, chronic treatment with metformin for 2 weeks affected hepatic AMPK activation rhythm but did not disrupt the circadian clocks in the liver and adipose tissues of the ob/ob mice. Conclusions: These results mitigate concerns that treatment with metformin impairs peripheral circadian clocks, although confirmation is needed in humans.

Influence of renal ischaemia-reperfusion injury on renal neutrophil gelatinase-associated lipocalin receptor (24p3R) in rats.

The neutrophil gelatinase-associated lipocalin (NGAL) receptor (24p3R) is expressed in distal nephron and contributes to the endocytosis of NGAL in urine. This study was undertaken to evaluate an influence of renal ischaemia-reperfusion injury on 24p3R. Unilateral renal pedicle was clamped for 0, 10, 20, 30, or 45 minutes in male Wistar rats. Urine was collected for 24 hours after reperfusion, and ischaemic kidney and blood sample were obtained. Apparent histological injury in the ischaemic kidney was detected in the 30 and 45 minutes-treated groups. Urinary NGAL excretion elevated in rats with renal ischaemia for more than 20 minutes, while serum creatinine increased in rats for more than 30 minutes of ischaemia. Renal protein expression of NGAL did not significantly change. Renal mRNA expressions of megalin and cubilin, which are expressed at renal proximal tubules and uptake NGAL, decreased in animals with renal ischaemia for more than 20 minutes. Renal protein expression of 24p3R, which is expressed at renal distal tubules and uptake NGAL, decreased in rats with renal ischaemia for 45 min. This study showed for the first time that renal 24p3R decreased in response to renal ischaemia. As relatively longer renal ischaemia (45 minutes) decreased renal 24p3R protein and increased urinary NGAL excretion, the down-regulation of 24p3R protein might contribute to the elevated urinary excretion of NGAL in rats with unilateral ischaemia-reperfusion injury.

Effects of ABCB1 and ABCG2 Polymorphisms on the Pharmacokinetics of Abemaciclib Metabolites (M2, M20, M18).

BACKGROUND/AIM: Abemaciclib, an oral anticancer drug used in the treatment of breast cancer, is metabolised to its active forms - M2, M20 and M18; these forms have a potency similar to that of the parent drug. Abemaciclib and its active metabolites are reportedly transported by P-glycoprotein and breast cancer resistance protein (BCRP). We previously reported that the ABCB1 2677G>T/A homozygous type is associated with a higher abemaciclib concentration leading to treatment withdrawal and/or dose reduction. However, the pharmacokinetics of its metabolites have not been investigated. The purpose of the present study was to evaluate the effects of ABCB1 and ABCG2 polymorphisms on the pharmacokinetics of the abemaciclib metabolites M2, M20 and M18. PATIENTS AND METHODS: We evaluated 40 patients with breast cancer who received 150 mg abemaciclib twice per day for 2 weeks at the Aichi Cancer Center Hospital, Japan. Peak areas (arbitrary unit) of abemaciclib metabolites were measured using liquid chromatography tandem with mass spectrometry and compared between ABCB1 1236T>C, 2677G>T/A, 3435C>T and ABCG2 421C>A gene polymorphisms. RESULTS: For ABCB1 2677G>T/A polymorphisms, exposure doses for the abemaciclib metabolites M2 and M20 were higher in the homozygous (TT + AT) group than in the wild-type and heterozygous (GG + GA + GT) groups (p=0.09 and p=0.06, respectively). No significant association was observed between abemaciclib metabolites and ABCB1 1236T>C, ABCB1 3435C>T and ABCG2 421C>A polymorphisms. CONCLUSION: The ABCB1 2677G>T/A polymorphism may influence tolerance to abemaciclib in breast cancer patients by affecting the pharmacokinetics of the agent and its active metabolites.

L-carnitine prevents lenvatinib-induced muscle toxicity without impairment of the anti-angiogenic efficacy.

Lenvatinib is an oral tyrosine kinase inhibitor that acts on multiple receptors involved in angiogenesis. Lenvatinib is a standard agent for the treatment of several types of advanced cancers; however, it frequently causes muscle-related adverse reactions. Our previous study revealed that lenvatinib treatment reduced carnitine content and the expression of carnitine-related and oxidative phosphorylation (OXPHOS) proteins in the skeletal muscle of rats. Therefore, this study aimed to evaluate the effects of L-carnitine on myotoxic and anti-angiogenic actions of lenvatinib. Co-administration of L-carnitine in rats treated with lenvatinib for 2 weeks completely prevented the decrease in carnitine content and expression levels of carnitine-related and OXPHOS proteins, including carnitine/organic cation transporter 2, in the skeletal muscle. Moreover, L-carnitine counteracted lenvatinib-induced protein synthesis inhibition, mitochondrial dysfunction, and cell toxicity in C2C12 myocytes. In contrast, L-carnitine had no influence on either lenvatinib-induced inhibition of vascular endothelial growth factor receptor 2 phosphorylation in human umbilical vein endothelial cells or angiogenesis in endothelial tube formation and mouse aortic ring assays. These results suggest that L-carnitine supplementation could prevent lenvatinib-induced muscle toxicity without diminishing its antineoplastic activity, although further clinical studies are needed to validate these findings.

Lenvatinib causes reduced expression of carnitine/organic cation transporter 2 and carnitine deficiency in the skeletal muscle of rats.

Lenvatinib, an oral tyrosine kinase inhibitor, is widely used to treat several types of advanced cancers but often causes muscular adverse reactions. Although carnitine supplementation may prevent these effects, the mechanism underlying lenvatinib-induced skeletal muscle impairment remains poorly understood. To this end, we aimed to investigate the impact of lenvatinib on carnitine disposition in rats. Once-daily administration of lenvatinib repeated for two weeks did not affect urinary excretion or serum concentration of carnitines throughout the treatment period but ultimately decreased the L-carnitine content in the skeletal muscle. The treatment decreased the expression of carnitine/organic cation transporter (OCTN) 2, a key transporter of carnitine, in skeletal muscle at the protein level but not at the mRNA level. In cultured C2C12 myocytes, lenvatinib inhibited OCTN2 expression in a dose-dependent manner at the protein level. Furthermore, lenvatinib dose-dependently decreased the protein levels of carnitine-related genes, adenosine triphosphate content, mitochondrial membrane potential, and markers of mitochondrial function in vitro. These results reveal the deleterious effects of lenvatinib on OCTN2 expression, carnitine content, and mitochondrial function in skeletal muscle that may be associated with muscle toxicity.

Sustained effect of habitual feeding time on daily rhythm of core body temperature in mice.

Background and aim: Circadian clocks in most peripheral tissues are entrained mainly by feeding. Therefore, this study aimed to investigate whether the daily rhythm of core body temperature (CBT), including the effect of diet-induced thermogenesis, varies according to habitual feeding time. Methods: Wild-type and uncoupling protein 1 (UCP1) knockout mice were fed only during the first 4 h (Breakfast group) or the last 4 h of the dark period (Dinner group) for 17 days. On day 18, both groups were fed twice for 2 h, at the same starting times. Locomotor activity and CBT were measured continuously during the experiment. Results: On day 18, CBT increased at the beginning of each feeding period, regardless of the group and strain. However, the CBT increase induced by the first meal decreased sharply in the Breakfast group and mildly in the Dinner group; the opposite was observed after the second meal. In UCP1 knockout, but not wild-type, mice, the total amount of CBT was significantly lower in the Dinner group than in the Breakfast group. These effects were mostly independent of the locomotor activity and food intake. Conclusion: These results reveal that the effect of habitual feeding time on the daily rhythm of CBT is sustained at least until the following day. These effects may be mediated by both UCP1-dependent and -independent mechanisms.

Mass Spectrometric Analysis of Sphingomyelin with N-α-Hydroxy Fatty Acyl Residue in Mouse Tissues.

Sphingomyelin (SM) with N-α-hydroxy fatty acyl residues (hSM) has been shown to occur in mammalian skin and digestive epithelia. However, the metabolism and physiological relevance of this characteristic SM species have not been fully elucidated yet. Here, we show methods for mass spectrometric characterization and quantification of hSM. The hSM in mouse skin was isolated by TLC. The hydroxy hexadecanoyl residue was confirmed by electron impact ionization-induced fragmentation in gas chromatography-mass spectrometry. Mass shift analysis of acetylated hSM by time of flight mass spectrometry revealed the number of hydroxyl groups in the molecule. After correcting the difference in detection efficacy, hSM in mouse skin and intestinal mucosa were quantified by liquid chromatography-tandem mass spectrometry, and found to be 16.5 ± 2.0 and 0.8 ± 0.4 nmol/µmol phospholipid, respectively. The methods described here are applicable to biological experiments on hSM in epithelia of the body surface and digestive tract.

Brown adipocyte-specific knockout of Bmal1 causes mild but significant thermogenesis impairment in mice.

OBJECTIVE: Impaired circadian clocks can cause obesity, but their pathophysiological role in brown adipose tissue (BAT), a major tissue regulating energy metabolism, remains unclear. To address this issue, we investigated the effects of complete disruption of the BAT clock on thermogenesis and energy expenditure. METHODS: Mice with brown adipocyte-specific knockout of the core clock gene Bmal1 (BA-Bmal1 KO) were generated and analyzed. RESULTS: The BA-Bmal1 KO mice maintained normal core body temperatures by increasing shivering and locomotor activity despite the elevated expression of thermogenic uncoupling protein 1 in BAT. BA-Bmal1 KO disrupted 24 h rhythmicity of fatty acid utilization in BAT and mildly reduced both BAT thermogenesis and whole-body energy expenditure. The impact of BA-Bmal1 KO on the development of obesity became obvious when the mice were fed a high-fat diet. CONCLUSIONS: These results reveal the importance of the BAT clock for maintaining energy homeostasis and preventing obesity.

A clean-up technology for the simultaneous determination of lysophosphatidic acid and sphingosine-1-phosphate by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry using a phosphate-capture molecule, Phos-tag.

Lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) are growth factor-like lipids having a phosphate group. The concentrations of these mediator lipids in blood are considered to be potential biomarkers for early detection of cancer or vascular diseases. Here, we report a method for simultaneous determination of LPA and S1P using Phos-tag, a zinc complex that specifically binds to a phosphate-monoester group. Although both LPA and S1P are hydrophilic compounds, we found that they acquire hydrophobic properties when they form complexes with Phos-tag. Based on this finding, we developed a method for the enrichment of LPA and S1P from biological samples. The first partition in a two-phase solvent system consisting of chloroform/methanol/water (1:1:0.9, v/v/v) is conducted for the removal of lipids. LPA and S1P are specifically extracted as Phos-tag complexes at the second partition by adding Phos-tag. The Phos-tag complexes of LPA and S1P are detectable by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) and quantifiable based on the relative intensities of ions using 17:0 LPA and C17 S1P as internal standards. The protocol was validated by analyses of these mediator lipids in calf serum, a rat brain and a lung. The clean-up protocol is rapid, requires neither thin-layer chromatography (TLC) nor liquid chromatography (LC), and is applicable to both blood and solid tissue samples. We believe that our protocol will be useful for a routine analysis of LPA and S1P in many clinical samples.

Elevated serum levels of arachidonoyl-lysophosphatidic acid and sphingosine 1-phosphate in systemic sclerosis.

Systemic sclerosis (SSc) is an often fatal disease characterized by autoimmunity and inflammation, leading to widespread vasculopathy and fibrosis. Lysophosphatidic acid (LPA), a bioactive phospholipid in serum, is generated from lysophospholipids secreted from activated platelets in part by the action of lysophospholipase D (lysoPLD). Sphingosine 1-phosphate (S1P), a member of the bioactive lysophospholipid family, is also released from activated platelets. Because activated platelets are a hallmark of SSc, we wanted to determine whether subjects with SSc have altered serum lysophospholipid levels or lysoPLD activity. Lysophospholipid levels were measured using mass spectrometric analysis. LysoPLD activity was determined by quantifying choline released from exogenous lysophosphatidylcholine (LPC). The major results were that serum levels of arachidonoyl (20:4)-LPA and S1P were significantly higher in SSc subjects versus controls. Furthermore, serum LPA:LPC ratios of two different polyunsaturated phospholipid molecular species, and also the ratio of all species combined, were significantly higher in SSc subjects versus controls. No significant differences were found between other lysophospholipid levels or lysoPLD activities. Elevated 20:4 LPA, S1P levels and polyunsaturated LPA:LPC ratios may be markers for and/or play a significant role in the etiology of SSc and may be future pharmacological targets for SSc treatment.

Production of bioactive lysophosphatidic acid by lysophospholipase D in hen egg white.

Lysophosphatidic acid (LPA), a lysophospholipid mediator, is produced extracellularly by lysophospholipase D (lysoPLD) secreted in several animal body fluids including blood plasma. Previously, we reported that hen egg white contains polyunsaturated fatty acid-rich LPA. In this study, we examined whether lysoPLD is involved in the production of LPA in hen egg white. LysoPLD activity was measured by determining LPA and choline by mass spectrometric and enzyme-linked fluorometric analyses, respectively. LysoPLD increased with increased dilution of egg white, indicating that one or more components of egg white strongly inhibit its lysoPLD activity. This dilution-dependent increase in the lysoPLD activity was masked by co-incubation of the egg white with lysozyme, a major protein in hen egg white. Furthermore, addition of Zn(2+), Mn(2+), Ni(2+), or Co(2+) to diluted egg white altered preference patterns of lysoPLD toward choline-containing substrates. In particular, the egg white lysoPLD activity was greatly increased when Co(2+) was added. The cation-requirement of lysoPLD activity in hen egg white resembled that of plasma autotaxin (ATX)/lysoPLD. Western blot analysis revealed that egg white contained a protein that was immunostained with anti-ATX antibody. These results suggested that LPA in hen egg white is produced from lysophospholipids, especially LPC, by the action of ATX/lysoPLD, possibly originating from hen oviduct fluid.

A Cleanup Method for Mass Spectrometric Analysis of Sphingosine- and Ceramide-1-Phosphate in Blood and Solid Tissue Using a Phosphate Capture Molecule.

Cleanup technology and mass spectrometric determination of sphingosine-1-phosphate (S1P) using a phosphate capture molecule are shown. The protocol is rapid, requires neither thin-layer chromatography nor liquid chromatography, and is applicable to both blood and solid tissue samples. The mass spectrometric method is also applicable to ceramide-1-phosphate.

Gadoxetic acid-enhanced magnetic resonance imaging to predict paritaprevir-induced hyperbilirubinemia during treatment of hepatitis C.

BACKGROUND: Paritaprevir inhibits organic anion-transporting polypeptide (OATP)1B1 and OATP1B3, which transport bilirubin. Hyperbilirubinemia is an adverse event reported during hepatitis C treatment. Gadoxetic acid is also transported by OATP1B1/1B3. We evaluated whether the enhancement effect in gadoxetic acid-enhanced magnetic resonance (MR) imaging could predict the plasma concentration of paritaprevir and might anticipate the development of hyperbilirubinemia. METHODS: This prospective study evaluated 27 patients with hepatitis C who underwent gadoxetic acid-enhanced MR imaging prior to treatment with ombitasvir, paritaprevir, and ritonavir. The contrast enhancement index (CEI), a measure of liver enhancement during the hepatobiliary phase, was assessed. Plasma trough concentrations, and concentrations at 2, 4, and 6 h after dosing were determined 7 d after the start of treatment. RESULTS: Seven patients (26%) developed hyperbilirubinemia (≥ 1.6 mg/dl). Paritaprevir trough concentration (Ctrough) was significantly higher in patients with hyperbilirubinemia than in those without (p = 0.022). We found an inverse relationship between CEI and Ctrough (r = 0.612, p = 0.001), while there was not a significantly weak inverse relationship between AUC0-6 h and CEI (r = -0.338, p = 0.085). The partial correlation coefficient between CEI and Ctrough was -0.425 (p = 0.034), while excluding the effects of albumin and the FIB-4 index. Receiver operating characteristic (ROC) curve analysis showed that the CEI was relatively accurate in predicting hyperbilirubinemia, with area under the ROC of 0.882. Multivariate analysis showed that the CEI < 1.61 was the only independent predictor related to the development of hyperbilirubinemia, with an odds ratio of 9.08 (95% confidence interval 1.05-78.86, p = 0.046). CONCLUSIONS: Hepatic enhancement with gadoxetic acid was independently related to paritaprevir concentration and was an independent pretreatment factor in predicting hyperbilirubinemia. Gadoxetic acid-enhanced MR imaging can therefore be useful in determining the risk of paritaprevir-induced hyperbilirubinemia.

Metabolic pathway that produces essential fatty acids from polymethylene-interrupted polyunsaturated fatty acids in animal cells.

Sciadonic acid (20:3 Delta-5,11,14) and juniperonic acid (20:4 Delta-5,11,14,17) are polyunsaturated fatty acids (PUFAs) that lack the Delta-8 double bond of arachidonic acid (20:4 Delta-5,8,11,14) and eicosapentaenoic acid (20:5 Delta-5,8,11,14,17), respectively. Here, we demonstrate that these conifer oil-derived PUFAs are metabolized to essential fatty acids in animal cells. When Swiss 3T3 cells were cultured with sciadonic acid, linoleic acid (18:2 Delta-9,12) accumulated in the cells to an extent dependent on the concentration of sciadonic acid. At the same time, a small amount of 16:2 Delta-7,10 appeared in the cellular lipids. Both 16:2 Delta-7,10 and linoleic acid accumulated in sciadonic acid-supplemented CHO cells, but not in peroxisome-deficient CHO cells. We confirmed that 16:2 Delta-7,10 was effectively elongated to linoleic acid in rat liver microsomes. These results indicate that sciadonic acid was partially degraded to 16:2 Delta-7,10 by two cycles of beta-oxidation in peroxisomes, then elongated to linoleic acid in microsomes. Supplementation of Swiss 3T3 cells with juniperonic acid, an n-3 analogue of sciadonic acid, induced accumulation of alpha-linolenic acid (18:3 Delta-9,12,15) in cellular lipids, suggesting that juniperonic acid was metabolized in a similar manner to sciadonic acid. This PUFA remodeling is thought to be a process that converts unsuitable fatty acids into essential fatty acids required by animals.

Movement of monoglyceride derived from hydrolysis of fluorescence-labeled lyso platelet-activating factor by lysophospholipase C through plasma membranes of porcine kidney epithelial cell line LLC-PK1.

To investigate the mechanisms of the release of lyso platelet-activating factor (PAF), an alkyl ether-linked lysophosphatidylcholine, from the kidney epithelial cell line LLC-PK1, the cell monolayer was incubated with a fluorescence-labeled lysoPAF analog, Bodipy-lysoPAF, on either the basolateral or apical side. The fluorescent lipids in the culture media mixed with or without bovine serum albumin at a final concentration of 2% were analyzed by thin layer chromatography. In both cases, two major bands, assignable to Bodipy-lysoPAF and Bodipy-monoglyceride (MG), were detected in the culture medium to which Bodipy-lysoPAF had been added, whereas the culture medium at the opposite side exhibited only the major band of Bodipy-MG. Our results suggest that lysoPAF was degraded by high ecto-lysophospholipase C activity. The possible physiological significance of this metabolic pathway is discussed.