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More accurate LDL-C calculation: Externally validated, guideline endorsed
Clinica Chimica Acta 506 (2020) 149–153
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Clinica Chimica Acta journal homepage: www.elsevier.com/locate/cca
More accurate LDL-C calculation: Externally validated, guideline endorsed Adam J. Brownstein, Seth S. Martin
⁎
T
Ciccarone Center for the Prevention of Cardiovascular Disease, Division of Cardiology, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, USA
A R T I C LE I N FO
A B S T R A C T
Keywords: Low density lipoprotein-cholesterol (LDL-C) Martin-Hopkins equation Friedewald equation Non-fasting lipid panel Cardiovascular disease Statins PCSK9 inhibitors
Low density lipoprotein-cholesterol (LDL-C) serves as the primary target of therapy for preventing atherosclerotic cardiovascular disease (ASCVD). Recently released European and American guidelines on lipid management recommend attaining very low LDL-C levels (< 1.8 mmol/L or even lower) in high and very-high risk patients. Therefore, utilizing an accurate means for determining LDL-C, especially at such low values, is of paramount importance to inform the best clinical decisions and use of effective therapies. This review compares the different methods of determining LDL-C, including the various forms of direct measurement and most commonly used calculations. This review discusses the evidence behind these methods in different populations of patients and in the fasting versus non-fasting state. The Martin/Hopkins method is the preferred method for determining LDL-C as it is the most accurate and widely applicable method. It is especially useful in patients with low LDL-C levels < 1.8 mmol/L (< 70 mg/dL) and high triglyceride levels between 1.7 and 4.5 mmol/L (150–399 mg/dL), and is reliable in the non-fasting state.
1. Introduction
2. Historical context and novel methods for calculating LDL-C
Our ability to calculate low-density lipoprotein cholesterol (LDL-C) concentrations without resorting to their direct measurement via the labor-intensive process of preparative ultracentrifugation has advanced the care of patients with atherosclerotic cardiovascular disease (ASCVD) and those at risk. While there are many potential targets to reduce the risk of ASCVD, such as LDL-C, non-high density lipoprotein cholesterol (non-HDL-C), apolipoprotein B (apoB), and Lipoprotein(a) (Lp(a)), major guidelines currently emphasize the importance of focusing our attention on LDL-C [1–3]. LDL-C is established as a causative agent of ASCVD and we have a wealth of evidence that specifically targeting LDL-C reduces risk of ASCVD [4]. As demonstrated by the Cholesterol Treatment Trialists’ Collaboration, the relative risk of major vascular events and all-cause mortality are reduced by 22% and 10%, respectively, for each 1 mmol/L (39 mg/dL) reduction in LDL-C [5]. Furthermore, prospective cohort studies have shown that exposure to elevated LDL-C levels during young adulthood confers increased risk for future ASCVD [6–9]. Given the significant relationship between LDL-C and cardiovascular health, and its prominence in guidelines and clinical practice, it is of the utmost importance that we have accurate means of determining patient LDL-C levels.
Friedewald and colleagues set the stage for future studies examining the relationship between LDL-C and ASCVD in their ground breaking 1972 publication of a formula to calculate LDL-C (Total Cholesterol (TC) – HDL-C – (Triglycerides (TG)/2.2) in mmol/L or (Triglycerides/5) in mg/dl) [10] derived from 448 individuals. However, as noted by the authors, the use of a fixed factor to estimate very low density lipoprotein cholesterol (VLDL-C) is problematic at low LDL-C values and high triglyceride values. Since this original publication, many groups have attempted to identify more accurate methods for calculating LDL-C. This has remained important in the absence of a cost-effective and accurate direct measurement. Although non-ultracentrifugation chemical based direct assays have emerged, these add expense and are not well validated for the measurement of LDL-C. Each direct chemical assay uses proprietary chemicals in an attempt to block non-LDL lipoproteins and facilitate measurement of LDL-C. However, these direct assays are not standardized, have limited data supporting their accuracy, and limited traceability, which lead to method dependent differences in LDL-C. As highlighted in the excellent summary article of the consensus statements of the European Atherosclerosis Society (EAS) and the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM) by
⁎ Corresponding author at: Ciccarone Center for the Prevention of Cardiovascular Disease, Johns Hopkins Hospital, 600 N. Wolfe St, Carnegie 591, Baltimore, MD 21287 USA. E-mail address: [email protected] (S.S. Martin).
https://doi.org/10.1016/j.cca.2020.03.030 Received 12 February 2020; Received in revised form 17 March 2020; Accepted 19 March 2020 Available online 20 March 2020 0009-8981/ © 2020 Elsevier B.V. All rights reserved.
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multiple studies from around the globe have since validated these results [23,24,26–30]. Importantly, this method has been validated in different subsets of patients. Whelton led a study in 2017 of NHANES, Johns Hopkins, and Mayo Clinic data showing that approximately 20% of individuals with LDL-C < 70 mg/dL (1.8 mmol/L) by the Friedewald equation actually had values ≥ 70 mg/dL (1.8 mmol/L) based on the Martin/Hopkins method [26]. Significance of the upward reclassification by Martin/Hopkins calculation was corroborated by LDLC measured by ultracentrifugation, as well as non-HDL-C and apoB levels. This method therefore improves LDL-C accuracy at low LDL-C levels (< 1.8 mmol/L), which has important implications given recent American College of Cardiology/American Heart Association (ACC/ AHA) and European Society of Cardiology/European Atherosclerosis Society (ESC/EAS) guidelines [2,3]. By correcting underestimation that would otherwise occur with the Friedewald equation, clinicians can avoid undertreatment of high risk patients. It has also been shown to be more accurate in patients taking proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors [23], patients with diabetes mellitus [27], patients with familial combined hyperlipidemia [31], and in nonfasting samples [25]. This method becomes especially important among patients with diabetes given that direct chemical LDL-C assays are even more discrepant among these patients [11]. With a wealth of data supporting the method, the clinical laboratory at Johns Hopkins and Quest Diagnostics have adopted the Martin/Hopkins method. It can be installed on the lab information system for automatic calculation of LDL-C. Laboratories can contact [email protected] for licensing. Of note, neither the Friedewald nor the Martin/Hopkins method is well suited to the setting of severe hypertriglyceridemia [14] where the presence of chylomicrons complicates LDL-C estimation. A summary of the strengths and limitations of different methods that are currently used clinically for determining patient LDL-C levels is depicted in Table 1. Recently, another method for calculating LDL-C was developed and compared to both the Friedewald and Martin/Hopkins calculations [32]. Data from 8,656 patients seen in the 1970–90s at the National Institutes of Health were used to derive the proposed equation: TC/ 0.948 minus HDL-C/0.971 minus (TG/8.56 plus [TG × non-HDL-C]/ 2140 minus TG2/16100) minus 9.44. Compared with the Martin/ Hopkins calculation, it was derived in a population < 1% the size and a population not reflective of routine contemporary clinical practice. The lipid profiles of the patient sample were skewed towards high TG and cholesterol. It is suggested that the new equation allows for more accurate calculation of LDL-C up to TG levels of 800 mg/dL, however the comparison was to Friedewald and Martin/Hopkins and these estimates are not currently limited to TG < 400 mg/dL and not intended for use up to TG levels of 800 mg/dL. The large mean absolute difference of 24.9 mg/dL reported for the proposed equation is concerning as this amount of error approaches the width of established clinical categories (30 mg/dL). Furthermore, the clinical priority in patients with TG ≥ 400 mg/dL is TG lowering to prevent pancreatitis. Overall, the proposed new equation requires further independent validation. In particular, dedicated testing is required in patients intensively treated to very low LDL-C levels < 40 mg/dL since the analyses provided in the report are incomplete and raise concern for underestimation of LDLC in such samples.
Langlois et al., LDL-C values obtained via direct chemical assays vary significantly among different manufacturers, most prominently among patients with hypertriglyceridemia (> 2 mmol/L), mixed dyslipidemia, and diabetes mellitus [11]. Since these direct LDL-C assays are not standardized, changes in LDL-C over time may not be reliable, rendering it challenging for a clinician to optimize lipid lowering therapy when using different manufacturers’ assays for therapeutic follow-up [11]. It should also be noted that calculated LDL-C values may be affected by (1) the biological variability of TG levels and (2) the variability of direct HDL-C assays among different manufacturers [12,13], with the largest discrepancy seen in dyslipidemic patients with low HDL-C levels [11]. All methods of LDL-C calculation are dependent in accuracy on the data used for calculation, namely total cholesterol, triglycerides, and HDL-C. The homogeneous HDL-C assays in particular may suffer from inaccuracy and insufficient traceability. Prior to publication of the novel Martin/Hopkins equation (TC – HDL-C – TG/adjustable factor) in 2013, the majority of previously published methods for calculating LDL-C failed to account for the interindividual variance in the TG:VLDL-C ratio, which serves as the backbone of this equation [14]. In 1986, DeLong et al. attempted to improve on the Friedewald equation by proposing to divide TG by 6 instead of 5 [15], yet this method still relied on a fixed factor. Chen et al. in 2010 [16] proposed the formula LDL-C (mg/dL) = non-HDLC × 90% - TG × 10%, while de Cordova et al. in 2013 [17] suggested LDL-C = ¾ (TC – HDL-C). Many other formulas have been published as well, but they all overlook this important interindividual variance [18–20] or utilize an adjustable factor that considers only TG levels as opposed to cholesterol concentrations [21]. A 2015 analysis of four formulas in hospitalized patients (not including the Martin/Hopkins equation) demonstrated that the Hattori formula (LDL-C = 0.94TC – 0.94HDL-C – 0.19TG) outperformed the Friedewald, Chen, and De Cordova formulas in hospitalized patients using a direct chemical LDL-C assay as the reference standard. The Hattori method [20], which was obtained via regression analysis, also is a fixed formula that does not account for the interindividual variance, as noted above. A recently developed equation in a Ghanian population also relied on a fixed factor [22]. Many of these studies evaluating different formulas for LDL-C calculation used direct LDL-C assays as the reference standard for comparison, which as mentioned above have significant shortcomings. Ideally, ultracentrifugation should be used as the reference standard, as was done for the DeLong [15], Hattori [20], Rao [21], and Martin [14] studies, as well as in analyses validating the Martin/Hopkins method [23–26]. The clinical relevance of estimating LDL-C accurately and the longstanding use of the Friedewald equation in clinical practice led Martin et al. in 2013 to develop an equation with a similar structure to Friedewald’s original formula [14]. However, it differs in a fundamental way in that it relies on distinct adjustable factors based on individual patient TG and non-HDL-C levels (174 different factors for TG < 400 mg/dL). The adjustable factors were obtained by taking the median ratio of TG to VLDL-C values stratified by TG and non-HDL-C levels in table format. A high level of confidence was generated for each cell in the table because of the very large population for derivation (~1 million) and demonstration that the dataset is nationally representative, with lipid distributions similar to the National Health and Nutrition Examination Survey (NHANES). Of note, in the middle of these tables were ratios that closely approximated the original fixed factor of 5 that Friedewald et al proposed for calculating in mg/dL (2.2 in mmol/L), indicating that this formula is generally accurate in the average patient. However, it is in the patients who are not average where using an adjusted factor increases accuracy in determining LDL-C concentrations since the majority of the variance of TG/VLDL-C was explained by TG and non-HDL-C levels. Since publication of these results based on the analysis of more than 1,350,000 patients from the Very Large Database of Lipids Study,
3. ACC/AHA and ESC/EAS guidelines on LDL-C The recent AHA/ACC and ESC/EAS guidelines both highlight the importance of focusing on LDL-C for primary and secondary prevention of ASCVD. Both sets of guidelines emphasize using maximally tolerated statin therapy to decrease LDL-C levels by > 50% followed by nonstatin therapy if the LDL-C is above a certain threshold (70 mg/dL or 1.8 mmol/L for the AHA/ACC guidelines and 55 mg/dL or 1.4 mmol/L for the ESC/EAS guidelines) in very high-risk patients [2,3]. Additionally, both guidelines emphasize that non-fasting samples are 150
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Recommended at TG levels > 4.5 mmol/L
√ – – √ √ – √ –
adequate for the vast majority of patients for risk stratification, as nonfasting samples have only mildly elevated TG levels compared to fasting samples (approximately 0.3 mmol/L or 27 mg/dL) [3,33] and have similar prognostic value compared to fasting samples [2,3,34]. However, they do differ in their LDL-C cutpoints and endorsement of novel methods for calculating LDL-C. On the one hand, the AHA/ACC Multi-society guidelines state that the Friedewald equation is generally sufficient but if TG levels are elevated (≥150 mg/dL or 1.7 mmol/L) or LDL-C is low (< 70 mg/dL or 1.8 mmol/L), then the Martin/Hopkins formula can be utilized (2). On the other hand, the ESC/EAS guidelines endorse use of the Friedewald equation and acknowledge its limitations, including at TG levels ≥2 mmol/L or 177 mg/dL and at very low LDL-C levels, but do not directly endorse novel LDL-C calculation methods (3). The ESC/EAS guidelines were released before the latest EAS/EFLM recommendations and it is anticipated that the next ESC/ EAS guidelines will be updated to endorse the Martin/Hopkins formula. Given the ESC/EAS guidelines’ emphasis on aggressively treating LDL-C to < 1.4 mmol/L or 55 mg/dL in very-high risk patients, use of a reliable method to calculate LDL-C at these low values is extremely important, as it has clinical ramifications for patient care. Use of the Friedewald equation at such low LDL-C levels may lead to underestimation of these values and would thus preclude very-high risk patients from being candidates for further lipid lowering therapy, such as PCSK9 inhibitors or ezetimibe, as shown in Fig. 1. The lower LDL-C goes, the more probability it is underestimated by the Friedewald equation, and the ESC/EAS guidelines also have an even more aggressive goal of < 1 mmol/L or < 40 mg/dL in very-high risk patients with progressive disease. To implement these recommendations well would require more accurate LDL-C assessment. 4. EAS/EFLM guidelines on LDL-C
√ – √ –
The guidelines released by EAS and EFLM and summarized by Langlois et al. endorse the Martin/Hopkins equation in patients with low LDL-C levels < 1.8 mmol/L (70 mg/dL) and/or TG levels between 2.0 and 4.5 mmol/L (175–400 mg/dL) [11] and make multiple important recommendations. Firstly, they highlight the major concerns with direct LDL-C assays, especially among patients with obesity, metabolic syndrome, and diabetes [11]. These assays can be used among patients with TG ≥ 4.5 mmol/L though are not particularly accurate in that setting and the clinical priority in severe hypertriglyceridemia becomes TG lowering to avoid pancreatitis. Secondly, they emphasize that using the Friedewald equation at low LDL-C values (< 1.8 mmol/ L) overestimates the VLDL-C component of cholesterol, thus underestimating the LDL-C fraction, which risks leading to underuse of guideline directed medical therapy and less favorable outcomes in high risk patients. Thirdly, they recommend using non-fasting lipid panels to assess patient risk for ASCVD, though one may consider fasting lipids if TG concentration is ≥4.5 mmol/L on the non-fasting sample. Lastly, they recommend primary emphasis on targeting LDL-C but that nonHDL-C and apoB can be used as secondary treatment targets in select patients, though recent evidence suggests that using these secondary targets may only be helpful in a small fraction of patients when using the Martin/Hopkins equation for calculating LDL-C [35].
√ √ √ – – √ √ √ √ √ √ – √ – – – Ultracentrifugation Friedewald Equation Martin/Hopkins Equation Direct LDL-C assays
Widely Clinically Available Well validated Gold Standard Method
Table 1 A comparison of methods to determine patient LDL-C levels.
Reliable in a majority of patients
Accurate at high TG (2.0–4.5 mmol/L) and/or low LDL-C levels (< 1.8 mmol/L)
Equivalent efficacy with fasting and nonfasting samples
A.J. Brownstein and S.S. Martin
5. Clinical implications Ever since publication of the Friedewald equation and the development of life-saving lipid lowering therapy, including statins, ezetimibe, and PCSK9 inhibitors, our ability to transform cardiovascular care for our patients has been remarkable. With the release of the AHA/ ACC, EAS/ESC, and EAS/EFLM guidelines and their emphasis on both the importance of LDL-C levels and employing accurate means of calculating them, clinicians now have the armamentarium to significantly improve the lives of our patients. Yet, recent research demonstrates that the medical community is not optimally screening high risk patients 151
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Fig. 1. Clinical Case: comparing the Friedewald and Martin/Hopkins Equations.
with lipid panels after myocardial infarctions and intensifying lipidlowering therapy to achieve optimal reductions in LDL-C [36–38]. It is imperative that we begin to implement decades of research demonstrating the importance of LDL-C targeted therapy on the front lines of clinical care for the health of our patients.
[5]
[6]
CRediT authorship contribution statement [7]
Adam J. Brownstein: Conceptualization, Writing - original draft. Seth S. Martin: Conceptualization, Writing - review & editing.
[8]
Declaration of Competing Interest [9]
SSM: consultant/advisory board for Sanofi, Regeneron, Amgen, Quest Diagnostics, Akcea, Novo Nordisk, Esperion; co-inventor for method to estimate LDL cholesterol levels, patent application by Johns Hopkins University pending and research support from Akcea, paid to the institution.
[10]
[11]
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Contents lists available at ScienceDirect
Clinica Chimica Acta journal homepage: www.elsevier.com/locate/cca
More accurate LDL-C calculation: Externally validated, guideline endorsed Adam J. Brownstein, Seth S. Martin
⁎
T
Ciccarone Center for the Prevention of Cardiovascular Disease, Division of Cardiology, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, USA
A R T I C LE I N FO
A B S T R A C T
Keywords: Low density lipoprotein-cholesterol (LDL-C) Martin-Hopkins equation Friedewald equation Non-fasting lipid panel Cardiovascular disease Statins PCSK9 inhibitors
Low density lipoprotein-cholesterol (LDL-C) serves as the primary target of therapy for preventing atherosclerotic cardiovascular disease (ASCVD). Recently released European and American guidelines on lipid management recommend attaining very low LDL-C levels (< 1.8 mmol/L or even lower) in high and very-high risk patients. Therefore, utilizing an accurate means for determining LDL-C, especially at such low values, is of paramount importance to inform the best clinical decisions and use of effective therapies. This review compares the different methods of determining LDL-C, including the various forms of direct measurement and most commonly used calculations. This review discusses the evidence behind these methods in different populations of patients and in the fasting versus non-fasting state. The Martin/Hopkins method is the preferred method for determining LDL-C as it is the most accurate and widely applicable method. It is especially useful in patients with low LDL-C levels < 1.8 mmol/L (< 70 mg/dL) and high triglyceride levels between 1.7 and 4.5 mmol/L (150–399 mg/dL), and is reliable in the non-fasting state.
1. Introduction
2. Historical context and novel methods for calculating LDL-C
Our ability to calculate low-density lipoprotein cholesterol (LDL-C) concentrations without resorting to their direct measurement via the labor-intensive process of preparative ultracentrifugation has advanced the care of patients with atherosclerotic cardiovascular disease (ASCVD) and those at risk. While there are many potential targets to reduce the risk of ASCVD, such as LDL-C, non-high density lipoprotein cholesterol (non-HDL-C), apolipoprotein B (apoB), and Lipoprotein(a) (Lp(a)), major guidelines currently emphasize the importance of focusing our attention on LDL-C [1–3]. LDL-C is established as a causative agent of ASCVD and we have a wealth of evidence that specifically targeting LDL-C reduces risk of ASCVD [4]. As demonstrated by the Cholesterol Treatment Trialists’ Collaboration, the relative risk of major vascular events and all-cause mortality are reduced by 22% and 10%, respectively, for each 1 mmol/L (39 mg/dL) reduction in LDL-C [5]. Furthermore, prospective cohort studies have shown that exposure to elevated LDL-C levels during young adulthood confers increased risk for future ASCVD [6–9]. Given the significant relationship between LDL-C and cardiovascular health, and its prominence in guidelines and clinical practice, it is of the utmost importance that we have accurate means of determining patient LDL-C levels.
Friedewald and colleagues set the stage for future studies examining the relationship between LDL-C and ASCVD in their ground breaking 1972 publication of a formula to calculate LDL-C (Total Cholesterol (TC) – HDL-C – (Triglycerides (TG)/2.2) in mmol/L or (Triglycerides/5) in mg/dl) [10] derived from 448 individuals. However, as noted by the authors, the use of a fixed factor to estimate very low density lipoprotein cholesterol (VLDL-C) is problematic at low LDL-C values and high triglyceride values. Since this original publication, many groups have attempted to identify more accurate methods for calculating LDL-C. This has remained important in the absence of a cost-effective and accurate direct measurement. Although non-ultracentrifugation chemical based direct assays have emerged, these add expense and are not well validated for the measurement of LDL-C. Each direct chemical assay uses proprietary chemicals in an attempt to block non-LDL lipoproteins and facilitate measurement of LDL-C. However, these direct assays are not standardized, have limited data supporting their accuracy, and limited traceability, which lead to method dependent differences in LDL-C. As highlighted in the excellent summary article of the consensus statements of the European Atherosclerosis Society (EAS) and the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM) by
⁎ Corresponding author at: Ciccarone Center for the Prevention of Cardiovascular Disease, Johns Hopkins Hospital, 600 N. Wolfe St, Carnegie 591, Baltimore, MD 21287 USA. E-mail address: [email protected] (S.S. Martin).
https://doi.org/10.1016/j.cca.2020.03.030 Received 12 February 2020; Received in revised form 17 March 2020; Accepted 19 March 2020 Available online 20 March 2020 0009-8981/ © 2020 Elsevier B.V. All rights reserved.
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multiple studies from around the globe have since validated these results [23,24,26–30]. Importantly, this method has been validated in different subsets of patients. Whelton led a study in 2017 of NHANES, Johns Hopkins, and Mayo Clinic data showing that approximately 20% of individuals with LDL-C < 70 mg/dL (1.8 mmol/L) by the Friedewald equation actually had values ≥ 70 mg/dL (1.8 mmol/L) based on the Martin/Hopkins method [26]. Significance of the upward reclassification by Martin/Hopkins calculation was corroborated by LDLC measured by ultracentrifugation, as well as non-HDL-C and apoB levels. This method therefore improves LDL-C accuracy at low LDL-C levels (< 1.8 mmol/L), which has important implications given recent American College of Cardiology/American Heart Association (ACC/ AHA) and European Society of Cardiology/European Atherosclerosis Society (ESC/EAS) guidelines [2,3]. By correcting underestimation that would otherwise occur with the Friedewald equation, clinicians can avoid undertreatment of high risk patients. It has also been shown to be more accurate in patients taking proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors [23], patients with diabetes mellitus [27], patients with familial combined hyperlipidemia [31], and in nonfasting samples [25]. This method becomes especially important among patients with diabetes given that direct chemical LDL-C assays are even more discrepant among these patients [11]. With a wealth of data supporting the method, the clinical laboratory at Johns Hopkins and Quest Diagnostics have adopted the Martin/Hopkins method. It can be installed on the lab information system for automatic calculation of LDL-C. Laboratories can contact [email protected] for licensing. Of note, neither the Friedewald nor the Martin/Hopkins method is well suited to the setting of severe hypertriglyceridemia [14] where the presence of chylomicrons complicates LDL-C estimation. A summary of the strengths and limitations of different methods that are currently used clinically for determining patient LDL-C levels is depicted in Table 1. Recently, another method for calculating LDL-C was developed and compared to both the Friedewald and Martin/Hopkins calculations [32]. Data from 8,656 patients seen in the 1970–90s at the National Institutes of Health were used to derive the proposed equation: TC/ 0.948 minus HDL-C/0.971 minus (TG/8.56 plus [TG × non-HDL-C]/ 2140 minus TG2/16100) minus 9.44. Compared with the Martin/ Hopkins calculation, it was derived in a population < 1% the size and a population not reflective of routine contemporary clinical practice. The lipid profiles of the patient sample were skewed towards high TG and cholesterol. It is suggested that the new equation allows for more accurate calculation of LDL-C up to TG levels of 800 mg/dL, however the comparison was to Friedewald and Martin/Hopkins and these estimates are not currently limited to TG < 400 mg/dL and not intended for use up to TG levels of 800 mg/dL. The large mean absolute difference of 24.9 mg/dL reported for the proposed equation is concerning as this amount of error approaches the width of established clinical categories (30 mg/dL). Furthermore, the clinical priority in patients with TG ≥ 400 mg/dL is TG lowering to prevent pancreatitis. Overall, the proposed new equation requires further independent validation. In particular, dedicated testing is required in patients intensively treated to very low LDL-C levels < 40 mg/dL since the analyses provided in the report are incomplete and raise concern for underestimation of LDLC in such samples.
Langlois et al., LDL-C values obtained via direct chemical assays vary significantly among different manufacturers, most prominently among patients with hypertriglyceridemia (> 2 mmol/L), mixed dyslipidemia, and diabetes mellitus [11]. Since these direct LDL-C assays are not standardized, changes in LDL-C over time may not be reliable, rendering it challenging for a clinician to optimize lipid lowering therapy when using different manufacturers’ assays for therapeutic follow-up [11]. It should also be noted that calculated LDL-C values may be affected by (1) the biological variability of TG levels and (2) the variability of direct HDL-C assays among different manufacturers [12,13], with the largest discrepancy seen in dyslipidemic patients with low HDL-C levels [11]. All methods of LDL-C calculation are dependent in accuracy on the data used for calculation, namely total cholesterol, triglycerides, and HDL-C. The homogeneous HDL-C assays in particular may suffer from inaccuracy and insufficient traceability. Prior to publication of the novel Martin/Hopkins equation (TC – HDL-C – TG/adjustable factor) in 2013, the majority of previously published methods for calculating LDL-C failed to account for the interindividual variance in the TG:VLDL-C ratio, which serves as the backbone of this equation [14]. In 1986, DeLong et al. attempted to improve on the Friedewald equation by proposing to divide TG by 6 instead of 5 [15], yet this method still relied on a fixed factor. Chen et al. in 2010 [16] proposed the formula LDL-C (mg/dL) = non-HDLC × 90% - TG × 10%, while de Cordova et al. in 2013 [17] suggested LDL-C = ¾ (TC – HDL-C). Many other formulas have been published as well, but they all overlook this important interindividual variance [18–20] or utilize an adjustable factor that considers only TG levels as opposed to cholesterol concentrations [21]. A 2015 analysis of four formulas in hospitalized patients (not including the Martin/Hopkins equation) demonstrated that the Hattori formula (LDL-C = 0.94TC – 0.94HDL-C – 0.19TG) outperformed the Friedewald, Chen, and De Cordova formulas in hospitalized patients using a direct chemical LDL-C assay as the reference standard. The Hattori method [20], which was obtained via regression analysis, also is a fixed formula that does not account for the interindividual variance, as noted above. A recently developed equation in a Ghanian population also relied on a fixed factor [22]. Many of these studies evaluating different formulas for LDL-C calculation used direct LDL-C assays as the reference standard for comparison, which as mentioned above have significant shortcomings. Ideally, ultracentrifugation should be used as the reference standard, as was done for the DeLong [15], Hattori [20], Rao [21], and Martin [14] studies, as well as in analyses validating the Martin/Hopkins method [23–26]. The clinical relevance of estimating LDL-C accurately and the longstanding use of the Friedewald equation in clinical practice led Martin et al. in 2013 to develop an equation with a similar structure to Friedewald’s original formula [14]. However, it differs in a fundamental way in that it relies on distinct adjustable factors based on individual patient TG and non-HDL-C levels (174 different factors for TG < 400 mg/dL). The adjustable factors were obtained by taking the median ratio of TG to VLDL-C values stratified by TG and non-HDL-C levels in table format. A high level of confidence was generated for each cell in the table because of the very large population for derivation (~1 million) and demonstration that the dataset is nationally representative, with lipid distributions similar to the National Health and Nutrition Examination Survey (NHANES). Of note, in the middle of these tables were ratios that closely approximated the original fixed factor of 5 that Friedewald et al proposed for calculating in mg/dL (2.2 in mmol/L), indicating that this formula is generally accurate in the average patient. However, it is in the patients who are not average where using an adjusted factor increases accuracy in determining LDL-C concentrations since the majority of the variance of TG/VLDL-C was explained by TG and non-HDL-C levels. Since publication of these results based on the analysis of more than 1,350,000 patients from the Very Large Database of Lipids Study,
3. ACC/AHA and ESC/EAS guidelines on LDL-C The recent AHA/ACC and ESC/EAS guidelines both highlight the importance of focusing on LDL-C for primary and secondary prevention of ASCVD. Both sets of guidelines emphasize using maximally tolerated statin therapy to decrease LDL-C levels by > 50% followed by nonstatin therapy if the LDL-C is above a certain threshold (70 mg/dL or 1.8 mmol/L for the AHA/ACC guidelines and 55 mg/dL or 1.4 mmol/L for the ESC/EAS guidelines) in very high-risk patients [2,3]. Additionally, both guidelines emphasize that non-fasting samples are 150
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Recommended at TG levels > 4.5 mmol/L
√ – – √ √ – √ –
adequate for the vast majority of patients for risk stratification, as nonfasting samples have only mildly elevated TG levels compared to fasting samples (approximately 0.3 mmol/L or 27 mg/dL) [3,33] and have similar prognostic value compared to fasting samples [2,3,34]. However, they do differ in their LDL-C cutpoints and endorsement of novel methods for calculating LDL-C. On the one hand, the AHA/ACC Multi-society guidelines state that the Friedewald equation is generally sufficient but if TG levels are elevated (≥150 mg/dL or 1.7 mmol/L) or LDL-C is low (< 70 mg/dL or 1.8 mmol/L), then the Martin/Hopkins formula can be utilized (2). On the other hand, the ESC/EAS guidelines endorse use of the Friedewald equation and acknowledge its limitations, including at TG levels ≥2 mmol/L or 177 mg/dL and at very low LDL-C levels, but do not directly endorse novel LDL-C calculation methods (3). The ESC/EAS guidelines were released before the latest EAS/EFLM recommendations and it is anticipated that the next ESC/ EAS guidelines will be updated to endorse the Martin/Hopkins formula. Given the ESC/EAS guidelines’ emphasis on aggressively treating LDL-C to < 1.4 mmol/L or 55 mg/dL in very-high risk patients, use of a reliable method to calculate LDL-C at these low values is extremely important, as it has clinical ramifications for patient care. Use of the Friedewald equation at such low LDL-C levels may lead to underestimation of these values and would thus preclude very-high risk patients from being candidates for further lipid lowering therapy, such as PCSK9 inhibitors or ezetimibe, as shown in Fig. 1. The lower LDL-C goes, the more probability it is underestimated by the Friedewald equation, and the ESC/EAS guidelines also have an even more aggressive goal of < 1 mmol/L or < 40 mg/dL in very-high risk patients with progressive disease. To implement these recommendations well would require more accurate LDL-C assessment. 4. EAS/EFLM guidelines on LDL-C
√ – √ –
The guidelines released by EAS and EFLM and summarized by Langlois et al. endorse the Martin/Hopkins equation in patients with low LDL-C levels < 1.8 mmol/L (70 mg/dL) and/or TG levels between 2.0 and 4.5 mmol/L (175–400 mg/dL) [11] and make multiple important recommendations. Firstly, they highlight the major concerns with direct LDL-C assays, especially among patients with obesity, metabolic syndrome, and diabetes [11]. These assays can be used among patients with TG ≥ 4.5 mmol/L though are not particularly accurate in that setting and the clinical priority in severe hypertriglyceridemia becomes TG lowering to avoid pancreatitis. Secondly, they emphasize that using the Friedewald equation at low LDL-C values (< 1.8 mmol/ L) overestimates the VLDL-C component of cholesterol, thus underestimating the LDL-C fraction, which risks leading to underuse of guideline directed medical therapy and less favorable outcomes in high risk patients. Thirdly, they recommend using non-fasting lipid panels to assess patient risk for ASCVD, though one may consider fasting lipids if TG concentration is ≥4.5 mmol/L on the non-fasting sample. Lastly, they recommend primary emphasis on targeting LDL-C but that nonHDL-C and apoB can be used as secondary treatment targets in select patients, though recent evidence suggests that using these secondary targets may only be helpful in a small fraction of patients when using the Martin/Hopkins equation for calculating LDL-C [35].
√ √ √ – – √ √ √ √ √ √ – √ – – – Ultracentrifugation Friedewald Equation Martin/Hopkins Equation Direct LDL-C assays
Widely Clinically Available Well validated Gold Standard Method
Table 1 A comparison of methods to determine patient LDL-C levels.
Reliable in a majority of patients
Accurate at high TG (2.0–4.5 mmol/L) and/or low LDL-C levels (< 1.8 mmol/L)
Equivalent efficacy with fasting and nonfasting samples
A.J. Brownstein and S.S. Martin
5. Clinical implications Ever since publication of the Friedewald equation and the development of life-saving lipid lowering therapy, including statins, ezetimibe, and PCSK9 inhibitors, our ability to transform cardiovascular care for our patients has been remarkable. With the release of the AHA/ ACC, EAS/ESC, and EAS/EFLM guidelines and their emphasis on both the importance of LDL-C levels and employing accurate means of calculating them, clinicians now have the armamentarium to significantly improve the lives of our patients. Yet, recent research demonstrates that the medical community is not optimally screening high risk patients 151
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Fig. 1. Clinical Case: comparing the Friedewald and Martin/Hopkins Equations.
with lipid panels after myocardial infarctions and intensifying lipidlowering therapy to achieve optimal reductions in LDL-C [36–38]. It is imperative that we begin to implement decades of research demonstrating the importance of LDL-C targeted therapy on the front lines of clinical care for the health of our patients.
[5]
[6]
CRediT authorship contribution statement [7]
Adam J. Brownstein: Conceptualization, Writing - original draft. Seth S. Martin: Conceptualization, Writing - review & editing.
[8]
Declaration of Competing Interest [9]
SSM: consultant/advisory board for Sanofi, Regeneron, Amgen, Quest Diagnostics, Akcea, Novo Nordisk, Esperion; co-inventor for method to estimate LDL cholesterol levels, patent application by Johns Hopkins University pending and research support from Akcea, paid to the institution.
[10]
[11]
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