Improvement in Lipid Profile after Long-term Consumption of Policosanol Accompanied by Reduced Oxidation of LDL and Aortic Stiffness via CETP Inhibition in Healthy Middle-aged Women
Kyung-Hyun Cho1, Dhananjay Yadav2, Yong-Bok Park3, Jae-Ryong Kim4
1 Department of Medical Biotechnology, Yeungnam University, Gyeongsan, Gyeongbuk, Korea; Research Institute of Protein Sensor, Yeungnam University, Gyeongsan, Republic of Korea; LipoLab, Yeungnam University, Gyeongsan, Republic of Korea.
2 Department of Medical Biotechnology, Yeungnam University, Gyeongsan, Gyeongbuk, Korea; Research Institute of Protein Sensor, Yeungnam University, Gyeongsan, Republic of Korea; LipoLab, Yeungnam University, Gyeongsan, Republic of Korea.
3 Major in Biotechnology, College of Natural Sciences, Kyungpook National University, Daegu, Republic of Korea.
4 Department of Biochemistry and Molecular Biology, Smart-Aging Convergence Research Center, College of Medicine, Yeungnam University, Daegu, Korea.
NAME, ADDRESS, E-MAIL ID OF THE CORRESPONDING AUTHOR: Dr. Kyung-Hyun Cho, Department of Medical Biotechnology, Yeungnam University, Gyeongsan, Gyeongbuk-712749, South Korea.
E-mail: chok@yu.ac.kr
Cuban Policosanol (PCO) was reported to lower serum Total Cholesterol (TC) as well as increase High-Density Lipoprotein Cholesterol (HDL-C) and enhance HDL functionality. In this study, we compared changes in the blood lipid and lipoprotein profiles in hypercholesterolemic women subjects (50-year-old) who consumed policosanol for 20 weeks. At week 0, subject had high TC but low HDL-C levels. After 20 weeks of policosanol consumption, serum TC and Triglyceride (TG) levels were reduced by 10% and 44%, respectively. HDL-C level was elevated 1.7-fold while Low-Density Lipoprotein (LDL) level was reduced by up to 20%. Serum Cholesteryl Ester Transfer Protein (CETP) activity was reduced from 33% CE-transfer at week 0 to 22% CE-transfer at week 20. Glycation extent was significantly reduced in all lipoprotein fractions, especially in LDL and HDL2. In Very Low-Density Lipoprotein (VLDL) and LDL, cholesterol and TG contents were reduced. In HDL2 and HDL3, cholesterol was more enriched and TG content reduced. LDL was more resistant to cupric ion-mediated oxidation and showed less atherogenic properties via phagocytosis into macrophages. Apolipoprotein A-I (apoA-I) was more enriched in HDL2 and HDL3 in a time-dependent manner. The antioxidant ability of HDL was enhanced by 25% in terms of ferric ion reduction ability and paraoxonase activity. In conclusion, 20 weeks of policosanol consumption improved the lipid profile by increasing HDL-C, and lipoprotein functionality to enhance antioxidant, anti-glycation, and anti-atherogenic properties via CETP inhibition.
Case Report
A 50-year-old female with hypercholesterolemia consumed policosanol for 20 weeks. She was an office worker for 22 years at Yeungnam University. She had taken daily 10 mg of policosanol (Rainbow & Nature Pvt., Ltd., Thornleigh, NSW, Australia) at the same time every evening with meals around 7-9 pm and consumed a typical Korean diet, which is enriched with rice and vegetables, and no changes in dietary patterns were observed in the past five years. The subject had maintained a sedentary lifestyle without exercise for the last five years. Before the study, the subject had suffered from cold hands and feet almost every night. Here, we investigated a case report of a hypercholesterolemic Korean middle-aged women (age) with low HDL-C who was treated with policosanol for 20 weeks to monitor the treatment efficacy in terms of lipid and lipoprotein functionality. Furthermore, blood pressure was monitored at four-week intervals for 20 weeks. Earlier the doctor had prescribed her to statin due to mild hypercholesterolemia but she quitted three months before policosanol consumption. No other medical record and family history was known.
Blood pressure was measured each visit for a total of three times, and the average was recorded at four-week intervals. Blood was obtained from the subject each visit following overnight fasting. Blood was collected using a vacutainer (BD Biosciences, Franklin Lakes, NJ, USA) containing EDTA (final concentration of 1 mM). TC, TG, HDL-C, and glucose were measured using commercially available kits (Cleantech TS-S; Wako Pure Chemical, Osaka, Japan). The Ferric Reducing Ability of Plasma (FRAP) was determined using the method described by Benzie and Strain [1]. The antioxidant activities of HDL fractions (20 μg each in PBS) were estimated by measuring increases in absorbance induced by generated ferrous ions. Paraoxonase-1 (PON-1) activity was determined by measuring the initial velocity of p-nitrophenol production at 37°C based on its absorbance at 405 nm (Microplate reader, Bio-Rad model 680; Bio-Rad, Hercules, CA, USA), as described [2]. Very Low-Density Lipoprotein (VLDL, d <1.019 g/mL), Low-Density Lipoprotein (LDL, 1.019 <d <1.063), High-Density Lipoprotein2 (HDL2, 1.063 <d <1.125), and High-Density Lipoprotein3 (HDL3, 1.125 <d <1.225) were isolated from the individual plasma of each group via sequential ultracentrifugation [3].
To differentiate the extent of glycation in the lipoproteins, it was determined by reading fluorometric intensities at 370 nm (excitation) and 440 nm (emission), as described [4] using a spectrofluorometer LS55 (Perkin-Elmer, Shelton, CT, USA).
Cholesteryl ester transfer protein assay was carried out according to our previous report [5]. Oxidized LDL (oxLDL) was obtained by incubation of the LDL fraction with CuSO4 (final concentration of 10 μM) for four hours at 37°C. The comparison of electromobility of LDL between weeks 0, 4, 8, 12, 16 and 20 with or without cupric ion in 0.5% agarose gel were measured [Table/Fig-1]. The extents of oxidized species in LDL and cell culture media were measured by thiobarbituric acid reactive substances (TBARS) assay [6]. Phagocytosis of LDL into macrophages was carried out in THP-1 cells, a human monocytic cell line, which were incubated in medium containing phorbol 12-myristate 13-acetate (PMA, 150 nM) in 24-well plates for 48 hours at 37oC in a humidified incubator (5% CO2, 95% air) in order to induce differentiation into macrophages. Cells were stained with oil-red O staining solution (0.67%) and washed with distilled water. THP-1 macrophage-derived foam cells were then observed and photographed using a Nikon Eclipse TE2000 microscope (Tokyo, Japan) at 400x magnification, as in our previous report [6,7]. After 20 weeks of policosanol consumption, glycation extent was significantly reduced in all lipoprotein fractions (VLDL, LDL, HDL2, and HDL3) Especially, LDL and HDL2 showed 45% and 40% reduction of glycation, respectively, at week 20 under the same protein content [Table/Fig-2]. [Table/Fig-3] displays the cholesterol and triglyceride contents of various lipoprotein fractions (VLDL, LDL, HDL2, and HDL3). Comparison of electromobility of native LDL or oxidized LDL in the presence of cupric ion during policosanol consumption is shown in [Table/Fig-4]. All LDL during policosanol consumption showed less mobility than LDL from week 0, except at 16 weeks. Clearer band intensity and slower electromobility indicate that LDL was less oxidized, and policosanol consumption protected LDL from oxidation. Quantification of oxidized species in cell culture media showed that LDL from week 0 resulted in a 10-fold higher level of MDA (around 4.5 μM) than PBS in media [Table/Fig-5]. However, LDL from week 20 resulted in a 54% lower MDA level (around 1.4 μM) in media. Agarose electrophoresis revealed that band intensities of HDL2 and HDL3 increased in a time-dependent manner [Table/Fig-6]. SDS-PAGE also revealed that increased apoA-I band intensities in HDL2 and HDL3 with policosanol consumption [Table/Fig-6]. At week 20, HDL2 showed 25% more enhanced FRAP ability than that at week 0 [Table/Fig-7a]. PON activity was enhanced 1.6-fold by policosanol consumption (week 20), as shown in [Table/Fig-7b].
Anthropometric parameters and biochemical analysis after 20 weeks of policosanol consumption.
| Parameters | Week 0 | Week 4 | Week 8 | Week 12 | Week 16 | Week 20 |
|---|
| BMI (kg/m2) | 17.8 | 17.7 | 18 | 17.6 | 18.2 | 17.9 |
| Body fat (kg) | 7.3 | 7.8 | 7.3 | 7.7 | 7.2 | 7.4 |
| Body moisture (AU) | 29.8 | 29.3 | 30 | 28.7 | 30.2 | 29.7 |
| Muscle mass (kg) | 38.5 | 37.9 | 38.8 | 37.1 | 39.1 | 38.4 |
| Visceral fat (kg) | 0.6 | 0.6 | 0.6 | 0.6 | 0.6 | 0.6 |
| Subcutaneous fat (kg) | 6.7 | 7.2 | 6.7 | 7.1 | 6.6 | 6.8 |
| SBP (mmHg) | 110 | 113 | 109 | 115 | 118 | 126 |
| DBP (mmHg) | 70 | 69 | 65 | 68 | 79 | 80 |
| Heart rate (BPM) | 62 | 65 | 69 | 65 | 62 | 65 |
| TC (mg/dL) | 238 | 245 | 240 | 235 | 228 | 216 |
| TG (mg/dL) | 99 | 54 | 38 | 46 | 75 | 56 |
| HDL-C (mg/dL) | 42 | 51 | 56 | 55 | 66 | 72 |
| % HDL-C in TC | 17 | 20 | 23 | 22 | 29 | 32 |
| TG/HDL-C | 2.4 | 1.1 | 0.7 | 0.8 | 1.1 | 0.7 |
| LDL-C (mg/dL) | 176 | 183 | 176 | 179 | 147 | 141 |
| CETP activity (CE-transfer, %) | 33 | 30 | 25 | 26 | 23 | 22 |
| Glucose (mg/dL) | 81 | 89 | 91 | 82 | 94 | 82 |
BMI: Body mass index; AU: Arbitrary units; SBP: Systolic blood pressure; DBP: Diastolic blood pressure; BPM: Beats per minutes; TC=Total cholesterol; TG: Triglyceride; HDL-C: High-density lipoprotein cholesterol; LDL-C: Low-density lipoprotein cholesterol; CETP: Cholesteryl ester transfer protein
Fluorometric determination of glycation extent of lipoproteins during policosanol consumption. VLDL, very low density lipoprotein; LDL, low density lipoprotein; HDL2 & HDL3, high density lipoprotein subfractions.
Cholesterol and triglyceride contents in various lipoproteins (VLDL, very low density lipoprotein; LDL, low density lipoprotein; HDL2 & HDL3, high density lipoprotein subfractions).
Comparison of electromobility of native LDL or oxidized LDL under the presence of cupric ion during policosanol consumption.
Uptake of LDL into macrophages and extent of oxidized species during policosanol consumption (quantification of oxidized species in cell culture media using the TBARS method, quantification of oil-red O-stained area by computer-assisted morphometry).
Electrophoretic patterns of HDL2 and HDL3 during policosanol consumption at weeks 0,4,8,12,16 and 20 (5 μg/lane, 15% SDS-PAGE).
(a) Antioxidant ability (ferric ion reduction ability of HDL2 from week 0 and week 20). (b) Paraoxonase activity of HDL2 and HDL3 during policosanol consumption.
Discussion
Policosanol might contribute to longevity via CETP inhibition [Table/Fig-1], elevation of HDL-C, and enhanced functionality of HDL, as in our previous report [8,9]. Low HDL levels (<40 mg/dL) are considered as an independent risk factor for CHD [10]. This current case report in one hypercholesterolemic female reveals that consumption of policosanol reduced the risk of glycation in all lipoproteins in a time-dependent manner [Table/Fig-2]. The triglyceride contents in lipoprotein fractions (VLDL, LDL, HDL2, and HDL3) were reduced after 20 weeks of policosanol consumption, and the cholesterol content was higher in good cholesterol (HDL2 and HDL3). Moreover, consumption of policosanol inhibited cupric ion-mediated oxidation of LDL and reduced cell-mediated (human monocytic cell line) generation of Malondialdehyde (MDA) by LDL. Although the female subject had higher cholesterol and lower HDL-C than normal, she showed normal ranges of blood pressure and body fat contents. TC gradually and slowly decreased while TG more rapidly decreased with a gradual decrease in CETP activity. The subject showed an almost 2-fold increase in %HDL-C in TC at week 0 and week 20, 17%, and 32%, respectively.
In the same context, apoA-I was elevated in HDL with enhanced antioxidant activity such as FRAP and paraoxonase activity. Moreover, enhanced antioxidant activity was connected to reduced oxidation of LDL [Table/Fig-4] and less uptake of LDL at week 20 [Table/Fig-5] since PON activity in HDL is involved in the removal of oxidized species in LDL. Higher apoA-I content is associated with stronger PON activity, which exerts anti-inflammatory activity against oxidation of LDL [11]. Elevated CETP activity is associated with increased serum TG and TG-enriched LDL levels. It is well established that serum TG level is an independent risk factor of inflammatory disease. TG levels are an important and independent predictor of Coronary Artery Disease (CAD) and stroke in the Asia-Pacific region [12]. In the current study, policosanol consumption caused a remarkable decrease in serum TG and increase in HDL-C via CETP inhibition accompanied by inhibition of LDL oxidation and phagocytosis into macrophages.
Conclusion
In a middle-aged woman, 20 weeks of policosanol consumption improved the lipid profile, including lowering of TC and TG and elevation of HDL-C. The pain of cold hands and feet every night disappeared after policosanol consumption. Lipoprotein functionality was also enhanced along with less oxidation and glycation, resulting in improved anti-atherogenic properties and vascular functions. These results suggest that Cuban policosanol can induce beneficial effects to suppress cardiometabolic risk.
BMI: Body mass index; AU: Arbitrary units; SBP: Systolic blood pressure; DBP: Diastolic blood pressure; BPM: Beats per minutes; TC=Total cholesterol; TG: Triglyceride; HDL-C: High-density lipoprotein cholesterol; LDL-C: Low-density lipoprotein cholesterol; CETP: Cholesteryl ester transfer protein
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