High-density lipoprotein (HDL) is known to have atheroprotective properties which could become dysfunctional under certain disease conditions, particularly during the atherosclerotic progression. The present study sought to assess HDL functionality in acute ischemic stroke (AIS) patients in comparison with controls and the functional alteration among the ischemic stroke subtypes. The HDL functionality was evaluated in 71 statin-naïve, adult patients of South Indian descent, admitted with AIS and were compared with that of 25 age- and sex-matched healthy volunteers. Functional assay of HDL was based on its antioxidant ability to inhibit low-density lipoproteins (LDL) oxidation by air using a dichlorodihydrofluorescein-based fluorescent assay and expressed as HDL oxidant index (HOI). The HOI was higher in ischemic stroke patients as compared with controls (1.07±0.32 vs. 0.51±0.12; P<0.001) indicating high oxidative stress and dysfunctionality in HDL. Regarding the stroke subtypes, HOI was >1 in all stroke subtypes: 1.05±0.301 for Cardioembolic subtype, 1.15±0.41 for large vessel disease, and 1.01±0.25 for small vessel disease when compared with controls. However, no significant difference was noted in HOI values among the three stroke subtypes in the post hoc analysis. It was found that HDL in all ischemic stroke subtypes had less antioxidant capacity, indicating dysfunctional HDL. Functional alteration occurred in HDL of patients even in the presence of normal HDL-cholesterol levels suggesting that dysfunctionality in HDL is unrelated to cholesterol content.
Atherothrombosis is one of the important causes of ischemic strokes (
We have previously identified HDL dysfunctionality in coronary artery disease (CAD) patients belonging to a South Indian cohort (
Our previous study observed a trend towards increased Apo B levels in intracranial atherosclerotic disease (ICAD) subtype of large vessel disease (LVD) despite showing no significant correlations between serum Apo B and A1 levels and the ischemic stroke subtypes (
Although several studies have reported the inverse association of HDL cholesterol with the risk of ischemic stroke (
The present cross-sectional study enrolled 75 consecutive, statin-naïve, first-ever ischemic stroke patients (age ≥18 years) of both sexes admitted to the Stroke unit of the Department of Neurology of Sree Chitra Tirunal Institute of Medical Science and Technology (SCTIMST), Trivandrum, India. Complete stroke etiologic workup was conducted to determine and classify stroke mechanism according to Trial of Org 10172 in Acute Stroke Treatment criteria (
The control group consisted of 25 age and sex-matched apparently healthy volunteers from the public and relatives of hospital staff without any history of co-morbidities, acute and chronic inflammatory disease, cancer, kidney or liver diseases and who were not on any anti-inflammatory medications or antioxidants. They were assessed based on a questionnaire and routine laboratory blood analysis for sugar and lipid profiles. The present study was approved by the institutional ethics committee at SCTIMST. Written informed consent was obtained from all participants enrolled in the study.
Blood samples were collected in EDTA-containing tubes after a 12 h period of overnight fasting from both patients and healthy volunteers. Plasma was separated by immediate centrifugation at 665 x g for 15 min at 25˚C, divided into aliquots and stored at -80˚C until further analysis.
Glucose, total cholesterol, HDL cholesterol, LDL cholesterol, and triglycerides were measured using enzymatic methods with the Dimension RxL Max Integrated Chemistry System (Siemens Healthcare Diagnostics Inc.). HDL was isolated from plasma by polyethylene glycol (PEG) precipitation method as previously described (
LDL was isolated from plasma of control subjects by standard sequential density gradient ultracentrifugation in Beckman Optima TLX 120 Ultracentrifuge with fixed angle rotor (Beckman Coulter, Inc.) (
HDL function can be measured by several assays including measuring its antioxidant capacity to prevent
For evaluating the HOI of the samples, LDL oxidation was performed in the presence and absence of test HDL and the change in fluorescence due to oxidation of DCFH was measured. The DCF fluorescence data was converted into an HOI that equaled the ratio of fluorescence of LDL in the presence of HDL divided by the fluorescence of LDL in the absence of HDL. An index <1.0 denoted protective anti-oxidant HDL, while an index >1.0 denoted pro-oxidant dysfunctional HDL. The assay distinguished the antioxidative potential of HDL taken from different participants. Each of the HOI values was given as the mean of duplicate measurements and the experiment was repeated twice to ensure its reproducibility.
Results of the demographic and baseline characteristics of the patient population were expressed as mean ± standard deviation along with frequency tabulation for variables with proportion or categorical distribution. The P-values were computed using Student's t-test for continuous variables, Mann-Whitney U Test, and Fischer's Exact test for categorical variables. Multiple comparisons of HOI were assessed in the patients, subtypes, and control groups using the analysis of variance and Tukey's honestly significantly difference post hoc test. Statistical analysis was performed with GraphPad Prism version 7.0 (GraphPad Software Inc.). P<0.05 was considered to indicate a statistically significant difference.
A total of 75 patients were enrolled for the present study, of which four patients were excluded due to the presence of high blood glucose levels at the time of enrollment. A total of 25 age- and sex-matched healthy volunteers were also included in the study for comparison. The baseline and biochemical characteristics of the study population are summarized in
HOI values of controls, total patients, and ischemic subtypes are given in
The main finding of the present study was that HDL in stroke patients including the LVD, SVD and cardioembolic subtypes had a noticeably increased oxidant capacity of HDL than that isolated from healthy subjects, thereby indicating the pathophysiological role of dysfunctional HDL in stroke. By measuring its antioxidant capacity in terms of its ability to inhibit LDL oxidation, which is the key mechanism in atherogenesis, it was found that functional alteration of HDL occurred in stroke patients, even in the presence of normal HDL cholesterol levels, suggesting that dysfunctionality in HDL is not related to its cholesterol content in stroke.
The association of dysfunctional HDL with stroke has been previously demonstrated in three other studies (
Studies conducted in CAD patients found dysfunctional HDL to be associated with poorer outcome and trigger inflammatory pathways in macrophages leading to atheroma formation (
Inflammation is a key mechanism causing structural variations and functional alterations in HDL (
In animal studies, on the other hand, HDL has been shown to exert a direct vascular and neuroprotective effect when administered during the acute phase of embolic stroke in rats, possibly by antioxidant or anti-inflammatory mechanisms (
The major strengths of the present study were thorough classification of the stroke subtypes and detailed ischemic stroke etiological evaluation with the documentation of clinical and imaging risk factors for all patients enrolled. The present study was the first to assess HDL function. However, it was not without limitations. Due to the small sample size, the present study may have been not strong enough, hence limiting the interpretation of the true difference in HDL oxidation across the stroke subtypes. The male ratio was higher because age-specific stroke rates are higher in men. The Hyderabad Stroke registry also found an increased incidence of stroke in men (78.3%) in India (
In conclusion, decreased antioxidant capacity in ischemic stroke subtypes, indicating dysfunctional HDL, may affect its protective functions in stroke.
Not applicable.
The datasets generated and/or analyzed during the current study are not publicly available due to compromising individual privacy but are available from the corresponding author on reasonable request.
DD, SK, PNS and SG contributed to the conception and design of the present study. Material preparation, data collection, and analysis were performed by DD and SK. The first draft of the manuscript was written by DD and all authors commented on previous versions of the manuscript. SG and DD confirm the authenticity of all the raw data. All authors read and approved the final manuscript.
The present study was performed in line with the principles of the Declaration of Helsinki. It was approved by the Institutional Ethics Committee of Sree Chitra Tirunal Institute for Medical Sciences and Technology (approval number IEC/1017). Informed consent was obtained from all individual participants included in the study.
Not applicable.
The authors declare that they have no competing interests.
Baseline and biochemical characteristics of study population.
Characteristic | Patients (n=71) | Control (n=25) | P-value |
---|---|---|---|
Age, median (interquartile range), years | 57 (23-85) | 53 (34-70) | 0.04 |
Male, n (%) | 47(66) | 15(60) | 0.60 |
BMI, (mean ± standard deviation) | 23.85±1.17 | 24.1±2.5 | 0.50 |
Smoking, (yes/no) | 27/44 | 0/25 | <0.0001 |
High blood pressure, (yes/no) | 39/32 | 0/25 | <0.0001 |
Coronary artery disease, (yes/no) | 6/65 | 0/25 | 0.30 |
Atrial fibrillation, (yes/no) | 12/59 | 0/25 | 0.03 |
Fasting glucose, (mg/dl) | 76.69±40.35 | 83±10 | 0.48 |
Total cholesterol, (mg/dl) | 195.2±46.81 | 197±31.1 | 0.86 |
HDL cholesterol, (mg/dl) | 45.56±12.22 | 49±10.78 | 0.22 |
LDL cholesterol, (mg/dl) | 129.19±43.68 | 131±26.5 | 0.85 |
Triglyceride, (mg/dl) | 100.6±60.01 | 89±31.5 | 0.36 |
Data are expressed as mean ± standard deviation or median (interquartile range). P-values are from Student's t-test for continuous variables, Mann-Whitney U test and Fischer's exact test for categorical variables. BMI, body-mass index; HDL, high-density lipoprotein; LDL, low-density lipoprotein.
HOI values in stroke patients, each ischemic stroke subtypes and in healthy controls.
Parameter | HOI |
---|---|
Total patients (n=71) | 1.07±0.32 |
Cardioembolism (n=25) | 1.05±0.30 |
Large vessel disease (n=22) | 1.15±0.41 |
Small vessel disease (n=24) | 1.01±0.25 |
Healthy controls (n=25) | 0.51±0.12 |
Data are given as mean ± standard deviation. HOI, high-density lipoprotein oxidant index.
Post-hoc analysis of the HOI values of the total patients, ischemic subtypes, and healthy controls.
Pairwise comparisons | HSD.05=0.2152, HSD.01=0.2581 | P-value |
---|---|---|
CE:LVD | 0.15 | 0.3 |
CE:SVD | 0.03 | 0.9 |
LVD:SVD | 0.17 | 0.1 |
CE:Total patients | 0.04 | 0.9 |
LVD:Total patients | 0.11 | 0.6 |
SVD:Total patients | 0.07 | 0.9 |
SVD:Controls | 0.49 | <0.001 |
CE:Controls | 0.52 | <0.001 |
Controls:Total patients | 0.56 | <0.001 |
LVD:Controls | 0.67 | <0.001 |
Data are given as mean ± standard deviation. P-values are from one-way analysis of variance followed by Tukey's HSD post hoc test. HSD, honestly significantly difference. HOI, high-density lipoprotein oxidant index; CE, cardioembolic disease; LVD, Large vessel disease; SVD, small vessel disease.