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- Indian Heart J
- v.76(Suppl 1); 2024 Mar
- PMC11019328
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Indian Heart J. 2024 Mar; 76(Suppl 1): S29–S32.
Published online 2024 Feb 29. doi:10.1016/j.ihj.2024.02.002
PMCID: PMC11019328
A.K. Pancholia,a,⁎ Nitin Kumar Kabra,b and Rajeev Guptac
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Abstract
Accurate measurement of various lipids- total cholesterol, cholesterol lipoproteins and triglycerides- is important for coronary artery disease (CAD) prevention and management. Over the years many technologies have developed for their accurate measurements and in recent years the assays have been internationally standardised. Most of the guidelines recommend measurement of non fasting levels of total cholesterol, low density lipoprotein cholesterol (LDL-C), high density lipoprotein cholesterol (HDL-C), non-HDL cholesterol (non-HDL-C) and triglycerides (TG) for risk estimation and guiding treatment. Measurement of lipid profile in clinics and emergency departments can lead to earlier estimation of CAD risk and rapid initiation of lipid lowering therapy. CAD risk and baseline levels of LDL-C guide intensity of lipid lowering therapies. The LDL-C targets according to CAD risk are detailed in this review. There is an urgent need for standardization of lipid estimation in medical laboratories across the country so that every eligible individual can receive evidence-based lipid lowering interventions.
Keywords: Laboratory, Lipid, Non fasting, NABL, Standardization
1. Introduction
Lipids including cholesterol are a class of molecules that are soluble in organic solvents and are essential to many body functions.1 They are transported in circulation as water soluble lipoproteins having two layers: (i) an amphiphilic surface comprising of proteins (apolipoproteins), cholesterol and phospholipids; and (ii) a hydrophobic core comprising of free fatty acids, cholesterol esters and triglycerides. Based on their density (which depends on their protein and triglyceride content) the lipoproteins are classified (in order of increasing density) into chylomicrons (CM), very-low-density lipoprotein (VLDL), intermediate density lipoprotein (IDL), low-density lipoprotein (LDL), and high-density lipoprotein (HDL).1 It is well-established that elevated atherogenic lipids are independent risk factors for coronary artery disease (CAD).1 All the international guidelines highlight importance of serum total cholesterol and its main subtypes- LDL cholesterol, HDL cholesterol, non-HDL cholesterol, VLDL cholesterol, lipoprotein(a) [Lp(a)]and triglycerides, in CAD pathophysiology.2, 3, 4 Accurate measurement of various cholesterol lipids and triglycerides is important to estimate CAD risk.5
2. Methodology
In this brief review we focus on methodology of cholesterol lipoprotein and triglyceride measurement and highlight controversies in the techniques and timing of their assessment (see Table 1).
Table 1
Measurement methods of commonly available cholesterol and lipid parameters.
| Lipid parameter | Method for measurement | NABL Approved |
|---|---|---|
| Total cholesterol | Cholesterol oxidase-peroxidase (CHO-POD) | Yes |
| LDL Cholesterol | Friedwald formula calculated: LDL C=Total-[HDL+(TG/5)] Direct: hom*ogenous assay | – Yes |
| Non-HDL cholesterol | Calculated: Non HDL C=Total-HDL cholesterol | – |
| HDL cholesterol | Enzymatic immune-inhibition | Yes |
| VLDL cholesterol | Calculated: VLDL C=TG/5 | – |
| Triglycerides | Glycerol-3 phosphate oxidase peroxidase (GPO-POD) | Yes |
| Remnant lipoproteins | Calculated: Remnant=total- (LDL+HDL) | – |
| Lipoprotein(a) | Immunoassays; Immunoturbidimetery; | NA |
| Apolipoprotein A | Immunoturbidimetery; Immunonephelometry | NA |
| Apolipoprotein B | Immunoturbidimetery; Immunonephelometry | NA |
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C cholesterol; HDL high density lipoprotein; LDL low density lipoprotein; NABL National Accreditation Board for Laboratories; TG triglycerides; VLDL very low density lipoprotein.
3. Results
3.1. Lipoprotein cholesterol measurements
3.1.1. Total cholesterol
This is recommended to be used to estimate the CV risk by all international guidelines for screening.1 It may be misleading in the individual case. To assess the adequate risk at least HDL-C and LDL-C should be analysed. Assessment of total risk does not include patients with familial hyperlipidaemia (including familial hypercholesterolemia or familial combined hypercholesterolemia) or those with total cholesterol >300mg/dl.
3.1.2. Low-density lipoprotein cholesterol (LDL-C)
There is a linear relation between LDL-C lowering and ASCVD risk reduction, supporting the fact that attaining low levels of LDL-C minimizes the risk of CV events.6,7 In the Cholesterol Treatment Trialists (CTT) collaborators, a reduction of (1mmol/L or 38.7mg/dl) in LDL-C levels showed a linear 21% reduction in the risk of major vascular events over 5 years.8 There is ample evidence to show that more-aggressive lipid-lowering therapy, more effectively reduces the incidence of CV events than does a less-aggressive lipid-lowering strategy.6,7,9 Subsequent studies and meta-analysis have revealed that achieving LDL-C levels even lower than these recommended targets further reduces ASCVD risk.10 Recently 2019 European Society of Cardiology (ESC) and European Atherosclerosis Society (EAS) dyslipidemia guidelines and Lipid Association of India consensus statement lowered the LDL-C target to <55mg/dl in very high risk groups.4,11
3.1.3. Calculated versus direct LDL cholesterol estimation
The Friedewald equation was developed in 1972 and estimates LDL-C as: total cholesterol (TC) minus high-density lipoprotein-cholesterol (HDL-C) minus triglycerides (TG)/5, with the latter term serving as an estimate for very low-density lipoprotein-cholesterol (VLDL-C).12 Originally developed for research purposes from a sample of just 448 individuals, the Friedewald equation has been widely adopted in clinical practice for several decades. However, the equation is prone to inaccuracy at low LDL-C and/or high TG levels, where errors in estimating VLDL-C are magnified given its use of a fixed factor of 5 to describe the relationship between TG and VLDL-C. This results in marked underestimation of LDL-C. Recently, with epidemics of obesity and diabetes resulting in hypertriglyceridemic states, and with novel therapeutics achieving historically low LDL-C levels, underestimation of LDL-levels with Friedewald's equation may lead to deferral or withdrawal of lipid-lowering therapies, potentially resulting in undertreated high-risk patients. At higher TG levels, chylomicrons accumulate and may alter the relationship between TG and VLDL-C; therefore, direct chemical assays are typically used to measure LDL-C. Several commercial direct assays have been developed for this purpose, but there remain substantial concerns regarding their standardization and accuracy, especially at high TGs and in those with known coronary disease.13
3.1.4. HDL cholesterol (HDL-C)
The inverse association between plasma HDL-C and the risk of ASCVD is among the most consistent and reproducible associations in observational epidemiology.13 Randomised trials showed no evidence that therapeutically increasing plasma HDL-C reduces the risk of CV events.1 There is currently no randomized trial or genetic evidence to suggest that raising plasma HDL-C is likely to reduce the risk of ASCVD events. HDL estimates in different labs using different reagents differ. This is a matter of concern and unfortunately, uniform testing and standards need to be suggested.
3.1.5. Non-HDL cholesterol (non-HDL-C)
Non-HDL-C is used as an estimation of the total number of atherogenic particles in plasma [VLDL+intermediate-density lipoprotein (IDL)+LDL] and relates well to apo B levels.1 Non-HDL-C is easily calculated from TC minus HDL-C (Fig. 1). Non-HDL cholesterol is a fairly reliable method of assessment and correlates very well with clinical events. Both being calculated by standard methods Non-HDL-C can provide a better risk estimation compared with LDL-C, in particular in raised TG combined with diabetes, the metabolic syndrome or chronic kidney disease (CKD). This is supported by a recent meta-analysis including 14 statin trials, seven fibrate trials, and six nicotinic acid trials.14,15
Fig. 1
Different plasma lipoproteins.
3.1.6. Lipoprotein (a)
Lp(a) is a genetically modified form of LDL-C particle and has greater propensity than LDL-C, to bind to oxidized lipoproteins.1 Lp(a) appears to be a particularly important ASCVD risk factor for Indians as Indians tend to have high prevalence of elevated Lp(a).16 Based on these evidences, estimation of Lp(a) levels is strongly recommended for ASCVD risk stratification in Indian subjects, particularly in those who have family history or premature CAD. High Lp(a) concentrations >30mg/dL indicate increased risk for incident myocardial infarction,15, 16, 17 although a European Atherosclerosis Society (EAS) consensus statement has proposed 50mg/dL concentration threshold for an increased risk.3,18 Methods recommended for measurement are different in Europe compared to India. In Indian scenario it is to be estimated when a personal or family history of premature atherosclerotic cardiovascular disease (<60 years of age), First degree relatives of those with high Lp(a) plasma concentrations (>200nmol/L) and Familial hypercholesterolaemia (FH), or other genetic forms of dyslipidaemia.
3.1.7. Triglycerides (TG)
Epidemiologic studies have suggested that TG is a relevant measure in evaluating CHD risk.19 In the largest population-based prospective study of more than 300,000 men and women, the strong linear association between TG levels and CHD risk persisted until adjustment for non HDL-C and HDL-C.20 The generally followed classification for TG values based on fasting lipid profile is: desirable <150mg/dL; high 150–499mg/dL; and very high ≥500mg/dL2,.3
3.1.8. Apolipoproteins
Apo B is the major apolipoprotein of the atherogenic lipoprotein families VLDL, IDL, and LDL. The concentration of apo B is a good estimate of the number of these particles in plasma. In several prospective studies Apo B has been shown to be equal to LDL-C in risk prediction.21 In statin trials Apo B has not been evaluated as a primary treatment target, but several post-hoc analyses of statin trials suggest that apo B may be not only a risk marker but also a better treatment target than LDL-C.1,6,7,21 It is also an important predictor of ASCVD risk. The use of Apolipoprotein B (ApoB) in risk stratification may be useful when LDL-c underestimates the risk as in high triglycerides (TG) conditions, diabetes mellitus, obesity, or very low LDL-C.21 The identification of ApoB as secondary goals <65, 80, and 100mg/dL for very-high, high-, and moderate-risk people, respectively, could become relevant in near future, particularly in the view of newer drugs targeting TG.22
3.2. Other issues
3.2.1. Non-fasting versus fasting concentrations
There is a minimal change in lipid parameters (+26mg/dl for triglycerides, −8mg/dl for total cholesterol, −8mg/dl for low-density lipoprotein cholesterol, +8mg/dl for remnant cholesterol, and −8mg/dl for non-high-density lipoprotein cholesterol) in the general population after food intake.4 Lipoprotein(a), apolipoprotein B, and high-density lipoprotein cholesterol are largely unaffected. Measuring lipid profile in the non-fasting state simplifies blood sampling for patients, laboratories, and clinicians.23 Non-fasting lipid profiles have been shown to predict increased risk of cardiovascular events.4 The benefit of reducing non fasting lipid levels to reduce the risk of CV disease has been proven in clinical trials. Before 2009 all cardiovascular societies, guidelines, and statements required fasting before measuring a lipid profile for cardiovascular risk prediction. But now several society guidelines and statements endorse non fasting lipid profiles.3, 4, 5
•
Non fasting is recommended as initial lipid profile in every patient, for cardiovascular risk assessment.
•
In most patients admitted with acute coronary syndromes, a non-fasting lipid testing is acceptable as it has no impact on LDL-C or HDL-C and apoB levels.
•
Non-fasting testing is easier to perform especially in children, elderly, diabetics and acute coronary syndrome patients. It gives reliable results. Non fasting testing can avoid early morning rush and thus more number of patients can get their testing done.
•
Fasting is required If non fasting triglyceride level is >400mg/dl, in known cases of hypertriglyceridemia, in those recovering from acute pancreatitis due to high TG or if another fasting test such as glucose is being performed.
3.2.2. Lipid measurement in clinics and emergency departments
It is need of hour to have point of care analysis for all clinics and emergency department. Patients of chest pain (typical/atypical) presenting to causality will require assessing risk factors like diabetes, BP, smoking which can be diagnosed historically but dyslipidemia cannot be diagnosed clinically without blood sampling. Hence we recommend point of care testing for immediate risk assessment and therapeutic monitoring of CVD.24 Portable accurate multi analysis meters are available that can give results in 3min and test for basic lipid profile (total, HDL, and LDL cholesterol), troponins, nt-ProBNP and HbA1c.25 The objective of point of care testing is to generate a result quickly so that appropriate treatment can be implemented, leading to an improved clinical or economic outcome.
3.3. Normal lipid levels
In the earlier years of development of guidelines, the US National Cholesterol Programs and other international agencies used a pragmatic approach to define normal levels of various cholesterol lipoproteins. These were based on median population levels which were considered normal and high and very high levels were determined using a 30mg/dl increment for high and very high levels. Consequent to availability of clinical trials focussing on cardiovascular outcomes, the levels have been changed to generate contemporary evidence-based guidelines. The levels of various cholesterol lipoproteins and triglycerides considered desirable are provided in various algorithms in this issue of the Indian Heart Journal. These are consensus driven guidelines that may be referred as CSI recommendations (Table 2).
Table 2
Standard lipid testing panels and targets for various risk groups (all values in mg/dl).
| Lipid parametera | Desirable levels of various lipid fractions | |||
|---|---|---|---|---|
| General | Low risk | Medium & High risk | Very High Risk | |
| LDL cholesterolb | <100 | <100 | <70 | <55 |
| Non-HDL cholesterol | <130 | <130 | <100 | <85 |
| HDL cholesterol | >40M, >50W | >40M, >50W | >40M, >50W | >40M, >50W |
| Triglycerides | <150 | <150 | <150 | <150 |
| Lipoprotein(a)a | <50 | <50 | <50 | <30 |
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General=no other cardiovascular risk factor.
Low risk=any one of major CAD risk factors (smoking/tobacco, hypertension, diabetes, family history of premature CAD).
Medium & high risk=2 or more risk factors with no manifest ASCVD, chronic kidney disease, long standing diabetes or heterozygous familial hypercholesterolemia.
Very high risk=clinical evidence of atherosclerotic CAD, atherosclerotic disease in other vascular beds, diabetes >20 years, heterozygous familial hypercholesterolemia with ASCVD, imaging showing >50% lesion in 2 coronary vessels.
ASCVD includes Coronary artery disease, cerebrovascular and peripheral vascular disease.
Extremely high-risk group has not been included in this Table.
aThe values may vary according to the laboratory-method used.
bDirect or calculated according to Friedewald formula.
4. Conclusion and recommendations
We believe that laboratory investigation of lipids plays an important role in risk assessment, screening, and management of cardiovascular disease (CVD) and in the diagnosis of dyslipidemias.26 it is well-established that elevated atherogenic lipids are an independent risk factor for CVD.1 We would recommend the following tests (Table 2). LDL cholesterol could be measured or calculated according to the Friedewals's formula. Considering the limitation of Friedewald formula in many clinic settings, we need to have a direct LDL measurement, wherever possible. Other formulas are also available such as Martin Hopkins formula, Sampson-NIH equation or an Indian version but are not recommended by international guidelines.2, 3, 4, 5 Extended lipid panel is presently not recommended by any international guideline for routine use but may be performed in case of family history of premature CAD or other specific clinical situations as risk enhancing factors.1,27 These include lipoprotein(a), apoprotein A1, and apolipoprotein B. Other lipid measurements are mainly employed as research tools and include measurement of LDL particle size and isoforms, HDL particle size and isoforms, extended lipoprotein panels for identification of Frederickson types. Specific genetic testing for LDLR, PCSK9, APOE and other genes are emerging and in future may occupy a centre stage in risk estimation.28
5. Standardization
National accreditation for biological laboratories (NABL) is the formal document available on www.nabl-india.org and is used for standardization of laboratories across the country.29 The document also provides a list of the 2509 labs that are NABL accredited. However, according to Foundation for Quality India there are more than 300,000 small and large labs in India and use different methodologies to measure various lipid parameters. It is recommended that all the medical laboratories, either in hospitals or stand-alone independent, should have uniform methodology and provide identical reference values and targets in risk groups (Table 2). This is a herculean task but is desirable if we wish to employ guideline-based treatment across the country.
Declaration of competing interest
The authors declare that they have no known competing financialinterests or personal relationships that could have appeared to influencethe work reported in this paper.
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