Hiromitsu Yokota

Serum autotaxin measurement in haematological malignancies: a promising marker for follicular lymphoma

Autotaxin (ATX) is a tumour cell motility‐stimulating factor originally isolated from melanoma cell supernatants. ATX is identical to lysophospholipase D, which produces a bioactive lipid mediator, lysophosphatidic acid (LPA), from lysophosphatidylcholine. ATX is overexpressed in various malignancies, including Hodgkin lymphoma, and ATX may stimulate tumour progression via LPA production. The present study measured the serum ATX antigen levels in patients with haematological malignancies using a recently developed automated enzyme immunoassay. The serum ATX antigen levels in patients with B‐cell neoplasms, especially follicular lymphoma (FL), were higher than those in healthy subjects. Serum ATX antigen levels in FL patients were associated with tumour burden and changed in parallel with the patients’ clinical courses. The serum ATX antigen levels were little affected by inflammation, unlike the soluble interleukin‐2 receptor and β2‐microglobulin levels. As expected, the plasma LPA levels in FL patients were correlated with the serum ATX antigen levels. Given that leukaemic tumour cells from FL patients expressed ATX, the shedding of ATX from lymphoma cells probably leads to the elevation of serum ATX antigen levels. Our results suggest that the serum ATX antigen level may be a promising and novel marker for FL.

Plasma sphingosine-1-phosphate measurement in healthy subjects: close correlation with red blood cell parameters

Abstract Background Since sphingosine-1-phosphate (Sph-1-P) plays an important role as an extracellular mediator through interaction with specific cell surface receptors, especially in the area of vascular biology and immunology/haematology, determination of its plasma concentration may become important from the clinical viewpoint. Thus, we attempted to develop a method of measuring the plasma Sph-1-P concentration for use in the clinical laboratory setting. Methods After two-step lipid extraction, Sph-1-P was coupled with o-phthaldialdehyde, and the resultant fluorescent derivative was separated by high-performance liquid chromatography. C17-Sph-1-P was used as the internal standard, instead of dihydrosphingosine-1-phosphate, which had been used previously for the same purpose but was actually detected in plasma. Results Our procedures for preparing the plasma samples and assay Sph-1-P were found to be satisfactory for clinical laboratory testing. The plasma Sph-1-P concentrations were significantly higher in men (413.1 ± 52.0 nmol/L; mean ± SD) than in women (352.4 ± 39.7 nmol/L). Unexpectedly, strong positive correlations were found between the plasma Sph-1-P concentration and red blood cell (RBC)-related parameters, rather than platelet-related parameters. Conclusions Our present study confirmed the possibility of the clinical introduction of plasma Sph-1-P measurement, and in addition, suggested that RBCs may be involved in the regulation of plasma Sph-1-P concentrations.

Stereospecific analysis of omeprazole in human plasma as a probe for CYP2C19 phenotype.

Omeprazole is a class referred to as proton pump inhibitor; it acts to regulate acid production in the stomach and is used to treat various acid-related gastrointestinal disorders. In the liver, it is metabolized to varying degrees by several cytochrome P-450 (CYP) isoenzymes which are further categorized into subfamilies of related polymorphic gene products. The metabolism of omeprazole is to a large extent dependent on CYP3A4 and CYP2C19. Omeprazole is metabolized to two major metabolites, 5-hydroxyomeprazole (CYP2C19) and omeprazole sulfone (CYP3A4). Minor mutations in CYP2C19 affect its activity in the liver and, in turn, the metabolic and pharmacokinetic profiles of omeprazole. The frequency of CYP2C19 poor metabolizers in population of Asian descent has been reported to range from 10 to 20%. Accordingly, results from population studies indicate that omeprazole can be used as a probe drug for phenotyping CYP2C19. The optical isomers of omeprazole show a clear difference in their metabolism by human liver microsomes. This study demonstrates the stereospecific analysis of omeprazole in human plasma as a probe drug of CYP2C19 phenotyping. The chiral separation of omeprazole was achieved on a chiral column with circular dichroism (CD) detection and LC/MS. A good resolution of enantiomers was obtained. The column used for chiral separation was CHIRALPAK AD-RH column (4.6 x 150 mm) using phosphate buffer and (or ammonium acetate) acetonitrile as an eluent. After a single oral dose of omeprazole (20 mg), the plasma concentrations of the separate enantiomers of omeprazole were determined for 3.5 h after drug intake. The present study is useful because of the part polymorphism plays in the therapeutic effectiveness of proton pump inhibitors during the treatment of acid-related diseases.

Itraconazole-Induced Cholestasis: Involvement of the Inhibition of Bile Canalicular Phospholipid Translocator MDR3/ABCB4

Biliary secretion of bile acids and phospholipids, both of which are essential components of biliary micelles, are mediated by the bile salt export pump (BSEP/ABCB11) and multidrug resistance 3 P-glycoprotein (MDR3/ABCB4), respectively, and their genetic dysfunction leads to the acquisition of severe cholestatic diseases. In the present study, we found two patients with itraconazole (ITZ)-induced cholestatic liver injury with markedly high serum ITZ concentrations. To characterize the effect of ITZ on bile formation in vivo, biliary bile acids and phospholipids were analyzed in ITZ-treated rats, and it was revealed that biliary phospholipids, rather than bile acids, were drastically reduced in the presence of clinically relevant concentrations of ITZ. Moreover, by using MDR3-expressing LLC-PK1 cells, we found that MDR3-mediated efflux of [14C]phosphatidylcholine was significantly reduced by ITZ. In contrast, BSEP-mediated transport of [3H]taurocholate was not significantly affected by ITZ, which is consistent with our in vivo observations. In conclusion, this study suggests the involvement of the inhibition of MDR3-mediated biliary phospholipids secretion in ITZ-induced cholestasis. Our approach may be useful for analyzing mechanisms of drug-induced cholestasis and evaluating the cholestatic potential of clinically used drugs and drug candidates.

Determination of omeprazole and its metabolites in human plasma by liquid chromatography-mass spectrometry.

Omeprazole is a benzimidazole compound that acts as a proton-pump inhibitor. Because the metabolism of omeprazole is mainly catalyzed by cytochrome P-450 (CYP) 3A4 and CYP2C19. the genetic polymorphism of CYP2C19 could be of clinical concern in the treatment of acid-related diseases with omeprazole. Therefore, a reliable method for omeprazole phenotyping is desirable in clinical situations. This study has demonstrated the determination of omeprazole and its metabolites in human plasma by liquid chromatography-three-dimensional quadrupole mass spectrometry with a sonic spray ionization interface. The analytical column was YMC-Pack Pro C18(50x2.0 mm I.D.) using acetonitrile-50 mM ammonium acetate (pH 7.25) (1:4) at a flow-rate of 0.2 ml/min. The drift voltage was 30 V. The sampling aperture was heated at 110 degrees C and Shield temperature was 230 degrees C. In the mass spectrum, the molecular ions of omeprazole, hydroxyomeprazole and omeprazole sulfone were clearly observed as base peaks. This method is sufficiently sensitive and accurate for pharmacokinetic studies of omeprazol.

Measurement of plasma lysophosphatidic acid concentration in healthy subjects: strong correlation with lysophospholipase D activity:

Abstract Background Lysophosphatidic acid (LPA) plays important roles in a variety of biological responses, especially in the area of vascular biology, and the determination of its plasma concentration is believed to be important. Several mechanisms are known to be involved in the metabolism of LPA. Methods To identify factors that may determine the plasma concentrations of this important bioactive lipid, we examined its concentrations using an enzymatic cycling assay and related parameters in 146 healthy subjects. Results The LPA concentration was significantly higher in women (mean ± SD, 0.103 ± 0.032 μmol/L; n = 47) than in men (0.077 ± 0.026 μmol/L; n = 99). A multiple regression analysis showed a strong positive correlation between the plasma LPA concentration and serum lysophospholipase D (lysoPLD) activity, while the LPA concentration was correlated with the plasma lysophosphatidylcholine (LPC) concentration only in men. Other lipid-related parameters were only slightly correlated or were not correlated with the LPA concentration. Conclusions Our findings suggested that conversion from LPC by lysoPLD might be the major route for LPA production in plasma.

Plasma concentration of bioactive lipid mediator sphingosine 1-phosphate is reduced in patients with chronic hepatitis C

BACKGROUND Bioactive lipid mediator S1P has been suggested to play pathophysiological roles in various fields of clinical science as a circulating paracrine mediator. We previously established a reliable method of measuring plasma S1P concentration, and reported that the one in healthy subjects has a gender difference and a correlation with red blood cell (RBC)-parameters, however, the reports of S1P measurements in the blood in patients with a specific disease have been scarce. Because our previous evidence suggests that S1P is involved in liver pathophysiology, we examined plasma S1P concentration in chronic hepatitis C patients. METHODS S1P assay was performed using a high-performance liquid chromatography system. RESULTS Plasma S1P concentrations were reduced in chronic hepatitis C patients compared with in healthy subjects with the same hemoglobin concentration, irrespective of gender. Among the blood parameters, serum hyaluronic acid concentration, a surrogate marker for liver fibrosis, was most closely and inversely correlated with plasma S1P concentration. Furthermore, plasma S1P concentration decreased throughout the progression of carbon tetrachloride-induced liver fibrosis in rats. CONCLUSIONS Plasma S1P concentration was reduced in chronic hepatitis C patients, and liver fibrosis might be involved, at least in part, in the mechanism responsible for this reduction.

Apolipoprotein A-I deficiency with accumulated risk for CHD but no symptoms of CHD

We evaluated a 69-year-old Japanese woman with apolipoprotein (apo) A-I deficiency, high levels of low-density lipoprotein (LDL)-cholesterol, hypertension and impaired glucose tolerance. The patient had corneal opacity, but neither xanthomas, xanthelasma, nor tonsillar hypertrophy. She was not symptomatic for coronary heart disease (CHD), and had normal electrocardiograms at rest and exercise using a cycle ergometer. She had severely reduced levels of high-density lipoprotein (HDL)-cholesterol (0.10-0.18 mmol/l) and no apo A-I (<0.6 mg/dl). LDL-cholesterol and apo B as well as apo E were increased even under treatment with 10 mg pravastatin per day. Gel filtration chromatography revealed that in addition to VLDL and LDL fractions, she had apo A-II rich and apo E rich fractions, which were present in the HDL fraction separated by ultracentrifugation. A cytosine deletion was identified by genomic DNA sequencing of the apo A-I gene of the patient at the third base of codon 184 in the fourth exon, which led to a frame shift mutation and early termination at codon 200. This patient is the oldest among those with apo A-I deficiency reported in the literature, and she had no symptoms of CHD despite the accumulated risk for the disease.

Hepatic stellate cell damage may lead to decreased plasma ADAMTS13 activity in rats.

ADAMTS13 is gaining attention, because its deficiency causes thrombotic thrombocytopenic purpura. Although its regulatory mechanism is not fully understood, we wondered if hepatic stellate cells (HSCs) play a role, because ADAMTS13 mRNA is exclusively expressed in the liver and primarily in HSCs. Plasma ADAMTS13 activity was markedly reduced in dimethylnitrosamine‐treated rats, where HSC apoptosis is an essential event, but not in carbon tetrachloride‐ or thioacetamide‐treated rats without HSC apoptosis. Furthermore, plasma ADAMTS13 activity was also reduced in 70% hepatectomized rats, where HSC loss occurs. These results suggest that HSC may be involved in the regulation of plasma ADAMTS13 activity.

Frequency distribution of thiopurine S-methyltransferase activity in red blood cells of a healthy Japanese population.

Thiopurine S-methyltransferase (TPMT), which exhibits a genetic polymorphism, plays an important role in the metabolism of thiopurine drugs such as mercaptopurine, thioguanine, and azathioprine. To determine the frequency distribution of TPMT activity in 157 Japanese subjects with different TPMT genotypes, ie, TPMT*1/*1 and TPMT*1/*3, the authors measured levels of 6-methylmercaptopurine formed from 6-mercaptopurine in red blood cells lysates by HPLC. The TPMT activities in our Japanese subjects ranged from 11.0 to 42.6 pmol/h/mgHb. Although the mean value of TPMT activities in 6 subjects with TPMT*1/*3C (20.3 ± 8.1 pmol/h/mgHb) was 25% lower than that in 151 subjects with TPMT*1/*1 (27.0 ± 5.1 pmol/h/mgHb), there was overlap. The ranges of TPMT activity in subjects with TPMT*1/*1 and those with TPMT*1/*3C were similar. The median values in TPMT*1/*3C and TPMT*1/*1 individuals were 20.1 (11.0–31.2) and 26.8 pmol/h/mgHb (15.7–42.7), respectively (Mann-Whitney U-test: median difference 6.7 pmol/h/mgHb, 95% CI 0–25.5, P < 0.05). This observation may have relevance for the use of 6-mercaptopurine and azathioprine as therapeutic agents in Japanese patients.