Kazuko Hiramatsu

Increased Superoxide Production by Mononuclear Cells of Patients With Hypertriglyceridemia and Diabetes

Diabetic patients with hypertriglyceridemia frequently develop atherosclerosis. Because superoxide (O−2) is suspected to play an important role in the initiation of atherosclerosis, we investigated whether an abnormal amount of O−2 was produced by circulating mononuclear cells of patients with both diabetes mellitus and hypertriglyceridemia. The rate of production of superoxide dismutase–inhibitable O−2 was measured when cells were stimulated by either 4β-phorbol 12β-myristate 13α-acetate (PMA) or by opsonized zymosan (OZ). In addition, the rates of O−2 production by mononuclear cells drawn from three other groups (normal, solely diabetic, and solely hypertriglyceridemic) were determined. We found that the rate of O−2 production by mononuclear cells from the diabetic hypertriglyceridemic group was significantly higher than that from normal, diabetic, and hypertriglyceridemic groups. When the rates of O−2 production by mononuclear cells were plotted against the levels of plasma triglyceride for all individuals tested, they correlated positively (r = .73 in PMA stimulation and r = .79 in OZ stimulation, P < .01). However, the rate of O−2 production did not relate to other parameters, i.e., plasma cholesterol level, hemoglobin A1 level in erythrocytes, and the molar ratio of free cholesterol to phospholipid in mononuclear cells. Thus, we concluded that the observed elevated rate of O−2 production in the diabetic hypertriglyceridemic mononuclear cells was a reflection of a hypertriglyceridemic condition and was not unique to the diabetic hypertriglyceridemic condition. Also, O−2 may be involved in the pathogenesis of atherosclerosis in diabetic hypertriglyceridemic patients when atherogenic factors specific to diabetes are concomitantly present.

Low superoxide scavenging activity associated with enhanced superoxide generation by monocytes from male hypertriglyceridemia with and without diabetes

To investigate the mechanism of increased superoxide (O2-) generation by monocytes from patients with hypertriglyceridemia, superoxide scavenging activity (SSA) and O2- generation by monocytes were determined concomitantly employing an electron spin resonance/spin trapping method and 2-methyl-6-[p-methoxyphenyl]-3,7-dihydroimidazo [1,2-a]-pyrazin-3-one (MCLA)-dependent chemiluminescence, respectively. Peripheral monocytes were separated by the adherent methods from the following four male groups: normal control, diabetes alone (DM), diabetes with hypertriglyceridemia (DM + HTG) and hypertriglyceridemia alone (HTG). Monocytes were stimulated by 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA) or opsonized zymosan (OZ). O2- generation by monocytes upon stimulation was enhanced in HTG and HTG + DM but not in DM as compared to that in normal controls. The mean value of SSA in monocytes was similar among the 4 groups. When the relationship was analyzed using various parameters, a significant positive relationship was found between O2- generation and the plasma triglyceride level; a significant negative correlation was found between SSA and both the O2- generation and the plasma triglyceride level. In the in vitro system, the SSA in monocytes decreased significantly after the the stimulation by either of PMA or OZ. The results indicate that the decrease of SSA in monocytes may originate from the enhanced in vivo O2- generation and is responsible for the enhanced O2- release against the stimuli in hypertriglyceridemia. These abnormal functions of monocytes may in part accelerate the development of atherosclerosis.

Reduction of platelet aggregation induced by euglycaemic insulin clamp

SummaryTo examine the effect of serum insulin independent of the level of blood glucose in vivo on platelet aggregation in healthy individuals, a euglycaemic insulin clamp was applied up to 4 h. During the clamp, blood glucose at 5.0 mmol/l and insulin levels at 100 μU/ml were maintained. Blood samples were drawn before, 2 and 4 h after the start of the insulin clamp. The platelet aggregation induced by 1 μmol/l and 2 μmol/l ADP, 1 μg/ml collagen and 2.7 μmol/l epinephrine was measured in the blood samples. Platelet aggregation induced by adenosine diphosphate, collagen and epinephrine in the 4 h sample was significantly reduced from the pre-clamp value of 8.4% to 3.9% (p<0.05), 26.2% to 7.0% (p<0.01) and 31.8% to 9.1% (p<0.01), respectively. On the other hand, when the same individuals were infused with physiological saline and blood glucose (4.4 mmol/l) and insulin level (10 mIU/l) were kept within normal values, there was no difference between the values of induced platelet aggregation in samples drawn before and during the insulin infusion. It was concluded that hyperinsulinaemia reduces platelet aggregation in vivo when euglycaemia was maintained.

Time course of superoxide generation by leukocytes-The MCLA chemiluminescence system

This study was performed to examine the pattern of Superoxide (O2−·) generation from leukocytes using the O2−· specific chemiluminescence (CL) method.Cypridina luciferin analog, 2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo[1,2-alpha]pyrazin-3-one (MCLA) was used as a CL probe. The appropriate conditions of the MCLA method was first determined for the evaluation of the time course of O2−· generation by leukocytes. The time course of O2−· generation obtained by the MCLA-CL system was compared with that by the luminol-dependent CL, electron spin resonance (ESR)/spin trapping, and cytochromec systems. Following stimulation by three different stimulants (PMA, OZ, FMLP), leukocytes continuously generated O2−· for up to 5 h in the MCLA-CL system, irrespective of the kind of stimulation. The curves obtained by generation ceased more rapidly in the luminol-CL, ESR/spin trapping, and cytochromec systems. A 50% activity of the initial value was observed at 70 min in the MCLA-CL system, but 30, 10 and 35 min in the other systems, respectively. The CL or O2−· generation value decreased to less than 1% (possible termination) at 300, 90, 120 and 180 min, respectively. With the exception of ESR studies with OZ, the cell viability was not significantly affected in any of the trials. These results indicate that leukocytes can generate O2−· much longer than previously estimated and that the MCLA-CL-system is the most suitable system for the measurement of the O2−· generation by leukocytes.

Rapid determination of lipids in healthy human lymphocytes

Lipids of human lymphocytes were determined from 20 ml of heparinized peripheral blood using thin-layer chromatography with a flame ionization detector, and gas chromatography. The weight per cent and microgram per 10(6) lymphocytes for cholesterol ester, triglyceride, free cholesterol and phospholipid were 11.1 and 5.2, 18.1 and 17.9, 15.1 and 8.5, and 55.7 and 44.2, respectively. Phospholipid was the major lipid component in human lymphocytes. Phospholipid was subfractionated into phosphatidylethanolamine, phosphatidylinositol plus phosphatidylserine, phosphatidylcholine and sphingomyelin in amounts of 25.2, 6.1, 46.9 and 22.9%, respectively. Total fatty acid composition was analyzed as: C14:0, 13.3%; C16:0, 20.9%; C16:1, 6.5%; C18:0, 19.6%; C18:1, 18.8%: C18:2, 7.1%; and C20:4, 12.3%. Higher cholesterol ester and triglyceride and lower C14:0 were characteristic of female lymphocytes when compared with male lymphocytes. The lipid composition quantitated by this method corresponded well with previously reported data. Thus, this method can be used clinically because of its simplicity and higher sensitivity.

Lipid content of human platelets quantitated by thin-layer chromatography in combination with flame ionization detection

The lipid contents of human platelets from twenty-one healthy adults were analysed using thin-layer chromatography in combination with flame ionization detection. The weight per cent of neutral lipids in human platelets was 14.6%, which consisted mainly of free cholesterol, that of phosphatidylethanolamine 24.9%, phosphatidylserine plus phosphatidylinositol 6.8%, phosphatidylcholine 35.2% and sphingomyelin 18.6%. Free cholesterol in 10(8) platelets was estimated as 7 micrograms and phospholipids as 46 micrograms from calibration standards. The reproducibility was satisfactory and the procedure could be performed quickly and simply.

Platelet aggregation and intraplatelet adenine nucleotides in diabetic retinopathy.

Twenty non-diabetic controls, 50 diabetics without retinopathy and 29 diabetics with retinopathy, all well-matched age groups, were studied in males and females, respectively. Moreover, diabetic patients with retinopathy were divided into the following two subgroups: mild retinopathy and severe retinopathy. Platelet aggregation was elevated in the male diabetics with mild or severe retinopathy. There was no difference in platelet volume between the groups. Intraplatelet ADP contents were significantly decreased in the female diabetics with severe retinopathy showing no high platelet aggregability. There was no significant difference in intraplatelet ATP contents between groups. These results indicated not only that platelet aggregation might be generally high in the diabetics with retinopathy, but also that dense granule release might occur readily in vivo in the female diabetics with severe retinopathy and therefore platelet aggregation was not elevated in the group.

SURFACE ULTRASTRUCTURE AND MICROVISCOSITY OF LYMPHOCYTE MEMBRANES IN MYASTHENIA GRAVIS

Myasthenia gravis (MG) is a disease caused by the impairment of neuromuscular junctions and characterized by high incidence of thymic involvement including thymoma or thymic hyperp1asia.l It has been demonstrated that the antibody against acetylcholine receptors (AChR) is present in sera of MG patienh2 The AChR has been found not only in synaptic membranes but also in thymic epithelial cells and thymic lymphocyte^.^ The antibody against AChR (anti-AChR antibody) is responsible for abnormality of neuromuscular j ~ n c t i o n . ~ Although direct evidence that peripheral lymphocytes of MG patients bear AChR is not found at present, abnormal findings such as decreased mitogenic activities,G higher response to AChR,7 and decreased T-cell in MG lymphocytes, and B-cell clone in the thymus were revealed. This evidence indicated that cell-mediated immune responses represented by lymphocytes play significant roles in the pathogenesis of MG. It is, therefore, of great interest to study the surface structures and functions of myasthenic lymphocytes resulting from the interaction between their receptors and anti-receptor antibodies since the impaired postsynaptic membrane is caused by the analogous interaction between AChR and the anti-AChR antibodies. The morphological alteration of cell surface can be observed by radioautography, ferritin or enzyme conjugated immunoelectron microscopy, immunoscanning electron microscopy and freeze-fractured technique. The functional changes in cell membranes can be evaluated by measuring the receptor activities and membrane microviscosity.8 We studied the relation between ultrastructural alterations and microviscosity of the cell membrane in the peripheral blood lymphocytes from MG patients, from the viewpoint that the alteration of MG lymphocyte membranes may represent the impairment of postsynaptic membranes in MG.