Shigemi Hosogaya

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.

Expression of the LIM Proteins Paxillin and Hic-5 in Human Tissues

The LIM domain is a protein-protein interaction motif critically involved in a variety of fundamental biological processes, including cytoskeletal organization, cell lineage specification, and organ development. In this study we examined the expression of the LIM proteins paxillin and Hic-5 in adult human tissues by immunohistochemistry and immunoblotting. Paxillin expression was widespread and observed both in non-muscle and muscle tissues. Of the latter, paxillin was mainly expressed in multinuclear striated muscle. In contrast, Hic-5 showed restricted expression and was expressed in muscle tissues, mainly in mononuclear smooth muscle. Taken together with previous findings, it appears likely that the counterbalance between paxillin and Hic-5 may be deeply involved in muscle differentiation.

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.

CETP is a determinant of serum LDL-cholesterol but not HDL-cholesterol in healthy Japanese

Cholesteryl ester transfer protein (CETP) is one of the factors that regulate plasma levels of HDL-cholesterol. To identify the factors that may regulate CETP activity, and to determine to what extent CETP is correlated with physiologic concentrations of lipoprotein, we performed an epidemiologic study in 586 healthy volunteers (317 males and 269 females mean age 52.2 +/- 10.9 years). CETP activity in these subjects was 192.96 +/- 48.73 (mean +/- S.D.) nmol/ml/h and distributed to a wide range (60-450 nmol/ml/h). Using multiple regression analysis, we found significant positive correlations between CETP activity and LDL-cholesterol (P < 0.03), apolipoprotein (apo) E (P < 0.005) and LCAT activity (P < 0.001). CETP activities showed significant negative correlation with apo A-I (P < 0.03). However, CETP activity showed no significant correlation either with HDL cholesterol or with apo B. One-way layout analysis of variance showed that alcohol drinking and cigarette smoking significantly reduced CETP activity, but there was no significant association between CETP activity and body mass index. Although CETP activities were significantly higher in females than in males (P < 0.001), multiple regression analysis showed no correlation between CETP activity and age in either the males or the females. Our results suggest that CETP activity regulates the concentration of apo A-I and LDL-cholesterol, and that such activity may be influenced by gender, alcohol consumption and cigarette smoking.

Nationwide multicenter study aimed at the establishment of common reference intervals for standardized clinical laboratory tests in Japan.

Abstract Background: The Japanese Association of Medical Technologists (JAMT) sought to establish common reference intervals (RIs) applicable nationwide in Japan for 27 serum constituent analytes for which certified reference materials are available and nine analytes frequently measured in routine tests. More than 100 laboratories certified for metrological traceability collaborated in the recruitment, sampling, and measurement of analytes for the establishment of RIs. No previous attempt has been made to establish RIs by such a large number of laboratories. The allowable limits of trueness and intermediate precision based on the JAMT criteria were applied to the reference values measured by these laboratories, and measured values within the allowance limits were used to establish RIs. Methods: Reference individuals included 5748 healthy volunteers aged 18–65 years who were engaged in medical care-related work based on the CLSI guidelines. After secondary exclusion of individuals in whom abnormal values were detected in basic routine test items and adjustment for the distribution of age and gender, 3371 reference individuals were chosen in the parametric determination of RIs. Employing the three-level nested ANOVA, between-laboratory, -region, -sex, and -age variations were evaluated. Results: No significant difference was noted in between-region variations in any item. Results of ANOVA revealed between-sex and -age variations in 14 and 15 analytes, respectively. Based on these results of variation, RIs were established with and without partition by sex. Conclusions: Since no between-region variation was detected in reference values among accuracy-certified core laboratories, RIs applicable nationwide were established.

Antibody studies of factor VIII inhibitor in a case with Waldenström's macroglobulinemia

We report a case of Waldenströms macroglobulinemia with prominent bleeding tendency; laboratory investigation revealed an elevated activated partial thromboplastin time. Further laboratory evaluation showed circulating factor VIII anticoagulant, deemed polyclonal IgG, with a titer of 700 Bethesda Units/ml. The factor VIII inactivation kinetics of the patient plasma were identical to those of a type II inhibitor, and the inhibitor was found to recognize the A2 domain of the factor VIII heavy chain. Apparently, paraprotein is not always the cause of reduced activity of coagulation factors in neoplastic dysproteinemias. Am. J. Hematol. 63: 145–148, 2000.

Modulation of sphingosine 1-phosphate/EDG signaling by tumor necrosis factor-α in vascular endothelial cells

Sphingosine 1-phosphate (Sph-1-P) is now regarded as a lysophospholipid mediator which is present in plasma and other body fluids, and exerts potent and pleiotropic biological effects [1,2]. Such extracellular mediator activities of Sph-1-P are mainly mediated by subfamilies of G proteincoupled receptors, of which the most completely characterized are those encoded by the endothelial differentiation genes (EDGs) [2,3]. Previously, we showed that in platelets, Sph-1-P is rapidly formed from sphingosine (Sph) by the action of Sph kinase, abundantly stored intracellularly, and released into the extracellular environment upon stimulation [1,4]. Furthermore, vascular endothelial cells express the EDG family Sph-1-P receptors, i.e., EDG-1 and EDG-3 [5]. Consistent with the expression of the high-affinity Sph-1-P receptors in these cells, nanomolar Sph-1-P reportedly induces a variety of vascular endothelial cell responses [5–9]. Sph-1-P stimulates endothelial cell survival or proliferation through a Gi-coupled receptor, probably EDG-1 [5,7]. Furthermore, Sph-1-P induces adherens junction assembly, migration, capillary tube formation, and promotion of angiogenesis [5,8,9]. In this case, the signals mediated via EDG-1 (coupled with Gi) and EDG-3 (coupled with G13 and Gq) are both necessary, and the small GTPase Rhoand Rac-mediated signalings are also involved [5,8]. Very recently, Sph-1-P has been shown to activate endothelial nitric oxide synthase through a novel mechanism involving Akt activation and produce nitric oxide, which plays an important role in endothelial survival and maintenance of the endothelial function [10]. Accordingly, Sph-1-P should be added to the list of platelet-derived bioactive mediators and may play an important role in platelet–endothelial cell interactions, which constitute a central part of vascular biology. To obtain insight into the physiological and pathophysiological role(s) of Sph-1-P in the vascular system, information about the regulation of endothelial cell expression of EDG family Sph-1-P receptors, i.e., EDG-1 and EDG-3, is very important. In fact, the EDG-1 cDNA was originally isolated as a phorbol ester-inducible immediate early transcript from vascular endothelial cells; morphogenetic differentiation of vascular endothelial cells into capillary-like tubules was induced under the conditions [11]. Furthermore, recent data have shown that the mechanical force associated with blood flow, i.e., the fluid shear stress, modulates vascular structure and function, and plays an important role in the pathogenesis of vascular diseases, including atherosclerosis, hypertension, and restenosis [12]. Shear stress has been reported to affect endothelial cell gene expression, and EDG-1 has been cloned as one of two cDNAs encoding a G protein-coupled receptor from a cDNA library of human umbilical vein endothelial cells (HUVECs) exposed to fluid shear stress; EDG-1 mRNA levels increase markedly in response to fluid flow [13]. Accordingly, the modulation of Sph-1-P/EDG signaling may be involved in important vascular responses related to angiogenesis and blood flow conditions. In this study, we examined the possibility that EDG expression, and hence, responsiveness to Sph-1-P in vascular endothelial cells, may be modulated by tumor necrosis factor-a (TNF-a), which is a pleiotropic cytokine that