Comparison of the modified polyacrylamide gradient gel electrophoresis and high-performance liquid chromatography methods in determining LDL size

Predictors of change in low-density lipoprotein size during lipid-lowering treatment in type 2 diabetes

Long-term efficacy of bezafibrate in reduction of small, dense low-density lipoprotein by hypotriglyceridemic action

Low density lipoprotein (LDL) is the main carrier of plasma cholesterol and a major component of atherosclerotic plaque [1]. Lowering LDL cholesterol reduces coronary events and mortality from coronary artery disease (CAD) [2–4], however, the relation between LDL cholesterol concentration and (CAD) is complex. Many patients with CAD have plasma LDL cholesterol concentrations in the normal rangefor the general population [5]. Thus, it could be that coronary risk goes beyond LDL cholesterol concentration to the characteristics of the LDL particles themselves. The purpose of this communication is to address the issue of whether LDL particle size and density influences its atherogenecity and how this might be modified by drug therapy.

Objective To make comparative analysis of the glycated hemoglobin A1c(HbA1c) values obtained by three HbA1c test methods in patients with variant hemoglobins. Methods Total of 50 blood samples of patients with different types of variant hemoglobin were collected from January 2012 to December 2012; 25 of them (14 males and 11 females) carried hemoglobin D, Q, G, J and E with a mean age of (24±3) years; the other 25 cases (11 males and 14 females) were blood samples with hemoglobin F from newborn infants. Meanwhile, 50 blood samples(25 males and 25 females) from people without variant hemoglobins were also collected as control(mean age (25±5) years). Three methods were used to test HbA1c, which were affinity high performance liquid chromatography (HPLC) method (Ultra 2 of American Primus), ion exchange HPLC method (VariantⅡ of American Bio-Rad and G8 of Japanese Tosoh) and immunization method (DCA Vantage of German Siemens). The statistic analysis were done with variance analysis and correlation analysis. Results The HbA1c of the group with normal HbA1c structure was 5.7%±1.1%, 5.7%±1.2%, 5.7%±1.2% and 5.7%±1.1% respectively when it was tested by affinity HPLC method (Ultra2), ion exchange HPLC method (G8), ion exchange HPLC (Variant Ⅱ) and immunization method (DCA Vantage), and there was no significant difference among the groups (F=0.023, P>0.05). In the 25 samples with hemoglobin F, HbA1c could not be detected by ion exchange HPLC or immunoassay method. The fasting blood glucose level correlated with HbA1c level tested by Ultra 2 method (r=0.647, P<0.05), but it didn't correlate with HbA1c level when tested with VariantⅡ and G8 as well as DCA Vantage method. The HbA1c result of affinity HPLC method was free from the disturbance of Hb D, Q, G, J and E, and had a obvious correlation with blood glucose (r=0.823, P<0.05). The HbA1c result of ion exchange HPLC method was disturbed by hemoglobin D, Q, G, J and E in varying degrees. The HbA1c measured by immunization method was associated with blood glucose (r=0.611, P<0.05). Conclusion The HbA1c value obtained by affinity HPLC method can accurately reflect the mean blood glucose level. Variant hemoglobins disturb the HbA1c result tested by ion exchange HPLC method, and the immunization test result is only disturbed by hemoglobin F. Key words: Glycated hemoglobin A1c; Variant hemoglobins; High performance liquid chromatography

Long-term effects of fish oil on lipoprotein subfractions and low density lipoprotein size in non-insulin-dependent diabetic patients with hypertriglyceridemia

High prevalence of small dense LDL in diabetic nephropathy is not directly associated with kidney damage: a possible role of postprandial lipemia

Aim: Hemoglobin A1c is a valuable parameter for the diagnosis and follow-up of its diabetes mellitus since its biological variation is low, does not require preparation before the test, is not affected by acute stress, and has high preanalytical stability. HbA1c measurement by HPLC has been determined as the reference method by National Glycohemoglobin Standardization Program (NGSP) in USA; after that The International Federation of Clinical Chemistry (IFCC) defined another reference method which could be related with NGSP. In our study, we aim to compare the two NGSP-certified methods of HbA1c, which are high-performance liquid chromatography (HPLC) and turbidimetric inhibition immunoassay (TINIA). Material and Method: HbA1c levels of the patients were measured using two HPLC and one TINIA method in three different hospitals (Lab A, Lab B (Both are HPLC), and Lab C (TINIA), in which Lab A was served as a reference). Because of the lower precision values of LabB, we firstly conducted a method comparison study of 40 volunteers (Group 1). After that, corrective and preventive activities carried out and the precision values in LabB reached the desired range. Following this, another method comparison study consisting of 60 new volunteers (Group 2) was conducted. The statistical flow of this study complied with Clinical Laboratory Standards Institute (CLSI) EP09-A3; Precision studies, Blant-Altman and Passing Bablok regression analysis were performed. Results: The percentage of the mean difference between the two HPLC methods (LabA and LabB) was 3.1%. After corrective and preventive actions had been taken, the mean difference between the two HPLC methods decreased to 2.0%. A decrease in systematic bias was found in our study. Two HPLC methods can be used interchangeably in both Group 1 and Group 2. In Group 1; 95% CI of intercept and slope were found as (-1.41 to -0.30) and (1.03 to 1.22), respectively. In Group 2; 95% CI of intercept and slope were found as (-1.33 to -0.31) and (1.01 to 1.17), respectively. HPLC and TINIA methods could not be used interchangeable without affecting patient results and outcome in both Group 1 and Group 2. Conclusion: Our study concluded that TINIA and HPLC methods could not be used interchangeably without affecting patient results and outcome. Because of the methodology that clinical laboratories are used to, clinicians and clinical biochemists should collaborate on managing diabetes mellitus regarding diagnosis, treatment, and follow-up.

To determine if obesity measures are related to measures of low-density lipoprotein (LDL) size and LDL subfractions in a population of Mexican-Americans with high prevalence of obesity. LDL size phenotypes, based on nondenaturing gradient gel electrophoresis and staining for cholesterol (using Sudan black B), were determined for 313 unrelated Mexican-American participants in the San Antonio Family Heart Study. LDL size measures included predominant particle diameter, median diameter (particle diameter, where half the LDL absorbance is on larger and half on smaller LDLs) and cholesterol level in various LDL subfractions. Adiposity traits included two measures of general body fatness (body-mass index (BMI) and fat mass determined with bioimpedance) and three measures of regional fat deposition (waist-to-hip ratio (WHR), waist circumference and subscapular-triceps skinfold ratio (STR)). Gender and diabetes were significantly associated with most LDL size measures. In addition, BMI, WHR, waist circumference and STR were significantly (P<0.05) associated with several LDL size measures. Stepwise regression analysis (including adjustment for age, gender and diabetes status) showed that in every case, the strongest adiposity correlate of LDL size, was WHR, which reflects deposition of visceral fat. If triglyceride (TG) concentration was also included in the models, no fat measure was independently correlated with LDL size, suggesting that elevation of TG, associated with increased adiposity, was more directly correlated with LDL size. Supporting this interpretation, we found that WHR was also the strongest correlate of TG among adiposity measures. Regression analysis of the LDL particle size cholesterol profile expressed in 0.1 nm increments revealed a positive correlation of WHR and LDLs in the interval 25.9-26.3 nm (P < 0.05) and a negative correlation of BMI with LDLs in the interval 27.3-28.1 nm (P < 0.05). These results suggest that different adiposity measures, reflecting different aspects of fat deposition, are related to specific LDL size intervals. We speculate that increased deposition of fat, particularly visceral fat, is associated with increased TG, which in turn is associated with decreases in LDL particle size.

High serum uric acid and low-grade inflammation are associated with smaller LDL and HDL particles

Post-prandial alterations in LDL size and subclasses in patients with growth hormone deficiency

Background: The 99mTc-tetrofosmin is a radiopharmaceutical used in oncology for scintigraphic quantification of the myocardial perfusion. The preparation of this drug is based on a complexation reaction of technetium 99 metastable (99mTc) with tetrofosmin. The reference method for quality control is thin layer chromatography, using TLC SA bands. This method is simple but only separates two types of impurities: free technetium and hydrolyzed technetium associated to hydrophilic impurities like gluconate-99mTc and takes from 30 to 35 minutes. This gluconate impurity gives a poor image quality and difficult interpretation issues. HPLC is a sensitive and specific method, it thus, has an interest in controlling RCP and identifying all impurities. Methods: The reference method is a method by planar chromatography TLC SA tape, size 1 cm x 20 cm. Two marks were scored: 3 cm from the edge to indicate the deposit (10 µL of the preparation) and 15 cm by the end of migration. Mobile phase was acetone: dichloromethane (65:35, v/v). The radioactive bands were quantified by counting the radioactivity using a radiochromatograph miniGITA® (Raytest) equipped with a scintillation probe. The chromatographic system consisted in a Symmetry Shield® column RP18 5μm 100Å (Waters) with a gamma detector Gammaram® (Lablogic). Empower® software (Waters) was used for peak integration. The mobile phase, at a rate flow of 1.0 mL / min, consisted of a mixture of acetonitrile (Waters) and titrisol® buffer (Waters) (40:60, v/v). The sample volumes injected were no more than 10 to 30 µL in order not to exceed 50,000 counts per second due to the risk of radioactive detector saturation. Results: The RCP was measured simultaneously by HPLC and reference method in 30 preparations. For HPLC, mean RCP = 97.21%, α= 2.178% [91.6%-99.63%]. For TLC SA, mean RCP = 97.99%, α= 1.135% [94.31%-99.86%]. The results obtained by both methods were compared using the Wilcoxon t test. The RCP obtained either by TLC SA or HPLC methods are not significantly different (p-value = 0,497, higher than in significance ≤ = 0.05) Conclusions: A new HPLC method was developed for the control of the RCP 99mTc-Tetrofosmin. This method is reliable, rapid, sensitive and easy to use when the equipment is available. It allows us to improve the detection of cardiotoxic side effects due to chemotherapy more quickly than TLC SA method and to prevent toxicity by dose adjustment of anticancer drugs. Although the HPLC method does not differ from TLC (reference method), HPLC provides additional information about the quality of the preparation (percentage of gluconate-99mTc). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2451. doi:1538-7445.AM2012-2451

Smaller low-density lipoprotein (LDL) size has recently been reported as a non-traditional lipid risk factor for coronary artery disease (CAD). Cholesteryl ester transfer protein (CETP) and the C/T hepatic lipase (HL) gene polymorphism may promote LDL size reduction via the CETP-mediated exchange of CE for triglyceride (TG) and subsequent HL-mediated TG hydrolysis in LDL. However, little is known about LDL size status and its relationship with CAD prevalence in haemodialysis (HD) patients who are at high risk for atherosclerosis. CETP levels, HL genotypes and LDL size were determined, and the determinants of LDL size and its association with CAD prevalence in HD patients (n = 236) aged over 30 years were investigated. The HD patients had a similar LDL size to the healthy subjects. In the HD group, high-density lipoprotein cholesterol was an independent positive determinant of LDL size, while log(10) (TG) was an independent negative determinant in the high (≥2.1 µg/mL) but not low (<2.1 µg/mL) CETP group. In the patients with hypertriglyceridemia, the high CETP group had a significantly smaller LDL size than the low CETP group. Among the patients with above-median TG levels, the CC genotype and CETP were independent negative determinants of LDL size. In the whole group and the high CETP group, the patients with CAD had a significantly smaller LDL size than those without CAD. Finally, DM and smaller LDL size were identified as independent risk factors for CAD prevalence. These suggest that a smaller LDL size, which is associated with higher levels of TG and CETP and the HL/CC genotype, may serve as a risk factor for CAD in HD patients.

The aim of this study was to investigate the relationship between endothelial dysfunction and low density lipoprotein (LDL) size and susceptibility to oxidation in nephrotic rats with or without deficiency of vitamin E and selenium. Four groups of male Wistar rats were studied: control (C), vitamin E and selenium deficient control (DefC), nephrotic (NS), and vitamin E and selenium deficient NS (DefNS). Nephrotic syndrome was induced by puromycin aminonucleoside. The molar ratio of vitamin E/LDL-cholesterol was significantly lower in DefNS, DefC rats, and NS vs. C rats. In comparison with control animals, vasodilation and LDL oxidability were significantly lower in nephrotic animals. LDL size was similar in all groups. Abnormal endothelial function in response to acetylcholine and carbachol was observed in NS animals compared to control rats. Relaxation response was inversely associated with an increase in LDL susceptibility to oxidation and with a lower molar ratio of vitamin E/LDL-c. LDL oxidability and LDL-c were the only variables independently associated with vasodilation. These results suggest that endothelial dysfunction of NS may be a consequence of the increased LDL susceptibility to oxidation, secondary to antioxidant deficiency.

Purpose: To develop a gradient high performance liquid chromatography (HPLC) method for the simultaneous determination of phenylephrine (PHE) and ibuprofen (IBU) in solid dosage form.Methods: HPLC determination was carried out on an Agilent XDB C-18 column (4.6 x 150mm, 5 μ particle size) with a gradient mobile phase composed of 0.1 % orthophosphoric acid and acetonitrile at a ratio of: 0.01/95/5, 2.5/95/5, 6/10/90, 8/10/90, 8.1/95/5 and 13/95/5 for time (min)/0.1 % orthophosphoric acid (%)/acetonitrile (%) at a flow rate of 1.0 mL/min. Column temperature was maintained at 30 °C and detection was carried out using a photodiode array (PDA) detector at 210 nm. Validation parameters, including system suitability, linearity, precision, accuracy, specificity, limit of detection (LOD), limit of quantification (LOQ), stability of sample and standard stock solutions as well as robustness were obtained as per International Conference on Harmonization (ICH) guidelines. The proposed method was applied to the determination of phenylephrine and ibuprofen in commercial tablets.Results: Retention time for phenylephrine and ibuprofen were 2.7 and 8.4 min, respectively while % recovery was 99.42 and 99.80 %, respectively. The relative standard deviation (% RSD) for assay of the tablets was &lt; 2 %.Conclusion: The method is fast, accurate, precise and sensitive, and hence it can be employed for routine quality control of tablets containing both drugs in quality control (QC) laboratories and pharmaceutical industry.Keywords: Phenylephrine, Ibuprofen, Simultaneous determination, Validation, Gradient HPLC.

In this research caffeine content in coffee sample from Abe Dongoro, Sasiga, Gida Ayana and Sibu Sire of Wollega administrative zone of Ethiopia were determined using High Performance Liquid Chromatography (HPLC) and UV-Vis Spectrophotometry methods. Caffeine in aqueous extract of coffee samples was extracted with dichloromethane prior to analysis by UV-Vis spectrophotometry method and dichloromethane was evaporated from the extract and the extract was dissolved in water (HPLC grade) to determine caffeine contents in coffee samples using HPLC method. The linearity of the HPLC and UV-Vis spectrophotometry methods were R² = 0.9999 and R² = 0.9997 respectively. HPLC and UV-Vis spectrophotometry methods were found to be accurate with recoveries of 97.5% and 117.4%, respectively. Limits of detection (LOD) obtained were 0.148 mg/L for HPLC method and 0.284 mg/L for UV-Vis spectrophotometric method. The caffeine contents in coffee samples obtained using UV-Vis spectrophotometry method was 3.42, 2.638, 2.207 and 2.986 mg/L for Abe Dongoro, Gida Ayana, Sasiga and Sibu Sire coffee samples respectively. Similarly, using HPLC method the caffeine contents in coffee samples obtained was 1.871, 1.601, 1.307, 1.83 mg/L for Abe Dongoro, Gida Ayana, Sasiga and Sibu Sire coffee samples. There is a significant difference between the caffeine contents in coffee samples obtained by the two methods.