Relationship between the cholesterol and triglyceride content of lipoprotein subclasses and carotid intima-media thickness: A cross-sectional population-based study.

BACKGROUND: The association between lipoprotein subclasses and carotid intima-media thickness (cIMT) progression has yet to be fully evaluated. We assessed which lipoprotein subclasses were associated with maximum cIMT levels in the general population. METHODS: In this study, cholesterol and triglyceride content of 20 lipoprotein subclasses were analyzed using gel permeation high-performance liquid chromatography (GP-HPLC) in 864 Japanese women and men (mean age 57 y, free of chronic liver or kidney diseases and off lipid-lowering, hormone replacement, or adrenocorticosteroid medications). Univariate and multivariate regression analyses and univariate and partial correlation analyses were performed to examine the relationships between lipoprotein subclasses and maximum cIMT levels. RESULTS: After adjusting for age, sex, systolic blood pressure, smoking, diabetes, and anti-hypertensive agents, elevated low-density lipoprotein (LDL)-2 and -3 cholesterol (particle diameter 25.5 nm and 23.0 nm, respectively; medium and small LDL) were associated with higher maximum cIMT levels in both women and men (all p for trend < 0.05). These associations were significant even after participants taking anti-diabetic or anti-hypertensive agents were excluded. No significant associations were found between any triglyceride subclasses and maximum cIMT levels. CONCLUSIONS: Smaller LDL particle cholesterol values are the most atherogenic lipoprotein parameter.

Development of internal standard for lipoprotein subclass analysis using dual detection gel-permeation high-performance liquid chromatography system.

The LipoSEARCH® System is an innovative lipoprotein class analysis method based on gel-permeation high-performance liquid chromatography (HPLC). This system uses a gel permeation column to separate the major lipoprotein subclasses (chylomicron, very low-density lipoprotein, low-density lipoprotein, and high-density lipoprotein) in serum according to particle size and splits them into two pathways to measure total cholesterol (TC; esterified + unesterified cholesterol) and triglyceride (TG) concentrations simultaneously to obtain chromatograms for each. These chromatograms were analyzed based on the results of the calibration serum by fitting Gaussian curves to profile the 20 lipoprotein subclasses defined in detail. An important assumption of this HPLC system is its simultaneous detection of two pathways to guarantee the accuracy of each analysis. Therefore, in the present study, we investigated the development of an internal standard that can guarantee the simultaneous detection of this system by adding a pigment to the serum. We focused on quinone pigments with absorption at 550 nm, which is the wavelength used for the enzymatic assay of TC and TG concentrations in the system. As a result, we succeeded in producing overlapping pigment peaks that appeared after the analytical chromatograms in two pathways. It is also suggested that the pigment solution as an internal standard is stable in freezing storage and has little effect on the analysis. The developed internal standard is expected to contribute to the accuracy assurance of lipoprotein analysis by this dual-detection HPLC system.

Distinct Differences in Lipoprotein Particle Number Evaluation between GP-HPLC and NMR: Analysis in Dyslipidemic Patients Administered a Selective PPARα Modulator, Pemafibrate.

AIM: We established a method to evaluate the lipid concentrations, size and particle numbers (PNs) of lipoprotein subclasses by gel permeation chromatography (GP-HPLC). Nuclear magnetic resonance (NMR) is widely used to analyze these parameters of lipoprotein subclasses, but differences of the two methods are unknown. Current study compared the PNs of each lipoprotein subclass measured by GP-HPLC and NMR, and assessed the effect of a selective PPARα modulator, pemafibrate. METHODS: Lipoprotein profiles of 212 patients with dyslipidemia who participated in the phase 2 clinical trial of a selective PPARα modulator, pemafibrate, were analyzed by two methods, GP-HPLC and NMR, which were performed with LipoSEARCH (Skylight Biotech) and LipoProfile 3 (LabCorp), respectively. GP-HPLC evaluated the PNs of 18 subclasses, consisting of CM, VLDL1-5, LDL1-6, and HDL1-6. NMR evaluated the PNs of 9 subclasses, consisting of large VLDL & CM, medium VLDL, small VLDL, IDL, large LDL, small LDL, large HDL, medium HDL and small HDL. RESULTS: Three major classes, total CM&VLDL, total LDL and total HDL were obtained by grouping of corresponding subclasses in both methods and PNs of these classes analyzed by GP-HPLC were correlated positively with those by NMR. The correlation coefficients in total CM&VLDL, total LDL and total HDL between GP-HPLC and NMR was 0.658, 0.863 and 0.798 (all p＜0.0001), respectively. The PNs of total CM&VLDL, total LDL and total HDL analyzed by GP-HPLC was 249.5±51.7nM, 1,679±359 nM and 13,273±1,564 nM, respectively, while those by NMR was 124.6±41.8 nM, 1,514±386 nM and 31,161±4,839 nM, respectively. A marked difference in the PNs between the two methods was demonstrated especially in total HDL. The number of apolipoprotein (Apo) B molecule per one ApoB-containing lipoprotein particle, total CM&VLDL plus total LDL, was 1.10±0.05 by GP-HPLC, while 1.32±0.18 by NMR. The number of ApoA-I per one HDL particle was 3.40±0.17 by GP-HPLC, but only 1.46±0.15 by NMR, much less than reported previously.From the phase 2 clinical trial, randomizing 212 patients to pemafibrate 0.025-0.2 mg BID, fenofibrate 100 mg QD, or placebo groups, pemafibrate reduced the PNs of CM, large VLDL1-VLDL3 and medium VLDL4, but not small VLDL5 by GP-HPLC. It significantly decreased the PNs of smaller LDL and larger HDL particles, but increased those of larger LDL and smaller HDL particles. In contrast, NMR showed marked variations in the effect of pemafibrate on lipoprotein PNs, and no significant size-dependent changes. CONCLUSIONS: GP-HPLC evaluates the lipoprotein PNs more accurately than NMR and can be used for assessing the effects of lipid-lowering drugs on lipoprotein subclasses.

A critical role of VMP1 in lipoprotein secretion.

Lipoproteins are lipid-protein complexes that are primarily generated and secreted from the intestine, liver, and visceral endoderm and delivered to peripheral tissues. Lipoproteins, which are assembled in the endoplasmic reticulum (ER) membrane, are released into the ER lumen for secretion, but its mechanism remains largely unknown. Here, we show that the release of lipoproteins from the ER membrane requires VMP1, an ER transmembrane protein essential for autophagy and certain types of secretion. Loss of vmp1, but not other autophagy-related genes, in zebrafish causes lipoprotein accumulation in the intestine and liver. Vmp1 deficiency in mice also leads to lipid accumulation in the visceral endoderm and intestine. In VMP1-depleted cells, neutral lipids accumulate within lipid bilayers of the ER membrane, thus affecting lipoprotein secretion. These results suggest that VMP1 is important for the release of lipoproteins from the ER membrane to the ER lumen in addition to its previously known functions.

Particle number analysis of lipoprotein subclasses by gel permeation HPLC in patients with cholesteryl ester transfer protein deficiency.

OBJECTIVE: We previously reported that patients with cholesteryl ester transfer protein (CETP) deficiency (CETP-D) have a higher prevalence of atherosclerotic cardiovascular disease, in spite of increased HDL-C levels. However, characterization of HDL in CETP-D has not been well described. Therefore, we examined HDL particle number (PN) rather than HDL-C level. APPROACH AND RESULTS: Nine patients with CETP-D and 9 normolipidemic subjects were enrolled. We performed gel permeation high-performance liquid chromatography (GP-HPLC) analysis, determined the cholesterol and triglyceride composition of all lipoprotein subclasses, and calculated the PN of each subclass, which consisted of 3 VLDL (large, medium, and small), 4 LDL (large, medium, small, and very small), and 5 HDL (very large, large, medium, small, and very small) subclasses. The PNs of large and medium LDL were significantly lower in CETP-D than that in healthy subjects (0.66- and 0.63-fold decrease, respectively; p<0.001), whereas the PN of very small LDL, which is known to be atherogenic, was significantly higher (1.36-fold increase, p = 0.016). The PNs of very large and large HDL in CETP-D were markedly higher than that in healthy subjects (19.9- and 4.5-fold increase, respectively; p<0.001), whereas the PNs of small and very small HDL, which have more potent anti-atherogenic functions, were significantly lower (0.76- and 0.61-fold decrease, respectively; p<0.001). CONCLUSION: We have assessed the PNs of detailed subclasses of patients with CETP-D for the first time. The PN of larger HDL was markedly increased, that of smaller HDL was decreased, and that of very small LDL was increased, suggesting that CETP-D has pro-atherogenic lipoprotein properties.

Association of High-Density Lipoprotein Subclasses with Chronic Kidney Disease Progression, Atherosclerosis, and Klotho.

BACKGROUND: Atherosclerosis is often a complication of chronic kidney disease (CKD) because of dyslipidemia and CKD-mineral and bone disorder. High-density lipoproteins (HDLs) are grouped into various subclasses composed of multiple proteins and lipids, and their transformation is altered in CKD. We investigated the roles of lipoprotein subclasses in CKD progression, and atherosclerosis, and the relationships with Klotho and fibroblast growth factor (FGF) 23. METHODS: Seventy-one CKD patients were enrolled in this prospective cohort study in Japan. The proportions of cholesterol level to total cholesterol level (cholesterol proportion) and lipoprotein particle numbers in 20 lipoprotein fractions were measured by a newly developed high-performance gel permeation chromatography. RESULTS: Diabetic nephropathy was observed in 23.9% of the patients. The mean age was 75.0 years and estimated glomerular filtration rate (eGFR) was 17.2 ml/min./1.73m2. The lipoprotein particle numbers in small HDLs were higher in Stage 4 group than in Stage 5 group (p = 0.002). Multivariate regression analysis adjusted for baseline characteristics showed that the cholesterol proportions in very small HDLs were associated with eGFR change rate [F19 ß = -17.63, p = 0.036] and ABI [F19 ß = 0.047, p = 0.047] in Stage 4 group, and that serum soluble α-Klotho level was associated with the lipoprotein particle numbers in very small HDLs [F19 ß = 0.00026, p = 0.012; F20 ß = 0.00041, p = 0.036] in Stage 5 group. CONCLUSIONS: This study showed that HDL subclasses are associated with CKD progression, ABI, and Klotho level in CKD-stage-specific manner.

Bioinformatic Analysis of Plasma Apolipoproteins A-I and A-II Revealed Unique Features of A-I/A-II HDL Particles in Human Plasma.

Plasma concentration of apoA-I, apoA-II and apoA-II-unassociated apoA-I was analyzed in 314 Japanese subjects (177 males and 137 females), including one (male) homozygote and 37 (20 males and 17 females) heterozygotes of genetic CETP deficiency. ApoA-I unassociated with apoA-II markedly and linearly increased with HDL-cholesterol, while apoA-II increased only very slightly and the ratio of apoA-II-associated apoA-I to apoA-II stayed constant at 2 in molar ratio throughout the increase of HDL-cholesterol, among the wild type and heterozygous CETP deficiency. Thus, overall HDL concentration almost exclusively depends on HDL with apoA-I without apoA-II (LpAI) while concentration of HDL containing apoA-I and apoA-II (LpAI:AII) is constant having a fixed molar ratio of 2 : 1 regardless of total HDL and apoA-I concentration. Distribution of apoA-I between LpAI and LpAI:AII is consistent with a model of statistical partitioning regardless of sex and CETP genotype. The analysis also indicated that LpA-I accommodates on average 4 apoA-I molecules and has a clearance rate indistinguishable from LpAI:AII. Independent evidence indicated LpAI:A-II has a diameter 20% smaller than LpAI, consistent with a model having two apoA-I and one apoA-II. The functional contribution of these particles is to be investigated.

Recent Advances in Analytical Methods on Lipoprotein Subclasses: Calculation of Particle Numbers from Lipid Levels by Gel Permeation HPLC Using "Spherical Particle Model".

Recently, we developed an analytical method for determining the lipid levels and particle numbers in lipoprotein subclasses covering a wide size range from chylomicrons to small high density lipoproteins, by using gel permeation high-performance liquid chromatography (GP-HPLC). The challenges in analytical methods on lipoprotein subclasses have been addressed from 1980 by Hara and Okazaki using commercial TSK gel permeation columns. Later, the improvements in the hardware, separation and detection of lipoproteins, and the data processing software, using a Gaussian distribution approximation to calculate lipid levels of lipoprotein subclasses, have been extensively utilized in these analytical methods for over thirty years. In this review, we describe on the recent advances in analytical methods on lipoprotein subclasses based on various techniques, and the calculation of particle numbers from lipid levels by GPHPLC using the "spherical particle model". Free/ester ratio of cholesterol in particular lipoprotein subclass was accurately estimated from triglyceride, total cholesterol (free and esterified) and the size of the particle based on this model originally proposed by Shen and Kezdy.

Selective evaluation of high density lipoprotein from mouse small intestine by an in situ perfusion technique.

The small intestine (SI) is the second-greatest source of HDL in mice. However, the selective evaluation of SI-derived HDL (SI-HDL) has been difficult because even the origin of HDL obtained in vivo from the intestinal lymph duct of anesthetized rodents is doubtful. To shed light on this question, we have developed a novel in situ perfusion technique using surgically isolated mouse SI, with which the possible filtration of plasma HDL into the SI lymph duct can be prevented. With the developed method, we studied the characteristics of and mechanism for the production and regulation of SI-HDL. Nascent HDL particles were detected in SI lymph perfusates in WT mice, but not in ABCA1 KO mice. SI-HDL had a high protein content and was smaller than plasma HDL. SI-HDL was rich in TG and apo AIV compared with HDL in liver perfusates. SI-HDL was increased by high-fat diets and reduced in apo E KO mice. In conclusion, with our in situ perfusion model that enables the selective evaluation of SI-HDL, we demonstrated that ABCA1 plays an important role in intestinal HDL production, and SI-HDL is small, dense, rich in apo AIV, and regulated by nutritional and genetic factors.

Serum cholesterol and triglyceride reference ranges of twenty lipoprotein subclasses for healthy Japanese men and women.

AIM: This epidemiological study was done to generate normal ranges for the cholesterol and triglyceride levels in serum lipoprotein subclasses isolated from healthy adults based on gender and menopausal status. METHODS: Cholesterol and triglyceride levels in 20 lipoprotein subclasses as separated by high performance liquid chromatography were measured in serum obtained from 825 fasting healthy subjects (267 men, 558 women). RESULTS: For serum cholesterol, 13.7% was found in very low density lipoprotein (VLDL) subclasses, 55.6% in low density lipoprotein (LDL) subclasses, and 30.4% in high density lipoprotein (HDL) subclasses. For serum triglycerides, these values were 52.1%, 27.9%, and 17.4%, respectively. Levels of cholesterol in some VLDL subclasses were inversely correlated with the levels of some HDL subclasses, while for triglycerides, elevated levels in any one subclass were generally strongly associated with elevated levels in all other subclasses. Men had significantly higher large VLDL-cholesterol levels than women (P < 0.05), while women had significantly higher small VLDL-cholesterol levels than men (P < 0.001). Women had significantly higher large LDL- and large and medium HDL-cholesterol levels than men (P < 0.001). Men had significantly higher chylomicron (CM), large and medium VLDL-, and small LDL-triglyceride levels than women (P < 0.001). Women had significantly higher very large and large HDL-triglyceride levels than men (P < 0.01). Postmenopausal women had significantly higher CM, all VLDL, and large, medium and small LDL-cholesterol levels, and significantly higher all VLDL, LDL, and HDL-triglyceride levels than premenopausal women (P < 0.001). CONCLUSIONS: Our data document important gender and menopausal status differences in cholesterol and triglyceride subclass levels, as well as significant correlations between values in the various serum lipoprotein subclasses.

Relationship between Icodextrin use and decreased level of small low-density lipoprotein cholesterol fractioned by high-performance gel permeation chromatography.

BACKGROUND: Because of the absorption of glucose in peritoneal dialysis (PD) solution, PD patients show an atherogenic lipid profile, which is predictive of poor survival in PD patients. Lipoprotein subclasses consist of a continuous spectrum of particles of different sizes and densities (fraction). In this study, we investigated the lipoprotein fractions in PD patients with controlled serum low-density lipoprotein (LDL) cholesterol level, and evaluated the effects of icodextrin on lipid metabolism. METHODS: Forty-nine PD patients were enrolled in this cross-sectional study in Japan. The proportions of cholesterol levels to total cholesterol level (cholesterol proportion) in 20 lipoprotein fractions were measured using an improved method of high-performance gel permeation chromatography (HPGPC). RESULTS: Twenty-six patients used icodextrin. Although no significant differences in cholesterol levels in LDL and high-density lipoprotein (HDL) were observed between the patients using icodextrin (icodextrin group) and control groups, HPGPC showed that the icodextrin group had significantly lower cholesterol proportions in the small LDL (t-test, p=0.053) and very small LDL (p=0.019), and significantly higher cholesterol proportions in the very large HDL and large HDL than the control group (p=0.037; p=0.066, respectively). Multivariate analysis adjusted for patient characteristics and statin use showed that icodextrin use was negatively associated with the cholesterol proportions in the small LDL (p=0.037) and very small LDL (p=0.026), and positively with those in the very large HDL (p=0.040), large HDL (p=0.047), and medium HDL (p=0.009). CONCLUSIONS: HPGPC showed the relationship between icodextrin use and the cholesterol proportions in lipoprotein fractions in PD patients. These results suggest that icodextrin may improve atherogenic lipid profiles in a manner different from statin.

The association of very-low-density lipoprotein with ankle-brachial index in peritoneal dialysis patients with controlled serum low-density lipoprotein cholesterol level.

BACKGROUND: Peripheral artery disease (PAD) represents atherosclerotic disease and is a risk factor for death in peritoneal dialysis (PD) patients, who tend to show an atherogenic lipid profile. In this study, we investigated the relationship between lipid profile and ankle-brachial index (ABI) as an index of atherosclerosis in PD patients with controlled serum low-density lipoprotein (LDL) cholesterol level. METHODS: Thirty-five PD patients, whose serum LDL cholesterol level was controlled at less than 120 mg/dl, were enrolled in this cross-sectional study in Japan. The proportions of cholesterol level to total cholesterol level (cholesterol proportion) in 20 lipoprotein fractions and the mean size of lipoprotein particles were measured using an improved method, namely, high-performance gel permeation chromatography. Multivariate linear regression analysis was adjusted for diabetes mellitus and cardiovascular and/or cerebrovascular diseases. RESULTS: The mean (standard deviation) age was 61.6 (10.5) years; PD vintage, 38.5 (28.1) months; ABI, 1.07 (0.22). A low ABI (0.9 or lower) was observed in 7 patients (low-ABI group). The low-ABI group showed significantly higher cholesterol proportions in the chylomicron fraction and large very-low-density lipoproteins (VLDLs) (Fractions 3-5) than the high-ABI group (ABI>0.9). Adjusted multivariate linear regression analysis showed that ABI was negatively associated with serum VLDL cholesterol level (parameter estimate=-0.00566, p=0.0074); the cholesterol proportions in large VLDLs (Fraction 4, parameter estimate=-3.82, p=0.038; Fraction 5, parameter estimate=-3.62, p=0.0039) and medium VLDL (Fraction 6, parameter estimate=-3.25, p=0.014); and the size of VLDL particles (parameter estimate=-0.0352, p=0.032). CONCLUSIONS: This study showed that the characteristics of VLDL particles were associated with ABI among PD patients. Lowering serum VLDL level may be an effective therapy against atherosclerosis in PD patients after the control of serum LDL cholesterol level.

Postprandial lipoprotein metabolism: VLDL vs chylomicrons.

Since Zilversmit first proposed postprandial lipemia as the most common risk of cardiovascular disease, chylomicrons (CM) and CM remnants have been thought to be the major lipoproteins which are increased in the postprandial hyperlipidemia. However, it has been shown over the last two decades that the major increase in the postprandial lipoproteins after food intake occurs in the very low density lipoprotein (VLDL) remnants (apoB-100 particles), not CM or CM remnants (apoB-48 particles). This finding was obtained using the following three analytical methods; isolation of remnant-like lipoprotein particles (RLP) with specific antibodies, separation and detection of lipoprotein subclasses by gel permeation HPLC and determination of apoB-48 in fractionated lipoproteins by a specific ELISA. The amount of the apoB-48 particles in the postprandial RLP is significantly less than the apoB-100 particles, and the particle sizes of apoB-48 and apoB-100 in RLP are very similar when analyzed by HPLC. Moreover, CM or CM remnants having a large amount of TG were not found in the postprandial RLP. Therefore, the major portion of the TG which is increased in the postprandial state is composed of VLDL remnants, which have been recognized as a significant risk for cardiovascular disease.

Measurement of serum remnant-like lipoprotein particle-triglyceride (RLP-TG) and RLP-TG/total TG ratio using highly sensitive triglyceride assay reagent.

BACKGROUND: Serum concentration of remnant-like lipoprotein particles (RLP) have been measured by cholesterol as RLP-C for clinical diagnostic purpose. However, the measurement of TG in RLP and the ratio of RLP-TG/total TG has not been well established. METHOD: Highly sensitive triglyceride assay reagent (TG-EX) was used for RLP-TG assay and compared with the previously used TG reagent (Determiner LTGII). Sera in health check-up populations, cardiovascular disease, diabetes and oral fat load cases were used for the evaluation of the new RLP-TG assay. Serum TC, TG, HDL-C, LDL-C and RLP-C concentrations were also determined in above cases. RESULTS: The detection limit of new RLP-TG using TG-EX was 2.0mg/dl. The within-run imprecision (n=10) was CV=3.0% (RLP-TG: 4.1 mg ± 0.7 mg/dl), CV = 1.4% (RLP-TG: 42.0 ± 0.6 mg/dl) and CV=0.5% (RLP-TG: 100.6 ± 0.6 mg/dl). Cut-off value (75 percentile) of RLP-TG determined in the fasting Japanese population was 13.1mg/dl in men and 9.9 mg/dl in women. In patients with metabolic syndrome, cardiovascular disease and diabetes, RLP-TG levels were significantly higher than those in normal control subjects. RLP-TG levels increased significantly after an oral fat load and the ratio of RLP-TG/total TG increased > 3-fold compared to the ratio in the fasting state. Approximately 80% of TG increased after an oral fat load was TG derived from remnant lipoproteins. CONCLUSION: Normal range of plasma RLP-TG in the fasting Japanese population was first determined using a highly sensitive TG assay reagent. RLP-TG was shown to be higher in cases with metabolic syndrome, cardiovascular disease, etc and a better marker than RLP-C for the measurement of postprandial remnant lipoproteins, together with total TG for RLP-TG/total TG ratio.

Particle size of apoB-48 carrying lipoproteins in remnant lipoproteins isolated from postprandial plasma.

BACKGROUND: Particle size of apoB-48 carrying lipoproteins in remnant-like lipoprotein particles (RLP) in postprandial plasma has not been well characterized. METHODS: Plasma lipids, lipoproteins and apolipoproteins in 12 healthy subjects were analysed after an oral fat load. RLP isolated by immunoaffinity gel from plasma of a normolipidaemic and a hyperlipidaemic subject in four hours after an oral fat load was fractionated by high-performance liquid chromatography (HPLC) and monitored by total cholesterol (TC), triglycerides (TG), apoB-48 and apoB-100. RESULTS: TC, low-density lipoprotein (LDL)-C and apoB did not change after an oral fat load, while TG, RLP-C, RLP-TG and apoB-48 increased significantly in postprandial plasma. HPLC profiles monitored by TC and TG revealed that major lipoproteins increased in RLP after an oral fat load was VLDL size particles. The percentage of RLP-TG in total TG and the ratio of RLP-TG/RLP-C were significantly increased in four hours after an oral fat load compared with the fasting state (P < 0.01). RLP in four hours after an oral fat load fractionated by HPLC and monitored by TC, TG, apoB-48 and apoB-100 revealed that VLDL size or smaller particles were the major lipoproteins. CONCLUSIONS: ApoB-48 carrying lipoproteins in RLP isolated from a normolipidaemic and a hyperlipidaemic subject after an oral fat load showed a similar particle size with apoB-100 carrying VLDL remnants. Therefore, the most apoB-48 carrying particles found in postprandial RLP can be classified as CM remnants. The majority of remnants in the postprandial state were not CM remnants, but VLDL remnants.

Ten-year evaluation of homogeneous low-density lipoprotein cholesterol methods developed by Japanese manufacturers. Application of the Centers for Disease Control and Prevention/Cholesterol Reference Method Laboratory Network lipid standardization protocol.

AIM: The risk index for atherosclerotic cardiovascular diseases in the Japanese metabolic syndrome-focused health checkup program was changed from total cholesterol (TC) to low-density lipoprotein cholesterol (LDL-C). We discuss the validity of this change with respect to standardization. METHODS: The beta-quantification procedure of the Centers for Disease Control and Prevention (CDC) uses the LDL-C reference value as a target. Clinical laboratories and commercial manufacturers use homogeneous LDL-C methods for standardization. (A) For clinical laboratories, LDL-C in 648 samples requested from 108 hospitals was analyzed. (B) Manufacturers participated in the CDC/Cholesterol Reference Method Laboratory Network LDL-C standardization protocol. The standardization was conducted with a performance follow-up for the 10-year period from 1998 to 2008 at 2-year intervals, 6 times. RESULTS: (A) In clinical laboratories, acceptable LDL-C levels within ±4% of the CDC's criteria remained 70.4%, 456 of 648 subjects. Negative maximum bias deviating from the LDL-C target value was -35.8%, -52.5 mg/dL, and positive maximum bias was +24.5%, +32.3 mg/dL. (B) For manufacturers, the standardization achievement rate of the analytical reagent/instrument/calibrator system in the last four standardizations from 2002 to 2008 remained on average 66.6%, far lower than the level required. CONCLUSIONS: The standardization achievement rate of homogeneous LDL-C methods was much low-er than that of TC. TC should still be used as a risk index for atherosclerotic cardiovascular diseases. The standardization achievement rate of homogeneous LDL-C should be maintained at 100%, at least using samples with normal lipoprotein profiles. The accuracy and specificity of LDL-C should be further improved before practical and clinical use.

Measurement performance of reagent manufacturers by Centers for Disease Control and Prevention/Cholesterol Reference Method Laboratory Network lipid standardization specified for metabolic syndrome-focused health checkups program in Japan.

AIM: This study was designed to clarify the current measurement performance of 7 reagent manufacturers for high-density lipoprotein cholesterol (HDLC), low-density lipoprotein cholesterol (LDLC) and triglycerides (TG) specified for the metabolic syndrome (MetS)-focused health checkups program in Japan. METHODS: Twenty HDLC, 21 LDLC and 9 TG analytical reagent/instrument/calibrator systems (system), and combinations of reagent lots, instrument models and calibrator lots, underwent Centers for Disease Control and Prevention (CDC)/Cholesterol Reference Method Laboratory Network (CRMLN) lipid standardization. Eighty and 100% systems were requested to achieve an accuracy of within +/-1% and +/-2% of the reference value, so that a clinical laboratory can meet the CDC criteria. RESULTS: The CDC performance criteria of HDLC, LDLC and TG require an accuracy of within +/-5%, +/-4% and +/-5%, respectively. For HDLC, all 20 systems met the criteria. Fourteen (70.0%) and 18 (90.0%) systems were within +/-1% and +/-2%, respectively. For LDLC, 14 (66.7%) of 21 systems met the criteria, but 7 (33.3%) failed. Five (23.8%) and 17 (81.0%) systems were within +/-1% and +/-2%, respectively. For TG, 8 of 9 systems met the criteria. Two (22.2%) and 4 (44.4%) systems were within +/-1% and +/-2%, respectively. The minimum and maximum differences of a specified sample among manufacturers were 1.6 and 11.0 mg/dL for HDLC, 7.8 and 33.0 mg/dL for LDLC, and 2.8 and 27.4 mg/dL for TG, respectively. CONCLUSION: Homogeneous HDLC methods are acceptable for MetS, but further accuracy improvement of homogeneous LDLC and TG methods will be needed because of their poor performance.

Evaluation of a gel-permeation high-performance liquid chromatography for determining triglyceride levels in serum major lipoproteins, compared with the ultracentrifugation/precipitation method.

OBJECTIVES: To evaluate the gel permeation high-performance liquid chromatography (GP-HPLC) method for determination of triglyceride (TG) levels in low-density lipoprotein (LDL) and high-density lipoprotein (HDL). DESIGN AND METHODS: The GP-HPLC and the ultracentrifugation (UC)/precipitation methods were used and compared. RESULTS: There was no significant difference in measured levels of LDL-triglyceride between the two methods, but the HDL-triglyceride levels measured by the GP-HPLC technique were significantly higher than the UC/precipitation one (145+/-47 mg/L and 121+/-45 mg/L respectively, n=38, p<0.0001). CONCLUSIONS: A GP-HPLC technique provides LDL-TG levels comparable to those obtained by the UC/precipitation method.

Antihypertensive effect of Pleurotus nebrodensis in spontaneously hypertensive rats.

In this study, we examined the effects of Pleurotus nebrodensis on systolic blood pressure of spontaneously hypertensive rats. Single-dose and continuous-dose tests with sample diets made from the fruit body of the mushroom, P. nebrodensis were carried out on SHR and control rats. Sample diets included 6% dry powder of fruit body (6% dry powder), hot water extract, polysaccharide fraction, protein fraction, dialyzable fraction and non-dialyzable fraction. Polysaccharide and protein fractions were obtained by hot water extraction. The protein fraction was separated to the dialyzable fraction and non-dialyzable fraction by dialysis. In the single-dose test, protein fraction, hot water extract and polysaccharide fraction decreased systolic blood pressure. Blood pressure was lowered after administration for 2 h, and it returned to the pre-administration blood pressure after 48 h. In the continuous-dose test, spontaneously hypertensive rats were fed each of the diets for 16 weeks. The 6% dry powder group showed significantly inhibited elevation of blood pressure compared with the control group and there was no influence on total cholesterol and triglyceride levels. The non-dialyzable fraction showed suppression of increase in blood pressure from the start of the continuous oral administration. Effects on the rennin angiotensin system and renal function were also indicated. The antihypertensive action effect of P. nebrodensis can be expected to not only prevent but also to improve hypertension.