Does Fredrickson Friedewald’s formula and direct assay of LDL concentration in serum give varying results? - A pilot retrospective study in a tertiary care hospital

Introduction

The link between various cardiovascular diseases(CVD) and lipoprotein has been found in various studies, which lead to formulation of the current national guidelines for cardiovascular risk assessment. These guidelines have used LDL-C for assessing the risk of CVD.^{1, 2} For estimating LDL-C, previously the methods used frequently were either Friedewald calculation or β-quantification method. The commonly used Friedewald calculation method uses triglycerides(TG), total cholesterol(TC), high-density lipoprotein (HDL) cholesterol levels for calculating LDL-C level.³ In-spite of being a routinely used method, Friedewald calculation has certain limitations i.e., it cannot be used in conditions of hypertriglyceridemia (>400 mg/dL), type III hyperlipoproteinemia and non-fasting patient’s sample. Moreover, there is reduced precision and accuracy as it a derived equation used to measure LDL-C. The newer methods like direct measurement method, also recommended by National Cholesterol Education Program (NCEP) expert laboratory panel has supplanted the older procedures for LDL-C estimation which depends on the physical separation of the different lipoprotein classes.^4,5 Major drawback of β-quantification method is being labour intensive involving manual pretreatment.⁵ Hence the direct measurement methods overcome this disadvantage as automation has been incorporated into it leading to improved imprecision over the older methods.

A few retrospective and cross-sectional studies have been conducted to explore the analytical equivalence between the older and newer methods. Therefore, we conducted this retrospective study to evaluate the comparison of baseline LDL-C concentrations as determined by a direct assay compared with Friedewald calculation.

Material and Methods:

The retrospective observational study was conducted in the Department of Biochemistry, Lady Hardinge Medical College and associated hospitals, New Delhi for a duration of 12 months from October 2022 to September 2023 with IEC registration no. LHMC/IEC/2023/PG Thesis/18. Serum samples of 216 adult patients (>18years) was collected, after 12-14hrs of overnight fast for analysing the lipid profile levels and processed in our clinical biochemistry lab. Hence, we extracted the data from report entry register of our clinical biochemistry lab. The subjects of this study included individuals whose LDL-C by direct homogeneous method and HDL-C, TC and TG which are variables of the Friedewald LDL-C estimation formula were measured. All the cases with TG >500 mg/dl were excluded from study sample. 216 study participants were placed into three groups based on their TG levels according to NCEP with TG <150mg/dl, 150-199mg/dl and > 199mg/dl as group 1(142 subjects), group 2(36 subjects) and group 3(38 subjects) respectively.

TC, TG, HDL-C and LDL-C levels were estimated by enzymatic methods on Beckman Coulter AU5800 and absorbance was measured spectrophotometrically. Serum HDL-C and LDL-C levels were estimated by direct homogenous method (two reagent assay), serum TC by enzymatic direct cholesterol oxidase peroxidase method and serum TG by enzymatic triglyceride glycerol phosphate oxidase method. For calculated LDL, Friedwald formula was used, i.e., LDL-C = TC-HDL- (TG/5). The other methods of cLDL are Modified Friedewald equation, LDL = TC-(TG/6 =HDL), Anandraja equation: LDL = (0.9TC)-(0.9TG/5)-28, Chen equation: LDL = (TC-HDL) x 0.9- (TG x 0.1), Hattori equation: LDL = (0.94 x TC)- (0.94 x HDL)- (0.19 x TG) and Vujovic equation: LDL = TC-HDL-(TG/6.58).⁶

Statistical analysis:

The data retrieved from the laboratory system was analysed using SPSS v23. Appropriate statistical tests were used, parametric data was expressed as mean and standard deviations. The Pearson test was used to find out correlation between the methods. Receiver operator curve analysis (ROC) was done to assess the diagnostic accuracy of dLDL and cLDL Intraclass correlation coefficient (ICC) was used to compare different methods of LDL estimation. p value <0.05 taken as statistically significant.

Results

The mean serum total cholesterol, triglycerides, dHDL, dLDL, cLDL of study population was 157 ± 41, 144 ± 84, 40 ±13.8, 104 ± 38.8 and 85.5 ± 35.6 mg/dl respectively. The mean values were comparable between male and female study participants. The mean serum total cholesterol, triglycerides, dHDL, dLDL, cLDL of study male and female population was 157 ± 43, 142 ± 79, 41.9 ±12.9, 108 ± 41.9 and 91.6 ± 35, and 156 ± 40.1, 154.9 ± 90, 39.3 ±14.5, 100 ± 35.5 and 82.3 ± 29 mg/dl respectively. When a comparison between dLDL-C and cLDL-C was done, the mean dLDL-C was found to be higher than mean cLDL-C. A strong positive correlation was seen between dLDL-C and cLDL-C as shown in figure 1. ICC was used to compare different methods of LDL estimation with varying levels of LDL among the three groups as shown in table 1. While all the methods of cLDL-C equations showed negative mean difference with dLDL-C, a positive mean difference of 45.7 was seen with Anandraja for group 3. The ICC showed excellent reliability for overall comparison between dLDL-C and cLDL-C by Friedewald equation, however reliability was found to be poor for group 2. Overall, Friedewald equation and Modified Friedewald equation showed near excellent reliability with ICC of 0.88(0.85-0.90) and 0.87(0.83-0.89) respectively, whereas the ICC of Hattori equation lied in poor reliability range in comparison to dLDL.

Discussion:

For formulating the diagnostic criteria for patients of hypercholesterolemia and further treating them the main target is LDL-C according to latest NCEP guidelines.⁷ The data available for assessing the predictive performance of various direct methods in relation to cardiovascular events is limited.⁸ In our study it was found that the mean dLDL-C was higher than mean cLDL-C. In a similar study conducted by Miler WG et al, two direct LDL assays viz., LDL-C(GZ,Genzyme) and LDL-C(RD, Roche Diagnostics) were compared with Friedwald calculation method.⁹ It was observed that both the direct assay methods were better in taking medical decision cut point range in comparison to Friedewald equation. The possible explanation for this observation may be due to dependency of Friedewald equation on other parameters (triglyceride, total cholesterol, and HDL-C). The dLDL-C method is having certain advantages over calculated methods as it is relatively unaffected by fasting status of patients and also the increased triglyceride(>400mg/dl) levels doesn’t affect the result.¹⁰ There are various studies which have found comparative results between direct and calculated methods of LDL-C estimation for the diagnostic and treatment assessment of hyperlipidaemic patients. 9-13

On the contrary there are studies which have questioned the credibility of direct assay methods in clinical decision making.¹⁴ Moreover, certain clinical trials have also proposed the role of estimating LDL-C by Friedewald calculation method over the direct method in treating hyperlipidaemic patients. 15, 16 Faas et al conducted a study on 464 patients to compare a direct LDL-C with the cLDL-C methods and found out that dLDL-C was 18% higher than the C-LDL-C which is consistent with our findings also. However, as treatment guidelines by NCEP-ATP III uses calculated LDL-C, whereas most commonly method nowadays is direct method which can lead to overtreatment of the hyperlipidaemic patients.¹⁵ Hence, more studies are required for establishing direct LDL-C in the treatment guideline goals before modifying them according to non-fasting sample values.

Limitations associated with this work could be that the gold standard method i.e., beta quantification could not be used to compare LDL-C levels. Another shortcoming could not retrieve adequate patient history including drug history like statins.

Conclusion:

A number of conventional calculated methods as well as newer direct methods are available to measure the LDL-C, however there is still paucity of data to establish their performance and use in clinical setup. In the study it was found that the mean dLDL-C value was higher than mean cLDL-C value with a strong positive correlation. It was also observed that Friedewald equation and Modified Friedewald equation showed an excellent performance among the calculated methods for estimating LDL-C. Hence, both Friedewald and Modified Friedewald equations can be used as a substitute for direct homogenous LDL estimation when there is scarcity of resources.

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