Shigeo Okubo

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.

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.

Stereospecific analysis of lorazepam in plasma by chiral column chromatography with a circular dichroism-based detector

The chiral separation of lorazepam was achieved on a chiral column with UV and circular dichroism (CD) detection. The good resolution of lorazepam enantiomers was obtained on the column of beta-cyclodextrin derivative immobilized silica gel under reversed-phase conditions. The enantiomeric separation and identification of lorazepam were succeeded by CD detector. The method described allows the quantitation of the stereoisomers of lorazepam in human plasma following the administration of a therapeutic dose of the racemic drug. Chiroptical detection is useful for the pharmacokinetic study of chiral drugs.

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.

Enantiomeric determination of L- and D-lactic acid in human cerebrospinal fluid by chiral ligand exchange high-performance liquid chromatography

Enantiomeric determination of L- and D-lactate in human cerebrospinal fluid (CSF) was achieved by HPLC on a chiral stationary phase with UV detection. Samples were submitted to a solid-phase extraction procedure using Oasis HLB Plus Extraction Cartridge and L- and D-lactate in the extract were separated by Shodex ORpac CRX-453 B column, a ligand exchange column for chiral separation, using a mobile phase containing copper (II) ion. L- and D-lactate were determined in 25 min. Intra-assay precision in CSF was 4.98% (mean 1.85 mmol/L) for L-lactate and 10.1% (mean 4.96 micromol/L) for D-lactate (n = 5). Detection limits were between 1.0 (L-lactate) and 1.5 (D-lactate) pmol. The mean values (n = 3) of analytical recovery for L- and D-lactate were 95% and 107%, respectively. The mean +/- SD of concentrations of L- and D-lactate in CSF (n = 20) were 1.52 +/- 0.54 mmol/L and 10.98 +/- 5.15 micromol/L, respectively.

Analysis of serum and urinary lysophospholipase D/autotaxin in nephrotic syndrome.

No Abstract available

Serum lysophospholipase D/autotaxin may be a new nutritional assessment marker: study on prostate cancer patients.

Background: It is now established that the bioactive lipid lysophosphatidic acid (LPA) contributes to cancer initiation, progression and metastasis, including those of prostate cancer. LPA is produced in the serum and plasma mainly by conversion from lysophospholipids through the action of lysophospholipase D (lysoPLD), which is identical to the soluble form of autotaxin (ATX) originally isolated as a tumour cell motility-stimulating factor. In this study, we evaluated the usefulness of lysoPLD/ATX activity as a diagnostic marker. Methods: The serum lysoPLD activity, assessed by measuring choline liberation from the substrate lysophosphatidylcholine, was measured in patients with prostate cancer and compared with the concentrations of prostate-specific antigen (PSA) and conventional nutritional assessment markers. Results: The serum lysoPLD activity in prostate cancer patients was not statistically different from that in the controls. Consistent with this, there was no correlation between the serum lysoPLD activity and the serum PSA concentrations. However, the lysoPLD/ATX activity did decrease after operation in the prostate cancer patients and seemed to reflect the postoperative damage or the nutritional status. Conclusions: We postulate that while the serum lysoPLD/ATX may not be a marker of prostate cancer, it promises to instead be a new marker of nutritional status.