K. Hirata

Serum Lipoprotein (a) Levels in Maintenance Hemodialysis Patients

To further understand lipoprotein (a) [Lp(a)] and atherosclerosis, we measured serum Lp(a), lipoprotein, and apolipoprotein levels in 55 patients (males, 24-73 years old) on maintenance hemodialysis, and compared them with those of 82 controls (males, 21-81 years old). The serum Lp(a) levels in patients on maintenance hemodialysis were significantly higher than those of the normal controls, while serum total cholesterol (TC), high-density lipoprotein-cholesterol, (HDL-C), HDL2-C, HDL3-C, apolipoprotein (apo) Al, apo All levels, and lecithin-cholesterol acyltransferase (LCAT) activities were significantly (p < 0.05) reduced in the patient group. The frequency distribution of serum Lp(a) levels in the patients was different from that in the control group, and no prognostic tendency of serum Lp(a) levels was noted by the etiology of renal failure as histologically determined by the renal biopsies. In the patient group, we also found that serum Lp(a) levels negatively correlated with serum triglycerides (TG) and total protein (TP) concentrations (p < 0.05), but no correlation was found between the duration of hemodialysis therapy or patient age and the serum levels of TC, TG, apo B and Lp(a) levels when tested for simple regression. Significant (p < 0.05) positive correlations were also found between TP and serum TG, apo B, and LCAT activities. These opposing tendencies of Lp(a) and serum TG, apo B, when measured against TP concentrations, indicate that serum TP levels may not affect serum lipoprotein and Lp(a) levels in the same direction. These data suggest that hemodialysis or end-stage renal disease itself, rather than hypoproteinemia, may hold the key to high serum Lp(a) levels in hemodialysis patients.

Quantitative characterization of insulin-glucose response in Watanabe heritable hyperlipidemic and cholesterol-fed rabbits and the effect of cilazapril.

A great deal of evidence suggests that insulin resistance, via hyperinsulinemia, contributes to hyperlipoproteinemia and coronary atherosclerosis. When Watanabe heritable hyperlipidemic (WHHL) rabbits, an animal model of familial hypercholesterolemia (FH), are compared with normolipidemic Japanese White (JW) rabbits, an elevated fasting plasma insulin level and a heightened plasma insulin response to an intravenous (i.v.) glucose challenge are found. To elucidate the mechanism behind this phenomenon, a two-compartment model of the glucose/insulin system was fitted to empirical time courses of glucose and insulin concentrations during an i.v. glucose tolerance test (IVGTT) by nonlinear least-square regression, and the model parameters such as the glucose utilization rate constant, insulin degradation rate constant, and pancreas sensitivity were determined. WHHL rabbits showed decreased values of glucose utilization and insulin degradation rate constants and slightly higher values of pancreas sensitivity. This suggests that insulin resistance occurs in extrapancreatic tissues, and that this may be attributable to insulin receptor and/or post-insulin receptor abnormalities. Cholesterol feeding did not significantly change glucose tolerance or insulin action in JW rabbits. The effects of an angiotensin-converting enzyme (ACE) inhibitor, cilazapril, on insulin resistance were also examined in WHHL and JW rabbits. A decreased insulin response to an i.v. glucose challenge and increased glucose utilization and insulin degradation rate constants were observed in WHHL rabbits that had been treated with cilazapril, indicating that cilazapril improved insulin resistance in WHHL rabbits, possibly by increasing the number of insulin receptors. No significant differences were found in glucose tolerance and insulin action in JW rabbits before and after cilazapril administration.(ABSTRACT TRUNCATED AT 250 WORDS)

In vivo conversion of recombinant human proapolipoprotein AI (rh-Met-proapo AI) to apolipoprotein AI in rabbits

In vivo conversion of recombinant human proapolipoprotein AI (rh-Met-proapo AI) from E. coli to apolipoprotein (apo) AI was investigated. rh-Met-proapo AI was labeled with 125I, and then administered intravenously to rabbits. Blood was sampled periodically for 6 days. The plasma decay curves of radioiodinated rt-Met-proapo AI were similar to those of human mature apo AI (fractional catabolic rate (FCR); 1.018 +/- 0.090/day vs. 0.976 1 0.031/day, respectively). In vivo conversion of rh-Met-proapo AI to mature apo AI was examined by autoradiography of the isoelectric focusing (IEF) slab gel, i.e., the HDL fraction from each sampling point was semiquantitatively applied to IEF. It was found that the radioactivity of rh-Met-proapo AI migrated to more acidic isoproteins, the conversion was complete within 24 h, and the FCR of rh-Met-proapo AI was 9.20 +/- 1.34/day. Although the plasma decay curves of both human pro (rh-Met-proapo AI) and mature apo AI were significantly steeper than those of rabbit mature apo AI4 and apo AI5 (FCR; 0.703 +/- 0.027/day and 0.795 +/- 0.031/day, respectively), the conversion rate of human rt-Met-proapo AI to mature apo AI in rabbit was assumed to be 1:1. In vitro incubation of rh-Met-proapo AI with rabbit serum produced mature apo AI isoproteins, as determined by the apo AI immunoblotting method. Prediction of the amino acid sequence at the NH2 terminus of rabbit proapo AI showed that the prosegment consisted of an alpha helix with a high probability of a beta turn at Pro9, which is close to that in humans. Thus, (1) the proteolytic cleavage of proapo AI is an extracellular event, (2) the converting enzyme in rabbits can also process human proapo AI, (3) this converting enzyme does not specifically and directly attack the Gln6-Asp7 bond which links the carboxyl-terminal residue of the hexapeptide to the amino-terminal residue of human mature apo AI. The conformation of proapo AI at the NH2 terminus (alpha helix of the prosegment and a beta turn at Pro9) may have a key role in this cleavage, and (4) the examination of rh-Met-proapo AI in rabbits helps to explain the early events of HDL biogenesis.

In vivo kinetics of lipoprotein(a) in homozygous Watanabe heritable hyperlipidaemic rabbits

Abstract. In vivo kinetics of lipoprotein(a) [Lp(a)] were investigated in homozygous Watanabe heritable hyperlipidaemic (WHHL) rabbits (an animal model of familial hypercholesterolemia (FH)), and in normo‐lipidemic Japanese White rabbits (controls). 1251‐labelled Lp(a) and 131I‐labelled LDL were simultaneously injected intravenously. Blood samples were then taken periodically. Kinetic parameters were cal culated from the plasma radioactivity decay curves. The fractional catabolic rates (FCRs) of both Lp(a) and LDL (1.355 ± 0.189 pools per day and 1.278 ± O.397 pools per day, respectively) in the WHHL rabbits were significantly (P<0.005) smaller than those in the control rabbits (2.008 ± 0.083 pools per day and 2.855 ± 0.759 pools per day, respectively). In WHHL rabbits, the FCRs of Lp(a) and LDL were similar. However, in control rabbits, the FCR of Lp(a) was significantly (P<0.01) smaller than that of LDL. In WHHL rabbit organs, the mean ratio of 125I‐Lp(a): 131I‐LDL, 48 h after injection, normalized to the corresponding isotope ratio in plasma, were 1.525, 1 020, 1.819 and 1.967, in liver, kidney, spleen and bile, respectively. These values were significantly higher than the corresponding values in control rabbits (0.590, 0.677, 0.862 and 0.766, respectively). Our data strongly suggest that Lp(a) clearance is not entirely dependent upon LDL receptors and may be mediated by some other mechanisms.

Serum lipoprotein(a) concentrations and apolipoprotein(a) phenotypes in the families of NIDDM patients

SummaryWe studied the quantitative and qualitative characteristics of lipoprotein(a) [Lp(a)] as a function of apolipoprotein(a) [apo(a)] phenotype in 87 members (42 males, 45 females) of 20 diabetic families, 26 of whom were diagnosed with non-insulin-dependent diabetes mellitus (NIDDM) with moderate glycaemic control (HbA1c7.1±1.2%). Apo(a) phenotyping was performed by a sensitive, high-resolution technique using SDS-agarose/gradient PAGE (3–6%). To date, 26 different apo(a) phenotypes, including a null type, have been identified. Serum Lp(a) levels of NIDDM patients and non-diabetic members of the same family who had the same apo(a) phenotypes were compared, while case control subjects were chosen from high-Lp(a) non-diabetic and low-Lp(a) non-diabetic groups with the same apo(a) phenotypes in the same family. Serum Lp(a) levels were significantly higher in NIDDM patients than in non-diabetic subjects (39.8±33.3 vs 22.3±19.5 mg/dl, p<0.05). The difference in the mean Lp(a) level between the diabetic and non-diabetic groups was significantly (p<0.05) greater than that between the high-Lp(a) non-diabetic and low-Lp(a) non-diabetic groups. An analysis of covariance and a least square means comparison indicated that the regression line between serum Lp(a) levels [log Lp(a)] and apo(a) phenotypes in the diabetic patient group was significantly (p<0.01) elevated for each apo(a) phenotype, compared to the regression line of the control group. These data, together with our previous findings that serum Lp(a) levels are genetically controlled by apo(a) phenotypes, suggest that Lp(a) levels in diabetic patients are not regulated by smaller apo(a) isoforms, and that serum Lp(a) levels are greater in diabetic patients than in non-diabetic family members, even when they share the same apo(a) phenotypes.

Combined therapy with probucol and pravastatin in hypercholesterolaemia. One year follow-up study.

SummaryThe effect of co-administration of low doses of pravastatin to hypercholesterolaemic patients already receiving long-term probucol treatment (mean 500–1,000 mg/day for 350 days) were investigated. Pravastatin 5 mg/day (Group 1; 12m, 13f; mean age 59.1 y) or 10 mg/day (Group 2; 8m, 11f; mean age 60.8 y) was administered, and blood was taken after 0, 3, 6, and 12 months.Both groups showed a significant reduction in serum total cholesterol (TC), phospholipid (PL), low density lipoprotein-cholesterol (LDL-C), LDL-triglyceride (TG), LDL-PL, apolipoprotein (apo) B, and apo E after the combined therapy. These levels were reduced more in Group 2 than in Group 1 subjects. In Group 2, significant falls in serum TG and apo CII were also observed. The changes in TC, PL, LDL-C, apo B, apo CII and apo E were dependent upon the dose of pravastatin, as assessed by two-way analysis of variance. Serum high density lipoprotein (HDL)3-C, apo AI and apo AII were slightly but significantly increased in both groups after 12 months of combined therapy, but the increase was not sufficient to reverse the probucol-induced lowering of the HDL level. We conclude that combined therapy resulted in a significant reduction in atherogenic lipoproteins and apolipoproteins, and an increasing dose of pravastatin (5 mg to 10 mg daily) made the lipid lowering effect more prominent. The reduction in serum HDL-C due to long-term probucol administration was not reversed by the addition of pravastatin.

In vitro conversion of recombinant human proapolipoprotein A-I to apolipoprotein A-I

We recently investigated the in vivo conversion of recombinant human proapolipoprotein A-I (rh-Met-proapo A-I) from E. coli to apolipoprotein (apo) A-I in rabbits. In vitro incubation of rh-Met-proapo A-I with rabbit serum produced mature apo A-I3 isoproteins, as determined by the immunoblotting method. However, at the time we were unable to chemically confirm a newly produced protein band which appeared at the position of human apo A-I3. Since then, we have confirmed the amino acid sequence of the protein using a membrane protein sequence technique, and have concluded that it corresponds to human apo A-I3.

High-density lipoprotein and apolipoprotein A-I deficiency induced by combination therapy with probucol and bezafibrate

The effects of the administration of slow-release bezafibrate to hypercholesterolaemic patients who were already receiving long-term probucol treatment (mean 865 days, 500–1000 mg·day−1) were investigated. Bezafibrate was administered at either 200 mg·day−1 (13 males, 13 females, mean age 55.2 years) or 400 mg·day−1 (11 males, 14 females, mean age 57.2 years), and blood was taken at 0, 3, 6 and 12 months after the beginning of combination therapy. Overall, serum total cholesterol (TC), triglyceride (TG), very low density lipoprotein (VLDL)-TC, high-density lipoprotein (HDL)-TG, VLDL-TG, VLDL-phospholipid (PL), lipoprotein (a) [Lp(a)], apolipoprotein (apo) C-III, apo E levels and LCAT activity decreased significantly with this combination therapy, while HDL cholesterol (C), HDL3-C, HDL-PL, apo A-I and apo A-II levels significantly increased, as assessed by analysis of variance (ANOVA). Five patients (one receiving 200 mg·day−1, four receiving 400 mg·day−1 bezafibrate) showed drastic reductions in HDL-C (HDL-C levels were reduced by a mean of 46.2%, 59.3% and 61.6% at 3, 6 and 12 months, respectively) after beginning combination therapy. These HDL-C reductions were maintained for the 1 year of combination therapy, but then returned to pre-combination treatment levels 1 month after discontinuation of bezafibrate. Serum probucol concentrations and cholesteryl ester transfer protein (CETP) mass were assayed at 6 months, and the probucol concentration was higher in the HDL-deficient group (56.2 vs 26.5 μg/ml). In contrast, CETP mass was significantly lower in HDL-deficient patients than in non-HDL-deficient patients (2.08 vs 2.87 mg·l−1). When the patients in the non-HDL-deficient group were divided into two groups, receiving low (200 mg·day−1, n−25) and high (400 mg·day−1, n−21) doses of bezafibrate, the former group showed a significant increase in probucol-lowered HDL-C and apo A-I, although these levels did not return to pre-probucol treatment levels, while the latter group showed no changes in HDL. These data suggest that the addition of a low dose of bezafibrate to probucol tended to reverse probucol-induced HDL lowering, while 9.8% (5 of 51 patients) of the patients exhibited a severe HDL deficiency. Since it is unclear whether or not such an extreme HDL reduction is harmful, HDL deficiency should be carefully monitored with this combination therapy.