Detectability of and interference by major and minor hemoglobin variants using a new-generation ion-exchange HPLC system with two switchable analysis modes.

Objectives: High-performance liquid chromatography (HPLC) is commonly used to measure hemoglobin A1c (HbA1c) levels and detect hemoglobin variants (Hb-Vars). HLC-723GR01 (GR01) is a new-generation automated ion-exchange HPLC system with two switchable analysis modes, namely short (30 s/test) and long modes (50 s/test). We evaluated the general performance of both analysis modes of GR01 for quantifying HbA1c and detecting Hb-Vars. Design and methods: We evaluated the instrument's precision based on CLSI protocol EP-05-A3. A comparison of the two analysis modes of GR01 against the standard mode of HLC-723G11 was performed on 100 whole blood samples. The GR01 long mode was compared with affinity HPLC (AF-HPLC) for detecting common Hb-Vars (HbE, HbD, HbS, and HbC, >20 samples). To examine the detection capability for minor Hb-Vars, we analyzed 26 Hb-Vars using multiple analyzers, including both analysis modes of GR01. Results: Both modes of GR01 had within-laboratory coefficients of variation of ≤1.0 % from four samples with HbA1c concentrations of 32-86 mmol/mol. Good correlation was observed between GR01 and HLC-723G11. The results for HbA1c detection in the presence of the major variants revealed a strong correlation between the long mode of GR01 and AF-HPLC (r = 0.986-0.998), and the difference biases ranged 0.1-1.9 mmol/mol. In the long mode, only one variant had a difference bias exceeding 14 % [10 % (%NGSP)]. Conclusion: The two analysis modes of GR01 were fast and had high accuracy and reproducibility, indicating their utility for routine clinical use in measuring HbA1c samples with Hb-Vars.

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.

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.

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.

[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.

Automated measurement method for the determination of vitamin E in plasma lipoprotein classes.

In a subendothelial space of atherosclerotic arteries, apolipoprotein B-containing lipoproteins are accumulated and oxidized, and the oxidized lipoproteins promote macrophage foam cell formation. Therefore, the analysis of vitamin E, a major antioxidant in lipoproteins, is important for understanding atherosclerotic pathogenesis. A new method for the automated measurement of vitamin-E (γ- and α-tocopherols) in plasma HDL, LDL, and VLDL was established by using anion-exchange-chromatography for separation of lipoproteins, reverse-phase-chromatography for separation of γ- and α-tocopherols in each of lipoproteins, and fluorescent detection. The within-day assay and between-day assay coefficients of variation for lipoprotein tocopherol levels were 4.73-12.84% and 7.00-14.73%, respectively. The γ- and α-tocopherol/cholesterol ratios of VLDL were higher in healthy plasma than in plasma of untreated patients with dyslipidemia, but the ratios of LDL and HDL were not different. This new estimated method can provide the reliable data of lipoprotein vitamin-E and would be useful for the clinical settings.

Relevance of intermediate-density lipoprotein cholesterol to Framingham risk score of coronary heart disease in middle-aged men with increased non-HDL cholesterol.

BACKGROUND: Cholesterol levels of non-high-density lipoprotein (non-HDL), which contains low-density lipoprotein (LDL), intermediate-density lipoprotein (IDL), very low-density lipoprotein (VLDL) and chylomicron (CM) remnant, have been proven to perform a significant predictor of coronary heart disease (CHD) better than LDL-cholesterol regardless of triglyceride (TG) levels. The present study investigated the relevance of TG-rich lipoproteins (IDL, VLDL, CM) to Framingham risk score (FRS) predictive of 10-year CHD risk. METHODS: Lipoprotein profiles (cholesterol levels of HDL, LDL, IDL, VLDL, CM) in Japanese men (n = 487) who underwent medical check-up were determined by using our developed anion-exchange high performance liquid chromatography (AEX-HPLC). Total-cholesterol (TC), TG, fasting blood sugar (FBS) and hemoglobin (Hb) A1c were measured by routine methods. The lipoprotein profiles, non-HDL-cholesterol, TC, and TG were examined on these associations with FRS. RESULTS: The lipid levels except for CM-cholesterol were significantly different between two groups (low FRS, < 10%; high FRS, ≥10%) (P < 0.0001), and body mass index (BMI), TC, TG, IDL-, and VLDL-cholesterol were significantly and positively correlated with FRS. Among them, the significant association of non-HDL-cholesterol to FRS was noted (r = 0.411, P < 0.0001). Multiple stepwise regression analysis shows that, in addition to non-HDL-cholesterol, IDL-cholesterol in TG-rich lipoproteins was significantly correlated with FRS in independently of BMI. These correlation results were similarly found even when the part of the study subjects (n = 348) without the drug therapy for hyperlipidemia, diabetes, and hypertension was investigated. CONCLUSIONS: These results suggest that IDL-cholesterol may serve as a useful marker for CHD risk in Japanese men with increased non-HDL-cholesterol.

Mapping a disordered portion of the Brz2001-binding site on a plant monooxygenase, DWARF4, using a quartz-crystal microbalance biosensor-based T7 phage display.

In small-molecule/protein interaction studies, technical difficulties such as low solubility of small molecules or low abundance of protein samples often restrict the progress of research. Here, we describe a quartz-crystal microbalance (QCM) biosensor-based T7 phage display in combination use with a receptor-ligand contacts (RELIC) bioinformatics server for application in a plant Brz2001/DWARF4 system. Brz2001 is a brassinosteroid biosynthesis inhibitor in the less-soluble triazole series of compounds that targets DWARF4, a cytochrome P450 (Cyp450) monooxygenase containing heme and iron. Using a Brz2001 derivative that has higher solubility in 70% EtOH and forms a self-assembled monolayer on gold electrode, we selected 34 Brz2001-recognizing peptides from a 15-mer T7 phage-displayed random peptide library using a total of four sets of one-cycle biopanning. The RELIC/MOTIF program revealed continuous and discontinuous short motifs conserved within the 34 Brz2001-selected 15-mer peptide sequences, indicating the increase of information content for Brz2001 recognition. Furthermore, an analysis of similarity between the 34 peptides and the amino-acid sequence of DWARF4 using the RELIC/MATCH program generated a similarity plot and a cluster diagram of the amino-acid sequence. Both of these data highlighted an internally located disordered portion of a catalytic site on DWARF4, indicating that this portion is essential for Brz2001 recognition. A similar trend was also noted by an analysis using another 26 Brz2001-selected peptides, and not observed using the 27 gold electrode-recognizing control peptides, demonstrating the reproducibility and specificity of this method. Thus, this affinity-based strategy enables high-throughput detection of the small-molecule-recognizing portion on the target protein, which overcomes technical difficulties such as sample solubility or preparation that occur when conventional methods are used.

The antitumor agent doxorubicin binds to Fanconi anemia group F protein.

Doxorubicin, a commonly used cancer chemotherapy agent, elicits several potent biological effects, including synergistic-antitumor activity in combination with cisplatin. However, the mechanism of this synergism remains obscure. Here, we employed an improved T7 phage display screening method to identify Fanconi anemia group F protein (FANCF) as a doxorubicin-binding protein. The FANCF-doxorubicin interaction was confirmed by pull-down assay and SPR analysis. FANCF is a component of the Fanconi anemia complex, which monoubiquitinates D2 protein of Fanconi anemia group as a cellular response against DNA cross-linkers such as cisplatin. We observed that the monoubiquitination was inhibited by doxorubicin treatment.

Estimation of lipoprotein profile in patients with type II diabetes and its relevance to remnant lipoprotein cholesterol levels.

BACKGROUND: Remnant lipoprotein (RLP), associated with atherosclerosis progression, is often elevated in diabetes mellitus. The RLP level is estimated by immune-separation method and agarose-gel electrophoresis (AGE). METHODS: The patients were grouped into three groups according to tertile of RLP-cholesterol (RLP-C) levels. The lipoprotein profiles of type II diabetic patients (T2DM) (n=194) were measured by an anion-exchange liquid chromatography (AEX-HPLC) and an AGE with lipid-staining or cholesterol-staining. RESULTS: IDL- and VLDL-cholesterol by the AEX-HPLC, and VLDL-levels by the AGE with lipid-staining and with cholesterol-staining were significantly different in the three groups. In all the subjects, IDL-cholesterol (r=0.531) and VLDL-cholesterol (r=0.880) by the AEX-HPLC method were strongly correlated with RLP-C, but only VLDL levels were correlated with RLP-C in AGE, respectively. CONCLUSION: These results suggest that the AEX-HPLC, which can provide cholesterol levels of not only VLDL but also IDL, is helpful for estimation of lipid profiles in T2DM with high RLP-C.

Camptothecin (CPT) directly binds to human heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) and inhibits the hnRNP A1/topoisomerase I interaction.

Camptothecin (CPT) is an anti-tumor natural product that forms a ternary complex with topoisomerase I (top I) and DNA (CPT-top I-DNA). In this study, we identified the direct interaction between CPT and human heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) using the T7 phage display technology. On an avidin-agarose bead pull down assay, hnRNP A1 protein was selectively pulled down in the presence of C20-biotinylated CPT derivative (CPT-20-B) both in vitro and in vivo. The interaction was also confirmed by an analysis on a quartz-crystal microbalance (QCM) device, yielding a K(D) value of 82.7 nM. A surface plasmon resonance (SPR) analysis revealed that CPT inhibits the binding of hnRNP A1 to top I (K(D): 260 nM) in a non-competitive manner. Moreover, an in vivo drug evaluation assay using Drosophila melanogaster showed that the knockout of the hnRNP A1 homolog Hrb87F gene showed high susceptibility against 5-50 µM of CPT as compared to a wild-type strain. Such susceptibility was specific for CPT and not observed after treatment with other cytotoxic drugs. Collectively, our data suggests that CPT directly binds to hnRNP A1 and non-competitively inhibits the hnRNP A1/top I interaction in vivo. The knockout strain loses the hnRNP A1 homolog as a both CPT-binding partner and naïve brakes of top I, which enhances the formation of the CPT-top I-DNA ternary complexes and subsequently sensitizes the growth inhibitory effect of CPT in D. melanogaster.

Novel terpenoids, trichoderonic acids A and B isolated from Trichoderma virens, are selective inhibitors of family X DNA polymerases.

Trichoderonic acids A (1) and B (2), novel terpenoids, and (+)-heptelidic acid (3) isolated from cultures of a fungus, Trichoderma virens, and their structures were identified by spectroscopic analysis. These compounds selectively and competitively inhibited the activities of mammalian DNA polymerases beta, lambda (pols beta, lambda), and terminal deoxynucleotidyl transferase (TdT) in family X of pols, and compound 2 was a stronger inhibitor than compound 1 or 3. On the other hand, compounds 1-3 did not influence the activities of the other families (A-, B-, and the Y-families) of the mammalian pols tested, and showed no effect on the activities of plant pol alpha, fish pol delta, prokaryotic pol, or the other DNA metabolic enzymes tested. Compound 2 suppressed the growth of two human cancer cell lines, cervix carcinoma cells (HeLa) and breast carcinoma cells (MCF-7). The results suggest that these compounds identified inhibition among the families of mammalian pols.

Identification of small molecule binding molecules by affinity purification using a specific ligand immobilized on PEGA resin.

We investigated the application of resins used in solid-phase synthesis for affinity purification. A synthetic ligand for FK506-binding protein 12 (SLF) was immobilized on various resins, and the binding assays between the SLF-immobilized resins and FK506-binding protein 12 (FKBP12) were performed. Of the resins tested in this study, PEGA resin was the most effective for isolating FKBP12. This matrix enabled the isolation of FKBP12 from a cell lysate, and the identification of SLF-binding peptides from a phage cDNA library. We confirmed the interaction between SLF and these peptides using a cuvette type quartz crystal microbalance (QCM) apparatus. Our study suggests that PEGA resin has great potential as a tool not only for the purification and identification of small-molecule binding proteins but also for the selection of peptides that recognize target molecules.