Remnant lipoprotein-cholesterol is a predictive biomarker for large artery atherosclerosis in apparently healthy women: usefulness as a parameter for annual health examinations.

BACKGROUND: Recently, remnant lipoprotein is expected to be a new therapeutic target in the age of 'beyond LDL-cholesterol'. The aim of this study was to clarify the clinical significance of remnant lipoprotein cholesterol (RemL-C) determination in annual health examinations with the focus on large artery atherosclerosis. Methods and results Subjects investigated were men (n = 528) and women (n = 318) who underwent annual health examinations at Osaka University. RemL-C was measured with a newly developed homogeneous assay. Carotid and aortic atherosclerosis was estimated by intima-media thickness (IMT) and cardio-ankle vascular index (CAVI), respectively. First, simple regression analysis revealed that the RemL-C levels positively correlated with maximum IMT, mean IMT and CAVI in the whole group (P < 0.05). Next, receiver operating characteristic curve analysis showed that the most effective levels of RemL-C for predicting carotid and aortic atherosclerosis were 0.21 mmol/L (P < 0.05) and 0.22 mmol/L (P < 0.01) or more, respectively. Odds ratios (ORs) of high RemL-C levels (0.21 mmol/L or more) for carotid and aortic atherosclerosis were significantly increased, especially in low-risk, apparently healthy women (OR: 4.20, P < 0.05 and 3.79, P < 0.01, respectively). Five out of 13 female low-risk cases (38%) with carotid atherosclerosis showed high serum RemL-C levels. It should be emphasized that conventional risk factors are still strong predictors for large artery atherosclerosis in the whole group. CONCLUSIONS: Our results indicate that high serum RemL-C level is a predictive hallmark for large artery atherosclerosis in apparently healthy women. Determination of RemL-C should be employed as one of the parameters in annual health examinations.

A novel apolipoprotein E mutation, ApoE Osaka (Arg158 Pro), in a dyslipidemic patient with lipoprotein glomerulopathy.

Lipoprotein glomerulopathy (LPG) is a rare disease characterized by the presence of thrombuslike deposition in markedly dilated glomerular capillaries and is often accompanied by an increased serum apolipoprotein E (apoE) level. Several gene mutations of apoE have been reported to be associated with LPG. In the current study, we report an LPG patient with a novel apoE mutation, apoE Osaka. The patient was a 45-year-old man who was hospitalized due to nephrotic syndrome. Light and electron microscopic observations of renal biopsy clearly showed characteristic findings of LPG, including lamellate thrombi in the lumen of dilated glomerular capillaries. His apoE phenotype was apoE3/2 and he had mild dyslipidemia with a mid-band on polyacrylamide gel electrophoresis. It is intriguing that the serum apoE level was within normal limits. We determined the sequence of the apoE gene using direct sequencing of the polymerase chain reaction (PCR) products. ApoE gene analysis showed a nucleotide substitution of G to C at codon 158 of exon 4. This mutation denoted an amino acid substitution of arginine residue for the proline residue at position 158 of apoE. The result of PCR associated with restriction fragment length polymorphism analysis also suggested that this mutation is heterozygous. It is possible that apoE Osaka mutation causes a conformational change of apoE protein and affects the interaction between abnormal apoE-containing lipoproteins and the endothelial cells of glomerular capillaries. The precise mechanism of LPG related with apoE Osaka, however, remains to be elucidated.

A case of adolescent hyperlipoproteinemia with xanthoma and acute pancreatitis, associated with decreased activities of lipoprotein lipase and hepatic triglyceride lipase.

Lipoprotein lipase (LPL) and hepatic triglyceride lipase (HTGL) enhance the hydrolysis of triglycerides (TG) transported by chylomicron (CM) and very-low-density lipoprotein (VLDL). We report a case of severe hyperchylomicronemia with high levels of remnant lipoprotein and total cholesterol (T-Chol) in a 15-year-old boy. Precise examination of the lipid profile showed decreased activities of both LPL and HTGL, although the protein mass for LPL and HTGL were maintained. In addition, bezafibrate treatment effectively ameliorated hypertriglyceridemia in this case. This is the first case of hyperchylomicronemia with decreased activities and unaffected protein masses for both LPL and HTGL, without overt immuno-dysfunction.

[Cutting-edge research on the metabolism of remnant lipoproteins].

Lipoproteins in the plasma transport lipids to various tissues through the bloodstream. In an analysis based on specific gravity using ultracentrifugation, these can be divided into 4 main fractions: chylomicrons, VLDL, LDL, and HDL. Metabolism of the triglyceride-rich (TG-rich) lipoproteins, chylomicrons and VLDL, begins with the hydrolysis of TGs by lipoprotein lipase (LPL) and proceeds through intermediate metabolites (remnants). Those resulting from the former are referred to as chylomicron remnants, and those from the latter as VLDL remnants. Both types of remnant are enriched in cholesteryl esters and apolipoprotein E (apoE), and, moreover, they are referred to as atherogenic lipoproteins that readily accumulate in the arterial walls. At the research level, the analytical methods for remnants such as electrophoresis, ultracentrifugation, gel filtration, etc., are not necessarily simple. One of the methods used as a clinical laboratory test for the quantification of remnant lipoproteins is the remnant-like particle-cholesterol (RLP-C) assay. The significance of the assay as an evaluation for arteriosclerosis is well recognized internationally. Recently, a new method for measuring remnant lipoprotein cholesterol (RemL-C) has been developed that uses a reagent consisting of enzymes and a surfactant. With this method, measurements simply involve an automated analyzer in the same manner as for the homogeneous assay for HDL-C or LDL-C and have been shown to correlate well with values using conventional RLP-C reagents. Measurements can be made simply without the need for special equipment in a short period of time, about 10 minutes. Furthermore, this means that measurements can be made in this way with higher reproducibility and precision. Nevertheless, where the principles behind the two assays are different, discrepancies in the measured values can be identified in some cases. It is also important to understand the characteristics of both methods when using them. After detailing lipoprotein remnant measurement methods, we point out the significance of lipoprotein remnant measurements under conditions that have been the focus of recent particular attention, such as postprandial hyperlipidemia and metabolic syndrome.

Update of Japanese common LDLR gene mutations and their phenotypes: Mild type mutation L547V might predominate in the Japanese population.

We investigated the LDLR gene mutations in 205 unrelated Japanese FH (familial hypercholesterolemia) heterozygotes to see if there is a prevalence of common mutations in the Japanese population. A total of 53 different small mutations (<25bp) and 10 kinds of large deletions (>25bp) were identified. Among them there were eight relatively frequent mutations: C317S, c.1845+2T>C, K790X, L547V, P664L, D412H, c.2312-3C>A and V776M. The patients with these mutations comprised 32% of the total FH heterozygotes investigated. Comparison of clinical phenotypes of the eight frequent mutations disclosed that a missense mutation, L547V, manifested a milder phenotype than the other mutations. The mild clinical phenotype was shown to be based on the high level of receptor activity remaining on the patient's cell surfaces. When we examined the presence of common mutations in a general population, the L547V mutation was detected with unexpectedly higher frequency than the other mutations, suggesting an underestimation of the frequency of this mutation in FH heterozygote patients. In conclusion, although there is a broad spectrum of LDLR gene mutations in the Japanese population, eight common mutations were observed. Among them, a mild phenotype mutation, L547V, might predominate in the Japanese population.

Genetic variants in PCSK9 in the Japanese population: rare genetic variants in PCSK9 might collectively contribute to plasma LDL cholesterol levels in the general population.

The aim of this study was to investigate whether plasma low-density lipoprotein cholesterol (LDL-C) levels in the general population are influenced by rare sequence variations in the PCSK9 gene. We sequenced the promoter and coding regions of the PCSK9 gene in individuals from the general population (n=3655) with the lowest (n=78) and highest (n=96) LDL-C levels and in individuals taking antihypercholesterolemia medication (n=96). We identified 33 sequence variants in the PCSK9 gene among which 24 were specific for Japanese. Statistical analysis showed that one missense mutation, R93C, was associated with low LDL-C levels. The other variants had no association with LDL-C levels or the numbers of individuals with the variants were too small for statistical analysis. A comparison of the numbers of individuals with nonsynonymous mutations between the low LDL-C and high LDL-C/treatment groups found that four missense mutations and one nonsense mutation were identified only in the low LDL-C group and six missense mutations were identified only in the high LDL-C/treatment group. As we have analyzed groups at opposite ends of the LDL-C spectrum, it is likely that some of these nonsynonymous mutations may be associated with either low or high LDL-C in the Japanese population. Based on the extremely high frequencies of the nonsynonymous mutations in PCSK9 compared with those of LDLR or apoB-100, PCSK9 mutations could be important factors that cumulatively influence plasma LDL-C levels in the general population.

Development of a homogeneous assay to measure remnant lipoprotein cholesterol.

BACKGROUND: Quantification of triglyceride-rich lipoprotein (TRL) remnants is useful for risk assessment of coronary artery disease and the diagnosis of type III hyperlipoproteinemia. Although an immunoseparation procedure for remnant-like particle cholesterol has been evaluated extensively in recent years, available methods for measuring TRL remnants have not achieved wide use in routine laboratory practice, suggesting a need for a homogeneous assay that can measure TRL remnant cholesterol in serum or plasma without pretreatment. METHODS: We screened for suitable surfactants that exhibited favorable selectivity toward the VLDL remnant (VLDLR) fraction, including intermediate-density lipoproteins (IDLs). We investigated the principal characteristics of this assay by gel filtration of lipoproteins and their particle size distribution. We developed a simple assay and evaluated its performance with the Hitachi-7170 analyzer. RESULTS: Polyoxyethylene-polyoxybutylene block copolymer (POE-POB) exhibited favorable selectivity toward VLDLR and IDL fractions. POE-POB removed apolipoprotein (apo) E and apo C-III from IDL particles in the presence of cholesterol esterase (CHER), and the particle size distribution of IDLs became smaller after the reaction. These results revealed that IDL particles are specifically modified in the presence of CHER and POE-POB, making their component cholesterol available for enzymatic assay. Addition of phospholipase D improved the reactivity toward chylomicron remnants (CMRs). We found a high correlation [y = 1.018x- 0.01 mmol/L, r = 0.962 (n = 160)] between the proposed assay and the immunoseparation assay in serum from healthy individuals. CONCLUSION: The homogeneous assay described in this report can measure TRL remnant cholesterol, including CMRs, VLDLRs, and IDLs, with high sensitivity and specificity.