Ir J Med Sci DOI 10.1007/s11845-014-1102-7
ORIGINAL ARTICLE
Chromium picolinate inhibits cholesterol-induced stimulation of platelet aggregation in hypercholesterolemic rats A. A. Seif
Received: 14 September 2013 / Accepted: 26 February 2014 Ó Royal Academy of Medicine in Ireland 2014
Abstract Background Hypercholesterolemia indirectly increases the risk of myocardial infarction by enhancing platelet aggregation. Chromium has been shown to lower plasma lipids. Aim This study was designed to investigate whether chromium inhibits platelet aggregation under hypercholesterolemic conditions. Methods Albino rats were divided into four groups: control rats fed with a normolipemic diet (NLD group), chromium-supplemented rats fed with NLD (NLD ? Cr group), rats fed with a high-fat diet (HF group), and chromium-supplemented rats fed with HF (HF ? Cr group). After 10 weeks, blood was collected to determine adenosine diphosphate and collagen-induced platelet aggregation and plasma levels of total cholesterol, triglycerides, high-density lipoprotein cholesterol, apolipoprotein A1, apolipoprotein B, and thromboxane B2. Lowdensity lipoprotein cholesterol was calculated by Friedewald formula. Results High-fat diet animals displayed significant elevation of plasma lipids and platelet aggregation which was normalized to control levels by chromium supplementation. Chromium supplementation in normolipemic (NLD ? Cr) rats did not produce significant changes in either plasma lipids or platelet activity. Conclusion Chromium supplementation to hypercholesterolemic rats improves the lipid profile and returns platelet
A. A. Seif (&) Physiology Department, Faculty of Medicine, Ain Shams University, Abbassyia District of Cairo Governorate, on Ahmed Lotfy Al-Sayed Street, Cairo, Egypt e-mail: [email protected]
hyperaggregability to control levels. This normalization is mostly due to a reduction in plasma cholesterol level. Keywords Chromium picolinate Cholesterol Platelet aggregation Rats
Introduction High cholesterol levels are a known risk factor for cardiovascular disease [1]. Among its detrimental effects, cholesterol augments arterial platelet accumulation [2] and platelet aggregation [3] which triggers the formation of blood clots and can lead to myocardial infarction and stroke. A study done by Ravindran and Krishnan [4] showed that hyperlipidemia increases the lipid content in platelets and enhances their reactivity which may contribute to accelerated atherogenesis associated with coronary artery disease. Moreover, reducing the level of low-density lipoprotein cholesterol (LDL-C) with statins has been shown to induce important pleiotropic effects such as platelet inhibition [5]. Chromium (Cr3?) is now recognized as an essential mineral that is thought to be necessary for normal glucose and lipid homeostasis [6]. Studies have revealed that Cr3? may also be beneficial in cardiovascular disease [7], suggesting its potential in lowering certain risk factors such as total serum cholesterol, LDL cholesterol, and serum triglycerides (TG) [8]. Chromium deficiency can also mimic many signs of cardiovascular disease, such as elevated serum cholesterol and TG, as well as decreased high-density lipoprotein cholesterol (HDL-C) [9]. Exposure of adipocytes to chromium picolinate (CrPic) induced a loss of plasma membrane cholesterol, and loading exogenous cholesterol back into the plasma membrane blocked the
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action of CrPic [10]. The beneficial effects of Cr3? due to its lipid-modulating effect on pathological platelet aggregation have not been studied. Given the prevalence of high blood cholesterol levels in the population [11], despite the availability of superior cholesterol lowering agents, and the potential importance of Cr3? in cholesterol-related disorders, this study was designed to test the ability of Cr3? to reduce platelet aggregation under hypercholesterolemic conditions where platelet aggregation might probably be induced.
evacuated from the capsule, dissolved in 2 mL distilled water, and given to each rat by gavages. At the end of 10 weeks (70 ± 3 days) and on the day of killing, after an overnight fast, rats were anaesthetized by injecting sodium thiopental (Nile Co.) in a dose of 40 mg/Kg BW, intraperitoneally. Through a median longitudinal incision, the abdominal cavity was opened and the aorta was dissected down to the aortic bifurcation from where blood was collected from all rats. Platelet aggregation analysis
All animal procedures and care were approved by the ethics committee of Faculty of Medicine, Ain Shams University (FMSU REC, Cairo, Egypt), which conforms to the Guide for Care and Use of Laboratory Animals published by the United States National Institutes of Health. Adult male albino rats (180–200 g) were obtained from commercial breeder and kept in the Physiology Department with 12-h periods of light and darkness at constant room temperature (20 °C). Rats were acclimatized for 5 days before beginning of the study. Four groups of 15 animals each were studied: control rats fed with a normolipemic diet (NLD group), rats fed with a HF group, chromiumsupplemented rats fed with NLD (NLD ? Cr group), and chromium-supplemented rats fed with HF (HF ? Cr group).
It was carried out following the turbidimetric method described by O’Brien [13]. Blood was collected into sodium citrate tubes which were gently inverted and then stored at room temperature for 30 min. Platelet-rich plasma was obtained by centrifugation of the samples at 100g for 15 min. Collection of the top layer from this step followed by subsequent centrifugation of the remaining sample at 2,400g for 15 min provided platelet-poor plasma. Equal volumes of platelet-rich and platelet-poor plasma fractions were aliquoted into separate cuvettes. Either adenosine diphosphate (ADP) or collagen was then added to the platelet-rich plasma such that the final concentrations were 7.5 lmol/L and 4 lg/mL, respectively. Upon addition of ADP or collagen aggregate, the sample was immediately measured in a Chrono-log aggregometer model 540. The platelet-poor plasma served as the blank and was treated in an identical fashion. The percentage of maximal aggregation was obtained by using the Aggro/Link (v.4.75) software.
Preparation of different dietary formulae
Plasma collection and lipid analysis
Control diet (carbohydrates 50–51 %, fats 4–5 %, and proteins 12–13 %) was prepared in our laboratory from three main natural sources, namely bread (balady bread), unsalted Karish cheese, and dry milk powder (284 K-Cal/ 100 g/day). HF group was prepared by adding butter to increase its fat content to 16–17 % (352 K-Cal/100 g/day). The preparation of different dietary formulae was achieved by calculations which depended upon the analytical composition of different food sources used in animal feeding in our laboratory based on what was described by Hallfrisch et al. [12]. Rats were fed with the diets for 10 weeks. Diets were freshly prepared every 2 days before consumption and stored at 4 °C until used. Rats were allowed free access to water. The diet consumption of all animals was monitored daily. CrPic (200 lg/day) was administered by gavages for 10 weeks. Chromium was obtained as capsules containing 200 lg CrPic supplied by Arab Co. for Pharm. and Med. Plants (MEPA Co., Egypt). Chromium powder was
Blood collected into sodium citrate tubes was centrifuged at 1,800g for 10 min. Plasma was stored at -80 °C. Plasma total cholesterol (Chol) and TG were measured by the enzymatic colorimetric method [14, 15]. HDL-C was determined by precipitation method [16]. Apolipoprotein A1 (Apo-A1) and apolipoprotein B (Apo-B) concentrations were measured by Turbidimetry [17]. LDL was calculated using Friedewald formula: LDL-C = TC-HDL-C-TG/5 (provided that triglyceride levels were lower than 350 mg/ dL). Plasma thromboxane B2 was determined by the technique of Chard [18].
Materials and methods Animal handling, diets, and treatment
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Statistical analysis Data were expressed as mean ± SD. Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by Tukey post hoc test for comparisons between individual groups. Differences were considered to be statistically significant when p was \0.05. Data
Ir J Med Sci Table 1 Plasma lipids in mg/dL observed in rats after 10 weeks of dietary feeding and chromium supplementation Parameter
NLD
NLD ? Cr
Chol
HF
HF ? Cr
172.66 ± 9.35
170.40 ± 14.71
249.13 ± 13.36**
179.53 ± 10.35
TG
96.26 ± 6.50
95.86 ± 7.39
155.46 ± 4.61**
100.33 ± 6.79
HDL-C
50.13 ± 3.66
51.80 ± 4.34
38.33 ± 2.55**
LDL-C
103.26 ± 8.72
99.40 ± 12.96
179.73 ± 13.96**
47.53 ± 4.74* 111.86 ± 9.44#
Apo-A1
139.13 ± 5.15
141.73 ± 6.36
123.33 ± 3.82**
137.80 ± 4.14
Apo-B
112.13 ± 4.45
109.66 ± 6.24
153.26 ± 8.94**
116.73 ± 8.69
Values represent mean ± SD for 15 animals NLD normolipemic diet, NLD ? Cr normolipemic diet ? chromium supplementation, HF high-fat diet, HF ? Cr high-fat diet ? chromium supplementation, Chol total cholesterol, TG triglycerides, HDL-C high-density lipoprotein cholesterol, LDL-C low-density lipoprotein cholesterol, Apo-A1 apolipoprotein A1, Apo-B apolipoprotein B * Significantly different from NLD group, p \ 0.05 ** Significantly different from NLD, NLD ? Cr, and HF ? Cr groups, p \ 0.0001 #
Significantly different from NLD ? Cr group, p \ 0.05
were analyzed using a statistical software package (SPSS for Windows, 10.0.1, 1999, SPSS Inc., Chicago, Ill, USA).
Results Plasma lipids Plasma was analyzed for TG, total cholesterol (Chol), HDLC, apo A1, and apo B (Table 1). Plasma LDL-C was calculated using Friedewald formula: LDL-C = TC-HDLC-TG/5 (Table 1). The HF group showed significant elevation of TG, Chol, LDL-C, and apo B levels with significant reduction in HDL-C and apo A1 levels compared to all other groups (p \ 0.0001). There was no significant difference between the NLD and NLD ? Cr groups regarding the lipid profile. Chromium supplementation in the HF ? Cr group improved the deteriorated lipid profile to control levels of NLD and NLD ? Cr groups except for HDL-C levels that were significantly lower in HF ? Cr group compared to NLD group (p \ 0.05) and LDL-C levels that were significantly higher in HF ? Cr group compared to NLD ? Cr group (p \ 0.05).
Fig. 1 ADP-induced platelet aggregation according to dietary intervention and chromium supplementation. NLD normolipemic diet, NLD ? Cr normolipemic diet ? chromium supplementation, HF high-fat diet, HF ? Cr high-fat diet ? chromium supplementation. Mean ± SD values (n = 15) are shown for each group. Significant at *p \ 0.0001 versus NLD, NLD ? Cr, and HF ? Cr groups
Platelet aggregation and plasma TX B2 Total platelet aggregation was measured after stimulation by either ADP (Fig. 1) or collagen (Fig. 2), and plasma TX B2 was determined (Fig. 3). The HF group had significantly increased ADP-induced aggregation compared with all other groups (P \ 0.0001). No significant difference in ADP-induced aggregation was encountered between NLD and NLD ? Cr groups. Chromium supplementation to hypercholesterolemic rats in HF ? Cr group normalized ADP-induced hyperaggregability to levels of NLD and
Fig. 2 Collagen-induced platelet aggregation according to dietary intervention and chromium supplementation. NLD normolipemic diet, NLD ? Cr normolipemic diet ? chromium supplementation, HF high-fat diet, HF ? Cr high-fat diet ? chromium supplementation. Mean ± SD values (n = 15) are shown for each group. Significant at *p \ 0.0001 versus NLD, NLD ? Cr, and HF ? Cr groups
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Fig. 3 Plasma thromboxane B2 according to dietary intervention and chromium supplementation. NLD normolipemic diet, NLD ? Cr normolipemic diet ? chromium supplementation, HF high-fat diet, HF ? Cr high-fat diet ? chromium supplementation. Mean ± SD values (n = 15) are shown for each group. Significant at *p \ 0.0001 versus NLD, NLD ? Cr, and HF ? Cr groups
NLD ? Cr groups. Similar trends between these groups were also observed with collagen-induced platelet aggregation. Plasma TX B2 (used to indirectly measure TX A2 production) was also significantly higher in HF group compared to all other groups (p \ 0.0001), whereas there was no significant difference between NLD and NLD ?Cr groups. Levels of TX B2 were normalized down to control levels in NLD and NLD ? Cr groups by chromium supplementation in HF ? Cr group with no significant difference between these groups.
Discussion The results of the present study revealed that a HF group produced significant elevation of plasma cholesterol, TG, LDL-C, apo B lipoprotein, and TX B2 as well as the % of platelet aggregation induced by ADP and collagen. This was accompanied by a significant reduction in plasma HDL-C and apo A-1 lipoprotein. Supplementation of hypercholesterolemic rats with CrPic in a dose of 200 lg/ 200 g rat/day (1 mg/kg/day), which is equivalent to a human dose of 0.16 mg/kg/day [19], administered by gavages for 10 weeks improved the deteriorated lipid profile, except for HDL-C levels that were significantly lower in HF ? Cr group compared to NLD group and LDL-C levels that were significantly higher in HF ? Cr group compared to NLD ? Cr group. Chromium supplementation also normalized ADP as well as collagen-induced hyperaggregability and levels of TX B2 to near control levels. Platelet aggregation has been shown to be enhanced by cholesterol, and 0.5 % cholesterol diet for 16 weeks induced platelet hyperaggregability to low doses of agonists as well as the development of hypercholesterolemic atherosclerosis in the rabbits [20]. In addition, a positive
123
correlation was found for serum lipids and all the platelet activation markers [21]. Another study indicated that the ADP-induced platelet aggregation increased by 71.67 % in hypercholesterolemia [22]. Plasma total and LDL-C, which are established risk factors for atherosclerotic vascular disease, might also contribute to a prothrombotic risk via enhanced platelet reactivity [23]. The circulating lipoproteins may cause some abnormalities in platelet composition and function in hypercholesterolemia. It seems that in hyperlipidemia, some platelets are in an activated state in circulation and that increased lipid peroxidation, early apoptosis, platelet–leukocytes aggregate formation, and platelet aggregation altogether accompany this process [24]. Thus, the significant increase in platelet aggregation in hypercholesterolemic rats encountered in the present study could be due to an alteration in platelet signaling, caused by elevated cholesterol levels. Moreover, it has been shown that cholesterol depletion impairs platelet aggregation by altering platelet ultrastructure critical in mediating secretion [25]. Cholesterol depletion impairs microtubule ring formation and aggregate size, besides diminishing the extent of the open canalicular system and collagen-induced platelet ATP release [25]. Confirming the relation between lipids and platelet function, lipid-lowering drugs, such as ezetimibe and simvaststin, have been shown to decrease ADP and collagen-induced platelet aggregation [26]. Combination of statins and N-3 fatty acid in hypercholesterolemic patients has been reported to inhibit platelet aggregation [27]. Chromium has been recently shown to be involved in cholesterol homeostasis. Aguilar et al. [28] showed that Cr3 ? at 5 mcg per gram of food given to 20 Wistar rats for 10 weeks resulted in a significant decrease in cholesterol levels. Chromium supplementation also lowered plasma triglyceride and cholesterol levels to near controls, normalized LDL cholesterol and VLDL cholesterol levels in hypercholesterolemic diabetic animals, and improved total cholesterol to HDL cholesterol and HDL cholesterol to LDL cholesterol ratios suggesting its anti-atherogenic effect [29]. The results of the present study are in agreement with previous studies, and the persistent significant elevation of LDL-C and decrease in HDL-C seen in chromium-supplemented rats (HF ? Cr) compared to control groups could be attributed to the need of a longer time or higher doses of chromium. Recent studies support a novel action of proteins involved in cellular cholesterol homeostasis in response to CrPic. Pattar et al. [10] examined SREBP, a sterol-regulating transcription factor that localizes to the ER/Golgi membranes in its immature form. When the cell is in the need of cholesterol, cleavage of immature ER/Golgilocalized SREBP results in the release of the mature form of SREBP from the ER/Golgi membrane and accumulation
Ir J Med Sci
of this active transcription factor in the nucleus where it binds to sterol response elements controlling the expression of proteins-regulating cholesterol homeostasis. For example, SREBP represses the expression of ABCA1, an ATPbinding cassette that mediates the cellular efflux of excess cholesterol. SREPB was upregulated by CrPic, whereas ABCA1 was decreased, consistent with the SREBP transcriptional repression of the ABCA1 gene [10]. Although the exact mechanism of Cr3?-induced cholesterol loss remains to be determined, these cellular responses highlight a novel and significant effect of chromium on cholesterol homeostasis. Furthermore, these findings provide an important clue to our understanding of how chromium supplementation might benefit hypercholesterolemia-associated disorders. The results of studies conducted on nondiabetic subjects with normal lipid profile, insulin sensitivity, and glucose tolerance showed that Cr3? supplements had only modest or no effects on the previous factors and that Cr3? supplements had positive effects in lowering elevated blood glucose, insulin, and lipid levels in subjects with insulin resistance and type 2 diabetes [30]. It was therefore unexpected to find a significant effect of chromium supplementation on lipid profile or platelet aggregation under normocholesterolemic conditions in the present study. In conclusion, the results of the present study show that CrPic might be an effective agent in inhibiting platelet hyperaggregability under hypercholesterolemic conditions. The extraordinary complexity of understanding chromium biology and toxicology certainly may involve an underappreciated cast of membrane lipids, including cholesterol. Given the enormous public health cost of cholesterolassociated diseases, the prospect of being able to use Cr3?—a relatively low-cost dietary supplement—as a therapy to correct cholesterol-dependent abnormalities merits further study. Conflict of interest
None.
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5. Piorkowski M, Fischer S, Stellbaum C et al (2007) Treatment with ezetimibe plus low-dose atorvastatin compared with higherdose atorvastatin alone: is sufficient cholesterol-lowering enough to inhibit platelets? J Am Coll Cardiol 49(10):1035–1042 6. Balk EM, Tatsioni A, Lichtenstein AH, Lau J, Pittas AG (2007) Effect of chromium supplementation on glucose metabolism and lipids: a systematic review of randomized controlled trials. Diabetes Care 30(8):2154–2163 7. Rajpathak S, Rimm EB, Li T et al (2004) Lower toenail chromium in men with diabetes and cardiovascular disease compared with healthy men. Diabetes Care 27(9):2211–2216 8. Rabinovitz H, Friedensohn A, Leibovitz A, Gabay G, Rocas C, Habot B (2004) Effect of chromium supplementation on blood glucose and lipid levels in type 2 diabetes mellitus elderly patients. Int J Vitam Nutr Res 74:178–182 9. Riales R, Albrink MJ (1981) Effect of chromium chloride supplementation on glucose tolerance and serum lipids including high-density lipoprotein of adult men. Am J Clin Nutr 34(12):2670–2678 10. Pattar GR, Tackett L, Liu P, Elmendorf JS (2006) Chromium picolinate positively influences the glucose transporter system via affecting cholesterol homeostasis in adipocytes cultured under hyperglycemic diabetic conditions. Mutat Res 610(1–2):93–100 11. America Heart Association (2006) Heart disease and stroke statistics, 2006 update: a report from the American Heart Association Statistics Committee and Stroke Statistics Subcommittee. Circulation 113:e85–e151 12. Hallfrisch J, Lazar FL, Reiser S (1979) Effect of feeding sucrose or starch to rats made diabetic with streptozotocin. J Nutr 109(11):1909–1915 13. O’Brien JR (1962) Platelet aggregation: Part I Some effects of the adenosine phosphates, thrombin, and cocaine upon platelet adhesiveness. J Clin Pathol 15(5):446–452 14. Allain CC, Poon LS, Chan CS (1974) Enzymatic determination of total serum cholesterol. Clin Chem 20(4):470–475 15. Bucolo G, David H (1973) Quantitative determination of serum triglycerides by the use of enzymes. Clin Chem 19(5):476–482 16. Demacker PN, Vos-Janssen HE, Hijmans AG, van’t Laar A, Jansen AP (1980) Measurement of high-density lipoprotein cholesterol in serum: comparison of six isolation methods combined with enzymic cholesterol analysis. Clin Chem 26(13):1780–1786 17. Sandkamp M, Tambyrajah B, Schriewer H, Assmann G (1988) Simplified turbidimetric determination of apolipoproteins A-I, A-II and B using a microtitre method. J Clin Chem Clin Biochem 26(11):685–688 18. Chard T (1990) An introduction to radioimmunoassay and related techniques, 4th edition. In: Burdon RH, van knippenberg PH (eds) Laboratory techniques in biochemistry and molecular biology vol 6, Part II. Elsevier Biomedical Press, Amsterdam 19. Reagan-Shaw S, Nihal M, Ahmad N (2008) Dose translation from animal to human studies revisited. FASEB J 22(3):659–661 20. Son DJ, Lee HW, Shin HW et al (2008) Enhanced release of sphingosine-1-phosphate from hypercholesterolemic platelets: role in development of hypercholesterolemic atherosclerosis. Prostaglandins Leukot Essent Fatty Acids 78(6):383–390 21. Go¨c¸men AY, Burgucu D, Gu¨mu¨s¸ lu¨ S (2011) Effect of resveratrol on platelet activation in hypercholesterolemic rats: CD40-CD40L system as a potential target. Appl Physiol Nutr Metab 36(3):323–330 22. Sengupta A, Ghosh M (2010) Modulation of platelet aggregation, haematological and histological parameters by structured lipids on hypercholesterolaemic rats. Lipids 45(5):393–400 23. Naqvi TZ, Shah PK, Ivey PA et al (1999) Evidence that highdensity lipoprotein cholesterol is an independent predictor of acute platelet-dependent thrombus formation. Am J Cardiol 84(9):1011–1017
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routine statin treatment inhibits platelet function, decreases patients’ daytime blood pressure, and improves inflammatory status. Eur J Clin Pharmacol 68(8):1139–1146 28. Aguilar MV, Martinez-para MC, Gonzalez MJ (1997) Effects of arsenic (V)-chromium (III) interaction on plasma glucose and cholesterol levels in growing rats. Ann Nutr Metab 41:189–195 29. Sundaram B, Singhal K, Sandhir R (2012) Anti-atherogenic effect of chromium picolinate in streptozotocin-induced experimental diabetes. J Diabetes. doi: 10.1111/j.1753-0407.2012. 00211.x 30. Althuis MD, Jordan NE, Ludington EA, Wittes JT (2002) Glucose and insulin responses to dietary chromium supplements: a meta-analysis. Am J Clin Nutr 76(1):148–155
ORIGINAL ARTICLE
Chromium picolinate inhibits cholesterol-induced stimulation of platelet aggregation in hypercholesterolemic rats A. A. Seif
Received: 14 September 2013 / Accepted: 26 February 2014 Ó Royal Academy of Medicine in Ireland 2014
Abstract Background Hypercholesterolemia indirectly increases the risk of myocardial infarction by enhancing platelet aggregation. Chromium has been shown to lower plasma lipids. Aim This study was designed to investigate whether chromium inhibits platelet aggregation under hypercholesterolemic conditions. Methods Albino rats were divided into four groups: control rats fed with a normolipemic diet (NLD group), chromium-supplemented rats fed with NLD (NLD ? Cr group), rats fed with a high-fat diet (HF group), and chromium-supplemented rats fed with HF (HF ? Cr group). After 10 weeks, blood was collected to determine adenosine diphosphate and collagen-induced platelet aggregation and plasma levels of total cholesterol, triglycerides, high-density lipoprotein cholesterol, apolipoprotein A1, apolipoprotein B, and thromboxane B2. Lowdensity lipoprotein cholesterol was calculated by Friedewald formula. Results High-fat diet animals displayed significant elevation of plasma lipids and platelet aggregation which was normalized to control levels by chromium supplementation. Chromium supplementation in normolipemic (NLD ? Cr) rats did not produce significant changes in either plasma lipids or platelet activity. Conclusion Chromium supplementation to hypercholesterolemic rats improves the lipid profile and returns platelet
A. A. Seif (&) Physiology Department, Faculty of Medicine, Ain Shams University, Abbassyia District of Cairo Governorate, on Ahmed Lotfy Al-Sayed Street, Cairo, Egypt e-mail: [email protected]
hyperaggregability to control levels. This normalization is mostly due to a reduction in plasma cholesterol level. Keywords Chromium picolinate Cholesterol Platelet aggregation Rats
Introduction High cholesterol levels are a known risk factor for cardiovascular disease [1]. Among its detrimental effects, cholesterol augments arterial platelet accumulation [2] and platelet aggregation [3] which triggers the formation of blood clots and can lead to myocardial infarction and stroke. A study done by Ravindran and Krishnan [4] showed that hyperlipidemia increases the lipid content in platelets and enhances their reactivity which may contribute to accelerated atherogenesis associated with coronary artery disease. Moreover, reducing the level of low-density lipoprotein cholesterol (LDL-C) with statins has been shown to induce important pleiotropic effects such as platelet inhibition [5]. Chromium (Cr3?) is now recognized as an essential mineral that is thought to be necessary for normal glucose and lipid homeostasis [6]. Studies have revealed that Cr3? may also be beneficial in cardiovascular disease [7], suggesting its potential in lowering certain risk factors such as total serum cholesterol, LDL cholesterol, and serum triglycerides (TG) [8]. Chromium deficiency can also mimic many signs of cardiovascular disease, such as elevated serum cholesterol and TG, as well as decreased high-density lipoprotein cholesterol (HDL-C) [9]. Exposure of adipocytes to chromium picolinate (CrPic) induced a loss of plasma membrane cholesterol, and loading exogenous cholesterol back into the plasma membrane blocked the
123
Ir J Med Sci
action of CrPic [10]. The beneficial effects of Cr3? due to its lipid-modulating effect on pathological platelet aggregation have not been studied. Given the prevalence of high blood cholesterol levels in the population [11], despite the availability of superior cholesterol lowering agents, and the potential importance of Cr3? in cholesterol-related disorders, this study was designed to test the ability of Cr3? to reduce platelet aggregation under hypercholesterolemic conditions where platelet aggregation might probably be induced.
evacuated from the capsule, dissolved in 2 mL distilled water, and given to each rat by gavages. At the end of 10 weeks (70 ± 3 days) and on the day of killing, after an overnight fast, rats were anaesthetized by injecting sodium thiopental (Nile Co.) in a dose of 40 mg/Kg BW, intraperitoneally. Through a median longitudinal incision, the abdominal cavity was opened and the aorta was dissected down to the aortic bifurcation from where blood was collected from all rats. Platelet aggregation analysis
All animal procedures and care were approved by the ethics committee of Faculty of Medicine, Ain Shams University (FMSU REC, Cairo, Egypt), which conforms to the Guide for Care and Use of Laboratory Animals published by the United States National Institutes of Health. Adult male albino rats (180–200 g) were obtained from commercial breeder and kept in the Physiology Department with 12-h periods of light and darkness at constant room temperature (20 °C). Rats were acclimatized for 5 days before beginning of the study. Four groups of 15 animals each were studied: control rats fed with a normolipemic diet (NLD group), rats fed with a HF group, chromiumsupplemented rats fed with NLD (NLD ? Cr group), and chromium-supplemented rats fed with HF (HF ? Cr group).
It was carried out following the turbidimetric method described by O’Brien [13]. Blood was collected into sodium citrate tubes which were gently inverted and then stored at room temperature for 30 min. Platelet-rich plasma was obtained by centrifugation of the samples at 100g for 15 min. Collection of the top layer from this step followed by subsequent centrifugation of the remaining sample at 2,400g for 15 min provided platelet-poor plasma. Equal volumes of platelet-rich and platelet-poor plasma fractions were aliquoted into separate cuvettes. Either adenosine diphosphate (ADP) or collagen was then added to the platelet-rich plasma such that the final concentrations were 7.5 lmol/L and 4 lg/mL, respectively. Upon addition of ADP or collagen aggregate, the sample was immediately measured in a Chrono-log aggregometer model 540. The platelet-poor plasma served as the blank and was treated in an identical fashion. The percentage of maximal aggregation was obtained by using the Aggro/Link (v.4.75) software.
Preparation of different dietary formulae
Plasma collection and lipid analysis
Control diet (carbohydrates 50–51 %, fats 4–5 %, and proteins 12–13 %) was prepared in our laboratory from three main natural sources, namely bread (balady bread), unsalted Karish cheese, and dry milk powder (284 K-Cal/ 100 g/day). HF group was prepared by adding butter to increase its fat content to 16–17 % (352 K-Cal/100 g/day). The preparation of different dietary formulae was achieved by calculations which depended upon the analytical composition of different food sources used in animal feeding in our laboratory based on what was described by Hallfrisch et al. [12]. Rats were fed with the diets for 10 weeks. Diets were freshly prepared every 2 days before consumption and stored at 4 °C until used. Rats were allowed free access to water. The diet consumption of all animals was monitored daily. CrPic (200 lg/day) was administered by gavages for 10 weeks. Chromium was obtained as capsules containing 200 lg CrPic supplied by Arab Co. for Pharm. and Med. Plants (MEPA Co., Egypt). Chromium powder was
Blood collected into sodium citrate tubes was centrifuged at 1,800g for 10 min. Plasma was stored at -80 °C. Plasma total cholesterol (Chol) and TG were measured by the enzymatic colorimetric method [14, 15]. HDL-C was determined by precipitation method [16]. Apolipoprotein A1 (Apo-A1) and apolipoprotein B (Apo-B) concentrations were measured by Turbidimetry [17]. LDL was calculated using Friedewald formula: LDL-C = TC-HDL-C-TG/5 (provided that triglyceride levels were lower than 350 mg/ dL). Plasma thromboxane B2 was determined by the technique of Chard [18].
Materials and methods Animal handling, diets, and treatment
123
Statistical analysis Data were expressed as mean ± SD. Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by Tukey post hoc test for comparisons between individual groups. Differences were considered to be statistically significant when p was \0.05. Data
Ir J Med Sci Table 1 Plasma lipids in mg/dL observed in rats after 10 weeks of dietary feeding and chromium supplementation Parameter
NLD
NLD ? Cr
Chol
HF
HF ? Cr
172.66 ± 9.35
170.40 ± 14.71
249.13 ± 13.36**
179.53 ± 10.35
TG
96.26 ± 6.50
95.86 ± 7.39
155.46 ± 4.61**
100.33 ± 6.79
HDL-C
50.13 ± 3.66
51.80 ± 4.34
38.33 ± 2.55**
LDL-C
103.26 ± 8.72
99.40 ± 12.96
179.73 ± 13.96**
47.53 ± 4.74* 111.86 ± 9.44#
Apo-A1
139.13 ± 5.15
141.73 ± 6.36
123.33 ± 3.82**
137.80 ± 4.14
Apo-B
112.13 ± 4.45
109.66 ± 6.24
153.26 ± 8.94**
116.73 ± 8.69
Values represent mean ± SD for 15 animals NLD normolipemic diet, NLD ? Cr normolipemic diet ? chromium supplementation, HF high-fat diet, HF ? Cr high-fat diet ? chromium supplementation, Chol total cholesterol, TG triglycerides, HDL-C high-density lipoprotein cholesterol, LDL-C low-density lipoprotein cholesterol, Apo-A1 apolipoprotein A1, Apo-B apolipoprotein B * Significantly different from NLD group, p \ 0.05 ** Significantly different from NLD, NLD ? Cr, and HF ? Cr groups, p \ 0.0001 #
Significantly different from NLD ? Cr group, p \ 0.05
were analyzed using a statistical software package (SPSS for Windows, 10.0.1, 1999, SPSS Inc., Chicago, Ill, USA).
Results Plasma lipids Plasma was analyzed for TG, total cholesterol (Chol), HDLC, apo A1, and apo B (Table 1). Plasma LDL-C was calculated using Friedewald formula: LDL-C = TC-HDLC-TG/5 (Table 1). The HF group showed significant elevation of TG, Chol, LDL-C, and apo B levels with significant reduction in HDL-C and apo A1 levels compared to all other groups (p \ 0.0001). There was no significant difference between the NLD and NLD ? Cr groups regarding the lipid profile. Chromium supplementation in the HF ? Cr group improved the deteriorated lipid profile to control levels of NLD and NLD ? Cr groups except for HDL-C levels that were significantly lower in HF ? Cr group compared to NLD group (p \ 0.05) and LDL-C levels that were significantly higher in HF ? Cr group compared to NLD ? Cr group (p \ 0.05).
Fig. 1 ADP-induced platelet aggregation according to dietary intervention and chromium supplementation. NLD normolipemic diet, NLD ? Cr normolipemic diet ? chromium supplementation, HF high-fat diet, HF ? Cr high-fat diet ? chromium supplementation. Mean ± SD values (n = 15) are shown for each group. Significant at *p \ 0.0001 versus NLD, NLD ? Cr, and HF ? Cr groups
Platelet aggregation and plasma TX B2 Total platelet aggregation was measured after stimulation by either ADP (Fig. 1) or collagen (Fig. 2), and plasma TX B2 was determined (Fig. 3). The HF group had significantly increased ADP-induced aggregation compared with all other groups (P \ 0.0001). No significant difference in ADP-induced aggregation was encountered between NLD and NLD ? Cr groups. Chromium supplementation to hypercholesterolemic rats in HF ? Cr group normalized ADP-induced hyperaggregability to levels of NLD and
Fig. 2 Collagen-induced platelet aggregation according to dietary intervention and chromium supplementation. NLD normolipemic diet, NLD ? Cr normolipemic diet ? chromium supplementation, HF high-fat diet, HF ? Cr high-fat diet ? chromium supplementation. Mean ± SD values (n = 15) are shown for each group. Significant at *p \ 0.0001 versus NLD, NLD ? Cr, and HF ? Cr groups
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Fig. 3 Plasma thromboxane B2 according to dietary intervention and chromium supplementation. NLD normolipemic diet, NLD ? Cr normolipemic diet ? chromium supplementation, HF high-fat diet, HF ? Cr high-fat diet ? chromium supplementation. Mean ± SD values (n = 15) are shown for each group. Significant at *p \ 0.0001 versus NLD, NLD ? Cr, and HF ? Cr groups
NLD ? Cr groups. Similar trends between these groups were also observed with collagen-induced platelet aggregation. Plasma TX B2 (used to indirectly measure TX A2 production) was also significantly higher in HF group compared to all other groups (p \ 0.0001), whereas there was no significant difference between NLD and NLD ?Cr groups. Levels of TX B2 were normalized down to control levels in NLD and NLD ? Cr groups by chromium supplementation in HF ? Cr group with no significant difference between these groups.
Discussion The results of the present study revealed that a HF group produced significant elevation of plasma cholesterol, TG, LDL-C, apo B lipoprotein, and TX B2 as well as the % of platelet aggregation induced by ADP and collagen. This was accompanied by a significant reduction in plasma HDL-C and apo A-1 lipoprotein. Supplementation of hypercholesterolemic rats with CrPic in a dose of 200 lg/ 200 g rat/day (1 mg/kg/day), which is equivalent to a human dose of 0.16 mg/kg/day [19], administered by gavages for 10 weeks improved the deteriorated lipid profile, except for HDL-C levels that were significantly lower in HF ? Cr group compared to NLD group and LDL-C levels that were significantly higher in HF ? Cr group compared to NLD ? Cr group. Chromium supplementation also normalized ADP as well as collagen-induced hyperaggregability and levels of TX B2 to near control levels. Platelet aggregation has been shown to be enhanced by cholesterol, and 0.5 % cholesterol diet for 16 weeks induced platelet hyperaggregability to low doses of agonists as well as the development of hypercholesterolemic atherosclerosis in the rabbits [20]. In addition, a positive
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correlation was found for serum lipids and all the platelet activation markers [21]. Another study indicated that the ADP-induced platelet aggregation increased by 71.67 % in hypercholesterolemia [22]. Plasma total and LDL-C, which are established risk factors for atherosclerotic vascular disease, might also contribute to a prothrombotic risk via enhanced platelet reactivity [23]. The circulating lipoproteins may cause some abnormalities in platelet composition and function in hypercholesterolemia. It seems that in hyperlipidemia, some platelets are in an activated state in circulation and that increased lipid peroxidation, early apoptosis, platelet–leukocytes aggregate formation, and platelet aggregation altogether accompany this process [24]. Thus, the significant increase in platelet aggregation in hypercholesterolemic rats encountered in the present study could be due to an alteration in platelet signaling, caused by elevated cholesterol levels. Moreover, it has been shown that cholesterol depletion impairs platelet aggregation by altering platelet ultrastructure critical in mediating secretion [25]. Cholesterol depletion impairs microtubule ring formation and aggregate size, besides diminishing the extent of the open canalicular system and collagen-induced platelet ATP release [25]. Confirming the relation between lipids and platelet function, lipid-lowering drugs, such as ezetimibe and simvaststin, have been shown to decrease ADP and collagen-induced platelet aggregation [26]. Combination of statins and N-3 fatty acid in hypercholesterolemic patients has been reported to inhibit platelet aggregation [27]. Chromium has been recently shown to be involved in cholesterol homeostasis. Aguilar et al. [28] showed that Cr3 ? at 5 mcg per gram of food given to 20 Wistar rats for 10 weeks resulted in a significant decrease in cholesterol levels. Chromium supplementation also lowered plasma triglyceride and cholesterol levels to near controls, normalized LDL cholesterol and VLDL cholesterol levels in hypercholesterolemic diabetic animals, and improved total cholesterol to HDL cholesterol and HDL cholesterol to LDL cholesterol ratios suggesting its anti-atherogenic effect [29]. The results of the present study are in agreement with previous studies, and the persistent significant elevation of LDL-C and decrease in HDL-C seen in chromium-supplemented rats (HF ? Cr) compared to control groups could be attributed to the need of a longer time or higher doses of chromium. Recent studies support a novel action of proteins involved in cellular cholesterol homeostasis in response to CrPic. Pattar et al. [10] examined SREBP, a sterol-regulating transcription factor that localizes to the ER/Golgi membranes in its immature form. When the cell is in the need of cholesterol, cleavage of immature ER/Golgilocalized SREBP results in the release of the mature form of SREBP from the ER/Golgi membrane and accumulation
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of this active transcription factor in the nucleus where it binds to sterol response elements controlling the expression of proteins-regulating cholesterol homeostasis. For example, SREBP represses the expression of ABCA1, an ATPbinding cassette that mediates the cellular efflux of excess cholesterol. SREPB was upregulated by CrPic, whereas ABCA1 was decreased, consistent with the SREBP transcriptional repression of the ABCA1 gene [10]. Although the exact mechanism of Cr3?-induced cholesterol loss remains to be determined, these cellular responses highlight a novel and significant effect of chromium on cholesterol homeostasis. Furthermore, these findings provide an important clue to our understanding of how chromium supplementation might benefit hypercholesterolemia-associated disorders. The results of studies conducted on nondiabetic subjects with normal lipid profile, insulin sensitivity, and glucose tolerance showed that Cr3? supplements had only modest or no effects on the previous factors and that Cr3? supplements had positive effects in lowering elevated blood glucose, insulin, and lipid levels in subjects with insulin resistance and type 2 diabetes [30]. It was therefore unexpected to find a significant effect of chromium supplementation on lipid profile or platelet aggregation under normocholesterolemic conditions in the present study. In conclusion, the results of the present study show that CrPic might be an effective agent in inhibiting platelet hyperaggregability under hypercholesterolemic conditions. The extraordinary complexity of understanding chromium biology and toxicology certainly may involve an underappreciated cast of membrane lipids, including cholesterol. Given the enormous public health cost of cholesterolassociated diseases, the prospect of being able to use Cr3?—a relatively low-cost dietary supplement—as a therapy to correct cholesterol-dependent abnormalities merits further study. Conflict of interest
None.
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