Novel concept of non-HDL cholesterol

- A predictor of cardiovascular disease risk in type II diabetes mellitus patients

 

 

Abstract               Background and Aim: Non-communicable diseases (NCDs) such as coronary artery disease (CAD) and diabetes mellitus (DM) are assuming importance due to an accelerating rate of morbidity and mortality. The overall mortality from acute myocardial infarction in the diabetic population is reported to be 4-7 times higher. Modern laboratory diagnosis of cardiovascular risk and dyslipidemia should be based on the use of indicators which present full impact of all plasma lipid components involved in atherosclerosis. One such marker is Non-HDL-C, the sum total of cholesterol accumulated in all lipoproteins, except HDL. Materials and Methods: The study population included 160 patients aged between 30 and 65 years with duration of diabetes more than five years. Age and gender matched 160 healthy subjects were enrolled as controls. Serum sample was used for the estimation of study parameters such as FBS, PPBS, Total Cholesterol, Triglycerides, HDL Cholesterol, LDL Cholesterol. Non-HDL cholesterol was calculated. Results: Total cholesterol, triglycerides, LDL-C, Non-HDL-C, TC/HDL-C and LDL-C/HDL-C were found to be significantly increased in type 2 diabetes mellitus patients when compared to controls. On the contrary, HDL-C was found significantly decreased in cases. Conclusion: Non-HDL cholesterol is a significant predictor of CVD risk in diabetic population. Because diabetic patients are at high risk for CVD, adequate risk assessment and appropriate management is imperative. Clinical Significance: Increased levels of Non-HDL-C, TC/HDL-C and LDL-C/HDL-C ratio indicate increased impending cardiovascular disease risk in diabetes mellitus.

Key Word: Cardiovascular disease, Dyslipidemia, LDL-C/HDL-C ratio, Non-HDL-Cholesterol, TC/HDL-C ratio, Type 2 Diabetes Mellitus

 

 

 

 

 

INTRODUCTION

The tag of importance attached to Non-communicable diseases (NCDs) such as coronary artery disease (CAD) and diabetes mellitus (DM) is due to an accelerating rate of morbidity and mortality, even in developing countries like India.¹ Deaths due to cardiovascular disease (CVD) account for up to 30% of all global deaths.^2,3 Compared to non-diabetic population, the overall mortality from acute myocardial infarction in the diabetic population is reported to be 4-7 times higher.³ Unfortunately, off late, the prevalence of these NCDs is becoming higher in middle- and lower-income groups in India.¹ The risk for CAD is two to four times higher in diabetic subjects ^1,3 and in Indians, CAD manifests a decade or two earlier. As type 2 diabetes shares several risk factors in common with CAD such as age, hypertension, dyslipidemia, obesity, physical inactivity and stress, an increase in the prevalence of diabetes indirectly implicates an escalating risk of CAD as well. Though these are not new risks, their ubiquitous presence is thought to be the driving force behind this epidemic.⁴ The main cause of CVD development is very well known to be atherosclerosis, mainly caused by dyslipidemia, characterized by elevated triglyceride (TG), low levels of HDL cholesterol (HDL-C) and a higher preponderance of LDL cholesterol (LDL-C). Abnormalities in lipid levels associated with diabetes mellitus are attributed to increased flux of free fatty acids secondary to insulin resistance.⁵ Modern laboratory diagnosis of cardiovascular risk and dyslipidemia should be based on the use of indicators which present full impact of all plasma lipid components involved in atherosclerosis. One such marker is Non-HDL-C, the sum total of cholesterol accumulated in all lipoproteins, except HDL i.e., chylomicrons, VLDL, their remnants IDL, LDL and Lp(a). Actually, not much attention is paid to the use of non-HDL-C but the latest guidelines from both European and American Cardiological Societies highlight the importance of this novel parameter for assessing the risk of atherosclerosis and CAD. Non-HDL-Cholesterol is considered as the second, just after the LDL-C goal of CVD therapy in patients with hypertriglyceridemia and should be calculated routinely in lipid profile.⁶ NCEP ATP III has stated that LDL-C alone is not a valid basis for therapeutic interventions at TG level over 200 mg/dL; rather Non-HDL cholesterol is identified as the therapeutic target at that level of TG. Non-HDL-C measurement could be more representative of all atherogenic apolipoprotein B (apoB) containing lipoproteins such as LDL-C, VLDL-C, IDL-C and lipoprotein (a). It has been suggested that this novel marker may prove to be a strong predictor of mortality due to CAD as well as non-fatal coronary events, than LDL-C in people with diabetes. Raised Non-HDL-C signifies accelerated cardiovascular disease risk even if LDL-C levels are at or below NCEP goal. Further, Non-HDL-C can also be used as a predictor of CAD in individuals with or without type 2 diabetes mellitus, metabolic syndrome or hypertriglyceridemia.^7,8 Although apolipoprotein B can be measured directly, quantifying the levels of Non-HDL cholesterol can be considered as a surrogate marker for apolipoprotein B, as it helps in evaluating the patient response during treatment. Further, LDL-C / HDL-C ratio is more accurate than either of the parameters alone since a ratio of > 4.1 is known to predispose to heart disease.^9,10 Interestingly, there is paucity of published literature on non-HDL-C from Indian population. These facts stimulated us to assess Non-HDL-Cholesterol level among patients with T2DM and to determine various lipid ratios. The very fact that glycaemic control in cardiovascular risk reduction has evolved as a paradigm in recent years stresses the importance of attending to the needs of the co-morbidities in overall reduction in morbidity and mortality.

 

MATERIALS AND METHODS

This hospital based study was conducted at a tertiary care hospital of Karnataka after obtaining institutional ethical clearance. The study population included 160 patients aged between 30 and 65 years with duration of diabetes more than five years. Age and gender matched 160 healthy subjects were enrolled as controls. All the subjects were explained about the study and informed written consent obtained well in advance. Patients with type 1 diabetes mellitus, gestational diabetes, Type 2 Diabetes Mellitus patients on hypolipidemic drugs, patients with thyroid disorders and/or obstructive liver disorders were excluded from the study. Patient history related to cardiovascular events was also recorded in addition to their clinical history and baseline demographic data. Following all standard protocols, aseptically collected venous blood (from ante-cubital vein) was used for biochemical analyses. Various methodologies followed for estimating our study parameters include: Glucose oxidase-peroxidase method (for FBS, PPBS), Urease method (for urea), Jaffe’s method (for creatinine), Uricase method (for uric acid), Cholesterol oxidase/peroxidase method (for total cholesterol), GPO-POD method (for triglycerides), enzymatic colorimetric methods (for HDL and LDL cholesterols). Non-HDL-C was obtained by subtracting HDL Cholesterol from total cholesterol levels. Statistical analysis was performed using SPSS 20. Data was expressed as mean ±SD. ANOVA was used for continuous variables and the chi-square test for categorical variables. P value <0.05 was considered statistically significant while those with ≤ 0.001 as highly significant.

OBSERVATIONS AND RESULTS

In this study, total cholesterol, triglycerides, LDL-C, Non-HDL-C, TC/HDL-C and LDL-C/HDL-C were found to be significantly increased in type 2 diabetes mellitus patients when compared to controls. On the contrary, HDL-C was found significantly decreased in cases. Results are summarized in table 1.

 

Table 1: Enumeration of demographics and lipid profile parameters of cases and controls

^a Statistically highly significant, ^bStatistically not significant

On performing correlation studies of blood sugar levels with other parameters, a very significant positive association, with a p value of 0.001 was obtained for the parameters: serum total cholesterol, serum triglycerides, LDL-C, Non-HDL cholesterol, TC/HDL-C and LDL-C/HDL-C ratios and a significant negative correlation with HDL-C amongst cases.

DISCUSSION

The life expectancy of people with diabetes is drastically reduced by almost eight years due to increased mortality. CAD accounts for > 80% of overall deaths and 75% of hospitalizations in diabetic subjects. Reports confirm that plaques are more vulnerable to rupture among patients with diabetes. The association between CAD and diabetes is strong despite the fact that there are wide geographic and ethnic variations in their prevalence^3,1. Broadly speaking, established CVD risk factors most often do not occur alone; and addition of associated morbidities results in multiplicative, rather than additive amplification of risk.⁴ Although several factors play a role in accelerated atherosclerosis associated with diabetes, lipoprotein abnormalities are the vital contributors. LDL, the main cholesterol-containing lipoprotein, is a major determinant of atherosclerosis in patients with diabetes. Whereas average LDL concentrations in diabetic patients may not be higher than those of their non-diabetic counterparts, changes in LDL particle composition, such as its density, oxidation potential and glycation render even normal LDL levels highly atherogenic. Other lipoprotein imbalances in patients with diabetes include changes in triglyceride-rich lipoproteins, leading to accumulation of VLDL remnants and IDL (both being highly atherogenic) and subsequent increase in foam cell formation, prothrombotic and procoagulant factors. Because of varied lipoprotein abnormalities in diabetes, an easily measurable but composite indicator may be useful to clinicians so that attentive management of all atherogenic subfractions of lipids becomes possible.^11,4 The ATP III of the NCEP recently recommended that non-HDL cholesterol could be used as a secondary target of therapy in people with TGs >200 mg/dl, especially in those with diabetes or metabolic syndrome. There are several advantages to it: First, it makes no assumption about the relationship between VLDL and triglycerides, since in patients with diabetes, this relationship can be altered, leading to false low LDL values as obtained by Friedewald formula, especially in association with elevated triglyceride levels. Second, non-HDL cholesterol includes an assessment of all apolipoprotein B-containing lipoproteins considered to be atherogenic, i.e., VLDL, IDL, LDL and even lipoprotein (a). Finally, non-HDL cholesterol has several practical advantages in a clinical setting, including the ability to be assessed in patients with triglyceride levels >400 mg/dl and in patients who are not fasting.¹¹ The usefulness of non-HDL-C in the prevention of CVD was confirmed in numerous clinical trials. Not lowering LDL-C alone, but of non-HDL cholesterol too is an important goal in prevention and treatment of cardiovascular diseases.⁶ Our study results presents with significant increase in total cholesterol, triglycerides, LDL-C, Non-HDL-C, TC/HDL-C and LDL-C/HDL-C ratios, but a significantly decreased HDL-C in cases. These observations are in accordance with results of Sapna S et al.¹⁰ In Diabetes Mellitus, skeletal muscle cells, heart and adipose tissue cells cannot take up and utilize glucose, instead obtain energy from oxidation of fatty acids, thus producing increased level of Acetyl Co-A which can be funneled for cholesterol synthesis, thus resulting in hypercholesterolemia. Similarly, in case of insulin resistance to fat cells, the activity of hormone sensitive lipase (HSL) increases resulting in enhanced lipolysis to release FFAs into circulation, which are taken up by various organs including liver (wherein FFAs leads to the synthesis of TG which along with cholesterol and apoproteins are incorporated into VLDL). Insulin deficiency is associated with decreased clearance of VLDL. Since, VLDL is rich in TG and cholesterol, it results in hypercholesterolemia and hypertriglyceridemia, together with decreased HDL and increased LDL. This can be better explained - VLDL transported TG is exchanged for HDL transported cholesteryl ester through the action of cholesteryl ester transfer protein (CETP), which results in increased amounts of both atherogenic cholesterol-rich VLDL remnant particles and TG-rich, cholesterol-deplete HDL particles. TG-rich HDL is subsequently hydrolysed by hepatic lipase or lipoprotein lipase; Apo A-I dissociates from small sized HDL, which is filtered by renal glomeruli and degraded in renal tubular cells.^{12, 13} Our study revealed that Non-HDL-C was significantly higher in diabetic mellitus patients than controls. It has been suggested that Non-HDL-C may be a strong predictor of CAD mortality and non-fatal coronary events than LDL-C in people with diabetes.¹⁴ Since, diabetic dyslipidemia is most commonly manifested as elevated TG and a decrease in HDL-C, with a predominance of low dense LDL-C particles and relatively normal LDL-C levels¹⁵, elevated Non-HDL-C implies an increased CVD risk even if LDL-C levels are at or below the NCEP goal or appear normal.¹⁶ Both NCEP and ADA recommend lowering LDL-C (primary target) and Non-HDL-C (secondary goal) to a goal of <100 mg/dL and <130mg/dL respectively in patients with diabetes.^{16, 17} Experimental evidence supports a more important role for apolipoprotein B (apoB) and apoB-containing lipoproteins than for LDL-C content in mediating atherogenesis. Lipoproteins containing apoB must first enter the arterial wall and undergo oxidation before they can contribute to atherogenesis. This modification not only affects the phospholipid layer of these lipoproteins, but also apo B’s structure, yielding ligands for scavenger receptors of macrophages in arterial wall.¹⁸ Subsequently, cholesterol accumulation and crystallization in macrophage cytoplasm leads to the formation of foam cells and progression to atherosclerotic plaque.¹⁹ Interestingly, measured apoB and Non HDL-C have been found to be highly correlated in a number of studies.^{20, 21} Recent studies involving subjects of different age groups have also shown the importance of Non-HDL-C as a reliable, less costly parameter that is strongly correlated with cardiovascular risk because Non-HDL-C includes all atherogenic lipid subfractions.²¹ Hypertension co-exists in a significant proportion of people with diabetes. Lowering blood pressure (BP) produces dramatic benefits in these subjects and BP targets have been modified specifically to avert disabling and fatal complications in the form of nephropathy, retinopathy, and vascular events.⁴ Currently available clinical data describe the relationship between the concentration of non-HDL-C and methods of imaging of atherosclerosis. The effect of serum lipids on the process of coronary arteries’ calcification (CAC), regarded as an early marker of subclinical atherosclerosis was described in a study by Orakzai et al and also many others.⁶ Hence, in patients with Diabetes, Non-HDL-C may be a stronger predictor of CVD than either LDL-C or TG. The present study showed significantly increased levels of TC/HDL-C and LDL-C/HDL-C ratios in type 2 diabetes mellitus patients compared to controls. Non-HDL-C and the ratios of TC to HDL-C and LDL-C to HDL-C may be superior to LDL-C alone in diabetic patients.

CONCLUSION

In brief, our results confirm that Non-HDL cholesterol is a significant predictor of CVD risk in diabetic population. Because diabetic patients are at high risk for morbidity and mortality due to CVD, adequate risk assessment and appropriate management is imperative.

 

CLINICAL SIGNIFICANCE

Simple measurement of non-HDL cholesterol, which can be conducted in the non-fasting state and regardless of triglyceride concentration, may be of immense help to the clinicians. The ATP-III of NCEP recommends a therapeutic goal of <130 mg/dl for non-HDL cholesterol.¹¹

 

LIMITATIONS

There is still much controversy about the use of non-HDL cholesterol in routine clinical / laboratory practice. Despite the numerous advantages it is not possible to exclude a higher diagnostic value of apolipoprotein B100 and apoB: apoAI ratio in primary CVD prevention.6 Estimated non-HDL-C value, combined with apolipoproteins, hsCRP and LDL particle number (LDL-P) assessment is suggested to be the most optimal solution. In a nutshell, routine use of simple non-HDL-C during lipid profile testing will nonetheless allow a better assessment of CVD risk.

 

REFERENCES

1. Mohan V, Venkatraman JV, Pradeepa R. Epidemiology of cardiovascular disease in type 2 diabetes: The Indian scenario. Journal of diabetes science and technology 2010; 4(1): 158-170
2. Chowdhury S, Chowdhury JR, Goswami S. The Importance of Non High Density Lipoprotein Cholesterol in Dyslipidemia Management. Diabetes and Metabolism 2015; 6(11): 1-5
3. Li YW, Aronow WS. Diabetes mellitus and cardiovascular disease. Journal of clinical and experimental cardiology 2011; 2(1): 114
4. Ali MK, Venkatnarayan KM, Tandon N. Diabetes and coronary heart disease: Current perspectives 2010 Nov, 132(5): 584-597
5. A Pandeya, Sharma M, Regmi P, Basukala A and Lamsal M. Pattern of dyslipidemia and evaluation of non-HDL cholesterol as a marker of risk factor for cardiovascular disease in type 2 diabetes mellitus. Nepal medical College Journal 2012; 14(4): 278-282
6. Bergmann K. Non HDL cholesterol and evaluation of cardiovascular disease risk. The journal of the international federation of clinical chemistry and laboratory medicine 2010 Oct; 21(3): 64-67
7. Kondru S, Thakur A. Role of Non-HDL Cholesterol and LDLc/HDLc ratio to assess cardiovascular risk in type II diabetes patients. International Research Journal of Medical Sciences 2015; 3(1): 23-28
8. Kumpatla S, Soni A, Narasingan SN, Viswanathan V. Presence of elevated Non-HDL among patients with T2DM with CV events despite of optimal LCL-C – A report from south India. Indian Heart J 2016 May-Jun; 68(3): 378-379
9. Natarajan S, Glick H, Criqui M, Horowitz D, Lipsitz SR and Kinosian B. Cholesterol measures to identify and treat individuals at risk for coronary heart disease. Am J Prev Med. 2003; 25: 50-57
10. Wilson PW, High-density lipoprotein, low-density lipoprotein and coronary artery disease. Am J Cardiol. 1990; 66(6): 7A-10A
11. Lu W, Resnick HE, Jablonski KA, Jones KL, Jain AK, Howard WJ et. al., Non-HDL cholesterol as a predictor of cardiovascular disease in type 2 diabetes. Diabetes care 2003 Jan; 26(1): 16-23
12. Sapna Smith and Alok M Lall. A study on lipid profile levels of diabetics and non-diabetics among Naini region of Allahabad, India. Turkish Journal of Biochemistry 2008; 33(4): 138-141
13. Mooradian AD, Haas MJ, Wehmeier KR, Wong NCW. Obesity-related changes in high-density lipoprotein metabolism. Obesity 2008; 16(6): 1152-1160
14. Indumathi V, Patil VS, Krishnaswamy D, Rajeshwari V. Non-HDL cholesterol and LDL-C/HDL-C ratio in type 2 diabetic patients. Int J Pharm Bio Sci.2011 Jan; 2(2): 71-77
15. Bittner V. Non-HDL Cholesterol: Measurement, interpretation and significance. John Hopkins Advance Studies Med. 2007; 7: 8-11
16. Peters AL. Clinical relevance of Non-HDL cholesterol in patients with diabetes. Clin Diabtes. 2008; 26: 3-7
17. Grundy SM. Low-density lipoprotein, non-high-density lipoprotein and apolipoprotein B as targets of lipid-lowering therapy. Circulation 2002; 285: 2486-2497
18. American Diabetes Association: Dyslipidemia management in adults with diabetes. Diabetes Care 2004; 27: S68-S71
19. Duewell P, Kono H, Rayner KJ, Sirois CM, Vladimer G, Bauernfeind FG et al., NLRP3 inflammasomes are required for atherogenesis and activated by cholesterol crystals. Nature 2010; 464(7293): 1357-1361
20. Grundy SM, Vega GL, Tomassini JE, tershakovec AM. Correlation of non-high-density lipoprotein cholesterol and low-density lipoprotein cholesterol with apolipoprotein B during simvastatin + fenofibrate therapy in patients with combined hyperlipidemia (a subanalysis of the SAFARI trial). Am J Cardiol. 2009; 104: 548-553
21. Wei fen Zhu, Liang L, Wang CL, fen FU J. Triglyceride and Non-High-Density-Lipoprotein Cholesterol as Preadictors of Cardiovascular Disease Risk Factors in Chinese Han Children. Indian Pediatrics 2013; 50: 394-398.