Study of serum non-HDL cholesterol in cerebrovascular disease

Background: Non-HDL cholesterol is a potential newer risk factor for cerebrovascular diseases (CVD). Objective: To explore the association of non-HDL cholesterol with cerebrovascular disease. Methods: This case control study was carried out in the Department of Biochemistry, BSMMU, Dhaka during the period of January to December 2007 to evaluate the association of non-HDL cholesterol with CVD in Bangladeshi population. A total number of 135 subjects of both sexes were grouped as Group(CVD cases) and Group-II (Healthy controls). Group-I include 85 cases of which 59 were ischaemic cerebrovascular diseases (ICVD) and 26 were haemorrhagic cerebrovascular diseases (HCVD). By taking the history and doing clinical examination and laboratory investigations, diabetes mellitus, malignant disease, renal disease, liver disease and diuretic medication were excluded from study subjects. Serum non-HDL cholesterol was measured in all study subjects. Statistical analysis was performed by using SPSS for windows version 12.0. Mean values of the findings were compared between groups. One way ANOVA test and multiple comparison (Bonferroni‘t’) test were used to see the level of significance. Results: Serum non-HDL cholesterol found significantly increased in CVD, ICVD and HCVD cases in comparison to control subjects. But ICVD and HCVD cases did not differ with respect to serum non-HDLcholesterol. Conclusion: The result shows that elevated non-HDL cholesterol is associated with CVD. Prospective study with large sample size is required to evaluate the elevated Non-HDL cholesterol as a risk factor of CVD.


Introduction
Cerebrovascular diseases (CVD) or strokes are one of the most common causes of mortality and long-term severe disability.It is the third leading cause of death after coronary heart disease (CHD) and malignancy and important cause of hospital admission in global perspective 1 .In Japan and other Asian countries including Bangladesh, CVD remains the most common disease.Despite the recent remarkable decrease in mortality, the proportion of patients treated for stroke as well as the prevalence of stroke has remained unchanged or even tended to increase 2 .Stroke accounts for 10-12% of all deaths in industrial countries.According to the consensus' statements on stroke, every 5 minutes someone in UK is having a stroke causing one in eight deaths and constitutes a formidable burden of disability and misery for patients, their caregivers and the wider community 3 .In USA, there are approximately 500,000 cases of stroke each year and of these 150,000 cases are left with mental and physical impairment requiring assistance for activities of daily living.The cost of acute and long term care for stroke patients is about 30 billion dollar per year in USA 4 .There are two classical type of CVD from pathophysiological point of view: Ischemic cerebrovascular diseases (ICVD) and Hemorrhagic cerebrovascular diseases (HCVD).In ICVD that is caused by occlusion of cerebral vessels thromboembolic phenomenon is primarily responsible, where hyperlipidemia and atherosclerosis plays a central role 5 .In HCVD that happens following the rupture of intracerebral or subarachnoid vessels chronic hypertension plays a central role 6 .Hypertension is the most consistently powerful predictor of stroke and it is a factor in nearly 70% stroke of both types 7,8 .
It is now apparently clear that hyperlipidemia leading to atherosclerosis predispose ICVD, although chronic hypertension may enhance the atherosclerotic plaque formation in hyperlipidemic subjects.In contrast, hypertension seems to be the sole factor to be incriminating in HCVD.Atherosclerosis is claimed to be involved with coronary artery disease (CAD), cerebrovascular disease (CVD) and peripheral vascular disease (PVD).Lipids and lipoprotein disorders are important metabolic risk factors of atherosclerosis.There is overwhelming convincing evidences relating hypercholesterolemia, increased LDL cholesterol, decreased HDL cholesterol and increased total cholesterol and HDL cholesterol ratio (TC/HDL) with CAD but their relation to CVD is controversial.Some studies showed positive correlation of ICVD with total cholesterol, LDL cholesterol, TAG and (TC/HDL) but negative correlation with HDL cholesterol 9,10,11 .In contrast, several other studies reported no association between stroke and total cholesterol, LDL cholesterol, HDL cholesterol, TAG 12,13,14  The only antiatherogenic lipoprotein is the HDL cholesterol.Plasma total cholesterol represent sum of the cholesterol content of all circulating lipoproteins irrespective of their atherogenic potential.So non-HDL cholesterol (total cholesterol minus HDL cholesterol) is more comprehensive measure of atherogenic lipoprotein than LDL cholesterol alone since it (non-HDL cholesterol) includes LDL cholesterol, VLDL, CMR, IDL and LP (a) .Therefore, measuring non-HDL cholesterol reflects atherogenic risk not captured by LDL cholesterol alone particularly in the context of hypertriglyceridemia, where there is increased concentration of CM, VLDL, CMR and IDL.Recently non-HDL cholesterol has shown to be a better predictor of cardiovascular death than LDL cholesterol even in patient with TAG concentrations <200mg/dl.So, although plasma LDL cholesterol is well established as a predictor of CAD, it may not be the best circulating marker, rather non-HDL cholesterol could be the right choice.To calculate non-HDL cholesterol, it is enough to measure total cholesterol and HDL cholesterol for which there is no need to put the patient in fasting state so as in LDL cholesterol measurement, which need fasting sample.So, non-HDL cholesterol is more universal and technically more compliant measure to assess atherogenic potential of an individual 15 .Relation of non-HDL cholesterol, LDL cholesterol and atherogenic risk was addressed by the NCEP-ATP III.When serum TAG is >150mg/dl, LDL cholesterol alone is not sufficient to define the risk associated with atherogenic lipoproteins, where non-HDL cholesterol is the best choice 16 .
In Bangladesh, CVD stands for quite a sizeable number of mortality and morbidity posing a major socioeconomic challenge in the rehabilitation of stroke survivours but the best option for stroke still remains in its prevention.So, we should take appropriate measures for prevention.Traditional modifiable risk factors are now being treated but there is a pressing need to identify additional treatable newer risk factors that are easily measured and highly prevalent in general population.Non-HDL cholesterol is such type of potentially modifiable newer risk factor.It is possible to reduce the probability of stroke by lowering down the raised non-HDL cholesterol, as it is related to stroke.Some limited studies of serum lipid and lipoprotein in CVD have been done but the studies involving non-HDL cholesterol are scanty in our population.So, the aim of this study is to evaluate the association between elevated non-HDL cholesterol with stroke in our population.

Materials and Methods
This case-control study was carried out in the Department of Biochemistry, Bangabandhu Sheikh Mujib Medical University (BSMMU), Dhaka during the period of January to December 2007.135 subjects of both sexes were grouped as Group-I (CVD cases) and Group-II (Healthy control).Group-I included 85 cases, of which 59 had ICVD and 26 had HCVD.Among ICVD cases, 42 were male and 17 were female and among HCVD cases, 19 were male and 7 were female.Group-II included 50 healthy controls of which 37 were male and 13 were female.By taking the history and doing clinical examination and laboratory investigations, diabetes mellitus, malignant disease, renal disease, thyroid disorder, liver disease and diuretic medication were excluded from study subjects.Ethical clearance for the study was taken from the Departmental and Central Ethical Committee, BSMMU, Dhaka.Permission for the study was taken from the concerned departments from where we collected our study subjects.
Informed written consent was taken from patient / attendants of all study subjects.5 ml fasting venous blood was collected from all study subjects with full aseptic precaution blood was allowed to clot and then centrifuged.Separated serum was then collected and preserved at -35°c and later on used for the measurement of lipid profile, creatinine and sugar concentration.All data were recorded systematically in a preformed data collection form and were expressed as mean (SD).Statistical analyses were performed by using SPSS for windows version 12.0.Mean values of the findings were compared between groups.One way ANOVA test and multiple comparison (Bonferroni't') test were used to see the level of significance.95% confidence limit (p<0.05) was taken as level of significance.

Results
Total 135 subjects of both sexes were grouped as Group-I (CVD cases) and Group-II (Healthy control).The serum non-HDL cholesterol concentration of all the study subjects was estimated and the results were expressed as mean(SD).The units of measurements were mg/dl.Group-I included 85 cases, 59 were ICVD and 26 were HCVD.Among ICVD cases 42 were male and 17 were female with mean age of 58.34(7.01)years and age range of 40-68 years and among HCVD cases 19 were male and 7 were female with mean age of 59.31(6.73)years and age range of 45-68 years.Group-II included 50 healthy controls, 37 were male and 13 were female with mean age of 50.48(5.50)years and age range of 40-62 years.The mean(SD) of non-HDL-c concentration of CVD, ICVD and HCVD cases and control subjects were 198.48(34.82)mg/dl with the range 129-358 mg/dl, 200.14(37.09)mg/dl with the range 152-358 mg/dl, 194.73(29.34)mg/dl with the range 129-258 mg/dl and 129.42 (27.46) mg/dl with the range 51-161 mg/dl, respectively.

Discussion
In this present case control study, the serum non-HDL cholesterol concentration was measured in 85 diagnosed CVD patients and 50 healthy control subjects to evaluate the association of non-HDL cholesterol with CVD.The mean non-HDL cholesterol level found to be significantly high (p 0.001) in CVD cases compared to their control value.This finding is consistent with other similar studies done abroad 17,18 .They concluded that elevated levels of serum triglycerides and non-HDL are associated with large arteries' atherosclerotic stroke.Those with the highest triglycerides were 2.7 times and those with highest non-HDL were 2.4 times more likely to have a large artery stroke.Recently, the use of non-HDL cholesterol level has been suggested as a better tool for risk assessment and treatment than LDL cholesterol level because non-HDL cholesterol includes all cholesterol present in lipoprotein particles considered to be atherogenic, including LDL, IDL, VLDL, CMR, LP (a) etc. and estimation of LDL cholesterol level using the formula can be inaccurate, when TAG is high.Despite the potential usefulness of non-HDL cholesterol level, only a few studies were done that have demonstrated that elevated non-HDL cholesterol level is associated with an increased risk for development of CVD 19 .In the present study, we also found high non-HDL cholesterol level in both ICVD and HCVD cases compare to control.But no statistically significant difference was found in between ICVD and HCVD cases with respect to non-HDL cholesterol.So, elevated non-HDL cholesterol level found to be associated with an increased risk for CVD irrespective of their clinical types.Serum TC, TAG & LDL cholesterol concentration were also found to be elevated significantly in CVD cases in comparison to control, which is consistent with that of other studies done abroad 20,21,22 .We have found serum HDL-C, significantly low in CVD cases in comparison to control which is supported by Sacco et al. 23 .Iso et al. 24 surprisingly found that the lowering of serum total cholesterol does not reduces the stroke mortality and morbidity, rather predispose HCVD, which is a consequence of weakening of intracerebral arterial endothelium.Sridharan 20 found low HDL cholesterol level and a high total cholesterol and HDL cholesterol ratio (TC/HDL) among the stroke patients.Sacco et al. 23 compared cholesterol levels in stroke patients with control subjects and found an inverse association between HDL cholesterol and the risk of ischemic stroke.Ebrahim et al. 25 studied blood cholesterol in a large cohort of young and middle aged Korean civil servants and found that low concentrations of cholesterol were associated with hemorrhagic stroke while high concentrations were associated with ischemic stroke.Higher total cholesterol and lower HDL cholesterol levels were associated with increased risk of ischemic stroke.The lowest levels of total cholesterol were associated with an increased risk of all hemorrhagic strokes 21.There is mounting epidemiologic evidence to support the relationship of lipids as a risk factor for ischemic stroke 26 .A 10 years follow up study of Japanese men and women demonstrated that lower HDL cholesterol levels were related significantly and independently to increased risk of all stroke incidences including the ischemic stroke incidence 27 .A new analysis of 61 prospective observational studies has failed to find any association of total cholesterol with stroke mortality 28 .LDL cholesterol was the common risk factor for ischemic stroke in men and women, whereas, non-HDL cholesterol, total cholesterol and their ratio were related to ischaemic stroke as risk factors only in women 22 .Another study done by Kurth et al. 39 revealed that total cholesterol, LDL cholesterol, TC/HDL, and non-HDL cholesterol are risk factors for ischaemic stroke only in women.These studies provide convincing evidence that atherogenic lipoproteins are strongly associated with atherogenicity, hypertension and CVD.Total cholesterol as well as LDL cholesterol is found to provide myopic view of the total atherogenic potential.Since non-HDL-c reflects the total plasma level of all atherogenic lipoproteins, it is especially promising as an acceptable, easy way to measure surrogate biomarker of atherogenic risk and thus, can be integrated into clinical practice as a comprehensive risk factor for CVD.______________

Table I :
Serum non-HDL cholesterol concentration among the groups of study subject *

Table - II
: Comparison of serum non-HDL cholesterol level between cases and controls**

Table III :
Comparison of serum non-HDL cholesterol level among the three groups of study subjects (ICVD, HCVD and controls) ***

Table IV :
Comparison of serum non-HDL cholesterol between different groups of study subjects****