A Dipeptidyl Peptidase-4 Inhibitor, Teneligliptin, Decreases Plasma Triglyceride-Rich Lipoprotein Remnants in Diabetic Patients with Chronic Kidney Disease Undergoing Hemodialysis.

OBJECTIVE: A high plasma level of remnant-like particle cholesterol (RLP-C), which is equivalent to triglyceride-rich lipoprotein remnant, is an important coronary risk marker. RLP-C level is high, independent of other plasma lipids, in patients with chronic kidney disease (CKD) undergoing hemodialysis. The effect of teneligliptin, a dipeptidyl peptidase (DPP)-4 inhibitor, on plasma levels of RLP-C in patients with diabetes mellitus and CKD under hemodialysis was studied. METHODS: Teneligliptin 20 mg/day was administered to 15 patients with diabetes and CKD undergoing hemodialysis for 12 weeks. Ten patients with diabetes and CKD undergoing hemodialysis were allocated to the control group. Blood was sampled following a 12-h fast. Fasting plasma glucose (FPG), C-peptide, triglyceride, low-density lipoprotein (LDL)-cholesterol (C), high-density lipoprotein (HDL)-C, RLP-C, apolipoprotein (apo) B, oxidized LDL, lipoprotein lipase, and glycated hemoglobin (HbA1c) were measured. RESULTS: HbA1c decreased in the teneligliptin group but significantly increased in the control group. FPG and RLP-C significantly decreased in the teneligliptin group. Plasma lipoprotein-related parameters except RLP-C were not affected by teneligliptin treatment. CONCLUSION: Teneligliptin treatment significantly reduced plasma levels of RLP-C, FPG, and HbA1c in patients with diabetes with CKD who are undergoing hemodialysis.

A Comparison of the Abdominal Fat Distribution and Coronary Risk Markers in Body Mass Index-matched Subjects with and without Fatty Liver.

Objective The close relationship between fatty liver and metabolic syndrome suggests that individuals with fatty liver may have multiple coronary risk factors. In the present study, we investigated the relationships among fatty liver, abdominal fat distribution, and coronary risk markers. Methods and Results Eighty-seven pairs of men and 42 pairs of women who were matched for age and body mass index were enrolled in the present study. The obesity-related markers, abdominal fat distribution (examined by CT), and coronary risk markers were compared in subjects with and without fatty liver. The visceral fat area was significantly larger in the men with fatty liver than in the men without fatty liver. The plasma levels of triglyceride and low-density lipoprotein cholesterol (LDL-C), as well as the homeostasis model assessment-insulin resistance level, were higher in both males and females with fatty liver than in those without fatty liver, while the plasma levels of high-density lipoprotein cholesterol (HDL-C) and adiponectin were lower in the males and females with fatty liver. The plasma levels of apolipoprotein B, remnant-like particle cholesterol (RLP-C), and oxidized LDL were higher in men with fatty liver, but not in women with fatty liver. Conclusion Both males and females with fatty liver had lower insulin sensitivity, lower plasma levels of HDL-C and adiponectin, and higher triglyceride and LDL-C levels. However, the plasma levels of apolipoprotein B, RLP-C, and oxidized LDL were only higher and closely associated with fatty liver in men. Men with fatty liver had a higher risk of coronary disease than women with fatty liver.

Comparison of the effects of low-dose rosuvastatin on plasma levels of cholesterol and oxidized low-density lipoprotein in 3 ultracentrifugally separated low-density lipoprotein subfractions.

BACKGROUND: Plasma-oxidized (ox) low-density lipoprotein (LDL) is an atherogenic lipoprotein. The distribution of ox-LDL in plasma LDL subfractions and the effect of statins on this distribution have not been investigated in detail. OBJECTIVE: We examined the distribution of cholesterol and ox-LDL in 3 ultracentrifugally separated plasma LDL subfractions and investigated the effects of a statin, rosuvastatin, on the levels of these lipoproteins. MATERIALS AND METHODS: Thirty-one polygenic hypercholesterolemic subjects were included in this study. Levels of cholesterol and ox-LDL in 3 plasma LDL subfractions and plasma levels of remnant-like particle cholesterol, ox-LDL, and adiponectin were measured after 0, 3, 6, and 12 months of treatment with rosuvastatin. Sequential ultracentrifugation was performed to subfractionate plasma lipoproteins. RESULTS: The mean daily dose of rosuvastatin over the 12 months of treatment was 2.9 ± 1.0 mg (mean ± standard deviation). The cholesterol subfraction distribution was 43 ± 10% as low-density LDL, 46 ± 8% as medium-density LDL, and 13 ± 5% as high-density LDL. Similarly, the distribution of ox-LDL was 31 ± 10% as low-density LDL, 48 ± 7% as medium-density LDL, and 22 ± 8% as high-density LDL. After 12 months of treatment with rosuvastatin, the level of cholesterol was significantly reduced in all 3 subfractions (P < .0001), as was the level of ox-LDL (P < .0001). Furthermore, the plasma cholesterol level in high-density lipoprotein2 increased significantly. CONCLUSIONS: The distribution of ox-LDL in plasma LDL subfractions was more skewed toward the denser subfractions, compared with cholesterol. Rosuvastatin treatment significantly reduced plasma levels of cholesterol and ox-LDL in all LDL subfractions.

Aggregation of rare/low-frequency variants of the mitochondria respiratory chain-related proteins in rheumatoid arthritis patients.

Exome sequencings were conducted using 59 patients having rheumatoid arthritis (RA) and 93 controls. After stepwise filtering, 107 genes showed less than 0.05 of P-values by gene-burden tests. Among 107 genes, NDUFA7 which is a subunit of the complex I in the mitochondrial respiratory chain was selected for further analysis based on previous reports. A case-control study was performed on the three single-nucleotide variants (SNVs) of NDUFA7 with 432 cases and 432 controls. An association was observed between NDUFA7 and RA with severe erosive arthritis. These results together with previous reports suggested the involvement of reactive oxygen species (ROS) in the pathogenesis of RA. In the next step, four SNVs from three genes related to the mitochondrial respiratory chain were selected, which is a major source of ROS, and conducted a case-control study. An association was observed based on a pathway-burden test comprising NDUFA7, SDHAF2, SCO1 and ATP5O: P=1.56E-04, odds ratio=2.16, 95% confidence interval=1.43-3.28. Previous reports suggested the involvement of ROS in the pathogenesis of RA. The aggregation of SNVs in the mitochondria respiratory chain suggests the pivotal role of those SNVs in the pathogenesis of RA with severe erosive arthritis.

Changes in ultracentrifugally separated plasma lipoprotein subfractions in patients with polygenic hypercholesterolemia, familial combined hyperlipoproteinemia, and familial hypercholesterolemia after treatment with atorvastatin.

BACKGROUND: Plasma levels of low-density lipoproteins (LDLs) are decreased through stimulation of their hepatic uptake by statins via an LDL receptor. However, it is unclear whether statins equally stimulate the hepatic uptake of all LDL subfractions. OBJECTIVE: We compared the effects of atorvastatin on 3 LDL subfractions, and their associations with LDL-receptor activities, in Japanese patients with polygenic hypercholesterolemia (PHC), familial combined hyperlipoproteinemia (FCHL), and familial hypercholesterolemia (FH). MATERIALS AND METHODS: Atorvastatin was administered to patients with PHC (n = 11), FCHL (n = 16), and FH (n = 13). We measured plasma levels of lipids, remnant-like particle cholesterol, apoproteins, and cholesterol in lipoprotein fractions. Sequential ultracentrifugation was performed to subfractionate the plasma lipoproteins, and lymphocyte LDL-receptor activities were estimated using flow cytometry. RESULTS: The average daily dosage of atorvastatin was 10, 27, and 40 mg in patients with PHC, FCHL, and FH, respectively; after 12 months of atorvastatin treatment, LDL cholesterol (LDL-C) plasma levels decreased by 44%, 50%, and 53%, respectively (all, P < .0001). Atorvastatin reduced low-density LDL-C plasma levels in patients with PHC (48% reduction), FCHL (53%), and FH (46%) (all, P < .0001). Plasma levels of medium-density and high-density LDL-C were also significantly reduced in the 3 patient groups (all, P ≤ .0147). LDL-receptor activity was negatively correlated with baseline levels of medium-density LDL-C and with the decreases in plasma md-LDL-C levels. CONCLUSION: Atorvastatin decreased the levels of the 3 LDL fractions. The md-LDL decrease appeared to be mainly because of stimulation of LDL-receptor activity.

Relationships between the visceral fat area on CT and coronary risk factor markers.

OBJECTIVE: The visceral fat area (VFA) was measured, and the relationships between the VFA and the body mass index (BMI), waist circumference, blood pressure, and indices of lipid and sugar metabolism were evaluated. METHODS: The subjects included 607 consecutive patients who underwent VFA examinations using computed tomography (CT) scans. In addition to the routine examination parameters, the levels of adiponectin and homeostasis model assessment as an index of insulin resistance (HOMA-IR) were measured in all subjects, and the levels of malondialdehyde-modified low-density lipoprotein (MDA-LDL), remnant-like particles (RLP), lipoprotein (a) (Lp(a)), apolipoprotein (Apo) AI, ApoB and ApoE were measured in 270 subjects. RESULTS: In both men and women, the VFA showed significant positive correlations with the age, BMI, waist circumference, subcutaneous fat area, visceral fat area/subcutaneous fat area (v/s) ratio, systolic blood pressure, diastolic blood pressure, the fasting blood sugar (FBS), the hemoglobin A1c (HbA1c), high-density lipoprotein cholesterol (HDLC), triglyceride (TG), uric acid, HOMA-IR and ApoB and the ApoB/LDLC ratio and significant negative correlations with the levels of HDLC and adiponectin. The levels of the total cholesterol (TC), low-density lipoprotein cholesterol (LDLC), non-HDLC, MDA-LDL and Lp(a) and the ApoB/ApoAI ratio were not correlated with the VFA in either men or women. The RLP exhibited a significant positive correlation with the VFA in women. CONCLUSION: The VFA exhibited high positive correlations with the waist circumference, blood pressure and TG level and a negative correlation with the HDLC level, regardless of gender, supporting the validity of the present diagnostic method for evaluating metabolic syndrome (MS). Although the LDLC level is not included in the diagnostic criteria for MS, the positive correlations between the VFA and the ApoB level and ApoB/LDLC ratio observed in both men and women indicate qualitative abnormalities of lipoproteins, such as an increase in the amount of small dense LDL. Measuring the levels of apolipoproteins in addition to lipoproteins during health screening is therefore useful for evaluating of atherogenicity.

Skew of plasma low- and high-density lipoprotein distributions to less dense subfractions in normotriglyceridemic chronic kidney disease patients on maintenance hemodialysis treatment.

BACKGROUND: Plasma levels of small, dense low-density lipoprotein (LDL) were reported to increase in chronic kidney disease (CKD) patients on hemodialysis (HD), but most of these patients were hypertriglyceridemic. Plasma levels of small, dense LDL are known to increase in hypertriglyceridemic subjects. Therefore, to investigate the direct effect of CKD on the distribution of LDL subfractions, we investigated the distribution of LDL subfractions in normotriglyceridemic CKD patients on HD. METHODS: The levels of plasma lipoprotein subfractions and lecithin:cholesterol acyltransferase (LCAT) and cholesteryl ester transfer protein (CETP), which markedly influence the distributions of plasma LDL and high-density lipoprotein (HDL) subfractions, were compared between 40 HD patients and 40 normolipidemic controls. Plasma lipoproteins were subfractionated into seven subfractions by ultracentrifugation. RESULTS: Plasma levels of cholesterol (C) in remnant-like particle, which is equivalent to the triglyceride (TG)-rich lipoprotein remnant, were twice as high in HD patients as those in controls with matched TG levels. Plasma levels of C and TG in VLDL and IDL (intermediate density lipoprotein) were slightly higher in HD patients than in controls. The C/TG ratio of VLDL was significantly higher in HD patients than in controls. In comparison with the corresponding values in controls, the C and TG levels in low-density LDL and HDL2 in HD patients were high, whereas those in medium-density LDL, high-density LDL, and HDL3 were low. Plasma LCAT activity and CETP mass were lower in HD patients than in controls. CONCLUSION: Distribution of LDL and HDL skewed toward less dense fractions in normotriglyceridemic CKD patients on HD. A decrease in reverse C transport likely played an important role in these changes in the patients.

Exome sequencing identifies novel rheumatoid arthritis-susceptible variants in the BTNL2.

The butyrophilin-like protein 2 gene (BTNL2) within the class III region of the major histocompatibility complex genomic region was identified as a rheumatoid arthritis (RA) susceptibility gene by exome sequencing (19 RA cases) with stepwise filtering analysis, and then validated by Sanger sequencing and association analysis using 432 cases and 432 controls. Logistic regression of the Sanger-sequenced single-nucleotide variants in an association study of 432 cases and 432 controls showed that 12 non-synonymous single-nucleotide polymorphisms (SNPs) in BTNL2 were significantly associated with RA. The lowest P-values were obtained from three SNPs, rs41521946, rs28362677 and rs28362678, which were in absolute linkage disequilibrium: P=4.55E-09, odds ratio=1.88, 95% confidence interval=1.52-2.33. The BTNL2 locates on chromosome 6 between HLA-DRB1 and NOTCH4, and is 170 kb apart from these two genes. Although DRB1 and NOTCH4 were reported to be RA-susceptible, the three BTNL2 SNPs retained significant association with RA when evaluated by the logistic regression with the adjustment for RA-susceptible HLA-DRB1 alleles in Japanese or rs2071282-T in NOTCH4: P=0.0156 and P=0.00368, respectively. These results suggest that the three non-synonymous SNPs in BTNL2 confer RA risk independently from HLA-DRB1 and NOTCH4.

Increase of electronegative-LDL-fraction ratio and IDL-cholesterol in chronic kidney disease patients with hemodialysis treatment.

BACKGROUND: It is known that the increased level of IDL and oxidized LDL are associated with risk of cardiovascular disease, and the lipoprotein abnormalities accelerate atherosclerosis. Cardiovascular disease is a major cause of mortality in chronic kidney disease patients with hemodialysis treatment (HD-Ps). Therefore, the estimation of lipoprotein profiles is important for prevention of cardiovascular disease in HD-Ps. We previously established an anion-exchange chromatographic method for measurement of cholesterol level in subclasses of HDL and LDL, IDL, VLDL, and chylomicron. An electronegative-LDL-fraction contained minimally oxidized-LDL. Lipoprotein profile can be accurately and conveniently determined by the new method. FINDING: In this study, lipoprotein profiles in HD-Ps and age-matched healthy subjects were estimated by using our established anion-exchange chromatographic method. The ratio of electronegative-LDL-cholesterol to total LDL-cholesterol and IDL-cholesterol in HD-Ps were significant higher than those in healthy subjects. CONCLUSIONS: The results suggest that the ratio of electronegative-LDL-cholesterol to total LDL-cholesterol and IDL-cholesterol obtained by the new method may serve as useful markers for risk of cardiovascular disease in HD-Ps.

A systems genetics approach provides a bridge from discovered genetic variants to biological pathways in rheumatoid arthritis.

Genome-wide association studies (GWAS) have yielded novel genetic loci underlying common diseases. We propose a systems genetics approach to utilize these discoveries for better understanding of the genetic architecture of rheumatoid arthritis (RA). Current evidence of genetic associations with RA was sought through PubMed and the NHGRI GWAS catalog. The associations of 15 single nucleotide polymorphisms and HLA-DRB1 alleles were confirmed in 1,287 cases and 1,500 controls of Japanese subjects. Among these, HLA-DRB1 alleles and eight SNPs showed significant associations and all but one of the variants had the same direction of effect as identified in the previous studies, indicating that the genetic risk factors underlying RA are shared across populations. By receiver operating characteristic curve analysis, the area under the curve (AUC) for the genetic risk score based on the selected variants was 68.4%. For seropositive RA patients only, the AUC improved to 70.9%, indicating good but suboptimal predictive ability. A simulation study shows that more than 200 additional loci with similar effect size as recent GWAS findings or 20 rare variants with intermediate effects are needed to achieve AUC = 80.0%. We performed the random walk with restart (RWR) algorithm to prioritize genes for future mapping studies. The performance of the algorithm was confirmed by leave-one-out cross-validation. The RWR algorithm pointed to ZAP70 in the first rank, in which mutation causes RA-like autoimmune arthritis in mice. By applying the hierarchical clustering method to a subnetwork comprising RA-associated genes and top-ranked genes by the RWR, we found three functional modules relevant to RA etiology: "leukocyte activation and differentiation", "pattern-recognition receptor signaling pathway", and "chemokines and their receptors".These results suggest that the systems genetics approach is useful to find directions of future mapping strategies to illuminate biological pathways.

Particular human leukocyte antigen alleles are associated with biochemical traits in the Japanese population.

We analyzed genetic associations among 7 biochemical traits (fasting plasma glucose, HbA1c, total cholesterol, low-density lipoprotein [LDL] cholesterol, high-density lipoprotein cholesterol, triglyceride, and uric acid) and 6 HLA loci using 1,616 individuals who visited the Health Evaluation and Promotion Center at Tokai University Hospital. Significant differences between the individuals carrying particular HLA alleles and those not carrying the alleles in certain biochemical traits were observed by Mann-Whitney U test. In female subjects, DPB1*03:01 was significantly associated with HbA1c (p = 0.0000665), and DRB1*14:03 was associated with total cholesterol concentration (p = 0.0015). In male subjects, C*14:02 demonstrated significant associations with fasting plasma glucose with p values of 0.0041. By contrast, Fisher's exact test indicated that female DRB1*14:03 was associated with a high concentration of total cholesterol (p = 000323, odds ratio [OR] = 4.32, 95% confidence interval [95% CI] = 1.83-10.36), whereas female DPB1*02:01 had a protective effect against a high concentration of LDL cholesterol (p =0.0043, OR = 0.41, 95% CI = 0.19-0.79). These associations have a statistical power of more than 0.8 and still retain significance after Bonferroni correction.

Exact break point of a 50 kb deletion 8 kb centromeric of the HLA-A locus with HLA-A*24:02: the same deletion observed in other A*24 alleles and A*23:01 allele.

In a structural aberration analysis of patients with arthritis mutilans, a 50 kb deletion near the HLA-A locus with HLA-A*24:02 allele was detected. It was previously reported that HLA-A*24:02 haplotype harbored a large-scale deletion telomeric of the HLA-A gene in healthy individuals. In order to confirm that the deletion are the same in patients with arthritis mutilans and in healthy individuals, and to identify the break point of this deletion, the boundary sequences across the deletion in A*24:02 was amplified by polymerase chain reaction (PCR) as a 3.7 kb genomic fragment and subjected to nucleotide sequence determination. A comparison of these genomic sequences with those of the non-A*24:02 haplotype revealed that the deleted genomic region spanning 50 kb was flanked by 3.7 kb repetitive element-rich segments homologous to each other on both sides in non-A*24. The nucleotide sequences of the PCR products were identical in patients with arthritis mutilans and in healthy individuals, revealing that the deletion linked to A*24:02 is irrelevant to the onset of arthritis mutilans. The deletion was detected in all other A*24 alleles so far examined but not in other HLA-A alleles, except A*23:01. This finding, along with the phylogenic tree of HLA-A alleles and the presence of the 3.7 kb highly homologous segments at the boundary of the deleted genomic region in A*03 and A*32, may suggest that this HLA-A*24:02-linked deletion was generated by homologous recombination within two 3.7 kb homologous segments situated 50 kb apart in the ancestral A*24 haplotype after divergence from the A*03 and A*32 haplotypes.

Effects of low-dose simvastatin on the distribution of plasma cholesterol and oxidized low-density lipoprotein in three ultra-centrifugally separated low-density lipoprotein subfractions: 12- month, open-label trial.

AIM: The effects of statins on the distribution of oxidized LDL in plasma LDL subfractions have not been well defined. Effects of 12-month treatment with low-dose simvastatin on the distribution of cholesterol and oxidized LDL in 3 ultracentrifugally separated plasma LDL subfractions were compared in patients with hypercholesterolemia. METHODS: Simvastatin was administered to 30 hypercholesterolemic subjects for 12 months at an initial dose of 5 mg/day, which was increased to 20 mg/day via 10mg/day to decrease plasma LDL-cholesterol (C) lower than 130 mg/dL. Simvastatin dose was fixed after 3 months of treatment. The amounts of cholesterol and oxidized LDL in 3 ultracentrifugally separated plasma LDL subfractions were compared between 0 and 12 months of treatment. RESULTS: The distribution of ox-LDL skewed to denser LDL fractions, compared with cholesterol in plasma LDL subfractions. Plasma cholesterol in low-density LDL, medium-density LDL and high-density LDL decreased significantly by 31%, 30%, and 25%, respectively (p<0.0001) after 12 months of simvastatin treatment. Plasma oxidized LDL was decreased from 70 U/L to 56 U/L in medium-density LDL (p=0.042). Oxidized LDL in low-density LDL and high-density LDL did not change significantly after 12 months of treatment. CONCLUSION: Treatment with low-dose simvastatin decreased plasma cholesterol in 3 LDL subfractions and oxidized LDL in medium-density LDL. The decrease of oxidized LDL seemed to be not due to the decrease of cholesterol in plasma LDL subfractions because the decreasing patterns of cholesterol and ox-LDL were different in 3 LDL subfractions.

Decrease in glomerular filtration rate by plasma low-density lipoprotein cholesterol in subjects with normal kidney function assessed by urinalysis and plasma creatinine.

OBJECTIVE: It has not been well defined whether plasma low-density lipoprotein cholesterol (LDL-C) progresses arteriolosclerosis (arteriosclerosis of small arteries) or not. Estimated glomerular filtration rate (e-GFR) is an indicator of the function of renal arterioles and capillaries of glomeruli. The relationship between e-GFR and plasma LDL-C was studied to estimate the effect of plasma LDL-C on the function of renal arterioles and capillaries of glomeruli to speculate the effect of plasma LDL-C on arteriolosclerosis. METHODS AND RESULTS: Major coronary risk factors; blood pressure, plasma lipids, and fasting plasma glucose were compared among 4 groups of examinees of a health evaluation and promotion center separated by e-GFR, namely, Control group, Group 1, 2, 3 from highest e-GFR to lowest e-GFR. Numbers of total male and female subjects were 4602 and 2920, respectively. Plasma LDL-C levels were significantly high in Group 2 and 3 in all male subjects and high in Group 1, 2, and 3 in male subjects with age of fifties, compared with Control group. Plasma LDL-C levels were significantly high in Group 1, 2, and 3 in all female subjects and high in Group 2 and 3 in female subjects with age of fifties, compared with Control group. Plasma levels of LDL-C were not significantly different at each years of age in subjects with age of fifties in both sex. BMI and waist circumference were higher in male subjects with low e-GFR but not in female subjects. Blood pressure and fasting plasma glucose were not high in subjects in Group 1, 2, and 3, compared with Control group in all subjects and subjects with age of fifties in both sex. CONCLUSIONS: We concluded that the high plasma level of LDL-C was the major risk factor among coronary risk factors to reduce GFR probably due to impairing the function of renal arterioles and capillaries of glomeruli in subjects with normal kidney function assessed by urinalysis and plasma creatinine.

Serum concentration of 27-hydroxycholesterol predicts the effects of high-cholesterol diet on plasma LDL cholesterol level.

AIM: The effect of dietary cholesterol on plasma cholesterol concentrations varies widely among individuals. Recent studies suggest that the synthesis of oxysterols is up-regulated when tissue cholesterol is saturated. The present study was undertaken to test the hypothesis that a serum high concentration of 27-hydroxycholesterol, one of the oxysterols, reflects positive cholesterol balance in the body and predicts intolerance to a high-cholesterol diet. METHODS: In 30 subjects, 750 mg/day of cholesterol was added for 4 weeks to the ordinary diet. Blood samples were collected at the start and finish of the supplementation. Serum sterol and oxysterol concentrations were measured by high-resolution GC-MS. RESULTS: A receiver operating characteristic curve was drawn and the cutoff point (80 ng/mg cholesterol) was chosen to maximize sensitivity (81.3%) and specificity (64.3%) for predicting a positive change of LDL cholesterol concentration after cholesterol loading. Subjects with higher serum 27-hydroxycholesterol concentrations (>/= 80 ng/mg cholesterol) showed significantly (P < 0.05) high values for the change of LDL cholesterol concentration (+7.4 +/- 3.4%, mean +/- SEM, n = 17) compared with those with lower 27-hydroxycholesterol levels (-5.3 +/- 2.7%, n = 13). CONCLUSIONS: In subjects with high serum 27-hydroxycholesterol concentrations were unable to adapt to a high-cholesterol diet. The concentration of serum 27-hydroxycholesterol appears to reflect cholesterol saturation in the body and predicts to some extent a responsiveness to dietary cholesterol.

Analyzing of high-density lipoprotein subfractions and low-density lipoprotein subfractions in human serum with anion-exchange chromatography.

OBJECTIVE: We previously established a separation method for five lipoprotein classes (HDL, LDL, IDL, VLDL and chylomicrons) in human serum by using anion-exchange high-performance liquid chromatography (AEX-HPLC). It was thought that analyzing of the subfractions of HDL or LDL is important for study of the atherogenic lipoproteins in atherosclerotic diseases, because a large number of studies were reported regarding them. Therefore, we estimated a separation mode of AEX-HPLC for analyzing cholesterol levels of two HDL-subfractions and two LDL-subfractions. METHODS: In this method, the lipoproteins were separated by a step gradient of sodium perchlorate concentration, and the separated lipoproteins were detected by using a post-column reactor with a reagent containing cholesterol esterase and cholesterol oxidase. RESULTS: The major component of earlier-eluting fraction in HDL (HDL-f) and later-eluting fraction in HDL (HDL-s) were HDL3 and HDL2, respectively. The retention time of peak of earlier-eluting fraction in LDL (LDL-f) was changed into that of later-eluting fraction in LDL (LDL-s) by the oxidization, incubating 2 micromol/l copper ion in PBS for 3h at 37 degrees C. The within- and between-day assay coefficients of variation (CV) for cholesterol levels of the lipoprotein subfractions were in the ranges of 0.85-19.23% and 2.55-23.50%, respectively. The cholesterol levels of HDL-f and HDL-s were lower in patients with coronary heart disease (CHD) than in healthy controls, while LDL-s cholesterol levels were higher in CAD patients than in healthy controls. CONCLUSIONS: This accurate and convenient assay of the lipoprotein subfractions may be useful for studying the atherogenic lipoproteins.

[Minimal effective dose on serum cholesterol concentration and the safety evaluation of dressing containing plant sterol in Japanese subjects].

We examined the minimal effective dose on serum cholesterol concentration and the safety of dressing containing plant sterol in humans. EXP.1: Sixty-eight healthy Japanese males (total cholesterol (TC) > or = 170 mg/dL) were randomly divided into four groups, and were given 0, 400, 800 or 1200 mg/day of plant sterol in 15 g dressing for 4 weeks followed by the washout period of 4 weeks. Although there were no significant differences in serum TC and low-density lipoprotein cholesterol (LDL-C) concentrations among all groups after feeding plant sterol for 4 weeks, in 36 subjects with TC > or = 220 mg/dL, serum LDL-C concentration tended to reduce when received 800 or 1200 mg of plant sterol, and the difference between 0 and 1200 mg groups was statistically significant. The difference between 0 and 800 mg groups was near significant (p=0.053). Intake of 400 mg of plant sterol did not change serum LDL-C concentration. EXP.2: Twenty-one healthy Japanese subjects (TC > or = 180 mg/dL, 10 men, 11 women) were given 2400 mg/day of plant sterol in 45 g dressing for 4 weeks. Clinical data were all remained normal. These results indicated that minimal effective dose of the plant sterol on serum cholesterol concentration in healthy male subjects is around 800 mg/day, and intake of 2400 mg/day of plant sterol is regarded to be safe.

[Effects of dressing containing plant sterol on serum cholesterol concentration and the safety evaluation in borderline or mildly hypercholesterolemic Japanese subjects].

In a placebo-controlled double-blind study, we examined the effects of dressing containing plant sterol (PS) on blood lipids and the safety in Japanese borderline or mildly hypercholesterolemic subjects. Fifty-nine subjects [total cholesterol (TC) concentration > or = 200 mg/dL] were randomly divided into two groups and were given daily 15 g of dressing containing 800 mg of PS [PS(+)-group] or without PS [PS(-)-group] for 12 weeks. Every 4 weeks, fasting blood was examined and subjective symptoms were analyzed. Serum TC, low-density lipoprotein cholesterol (LDL-C) and apolipoprotein B (ApoB) concentrations did not change in the PS(-)-group, while TC and ApoB significantly decreased in the PS(+)-group at 8 and 12 weeks and LDL-C at 4, 8 and 12 weeks. Moreover, serum TC, LDL-C and ApoB concentrations were significantly lower than those of PS(-)-group at 8 and 12 weeks. Other laboratory tests were all in normal ranges and no adverse events were observed. The results indicated that PS-containing dressing decreased serum TC, LDL-C and ApoB concentrations in borderline or mildly hypercholesterolemic subjects. It is therefore proved that the dressing containing PS is helpful in maintaining blood cholesterol level normal and hence, the health of Japanese.