Direct Homogeneous Assay Research Articles

A-214 Evaluate LDL-C using NIH equation 2, Friedewald equation, and direct homogeneous measurement

Abstract Background It is essential to accurately assess Low-density lipoprotein cholesterol (LDL-C) levels, a key marker for cardiovascular disease often used for treatment recommendations. The Friedewald equation has been used for many years to calculate plasma LDL-C levels, but it has limitations. Therefore, before implementing the NIH equation, we compared LDL-C results from NIH Equation 2 to the Friedewald formula and then to Atellica direct homogeneous assay LDL-C results. Methods A retrospective study of 1200 randomly selected patients out of 53,258 data points pulled from the laboratory information (LIS) system between November 2021 and October 2023. The study evaluated triglycerides, LDL-C, total cholesterol, and HDL-C measurements. The LDL-C calculated using NIH equation 2 has been set in the LIS as a non-reportable test component since November 1, 2021. The results obtained from NIH equation 2 and Friedewald equation were compared. LDL-C levels were measured using an Atellica direct homogeneous assay for an additional 75 specimens. Deming regression analysis compared measured and calculated LDL-C using both formulae. Results The LDL-C calculated using the Friedewald equation has a mean of 106.1 mg/dL and SD 40.1 mg/dL, while the NIH equation has a mean of 108.5 mg/dL and SD 39.9 mg/dL. The average difference between both equations is 2.5 mg/dL, and there is a significant difference between both calculations using paired t-tests (p-value<0.0001). We first compared the calculated LDL-C derived from the Friedewald formula to the NIH equation 2. The regression analysis equation for the Friedewald formula was y = -2.7 + 1.001 X (r = 0.99). When the Triglyceride values <150 mg/dL (N=879), the average difference is 1.3, and the regression equation is -0.45+1.01X(r=0.99). However, when the Triglyceride value >= 150 mg/dL (N=321), the average bias is 5.4 mg/dL, and the regression equation is -11.2+1.05X(r=0.99). The Friedewald formula was compared to the measured LDL-C in 75 specimens; the regression analysis for the Friedewald formula was y = -19.6 + 1.06 X (r = 0.91, p-value <0.0001). The 75 measured LDL-C results were also compared with the NIH formula. The regression analysis of NIH LDL-C with measured LDL-C was y = -5.6 + 0.981 x (r = 0.90, p-value = 0.0002). Both formulae showed a linear relationship against measured LDL-C. The NIH formula outperformed the Friedewald formula; bias difference -7.8(-6.8%) compared to the Friedwald equation bias -12.9(-12.0%). If the NIH equation had been used during this period, an additional 1,116 patients would have had LDL-C results. This is because the Friedewald equation should not be used if the triglyceride value is above 400 mg/dL. Out of the additional patients, 513 (46%) had a high LDL (>100 mg/dL). Conclusions The NIH formula performed better than the Friedewald formula, with a less negative bias when both calculations were compared to direct homogenous measurement. The average difference between the two formulas showed the lowest value when triglyceride < 150 mg/dL. Moving to NIH equation 2 will benefit our population as we had 2% of our patients in the past two years have triglyceride >400 mg/dL.

Correlation of extended Martin/Hopkins equation with a direct homogeneous assay in assessing low-density lipoprotein cholesterol in patients with hypertriglyceridemia.

The Friedewald or Martin/Hopkins equation is widely used to estimate low-density lipoprotein cholesterol (LDL-C) at triglyceride (TG) levels <400 mg/dL. In this study, we aimed to validate the recently developed Sampson and extended Martin/Hopkins equations intended for use in patients with TG levels up to 800 mg/dL by comparing them to a direct homogenous assay. In total, 8676 participants with serum TG levels <800 mg/dL were enrolled in this study. LDL-C was directly measured using Abbott homogeneous assay (DLDL) and estimated using the Friedewald (FLDL), Martin/Hopkins (MLDL), extended Martin/Hopkins (EMLDL), and Sampson equations (SLDL). The overall concordance between the DLDL and LDL-C estimates was calculated. The performance of the four equations was also compared using Bland-Altman plots and mean absolute difference (MAD). The EMLDL was more accurate than other LDL-C equations particularly for patients with TG≥400 mg/dL (MAD = 10.43; vs. FLDL: MAD = 21.1; vs. SLDL: MAD 11.62). The overall concordance of FLDL, MLDL, EMLDL, and SLDL with DLDL in TG values ranging from 200 to 799 mg/dL were 52.2, 70.5, 71.6, and 65.7%, respectively (p < 0.001), demonstrating the EMLDL as the most optimal estimation method, particularly for high TG levels (≥200 mg/dL). Both the original and extended Martin/Hopkins method are optimal in estimating LDL-C levels in clinical laboratories using the Abbott analyzer in patients with TG levels of 200-399 and 400-799 mg/dL, respectively. Meanwhile, caution is need that considerable underestimation of Friedewald and Sampson equation could lead to undertreatment in hypertriglyceridemia.

Evaluation of equations for calculating LDL-cholesterol in patients with concomitant hypertriglyceridemia and low LDL-cholesterol: is there a preferred equation?

It is critical to reliably estimate Low Density Lipoprotein Cholesterol (LDL-C) in patients with concomitant hypertriglyceridemia and low LDL-C. We retrospectively compared the performances of the Friedewald (LDL-F), Martin-Hopkins (LDL-MH) and Sampson (LDL-SA) equations against a direct homogeneous LDL-C assay (dLDL-C) on observations presenting mild hypertriglyceridemia (triglycerides between 1.69 and 3.9 mmol/L) and low LDL-C (< 2.58 mmol/L). Observations were stratified according to their LDL-C. Agreement of the equations with dLDL-C was assessed using Intraclass Correlation Coefficients (ICC) with an agreement cut-off of 0.9, and analysis of Bland-Altman plots. Independently of the LDL-C stratum evaluated, the three equations failed to meet the 0.9 ICC cut-off, although their agreement with dLDL-C improves as LDL-C increases. Analysis of Bland-Altman plots shows a downwards discordance of LDL-F with dLDL-C, and an upwards discordance of LDL-MH and LDL-SA with direct LDL-C. LDL-MH resulted in the least observations outside the Bland-Altman limits of agreement. While no equation can be deemed satisfactory enough to replace direct assays in patients with low LDL-C and concomitant hypertriglyceridemia, LDL-MH seems to perform better than the other equations in estimating LDL-C in these patients.

Is Machine Learning-derived Low-Density Lipoprotein Cholesterol estimation more reliable than standard closed form equations? Insights from a laboratory database by comparison with a direct homogeneous assay

BackgroundThere is no consensus on the best method to estimate Low Density Lipoprotein-Cholesterol (LDL-C) in routine laboratories. MethodsWe conducted a retrospective study to compare the performances of a Machine Learning (ML) algorithm using the K-Nearest Neighbors (LDL-KNN) method with that of the Friedewald formula (LDL-F), the Martin-Hopkins equation (LDL-NF), the de Cordova equation (LDL-CO) and the Sampson equation (LDL-SA) against direct homogeneous LDL-C assay (LDL-D) in patients who presented to the Laboratories of Hôtel Dieu de France university hospital in Beirut, Lebanon, from September 2017 to July 2020. Agreements between methods were analyzed using Intraclass Correlation Coefficients (ICC) and the Bland-Altman method of agreement. Results31,922 observations from 19,279 subjects were included, with a mean age of 52 ± 18 years and 10,075 (52.3%) females. All methods except LDL-F and LDL-CO exhibited an overall ICC beyond the 0.9 cut-off. LDL-SA, LDL-NF and LDL-KNN were less susceptible to triglyceridemia than LDL-F and LDL-CO, with LDL-KNN resulting in the lesser fraction of points beyond the Bland-Altman limits of agreement. ConclusionAn ML algorithm using LDL-KNN is promising for the estimation of LDL-C as it agrees better with LDL-D than closed form equations, especially in mild and severe hypertriglyceridemia.

Evaluation of twelve formulas for LDL-C estimation in a large, blinded, random Italian population.

Background and aimsLow-density lipoprotein-cholesterol (LDL-C) is the major determinant of cardiovascular disease (CVD) burden. Being the direct assays time consuming, expensive, not fully standardized and not worldwide available, indirect formulas represent the most used laboratory estimation of LDL-C. In this study we analyzed the accuracy of twelve formulas for LDL-C estimation in an Italian population of 114,774 individuals. MethodsAll lipid samples were analyzed using direct homogeneous assay. The population was divided into various subgroups based on triglycerides and directly dosed LDL-C (D-LDL) levels. Twelve formulas (Friedewald, DeLong, Hata, Hattori, Puavillai, Anandaraja, Ahmadi, Chen, Vujovic, de Cordova, Martin, and Sampson) were compared in terms of their mean absolute deviations and the correlation and concordance of their estimated LDL-C with the respective D-LDL values. ResultsLCL-C measured by Friedewald formula and direct assay differed by more than 9 mg/dL. For D-LDL>115 mg/dl, we observed a concordance rate of only 55% between Friedewald and the respective D-LDL values. For TG<250 mg/dl, the proportion of reclassification between the different formulas and D-LDL was 14.1% with Vujovic, 14.4% Sampson, 15.9% DeLong, 16.5% Puavilai, 19.9% Martin, 21.9% Friedewald, 23.5% Chen, 29% Anandaraja, 31.1% Ahmadi, 31.5% Hata, 33.2% Hattori, and 44.4% with De Cordova formula. ConclusionsOur study compared for the first time 12 different LDL-C formulas on a Southern European population of more than 100,000 people. 'Several formulas showed better accuracy compared to Friedewald. Sampson, Martin and Vujovic resulted the most accurate formulas.

Small Dense Low-Density Lipoprotein Cholesterol: A Residual Risk for Rapid Progression of Non-Culprit Coronary Lesion in Patients with Acute Coronary Syndrome.

Aim: This study investigated whether the small dense low-density lipoprotein cholesterol (sd-LDL-c) level is associated with the rapid progression (RP) of non-culprit coronary artery lesions and cardiovascular events (CE) after acute coronary syndrome (ACS). Methods: In 142 consecutive patients with ACS who underwent primary percutaneous coronary intervention for the culprit lesion, the sd-LDL-c level was measured using a direct homogeneous assay on admission for ACS and at the 10-month follow-up coronary angiography. RP was defined as a progression of any pre-existing coronary stenosis and/or stenosis development in the initially normal coronary artery. CEs were defined as cardiac death, myocardial infarction, stroke, or coronary revascularization. Results: Patients were divided into two groups based on the presence ( n =29) or absence ( n =113) of RP after 10 months. The LDL-c and sd-LDL-c levels at baseline were equivalent in both the groups. However, the sd-LDL-c, triglyceride, remnant lipoprotein cholesterol (RL-c), and apoC3 levels at follow-up were significantly higher in the RP group than in the non-RP group. The optimal threshold values of sd-LDL-c, triglyceride, RL-c, and apoC3 for predicting RP according to receiver operating characteristics analysis were 20.9, 113, 5.5, and 9.7 mg/dL, respectively. Only the sd-LDL-c level (≥ 20.9 mg/dL) was significantly associated with incident CEs at 31±17 months (log-rank: 4.123, p =0.043). Conclusions: The sd-LDL-c level on treatment was significantly associated with RP of non-culprit lesions, resulting in CEs in ACS patients. On-treatment sd-LDL-c is a residual risk and aggressive reduction of sd-LDL-c might be needed to prevent CEs.

Comparison of low-density lipoprotein cholesterol level calculated using the modified Martin/Hopkins estimation or the Friedewald formula with direct homogeneous assay measured low-density lipoprotein cholesterol.

IntroductionLow-density lipoprotein cholesterol (LDL-C) represents the primary lipoprotein target for reducing cardiovascular risk (CV). The aim of our study is to compare the direct and the calculated LDL-C levels in the range below 1.8 mmol/l and 2.6 mmol/l depending on triglycerides, and to evaluate the variation in remnant lipoprotein cholesterol.Material and methodsWe investigated 14 906 lipid profiles from fasting blood samples of Hungarian individuals with triglycerides < 4.5 mmol/l. Total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), triglycerides (TG) and direct LDL-C were measured by the enzymatic assay. We calculated LDL-C by Friedewald’s formula (F-LDL-C) and by using the new Martin/Hopkins estimation (MH-LDL-C).ResultsFor F-LDL-C below 1.8 mmol/l, MH-LDL-C was 58% between 1.8 and 2.59 mmol/l when TG was in the range 2.3–4.5 mmol/l. For F-LDL-C below 2.6 mmol/l, the MH-LDL-C concordance was 73% in the same TG range (2.3–4.5 mmol/l. If MH-LDL-C was less than 1.8 mmol/l or between 1.8 and 2.59 mmol/l, the difference between non-HDL-C (TC – HDL-C = AC: atherogenic cholesterol) and (MH)LDL-C was less than 0.8 mmol/l in the TG range below 2.3 mmol/l. The remnant lipoprotein cholesterol values were on average 0.5 mmol/l lower by the Martin/Hopkins estimation compared to the Friedewald’s calculation if the TG was above 2.3 mmol/l.ConclusionsThe Friedewald equation tends to underestimate LDL-C levels in very high and high-risk settings. Our analysis supports the conclusion that in Hungarian patients, LDL-C estimation using the Martin/Hopkins formula, which is validated by the beta-quantification method, yields a more accurate LDL-C value than that calculated by the Friedewald formula.

Evaluation of eight formulas for LDL-C estimation in Iranian subjects with different metabolic health statuses

BackgroundConsidering the crucial role of low-density lipoprotein-cholesterol (LDL-C) concentration in determining cardiovascular risk, the accuracy of LDL-C estimation is essential. To date, various types of formulae have been introduced, albeit their accuracy has not been assessed in varied populations. In this study, the accuracy of eight formulae for LDL-C estimation was evaluated in an Iranian population.MethodsA data set of 2752 individuals was included in the study and all samples were analyzed in term of lipid profiles using direct homogeneous assay. The population was divided into various subgroups based on the triglyceride (TG), high-density lipoprotein- cholesterol (HDL-C), total cholesterol (TC), fasting blood sugar (FBS) and age values and estimated LDL-C values by Friedewald, Chen, de Cordova, Vujovic, Anandaraja, Hattori, Ahmadi, and Puavillai equations were compared to the directly measured LDL-C in each subgroup.ResultsEstimated LDL-C values by Puavillai formulae showed an insignificant difference compared to the directly measured LDL-C in subjects with high level of TG. However, for TG range < 3.38 mmol/L and high levels of HDL-C, the difference between the means of estimated LDL-C by Hattori and de Cordova formulas, and directly measured LDL-C was relatively lower than other equations. In addition, estimated LDL-C by Hattori and de Cordova formulae had insignificant differences as compared to the direct LDL-C at some levels of cholesterol, the normal level of FBS and some age ranges.ConclusionsTherefore, it seems that Hattori and de Cordova formulas can be considered as the best alternatives for LDL-C direct measurement in the Iranian population, especially for healthy subjects.

Usefulness of the Martin Method for Estimation of Low-Density Lipoprotein Cholesterol in Coronary Atherosclerosis

Objective: This study was conducted to validate the Martin method in coronary atherosclerosis in comparison with the Friedewald equation. Subjects and Methods: A total of 299 participants with a coronary artery calcium score (CACS) ≥300 and a serum triglyceride (TG) level <400 mg/dL at Seoul National University Hospital Healthcare System Gangnam Center, Seoul, Korea, were enrolled in this study. Low-density lipoprotein cholesterol (LDL-C) was directly measured with a homogeneous assay (DLDL) and estimated by both the Friedewald equation (FLDL) and Martin method (MLDL). Overall concordances between DLDL and LDL-C estimates were calculated as the percent agreement. The McNemar test was used to compare the rate of reclassification of participants with FLDL and MLDL, and to determine which differed significantly from each other. Results: Overall concordance between DLDL and MLDL was slightly higher than that between DLDL and FLDL (73.2 vs. 70.9%, p < 0.001). The FLDL showed poor performance when the TG level was ≥200 mg/dL, mostly by underestimation, which represented a 64.7% discordance with DLDL. The reclassification rate by MLDL, however, did not exceed 35.3% in all of the TG groups. Conclusions: The Martin method to estimate LDL-C using the strata-specific TG:VLDL ratio showed a 2-fold better concordance with LDL-C measured with a direct homogeneous assay in coronary atherosclerosis compared to the Friedewald equation when the TG level was ≥200 mg/dL. This finding suggests that MLDL could be a better alternative for estimating LDL-C compared to FLDL when the TG level is ≥200 mg/dL in coronary atherosclerosis.

Homogeneous assay for whole blood folate using photon upconversion.

Red blood cell folate is measured for folate deficiency diagnosis, because it reflects the long-term folate level in tissues, whereas serum folate only represents the dietary intake. Direct homogeneous assay from whole blood would be ideal but conventional fluorescence techniques in blood suffer from high background and strong absorption of light at ultraviolet and visible wavelengths. In this study, a new photon upconversion-based homogeneous assay for whole blood folate is introduced based on resonance energy transfer from upconverting nanophosphor donor coated with folate binding protein to a near-infrared fluorescent acceptor dye conjugated to folate analogue. The sensitized acceptor emission is measured at 740 nm upon 980 nm excitation. Thus, optically transparent wavelengths are utilized for both donor excitation and sensitized acceptor emission to minimize the sample absorption, and anti-Stokes detection completely eliminates the Stokes-shifted autofluorescence. The IC50 value of the assay was 6.0 nM and the limit of detection (LOD) was 1 nM. The measurable concentration range was 2 orders of magnitude between 1.0-100 nM, corresponding to 40-4000 nM folate in the whole blood sample. Recoveries of added folic acid were 112%-114%. A good correlation was found when compared to a competitive heterogeneous assay based on the DELFIA-technology. The introduced assay provides a simple and fast method for whole blood folate measurement.

Status of Dyslipidemia Treatment in Japanese Adults: An Analysis of the 2009 Japan Society of Ningen Dock Database

The Japan Atherosclerosis Society (JAS) has recommended serum lipid management goals (SLMGs) based on the coronary heart disease (CHD) risk classification included in its 2007 guidelines for the diagnosis and prevention of atherosclerotic cardiovascular disease in the Japanese population (JAS GL 2007). The Japan Society of Ningen Dock created a database of subjects receiving annual health examinations at 21 institutes nationwide. Using this database, we evaluated the efficacy of current treatment for patients with dyslipidemia by identifying risk factors for CHD development, based on the JAS recommendations. This multicenter, retrospective study was conducted using data obtained from 21 institutions across Japan. 17,991 adults taking dyslipidemia medications were enrolled. The JAS GL 2007 was used for evaluation. Since the guideline indicated separate goals (secondary prevention for subjects with a prior history of CHD and primary prevention for those with other CHD risk factors), we evaluated the percentages of goals met. The serum low-density lipoprotein cholesterol (LDL-C) levels were calculated using the Friedewald formula. The LDL-C level was measured using a direct homogeneous assay if the triglycerides (TG) level was 400 mg/dL or higher. The achievement rates of the SLMGs were as follows: LDL-C, 72.3%; high-density lipoprotein cholesterol (HDL-C), 94.6%; and TG, 69.7%. Our results regarding Japanese patients receiving dyslipidemia treatment for CHD prevention identified insufficient reductions in the levels of LDL-C and TG in those at high risk for CHD and suggest the need for more aggressive lipid-lowering therapy.

Status of Lipid Management Using Lifestyle Modification in Japanese Adults: An Analysis of the 2009 Japan Society of Ningen Dock Database

The Japan Atherosclerosis Society (JAS) has recommended serum lipid management goals (SLMGs) based on the coronary heart disease (CHD) risk classification included in its 2007 guidelines for the diagnosis and prevention of atherosclerotic cardiovascular disease in the Japanese population (JAS GL 2007). The Japan Society of Ningen Dock created a database of subjects receiving annual health examinations. Using this database, we evaluated the lifestyles of patients with dyslipidemia by identifying risk factors for CHD development based on the JAS recommendations. A total of 223,407 adults (men: 138,435; women: 84,972) aged between 20 and 79 years were enrolled in the analysis. Those who were already being treated for dyslipidemia and had a history of CHD were excluded. CHD risk factors in the JAS GL 2007, such as an advanced age, hypertension, diabetes mellitus, smoking habits, a family history of coronary artery disease, and low high-density lipoprotein cholesterol levels, were used for the evaluation. The subjects were categorized into three groups (Categories I, II and III) according to the number of risk factors other than the low-density lipoprotein cholesterol (LDL-C) level. We evaluated the percentage of goals met during primary prevention in each group. The serum LDL-C levels were calculated using the Friedewald formula. The LDL-C levels were measured using a direct homogeneous assay if the triglyceride level was ≥400 mg/dL. Overall, 72.9% of the subjects achieved their SLMGs. Most subjects (>90%) with no CHD risk factors other than the LDL-C level in Category I achieved their SLMGs, while less than half of the subjects in Category III achieved their goal. Smoking cessation and medication administration should be considered in patients in Categories II and III.

Agreement Between Fasting and Postprandial LDL Cholesterol Measured with 3 Methods in Patients with Type 2 Diabetes Mellitus

LDL cholesterol (LDL-C) is a modifiable cardiovascular disease risk factor. We used 3 LDL-C methods to study the agreement between fasting and postprandial LDL-C in type 2 diabetes (T2DM) patients. We served 74 T2DM patients a standardized meal and sampled blood at fasting and 1.5, 3.0, 4.5, and 6.0 h postprandially. We measured LDL-C by use of modified β quantification (MBQ), the Friedewald equation (FE), and a direct homogeneous assay (DA). We evaluated agreement using 95% limits of agreement (LOA) within ±0.20 mmol/L (±7.7 mg/dL). LDL-C concentrations at all postprandial times disagreed with those at fasting for all methods. In 66 patients who had complete measurements with all LDL-C methods, maximum mean differences (95% LOA) in postprandial vs fasting LDL-C were -0.16 mmol/L (-0.51; 0.19) [-6.2 mg/dL (-19.7; 7.3)] with MBQ at 3 h; -0.36 mmol/L (-0.89; 0.17) [-13.9 mg/dL (-34; 6.6)] with FE at 4.5 h; and -0.24 mmol/L (-0.62; 0.05) [-9.3 mg/dL (-24; 1.9)] with DA at 6.0 h. In postprandial samples, FE misclassified 38% of patients (two-thirds of statin users) into lower Adult Treatment Panel III (ATP III) risk categories. Greater disagreement between fasting and postprandial LDL-C was observed in individuals with postprandial triglyceride concentrations >2.08 mmol/L (>184 mg/dL) and in women (interactions: P ≤ 0.038). Differences up to 0.89 mmol/L (34 mg/dL) between fasting and postprandial LDL-C concentrations, with postprandial LDL-C concentrations usually being lower, were found in T2DM by 3 different LDL-C methods. Such differences are potentially relevant clinically and suggest that, irrespective of measurement method, postprandial LDL-C concentrations should not be used to assess cardiovascular disease risk.

Two-photon excited fluorescent chemosensor for homogeneous determination of copper(ii) in aqueous media and complicated biological matrix

In the present work, a two-photon excited fluorescent chemosensor for Cu(2+) was prepared. The probe was constructed on the basis of internal charge transfer (ICT) principle with macrocyclic dioxotetraamine as the Cu(2+) receptor. The good water-solubility of the molecule enabled recognition and assay of Cu(2+) ions in biological media. The photophysical properties of the chemosensor were investigated in detail, exhibiting favorable fluorescence quantum yield and moderate two-photon absorption cross-section. The studies on binding thermodynamics demonstrated the formation of 1 : 1 complex between the chemosensor and Cu(2+) and an association constant of ca. 1.04 × 10(5) M(-1). Due to the rational design of the molecular structure, the sensor was highly specific to Cu(2+), which ensured high selectivity in Cu(2+) determination. Upon Cu(2+) binding, the intramolecular charge-transfer extent within the chromophore was weakened resulting in a remarkable quenching of fluorescence, based on which quantitative determination of Cu(2+) was performed. Good linearity was obtained between the fluorescence quenching value and Cu(2+) concentration ranging from 0.04 to 2.0 μM in aqueous solution. Benefiting from the merits of two-photon excitation, the chemosensor was free of interference from background luminescence in serum. A homogeneous quantitative determination of Cu(2+) was achieved in the serum medium with a linear range of 0.04 to 2.0 μM. Considering the structural flexibility of the sensor, this work also opens up the possibility to construct other two-photon excited chemosensors for direct homogeneous assay of various molecules/ions in complicated biological sample matrices.

Comparison of low-density lipoprotein cholesterol concentrations measured by a direct homogeneous assay and by the Friedewald formula in a large community population

Background We compare the direct homogeneous low-density lipoprotein cholesterol (LDL-C) assay with the Friedewald formula (FF) for determination of LDL-C in a large community-dwelling population. Methods A total of 21,194 apparently healthy subjects aged 40 to 79 years with triglyceride (TG) concentrations < 4.52 mmol/l were enrolled. LDL-C were directly measured by the enzymatic homogeneous assay (LDL-C (D)) and also estimated by the FF (LDL-C (F)). Paired t-test, Pearson's correlation coefficient and linear regression analysis were performed and the concordances of the National Cholesterol Education Program (NCEP) risk category were estimated. Results Both in fasting (n = 3270) and nonfasting samples (n = 17,924), LDL-C (D) highly correlated with LDL-C (F): r = 0.971 and 0.955, respectively. Concordant results for NCEP categories were 84.8% for fasting samples and 80.1% for nonfasting samples. However, the bias between the 2 measurements increased in samples with TG concentrations > 1.69 mmol/l, especially in nonfasting samples. Conclusions The results showing less variability of the direct LDL-C assay than that of the FF in nonfasting samples suggest that epidemiological studies can use LDL-C measured by the direct assay both in fasting and nonfasting samples.

Otoimmün Hepatitli Bir Olguda Yanlış-Negatif Yüksek Yoğunluklu Lipoprotein-Kolesterol

False-negative high density lipoprotein-cholesterol (HDL-C) detection is a remarkable condition in clinical laboratory practice. We report an 18-year-old female who admitted to transplantation division, with cirrhotic process of autoimmune hepatitis in which polyclonal hypergammaglobulinemia negatively interfered with HDL-C. The HDL-C and immunoglobulin G (IgG) levels were measured before and after liver transplantation. The patient had high IgG with polyclonal hypergammaglobulinemia and an undetectable HDL-C before transplantation. Falsely undetectable HDL-C levels were ruled out by two easy methods: sample dilution and back calculation based on Friedewald formula. This false negative value of HDL-C was also confirmed with lipoprotein electrophoresis. One must be careful in hyperimmunglobulinemic patients when using direct homogeneous HDL-C assay.

A novel direct homogeneous assay for ATP citrate lyase

ATP citrate lyase (ACL) is a cytosolic enzyme that catalyzes the synthesis of acetyl-CoA and oxaloacetate using citrate, CoA, and ATP as substrates and Mg(2+) as a necessary cofactor. The ACL-dependent synthesis of acetyl-CoA is thought to be an essential step for the de novo synthesis of fatty acids and cholesterol. For this reason, inhibition of ACL has been pursued as a strategy to treat dyslipidemia and obesity. Traditionally, ACL enzyme activity is measured indirectly by coupling to enzymes such as malate dehydrogenase or chloramphenicol acetyl transferase. In this report, however, we describe a novel procedure to directly measure ACL enzyme activity. We first identified a convenient method to specifically detect [(14)C]acetyl-CoA without detecting [(14)C]citrate by MicroScint-O. Using this detection system, we devised a simple, direct, and homogeneous ACL assay in 384-well plate format that is suitable for high-throughput screening. The current assay consists of 1) incubation of ACL enzyme with [(14)C]citrate and other substrates/cofactors CoA, ATP, and Mg(2+), 2) EDTA quench, 3) addition of MicroScint-O, the agent that specifically detects product [(14)C]acetyl-CoA, and 4) detection of signal by TopCount. This unique ACL assay may provide more efficient identification of new ACL inhibitors and allow detailed mechanistic characterization of ACL/inhibitor interactions.

Measured versus calculated LDL-cholesterol in subjects with type 2 diabetes.

Objective:There is a strong positive association between increased low-density lipoprotein cholesterol (LDL-C) and coronary heart disease (CHD). The accuracy of LDL-C estimation is essential and critically important. The aim of present study was to compare calculated LDL-C with direct homogeneous assay in patients with type 2 diabetes.Methods:This observational study was carried out at Baqai Institute of Diabetology and Endocrinology (BIDE) from January 2011 to December 2013. A total of 9620 patients with type 2 diabetes were included in the study. Fasting blood glucose, total Cholesterol, triglyceride, HDL cholesterol and LDL cholesterol were obtained using standard methods. Calculated LDL-C was obtained by Friedewald formula.Results:Mean difference of measured and calculated LDL-C was found to be -0.25, 6.63 and 46.55 mg/dl at triglyceride levels < 150 mg/dl, 150 - 400 mg/dl and ≥ 400 mg/dl, respectively. The result shows that the difference between measured and calculated LDL-C increases as the triglyceride level increases.Conclusions:The findings of our study suggested that calculated LDL-C was lower, as compared to measured LDL-C, which may cause misclassifications that may have an impact on therapeutic decisions in patients with diabetes. Calculated LDL-C may depend on triglyceride levels so LDL-C should be measured by direct assay in routine clinical laboratories.