comparative study of low density lipoprotein by estimation and calculation from lipid profile in a tertiary hospital

introduction: Low-density lipoprotein cholesterol (LDL-C) is widely recognized as an established cardiovascular risk marker. Recent studies have shown that Friedewald underestimates LDL-C at lower levels, which could result in under treatment of high-risk patients of Atherosclerotic cardiovascular disease (ASCVD). A novel method Martin/Hopkins (LDL-CN) using a patient-specific conversion factor provides more accurate calculated LDL-C levels, LDL-C estimation is very important in high-risk patients to consider therapy. aims: To compare LDL-C values by using two formulae and direct estimation. Materials and methods: A prospective study was undertaken in Department of Biochemistry in 1393 subjects from Dec 2018 to March 2019 with mean age of (53.23±13.36). LDL-C was calculated by using Friedewald formula LDL-C (LDL-CF) and Martin/Hopkins equation (LDL-CN) in 1000 subjects with Triglycerides (TG) <400mg/dl and for 393 patients with TG>400 ldl was directly estimated (LDL-CD) with kit in Beckman coulter Dxc860i. results: For this analysis, the mean age was (53.23±13.36). In 1000 patients, with TG<400mg/dl the mean LDL-CF (95.13±36.16mg/dl) was significantly lower compared to LDL-CN (98.90±35.88mg/dl) with p value (0.01). Thus, LDL-CN yielded a better diagnostic value than LDL-CF with regression of r0.94. In 393 cases with Tg >400 mg/dl, the mean of LDL-CF (71.83±55.93mg/dl), was significantly lower compared to LDL-CD (104.17±41.6mg/dl) and LDL-CN (107.37±42.73mg/dl). p value was significant between Martin/Hopkins and Friedewald (<0.0001) and it is significant between LDL-CD and LDL-CF (<0.0001) but not between LDL-CD and LDL-CN (0.28). conclusion: This data suggest that Martin/Hopkins equation should be the preferred method to calculate LDL-C levels in with TG<400mg/dl and either direct measurement or Martin/Hopkins are the preferred methods in patients with TG>400mg/dl to treat high risk cases of ASCVD.

introduction Low-density lipoprotein cholesterol (LDL-C) is a major modifiable risk factor for cardiovascular disease (CVD) [1], its accurate assessment is important for therapeutic decisions. In routine clinical practice worldwide, it is typically calculated using the Friedewald formula [2]. From the outset, the formula's inaccuracies at triglyceride levels at 400 mg/dl were recognized by Friedewald et al. [3]. However, even when triglyceride levels are under 400 mg/dl, a number of studies have suggested that LDL-C estimated by the Friedewald formula (LDL-CF) underestimate LDL-C and misclassify CVD risk [4][5][6][7], particularly in individuals with high levels of triglycerides [4][5][6] and LDL-C less than 70 mg/dl [7].
β-quantification, a time consuming and expensive technique, requires ultracentrifugation and large volume of sample is the reference method for the quantitative estimation of LDL-C in circulation. Therefore, this method is not suitable for routine laboratory testing [8].
A less expensive and easy approach for the estimation of LDL-C include homogeneous direct measurement [9,4] and Friedewald formula [3] worldwide.
In a study by Martin et al. [10], an alternative to the Friedewald formula was proposed to improve estimation of LDL-C at triglyceride levels under 400 mg/dl. The Friedewald equation calculates LDL-C as LDL-CF = [total cholesterol]-[high-density lipoprotein cholesterol (HDL-C)] -[triglycerides/ 5], where the final term is the estimate of verylow-density lipoprotein cholesterol (VLDL-C). This equation therefore uses a fixed factor of 5 for the ratio of triglycerides to VLDL-C (TG: VLDL-C); however, the Martin equation applies an adjustable factor determined as the N-strata-specific median TG: VLDL-C ratio based on triglyceride and non-HDL-C concentrations to estimate the novel LDL-C (LDL-CN). Compared with LDL-CF, LDL-CN was reported to be closer to directly measured LDL-C (LDL-CD) and improved concordance in guideline risk classification with LDL-CD especially at LDL-C less than 70mg/dl. The present study was designed to compare the LDL-C calculated by formulae which use HDL-C, total cholesterol (TC) and TG to calculate LDL-C with directly measured LDL-C with TG levels <400mg/ dl and >400mg/dl in Indian population with the assumption that the results obtained by direct assays are the most accurate.

Materials and methods
A prospective cross sectional study was undertaken for the out-patients and in-patients samples which had come for LDL-C estimation from the month of Dec 2018 to march 2019 at Krishna Institute of Medical Sciences (KIMS) Hospital, Secunderabad, India (n =1393). Samples of all age groups were taken into the study.
Serum lipid concentrations (Total cholesterol, HDL, TG, LDL-C) were directly measured using Beckman coulter in DXC 860i, an automated analyser. Standard operative procedures and quality control as described by the manufacturer were followed.
LDL-C was calculated with Friedewald formula and Martin/Hopkins formula for samples with TG<400mg/dl. LDL was measured and calculated with above formulae for TG>400 mg/dl. All the values were analysed and recorded.

Principle of directly measured lDl
It is a homogeneous assay without the need for any off-line pre-treatment or centrifugation steps.

Cholesterol oxidase
Peroxidase The method depends on a unique detergent which solubilises only the non-LDL lipoprotein particles and releases cholesterol to react with cholesterol esterase and cholesterol oxidase to produce a non-colour forming reaction. A second detergent solubilises the remaining LDL particles, and a chromogenic coupler allows for colour formation.

statistical analysis
The data was entered into an excel sheet and analysed. Descriptive statistics such as tables were used to describe the data. p value were calculated wherever necessary for LDL estimated by Friedwalds formula and Mortin/Hopkins formula and direct estimation.
The aim was to compute the levels of differences between the methods. The probability of significance (p value) is considered significant less than 0.05 i.e., considering α =5. The values are analysed in terms of mean, standard deviation, standard error of mean, paired sample t test.   In Table 3, of 393 cases with TG >400 mg/dl, the mean of LDL-CF (71.83±55.93mg/dl), was significantly lower compared to LDL-CD (104.17±41.6mg/dl). p value was significant between direct estimation and Friedewald (<0.0001) with mean difference of 32.34. In Table 4, of 393 cases, p value was not significant between LDL-CD and LDL-CN (0.28) as the difference in mean value is very less, stating that either Martin/ Hopkins formula or direct measurement can be used for measuring LDL-C in TG>400mg/dl.

Discussion
Friedewald formula is the formula of choice for LDL-C calculation in most laboratories across the world. Many studies have shown its limitation and some have shown that other equations perform better for certain groups of populations. Measurement of LDL-C is of wide interest and deeply ingrained in practice. Guidelines around the globe focus on the LDL-C cut points, including guidelines from the National heart, lung, and blood institute [1, 11,12]. Canadian Cardiovascular Society [13], European Society of Cardiology and European Atherosclerosis Society [14] and the American Heart Association and American College of Cardiology [12,15]. Some of these guidelines [13,14] assign the highest level of evidence (Class 1A) to LDL-C treatment goals. LDL-C has been a focus in the inclusion criteria of numerous clinical trials, serially quantified during trials, and used as a target for drug titration in some trials [16,17]. The Cholesterol Treatment Trialists summarize the totality of evidence for statin therapy as the risk reduction indexed to a 39 mg/dl lowering of LDL-C [16,17].
In our study, with TG<400mg/dl and TG>400mg/ dl p value is significant with Martin/Hopkins when compared to Friedewald formula (p≤0.0001) which is in accordance with Martin et al. [19], showed greater accuracy with the novel calculation method (Martin/Hopkins) for all clinical LDL-C categories (87-94%) vs Friedewald estimation (71-93%) in both fasting and non-fasting samples (p≤0.001).
In the derivation study, overall accuracy of the Martin/Hopkins equation compared to direct ultracentrifugation was 92% in contrast to 85% accuracy for Friedewald estimation (p<0.001) [12] as same as our study.
Our results indicate that Friedewald formula fails to provide a good result at TG>400mg/dl. This is contradictory to the study done by Sha et al. [18], in Bangladeshi population which concluded that Friedewald formula can be used up to serum TG concentration of 700mg/dl. In a study [19], the Martin/Hopkins method provides a more accurate estimate of LDL-C levels than Friedwalds formula in LDL<40mg/dl is in accordance with our study.
In a study [20], the novel equation provides a higher estimation of exact LDL-C values than the Friedewald equation, particularly in patients with low LDL-C levels, which may result in under treatment of some patients whose LDL-C was calculated using the Friedewald method. However, neither may be suitable for patients with TG≥400 mg/dl is in accordance with our study saying direct measurement provides better results of LDL-C than calculations.

study limitations
This study compares calculated LDL-C with direct LDL-C assay and not with the reference method i.e., ultracentrifuge and precipitation for comparison. Also, the study uses only one assay for TG, TC, LDL-C and HDL-C and other assay methods have not been considered. Another limitation is that the number of samples with TG>400mg/dl was small. Factors such as race/ethnicity, obesity, diabetes, and insulin resistance, which may affect variance in the TG: VLDL-C ratio, were not available for analysis. It is unknown to what extent patients in our study sample were treated with lipid-modifying drug therapies. A multicentre, prospective study should be conducted to expand the knowledge gained from this study. Lifestyle modification and public education are recommended for prevention of CVD.