Kazuhiko Matsuno

Effects of Midstream Collection and the Menstrual Cycle on Urine Particles and Dipstick Urinalysis among Healthy Females

For urinalysis, midstream collection is recommended (1)(2)(3). Health-associated reference limits for leukocyte and erythrocyte counts in female urine are important for detecting hematuria, pyuria, and urinary tract infection (3). To understand the effects of urinary collection and the menstrual cycle on urinalysis, we examined first-stream and midstream urine samples from healthy female students with use of an automated dipstick reader and the fully automated urine cell analyzer, UF-100. Specimens were obtained from 64 healthy female students (age range, 18–20 years) at the College of Medical Technology. All were asymptomatic and had no extant urologic disease. They were instructed to collect only first and midstream urine samples in sterile containers at the same time and not to wipe or spread the labia. The volume of the first urine was measured, and the specimen was analyzed within 2 h. The students provided written, informed consent to participate in the study as well as information about their menstrual cycles. Specimens were classified into four groups according to the number of days after menstruation as follows: menstrual (1–7 days after menstruation; n = 15), follicular (8–15 days after menstruation; n = 21), ovulatory (16–19 days after menstruation; n = 6), and luteal (20–31 days after menstruation; n = 22). Particles in the urine were analyzed by use of a fully automated urine cell analyzer, UF-100 (Sysmex Corporation). We used the manufacturer-defined review …

Human CD36 deficiency is associated with elevation in low-density lipoprotein-cholesterol.

To find out whether CD36 plays a role in the human lipoprotein metabolism, we studied lipoprotein profiles in subjects with CD36 deficiency. Apparently healthy Japanese volunteers (n = 790) were classified by flow cytometry into three groups of normal (platelet and monocyte CD36+, n = 741, 93.8%), type-II deficiency (platelet CD36- and monocyte CD36+, n = 45, 5.7%), and type-I deficiency (platelet and monocyte CD36-, n = 4, 0.5%). At least one of reported mutations in the CD36 gene was found in all four subjects with type-I deficiency and in 23 of the 45 subjects with type II. Among 779 subjects (731 normals, 44 type II, and four type I) with serum triglyceride levels of <400 mg/dL, serum total cholesterol and low-density lipoprotein (LDL) cholesterol were significantly elevated in type-II deficiency (P = 0.0095 and 0.0382 versus normal, respectively, Scheffes F-test), while differences were not significant in triglyceride and high-density lipoprotein-cholesterol. Similar tendency was observed in type-I deficiency, although the differences were not statistically significant because of small sample size. We conclude that CD36 deficiency elevates LDL cholesterol, indicating a contribution of CD36 to LDL metabolism.

Release reaction of brain-derived neurotrophic factor (BDNF) through PAR1 activation and its two distinct pools in human platelets

Brain-derived neurotrophic factor (BDNF) is a cytokine that plays important roles in the survival, development, and plasticity of neurons. BDNF is also expressed in peripheral tissues and cells. In this article, we report the BDNF release reaction through thrombin stimulation and its localization in human platelets. Platelets from healthy volunteers were subjected to PAR1-AP or PAR4-AP stimulation. Release of BDNF was measured by ELISA. Localization of BDNF in resting and thrombin-activated platelets was examined by immunoelectron microscopy and sucrose gradient ultracentrifugation following western blotting. BDNF was released dose-dependently with PAR1-AP concentrations with drastic release at low PAR1-AP concentrations and gently release at high PAR1-AP concentrations. Maximum BDNF release was approximately 37% at 132 μM PAR1-AP. In contrast, 3.8% BDNF was released with 1.13 mM PAR4-AP stimulation. In immunoelectron microscopy and sucrose gradient ultracentrifugation analyses, BDNF was detected not only in α-granules but also cytoplasm in of the resting platelets, and it was distributed in the swollen open canalicular system fused to α-granules at 1 min and disappeared at 5 min after stimulation by thrombin. However, BDNF in cytoplasm remained throughout platelet activation. In conclusions, we demonstrate that BDNF is released from platelets through predominately PAR1 regulation. Furthermore, we identified two pools of BDNF in the α-granules and cytoplasm of human platelets, and only BDNF in α-granules is released through platelet activation.

A rapid and simple quantification of human apolipoprotein E-rich high-density lipoproteins in serum

A rapid and simple method for the quantitative determination of human serum apo E-rich high-density lipoproteins is described. A sample was divided into two parts; one part was mixed with an equal volume of 13% polyethylene glycol 6000, and the other part was mixed with a solution containing dextran sulfate, sodium phosphotungstate, and Mg2+, respectively. The mixed solutions were centrifuged (2000 g; 15 min). The supernate obtained by the former procedure contained both apo E-rich HDL and apo E-poor HDL, but that obtained by the latter procedure contained solely apo E-poor HDL. The serum apo E-rich HDL concentration in terms of apo E (E) and cholesterol (C), was given by the following equations: E = EP x 2, and C = (CP - CD) x 2, where EP and CP were the concentrations of apo E and cholesterol, respectively, in the supernate obtained with 13% polyethylene glycol, and CD was the concentration of cholesterol in the supernate obtained with the mixture solution of dextran sulfate, sodium phosphotungstate, and Mg2+. Normal serum apo E-rich HDL concentrations were 2.6 +/- 1.5 and 6.7 +/- 2.3 mg/dl (means +/- SD, n = 38) in terms of apo E and cholesterol, respectively. Apo E-rich HDL was increased strikingly in the sera from three patients with hepatobiliary diseases.

Spatial distribution and temporal change of cytoplasmic free calcium in human platelets

Our digital imaging microscope equipped with a microspectrofluorometer revealed in single resting human platelets the existence of continuous Ca2+ gradient increasing towards the plasma membrane (frequency; 100%) and discontinuous ones (Ca2+ plateaus) in the endoplasmic regions (frequency: 70%). An average cytoplasmic free Ca2+ concentration ([ Ca2+]i) in a whole cytoplasm was 72 +/- 7 nM, ranging from 30 nM in the lowest to 150 nM in the highest region just beneath the plasma membrane. When stimulated with thrombin, [Ca2+]i uniformly increased to the average [Ca2+]i of 300 nM and these gradients disappeared. This [Ca2+]i transient was followed by the sustained increase in [Ca2+]i in both single cells and cell suspension.

Dipyridamole potentiates the anti-aggregating effect of endothelium-derived relaxing factor

One effector of the anti-aggregatory property of endothelium is thought to be endothelium-derived relaxing factor. The best characterized of these, nitric oxide, inhibits platelet aggregation by increasing cyclic GMP levels. The effects of nitric oxide and dipyridamole (a cyclic GMP phosphodiesterase inhibitor), alone and in combination, on in vitro platelet aggregation were evaluated. Dipyridamole had no effect per se on platelet aggregation but potentiated the inhibition of aggregation due to nitric oxide. This was concomitant with an increase in platelet cyclic GMP concentration. The author suggests an alternative mechanism for the clinical efficacy of dipyridamole as an antiplatelet agent.

Inhibition of Tumor Cell Arrest in Lungs by Antimetastatic Chitin Heparinoid

We have investigated the effect of sulfated chitin derivatives on the intravascular events in the metastatic cascade. 6‐O‐Sulfated carboxymethyl chitin (SCM‐chitin III), as well as heparin, significantly inhibited the arrest of B16‐BL6 cells in lungs after co‐injection with radiolabeled tumor cells, but carboxymethylated chitin (CM‐chitin) had no effect. Heparin showed a potent inhibitory effect on tumor cell‐elicited platelet aggregation and on blood coagulation, which can subsequently enhance the survival, arrest and invasiveness of tumor cells, whereas SCM‐chitin III showed much weaker properties. In contrast, SCM‐chitin III was found to inhibit the adhesion of tumor cells to subendothelial matrix, while heparin did not. SCM‐chitin III was still active in inhibiting experimental lung metastasis even in mice which had been pretreated with anti‐asialo GM1 serum or carrageenan to eliminate NK cells or macrophages. Thus, these results suggest that SCM‐chitin‐mediated inhibition of tumor metastases is distinct from that by heparin and may be due to interference with tumor cell arrest in the capillaries and consequently to the inhibition of tumor cell adhesion to subendothelial matrix.

Evaluation of G-to-A substitution in the apolipoprotein A-I gene promoter as a determinant of high-density lipoprotein cholesterol level in subjects with and without cholesteryl ester transfer protein deficiency

The effect of a polymorphism, guanine (G) to adenine (A) substitution in the promoter of apolipoprotein A-I gene at a position 78 bp upstream of the transcription initiation site, on the serum high-density lipoprotein (HDL)-cholesterol level was studied in 168 Japanese subjects with HDL-cholesterol levels ranging from 26 to 171 mg/dl. Considering the significant effect of cholesteryl ester transfer protein (CETP) on the HDL-cholesterol level and the common occurrence of its deficiency, we performed statistical analyses separately for two groups: one without CETP deficiency (n=126) and the other with CETP deficiency (n=42). In the group without CETP deficiency, in which the numbers of G/G, G/A, and A/A genotypes were 92 (73.0%), 28 (22.2%), and 6 (4.8%), respectively, the frequency of the A allele in the subjects with HDL-cholesterol levels of ≥70 mg/dl did not differ from subjects with HDL-cholesterol levels of ≤69 mg/dl, irrespective of gender: 0.154 and 0.145 in males, and 0.182 and 0.174 in females, respectively, for the ≥70 mg/dl and ≤69 mg/dl groups. Additionally, the HDL-cholesterol levels for the subjects with the G/G genotype did not differ from those for the subjects with the A allele: 64 ±22, 58±14, 77±14 and 62±16 mg/dl, respectively, for the G/G, G/A, A/A, and G/A+A/A in males, and 72 ±18, 74±24, 63±4, and 73±23 mg/dl in females. For the group with CETP deficiency, in which the numbers of G/G and G/A+A/A genotypes were 25 (59.5%) and 17 (40.5%), the HDL-cholesterol levels also did not differ: 98±24 mg/dl and 99±30 mg/dl, respectively, for the G/G and G/A+A/A genotypes. Thus, there is no evidence that the polymorphism has any effect on serum HDL-cholesterol levels regardless of CETP status. We conclude that the G-to-A substitution in the promoter of apolipoprotein A-I gene does not significantly alter serum HDL-cholesterol level.

Spurious elevation of serum high-density lipoprotein cholesterol in patients with cholestatic liver diseases.

Strikingly discrepant values were obtained by two commercial precipitating reagents for serum HDL cholesterol determination in three patients with cholestatic liver diseases (two patients with primary biliary cirrhosis and one patient with chronic hepatitis). An abnormal alpha 2-migrating lipoprotein (slow alpha-lipoprotein) was observed in agarose gel electrophoresis for each serum. The slow alpha-lipoprotein was partly recovered in the supernatant by precipitation with polyethylene glycol, and was completely precipitated with a polyanion-containing reagent, which well explains the discrepancy. The slow alpha-lipoprotein isolated from one of the cases is notable for (1) having an intermediate particle size between normal LDL and normal HDL, (2) containing apo E as the major apolipoprotein, and (3) being enriched with cholesterol (esterified and free) and phospholipid. Cholesterol accumulation was also found in another HDL subclass, alpha 1-migrating HDL. A severe impediment in the clearance of cholesterol-loaded HDL particles from plasma was implied. Electrophoresis of serum lipoproteins and/or the measurement of serum apo E concentrations are necessary to avoid an erroneous estimation of HDL cholesterol in patients with hepatobiliary diseases.

Cytosolic free magnesium concentration in human platelets.

Although the magnesium ion (Mg2+) is considered to play an important role in cell activation, information is limited by the lack of suitable methods for measuring cytosolic free Mg2+ concentration ([Mg2+]i). We measured [Mg2+]i in resting and activated human platelets using a new fluorescent Mg(2+)-indicator, mag-fura-2. [Mg2+]i was 0.54 +/- 0.14 mM in resting platelets from 15 healthy volunteers. [Mg2+]i was elevated to 1.33 +/- 0.44 mM and 0.92 +/- 0.37 mM in platelets stimulated with thrombin and collagen, respectively. Increased Mg2+ was considered to be derived chiefly from intracellular Mg2+ mobilization. These results suggest that platelet activation is associated with the increase in [Mg2+]i. The estimation of [Mg2+]i using this method is useful for the investigation of mechanism for various cell activation.