Development of blood collection tubes for glucose measurement using adenosine 3-phosphate and sodium fluoride as glycolytic inhibitors.

BACKGROUND: Blood collection tubes with sodium fluoride (NaF) added as a glycolytic inhibitor are widely used for glucose measurement. However, the glycolytic inhibitory effects of NaF are insufficient, and decreases in glucose levels over time after blood collection have become a problem. METHODS: Blood from a volunteer collected using an NaF tube was used to compare the glycolysis inhibitory abilities of ATP and ADP. Blood samples from 10 volunteers were collected in NaF tubes and NaF tubes with added ATP (NaF-ATP tubes). The stability of glucose and haemoglobin (Hb)A1c after whole-blood storage from immediately after blood collection to 24 h later was compared. RESULTS: ATP and ADP had similar inhibitory effects on glycolysis, but ATP was selected as an additive for blood collection tubes because ADP was more haemolytic than ATP. We verified the ability of NaF blood collection tubes supplemented with ATP to inhibit glycolysis. Mean (± standard deviation) glucose levels (n=10) after storage for 24 h after blood collection decreased to -9.0 ± 2.7 mg/dL (-0.50 ± 0.15 mmol/L) in conventional NaF tubes. NaF-ATP(20) tubes with 20 mg (0.036 mmol) ATP added showed a reduced decrease, with a mean of -5.8 ± 2.9 mg/dL (-0.32 ± 0.16 mmol/L). NaF-ATP tubes also had no effect on HbA1c measurement. CONCLUSION: This study reports on a blood collection tube that enables the measurement of glucose and HbA1c. Based on the results of validation, we conclude that NaF-ATP tubes can reduce decreases in glucose over time in stored whole blood compared to conventional NaF tubes.

Measurement of cholesterol levels of lipoprotein subclasses in human serum using anion-exchange high-performance liquid chromatography with a linear concentration gradient of sodium perchlorate.

BACKGROUND: Relationships between the subclasses of high-density lipoprotein (HDL) or low-density lipoprotein (LDL) and the risk of atherosclerotic cardiovascular disease have been studied, and using various methods, such as ultracentrifugation, electrophoresis, and nuclear magnetic resonance, for analysing lipoprotein subclasses. We established a method for HDL and LDL subclasses using anion-exchange high-performance liquid chromatography (AEX-HPLC) with a linear concentration gradient of sodium perchlorate (NaClO4). METHOD: In the AEX-HPLC, the subclasses of HDL and LDL were separated, and detected using a post-column reactor with an enzymatic cholesterol reagent, that contained cholesterol esterase, cholesterol oxidase, and peroxidase as major ingredients. LDL subclasses were divided based on the absolute value of first-derivative chromatogram. RESULT: Three HDL subclasses, HDL-P1, HDL-P2, and HDL-P3, and three LDL subclasses, LDL-P1, LDL-P2, and LDL-P3, were separated by AEX-HPLC, and detected in order. The major components of HDL-P2 and HDL-P3 were HDL3 and HDL2, respectively. The linearity was determined for each lipoprotein subclass. The coefficients of variation of cholesterol concentration of the subclasses for within-day assay (n = 10) and between-day assay (n = 10) ranged between 3.08-8.94% and 4.52-9.97%, respectively. Cholesterol levels in HDL-P1 of diabetic patients were positively correlated with oxidized LDL levels (r = 0.409, p = 0.002). Moreover, cholesterol levels in LDL-P2 and LDL-P3 were positively correlated with oxidized LDL levels (r = 0.393, p = 0.004 and r = 0.561, p < 0.001, respectively). CONCLUSION: AEX-HPLC may be highly suitable as an assay to clinically assess lipoprotein subclasses.

Evaluation of the two-point calibration bromocresol green method, showing reduced deviation from the bromocresol purple method in sera from patients with hypoalbuminemia.

BACKGROUND: The bromocresol green (BCG) and bromocresol purple (BCP) methods are widely used for albumin measurements in routine testing, but the BCG method is known to react with globulin fractions and to have low specificity for albumin. We evaluated a calibration method using different concentrations of human serum albumin standards (two-point calibration BCG method) with the aim of reducing the effect of globulin fractions on the BCG method in patients with hypoalbuminemia. METHOD: In the two-point calibration BCG method, two concentrations of standard solutions and their calibration values are set based on the difference in albumin concentrations measured by the BCG method (BCG-HSA method) and the modified BCP (modified BCP-HSA method) calibrated with human serum albumin standard solution (HSA). Albumin concentrations were measured in 136 patient serum samples (healthy group: 52, hypoalbuminemic group: 84) by the two-point calibrated BCG method and compared with those obtained using the modified BCP-HSA method. RESULTS: The mean albumin concentrations obtained using the two-point calibrated BCG and modified BCP-HSA methods were 39.18 ± 3.42 g/L and 39.37 ± 3.14 g/L (healthy group) and 26.20 ± 6.23 g/L and 26.23 ± 5.67 g/L (hypoalbuminemia group), respectively. The results of the two-point calibration BCG method were in a close agreement over the entire concentration range tested compared to the modified BCP-HSA method. CONCLUSIONS: Based on these results, this calibration method reduces the influence of the globulin fraction on the BCG method. In the hypoalbuminemic group, the calibration method was shown to provide results consistent with the BCP method, which is highly specific for albumin.

Postprandial fatty acid metabolism with coconut oil in young females: a randomized, single-blind, crossover trial.

BACKGROUND: Approximately 84% of the fatty acids contained in coconut oil (CO) are saturated fatty acids (SFAs), and approximately 47% of the SFA are lauric acid with 12 carbon atoms. Lauric acid carbon chain length is intermediate between medium and long-chain fatty acids (LCFAs). We examined how CO acts on lipid-related substances in the blood to determine whether its properties were similar to medium-chain fatty acids (MCFAs) or LCFAs. METHODS: This is a randomized controlled, single-blind, crossover study. Fifteen females were enrolled, using 3 test meals containing 30 g each of 3 different oils: CO (CO-meal), medium-chain triacylglycerol oil (MCT-meal), and long-chain triacylglycerol oil (LCT-meal). Blood samples were collected at fasted baseline and every 2 h for 8 h after the intake of each test meal. RESULTS: Repeated measures ANOVA of the ketone bodies and triglyceride (TG) showed an interaction between time and the test meal (P < 0.01 and P < 0.001, respectively). In subsequent Tukey's honestly significant difference (HSD) test of the ketone bodies, statistically significant differences were observed between the CO-meal and the LCT-meal (P < 0.05) 83.8 (95% CI, 14.7, 153.0) and between the MCT-meal and the LCT-meal (P < 0.05) 79.2 (95% CI, 10.0, 148.4). The incremental area under the curve (iAUC) and maximum increase in very low-density lipoprotein cholesterol (VLDL-C) and intermediate-density lipoprotein cholesterol (IDL-C) were the lowest for CO-meal intake. CONCLUSIONS: The characteristics of lauric acid contained in CO, including the kinetics of ß-oxidation and effects on blood TG, were very similar to those of MCFA. Moreover, regarding the iAUC and peak increment, VLDL-C and IDL-C were the lowest with the CO-meal. These results suggest that the intake of CO after fasting does not increase the TG, VLDL-C, and IDL-C, and may help prevent dyslipidemia. This trial was registered at UMIN (URL of registration: https://www.umin.ac.jp) as UMIN000019959.

Clinical Significance of Intermediate-Density Lipoprotein Cholesterol Determination as a Predictor for Coronary Heart Disease Risk in Middle-Aged Men.

Background: Not only low-density lipoprotein (LDL) cholesterol but also non-high-density lipoprotein cholesterol (non-HDL-C), very low-density lipoprotein (VLDL) cholesterol (VLDL-C), and intermediate-density lipoprotein (IDL) cholesterol (IDL-C) are reported to be significant risk markers for coronary heart disease (CHD). We reported the relevance of IDL-C to Framingham risk score (F-score), but the present study addressed the relevance of IDL-C to Suita score (S-score), a risk score for coronary heart disease (CHD) developed for the Japanese individuals in addition to F-score. Methods: The cholesterol levels of lipoproteins, including triglyceride (TG)-rich lipoproteins (IDL and VLDL), were measured by an anion exchange high-performance liquid chromatography (AEX-HPLC). This study enrolled 476 men, aged mean 51 years and free of CHD and stroke. Results: Non-HDL-C, IDL-C, and VLDL-C significantly correlated with F-score and S-score. In the multiple stepwise regression analysis, IDL-C as well as body mass index (BMI) significantly correlated with both F-score and S-score in both the total subjects and the subjects without drug therapy. The multivariate logistic analysis with the model composed of BMI and IDL-C as the predictor variables demonstrated that 1 SD increase in IDL-C was an independent predictor for 10-year CHD risk >10% of F-score (OR 1.534, 95% CI 1.266-1.859, p < 0001) and that of S-score (OR 1.372, 95% CI 1.130-1.667, p = 0.0014) in the total subjects. Even in the subjects without the drug therapy, the increased IDL-C, as well as BMI, were significant predictors for 10-year CHD risk >10% of S-score as well as F-score. Conclusion: These results suggest the significant relevance of the increased IDL-C for CHD risk scores in middle-aged men free of CHD and stroke. Further investigations are needed in women and elderly subjects.

Verification of Low-Density Lipoprotein Cholesterol Levels Measured by Anion-Exchange High-Performance Liquid Chromatography in Comparison with Beta Quantification Reference Measurement Procedure.

BACKGROUND: A new lipoprotein testing method based on anion-exchange HPLC (AEX-HPLC) was recently established. We verified the accuracy of LDL-C levels, a primary therapeutic target for the prevention of cardiovascular disease (CVD), measured by AEX-HPLC comparing with LDL-C levels measured by beta quantification-reference measurement procedure (BQ-RMP), homogenous assays, and calculation methods. METHODS: We compared LDL-C levels measured by AEX-HPLC (adLDL-Ch: LDL-Ch and IDL-Ch) and BQ-RMP using blood samples from 52 volunteers. AdLDL-Ch levels were also compared with those measurements by homogeneous assays and calculation methods (Friedewald equation, Martin equation, and Sampson equation) using blood samples from 411 participants with dyslipidemia and/or type 2 diabetes. RESULTS: The precision and accuracy of adLDL-Ch were verified by BQ-RMP. The mean percentage bias [bias (%)] for LDL-C was 1.2%, and the correlation was y = 0.990x + 3.361 (r = 0.990). These results met the acceptable range of accuracy prescribed by the National Cholesterol Education Program. Additionally, adLDL-Ch levels were correlated with LDL-C levels measured by the 2 homogeneous assays (r > 0.967) and the calculation methods (r > 0.939), in serum samples from patients with hypertriglyceridemia. CONCLUSIONS: AEX-HPLC is a reliable method for measuring LDL-C levels for CVD risk in daily clinical laboratory analyses.

Innovatively Established Analysis Method for Lipoprotein Profiles Based on High-Performance Anion-Exchange Liquid Chromatography.

Separation analysis of lipoprotein classes have various methods, including ultracentrifugation, electrophoresis, and gel permeation chromatography (GPC). All major lipoprotein classes can be separated via ultracentrifugation, but performing the analysis takes a long time. Low-density lipoprotein (LDL), intermediate-density lipoprotein (IDL), and very low-density lipoprotein (VLDL) in patient samples cannot be sufficiently separated via electrophoresis or GPC. Thus, we established a new method [anion-exchange high-performance liquid chromatography (AEX-HPLC)] by using HPLC with an AEX column containing nonporous gel and an eluent containing chaotropic ions. AEX-HPLC can separate five lipoprotein fractions of high-density lipoprotein (HDL), LDL, IDL, VLDL, and others in human serum, which can be used in substitution for ultracentrifugation method. The method was also approved for clinical use in the public health-care insurance in Japan in 2014. Furthermore, we developed an additional method to measure cholesterol levels of the four leading lipoprotein fractions and two subsequent fractions (i.e., chylomicron and lipoprotein(a)). We evaluated the clinical usefulness of AEX-HPLC in patients with coronary heart disease (CHD), diabetes, and kidney disease and in healthy volunteers. Results indicate that the cholesterol levels in IDL and VLDL measured by AEX-HPLC may be useful risk markers of CHD or diabetes. Furthermore, we developed another new method for the determination of alpha-tocopherol (AT) in lipoprotein classes, and this method is composed of AEX-HPLC for the separation of lipoprotein classes and reverse-phase chromatography to separate AT in each lipoprotein class. The AT levels in LDL were significantly correlated with the lag time to copper ion-induced LDL oxidation, which is an index of oxidation resistance. The application of AEX-HPLC to measure various substances in lipoproteins will be clinically expected in the future.

Diabetic dyslipidemia: evaluation and mechanism.

Diabetes is one of the well-established independent risk factors for cardiovascular diseases. Diabetes induces dyslipidemia which is characterized by elevated fasting triglyceride (TG) and reduced high-density lipoprotein-cholesterol (HDL-C), and such diabetic dyslipidemia is a crucial determinant for atherogenesis and atherosclerotic progression in patients with diabetes. Previous measurement methods of lipoproteins have problems including time-consuming (ultracentrifugation) and inaccurate and impossible measurements of TG-rich lipoproteins such as chylomicron, intermediate-density lipoprotein (IDL) and very low-density lipoprotein (VLDL). Our developed anion-exchange high-performance liquid chromatography (AEX-HPLC) can measure all fractions of lipoproteins accurately. Our studies using AEX-HPLC showed that IDL and VLDL in type 2 diabetes were higher than non-diabetic subjects, and IDL and VLDL were higher in the order of type 2 diabetic patients with obesity, type 2 diabetic patients without obesity, and non-diabetic subjects. Here, we also describe the underlying mechanisms for development of diabetic dyslipidemia.

Cholesterol concentrations in lipoprotein fractions separated by anion-exchange-high-performance liquid chromatography in healthy dogs and dogs with hypercholesterolemia.

Anion-exchange (AEX)-high-performance liquid chromatography (HPLC) for measurement of cholesterol can be used to separate serum lipoproteins (high-density lipoprotein (HDL); low-density lipoprotein (LDL); intermediate-density lipoprotein (IDL); very-low-density lipoprotein (VLDL)) in humans. However, AEX-HPLC has not been applied in veterinary practice. We had three objectives: (i) the validation of AEX-HPLC methods including the correlation of serum cholesterol concentration in lipoprotein fraction measured by AEX-HPLC and gel permeation-HPLC (GP-HPLC) in healthy dogs and those with hypercholesterolemia was investigated; (ii) the reference intervals of lipoprotein fractions measured by AEX-HPLC from healthy dogs (n=40) was established; (iii) lipoprotein fractions from the serum of healthy dogs (n=12) and dogs with hypercholesterolemia (n=23) were compared. Analytic reproducibility and precision of AEX-HPLC were acceptable. Positive correlation between serum concentrations of total cholesterol (Total-Chol), HDL cholesterol (HDL-Chol), LDL cholesterol (LDL-Chol)+IDL cholesterol (IDL-Chol), and VLDL cholesterol (VLDL-Chol) was noted for AEX-HPLC and GP-HPLC in healthy dogs and dogs with hypercholesterolemia. Reference intervals measured by AEX-HPLC for serum concentrations of Total-Chol, HDL-Chol, and LDL-Chol were determined to be 2.97-9.32, 2.79-6.57, 0.16-3.28mmol/L (2.5-97.5% interval), respectively. Furthermore, there was significant difference in lipoprotein profiles between healthy and dogs with hypercholesterolemia. These results suggest that AEX-HPLC can be used to evaluate lipoprotein profiles in dogs and could be a new useful indicator of hyperlipidemia in dogs.

Cholesterol Levels of Six Fractionated Serum Lipoproteins and its Relevance to Coronary Heart Disease Risk Scores.

AIM: Evaluation of serum lipoprotein profiles including triglyceride (TG)-rich lipoprotein, that is, intermediate-density lipoprotein (IDL), very low-density lipoprotein (VLDL), and chylomicron (CM) remnant is important to manage coronary heart disease (CHD) risk. The purpose of this study was to investigate CHD or cardiovascular disease (CVD) risk scores with cholesterol levels of six fractionated lipoprotein classes {high-density lipoprotein [HDL], low-density lipoprotein [LDL], IDL, VLDL, CM including CM remnant, and lipoprotein (a) [Lp (a)]} in Japanese healthy men. METHODS: The present study enrolled 161 healthy men without any medications. Lipoprotein profiles (fractionated lipoprotein cholesterol levels) were measured by anion-exchange high-performance liquid chromatography (AEX-HPLC) method and were compared with age, estimated glomerular filtration rate (eGFR), and three risk scores, that is, NIPPON DATA, Hisayama risk predicting model, and Suita score. RESULTS: Levels of LDL-cholesterol (C), VLDL-C, and CM-C significantly differed with age, while values of HDL-C, IDL-C, and Lp(a)-C were not different. The eGFR inversely correlated with LDL-C, IDL-C, VLDL-C, and CM-C. In a stepwise multiple logistic regression analysis, VLDL-C only correlated independently with eGFR. Three risk scores significantly correlated with CM-C. CONCLUSIONS: These results suggested that VLDL-C concentration contributes to an increased risk at early stages of renal dysfunction, and CM-C may serve as a marker for estimating CHD risk in Japanese healthy men.

Plasma concentration of serotonin is a novel biomarker for coronary microvascular dysfunction in patients with suspected angina and unobstructive coronary arteries.

AIMS: Although the importance of coronary microvascular dysfunction (CMD) has been emerging, reliable biomarkers for CMD remain to be developed. We examined the potential usefulness of plasma concentration of serotonin to diagnose CMD in patients with suspected angina and unobstructive coronary arteries. METHODS AND RESULTS: We enrolled 198 consecutive patients (M/F 116/82, 60.2 ± 13.3 years old) who underwent acetylcholine provocation test and measured plasma serotonin concentration. Coronary microvascular dysfunction was defined as myocardial lactate production without or prior to the occurrence of epicardial coronary spasm during acetylcholine provocation test. Although no statistical difference in plasma concentration of serotonin [median (inter-quartile range) nmol/L] was noted between the vasospastic angina (VSA) and non-VSA groups [6.8 (3.8, 10.9) vs. 5.1 (3.7, 8.4), P = 0.135], it was significantly higher in patients with CMD compared with those without it [7.7 (4.5, 14.2) vs. 5.6 (3.7, 9.3), P = 0.008]. Among the four groups classified according to the presence or absence of VSA and CMD, serotonin concentration was highest in the VSA with CMD group. Importantly, there was a positive correlation between plasma serotonin concentration and baseline thrombolysis in myocardial infarction frame count (P = 0.001), a marker of coronary vascular resistance. The classification and regression trees analysis showed that plasma serotonin concentration of 9.55 nmol/L was the first discriminator to stratify the risk for the presence of CMD. In multivariable analysis, serotonin concentration greater than the cut-off value had the largest odds ratio in the prediction of CMD [odds ratio (95% confidence interval) 2.63 (1.28-5.49), P = 0.009]. CONCLUSIONS: Plasma concentration of serotonin may be a novel biomarker for CMD in patients with angina and unobstructive coronary arteries.

Effect of dietary modification by calorie restriction on cholesterol levels in lipoprotein(a) and other lipoprotein classes.

Background Dietary habits are associated with obesity which is a risk factor for coronary heart disease. The objective is to estimate the change of lipoprotein(a) and other lipoprotein classes by calorie restriction with obesity index and Framingham risk score. Methods Sixty females (56 ± 9 years) were recruited. Their caloric intakes were reduced during the six-month period, and the calorie from fat was not more than 30%. Lipoprotein profiles were estimated at baseline and after the six-month period of calorie restriction. Cholesterol levels in six lipoprotein classes (HDL, LDL, IDL, VLDL, chylomicron and lipoprotein(a)) were analysed by anion-exchange liquid chromatography. The other tests were analysed by general methods. Additionally, Framingham risk score for predicting 10-year coronary heart disease risk was calculated. Results Body mass index, waist circumference, insulin resistance, Framingham risk score, total cholesterol, LDL-cholesterol and IDL-cholesterol were significantly decreased by the calorie restriction, and the protein and cholesterol levels of lipoprotein(a) were significantly increased. The change of body mass index was significantly correlated with those of TC, VLDL-cholesterol and chylomicron-cholesterol, and that of waist circumference was significantly correlated with that of chylomicron-cholesterol. The change of Framingham risk score was significantly correlated with the change of IDL-C. Conclusion Obesity indexes and Framingham risk score were reduced by the dietary modification. Lipoprotein profile was improved with the reduction of obesity indexes, but lipoprotein(a) was increased. The changes of obesity indexes and Framingham risk score were related with those of triglyceride-rich lipoproteins, e.g. IDL, VLDL and CM.

Serotonin in peripheral blood reflects oxidative stress and plays a crucial role in atherosclerosis: Novel insights toward holistic anti-atherothrombotic strategy.

We enrolled 132 outpatients with cardiovascular risk factors to evaluate the serotonin levels in platelet-poor plasma (PPP) and whole blood (WB). PPP serotonin levels and PPP/WB serotonin ratio were significantly correlated with levels of oxidative stress measured by derivative reactive oxygen metabolites (d-ROM). Twenty-five subjects were revealed to have stable coronary artery disease (CAD), and the levels CRP, d-ROM, and PPP/WB serotonin ratio were significantly higher in subjects with CAD than in those without CAD. Logistic regression analysis performed with the endpoint of having CAD revealed that the PPP/WB serotonin ratio was independently associated with CAD (odds ratio 3.37, 95% confidence interval 1.04-10.9, P = 0.04). Receiver operating characteristic (ROC) curve analyses to discriminate subjects with CAD from those without CAD indicated that combining PPP/WB serotonin ratio and d-ROM improved diagnostic utility. Targeting the serotonin-oxidative stress axis as part of a holistic anti-atherothrombotic strategy could be beneficial for patients with atherosclerosis.

Blood levels of serotonin are specifically correlated with plasma lysophosphatidylserine among the glycero-lysophospholipids.

BACKGROUNDS: Glycero-lysophospholipids (glycero-LPLs), which are known to exert potent biological activities, have been demonstrated to be secreted from activated platelets in vitro; however, their association with platelet activation in vivo has not been yet elucidated. In this study, we investigated the correlations between the blood levels of each glycero-LPL and serotonin, a biomarker of platelet activation, in human subjects to elucidate the involvement of platelet activation in glycero-LPLs in vivo. METHODS AND RESULTS: We measured the plasma serotonin levels in 141 consecutive patients undergoing coronary angiography (acute coronary syndrome, n = 38; stable angina pectoris, n = 71; angiographically normal coronary arteries, n = 32) and investigated the correlations between the plasma levels of serotonin and glycero-LPLs. The results revealed the existence of a specific and significant association between the plasma serotonin and plasma lysophosphatidylserine (LysoPS) levels. On the contrary, regular aspirin intake failed to affect the plasma LysoPS levels despite the fact that the plasma lysophosphatidic acid, lysophosphatidylethanolamine, lysophosphatidylglycerol, and lysophosphatidylinositol levels were lower in those who had taken aspirin regularly. CONCLUSION: We found a specific positive correlation between the blood levels of serotonin and LysoPS, a new lipid mediator. Thus, LysoPS might be specifically involved in strong platelet activation, which is associated with the release of serotonin. GENERAL SIGNIFICANCE: Our present results suggest the possible involvement of LysoPS in the pathogenesis of atherosclerotic diseases.

[Differences in Measured Values among Homogenous Assay Reagents of LDL-C in LP-X Positive Serum Samples].

The LDL-C level measures with homogeneous (direct) assays in almost of clinical laboratories. Several reports however showed differences in measured values among the assay reagents. We investigated the differences in LDL-C values among direct assays and Friedewald formula (F-f) in 58 LP-X positive serum samples from jaundice patients by comparing LDL-C values measured by anion-exchange chromatography (AEX-HPLC), largely comparable to ultracentrifugation method. Changes in LDL-C values during the treatment of 8 patients were also investigated. Direct assay reagents from Sekisui Medical (S-r), Denka-Seiken (D-r), Wako Chemical (W-r), and Kyowa Medics (K-r) were used for comparison. F-f, S-r, and D-r correlated with AEX-HPLC with r values < 0.6 while W-r and K-r correlated with AEX-HPLC with r-vales > 0.6. Two samples in which F-f values provided 500 mg/dL plus bias to AEX-HPLC (LDL-C value of 220 mg/dL) demonstrated increased levels of IDL-C before treatment. LDL-C values (S-r and D-r) of the 2 samples were relatively high and near to F-f data while LDL-C values (W-r and K-r) were relatively low and close to AEX-HPLC data. The jaundice treatment decreased LDL-C values (S-r and D-r) and converged to 220 mg/dL, indicating that S-r and D-r might react markedly to IDL. These changes were consistent with decreases in serum free cholesterol and phospholipid in support of LP-X. By contrast, W-r and K-r data showed upward tendency and also converged to 220 mg/dL. These results suggest that LDL-C direct assay reagents would be classified into 2 groups with respect to the reagent reactivity to LP-X.

A rapid anion-exchange chromatography for measurement of cholesterol concentrations in five lipoprotein classes and estimation of lipoprotein profiles in male volunteers without overt diseases.

BACKGROUND: Analysis of lipoprotein profile gives important clinical information for lipid-lowering therapy which prevents atherosclerotic diseases. The lipoprotein classes can be isolated from serum with ultracentrifugation, which inevitably consumes a long time and needs large serum volume. We have established a method with anion-exchange chromatography with 1.0 µL of the injected volume in 5.2 min for assay of one sample. METHODS: One-hundred-forty-one male volunteers without overt diseases were divided three groups (Group 1, non-dyslipidemia with LDL-cholesterol [LDL-C] <120 mg/dL and HDL-cholesterol (HDL-C) ≥40 mg/dL; Group 2, borderline dyslipidemia with 120 ≤ LDL-C < 140 mg/dL and HDL-C ≥40 mg/dL; Group 3, dyslipidemia with LDL-C ≥ 140 mg/dL or HDL-C < 40 mg/dL). Their lipoprotein profiles were evaluated by rapid anion-exchange chromatography, which measured concentrations of HDL-C, LDL-C, IDL-cholesterol, VLDL-cholesterol, and other fraction (chylomicron + lipoprotein [a])-cholesterol (other-C). RESULTS: The within-day and between-day assay coefficients of variation of lipoprotein cholesterol values were 0.33-4.31% and 2.37-9.19%, respectively. The correlation coefficients between values of HDL-C, LDL-C, IDL-C and VLDL-C by the anion-exchange chromatography and those by ultracentrifugal method were 0.97, 0.92, 0.58 and 0.94, respectively. Group 3 had significantly lower HDL-C and higher concentrations of IDL-C, VLDL-C and other-C than did Group 1. Group 2, borderline dyslipidemia, had significantly higher concentrations of IDL-C and VLDL-C than did Group 1. CONCLUSION: The rapid anion-exchange chromatography assay may be sufficiently applied to the assessment of borderline dyslipidemia.

[Fundamental Evaluation and Clinical Usefulness of Lipoprotein Analysis System with Anion-Exchange Chromatography].

We established a method for measurement of the cholesterol concentrations of high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C), intermediate density lipoprotein cholesterol (IDL-C) and very low density lipoprotein cholesterol (VLDL-C) by using high performance liquid chromatography (HPLC) with anion-exchange column. The HPLC method has been covered by insurance in 2013, and HLC-729LPII (LPII) system constructed by this method has come on the market in 2014. We evaluated the fundamental precision data of lipoprotein cholesterol values measured by HLC-729 LPII. The within-day and between-day assay coefficients of variation of lipoprotein cholesterol values were 1.4-10.7 (%CV). The lipoprotein profiles of patients with type 2 diabetes mellitus (T2DM, n = 60) without dialysis therapy were measured by LPII. HDL-C obtained by LPII was highly correlated with that obtained by direct assay. LDL-C obtained by LPII was highly correlated with those obtained by direct assay and calculated by Friedewald's formula. In addition, IDL-C obtained by LPII was negatively correlated with estimated Glomerular Filtration Ratio (eGFR). These results suggest that the new HPLC method can be applied to estimate lipoprotein profile of T2DM patients. Particularly, IDL cholesterol may be useful for the evaluation of impaired lipid metabolism in T2DM patients without dialysis therapy, but it remains to be cleared.