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Department of Applied Life Science, Faculty of Applied Biological Sciences, Gifu University, Gifu 501-1193, Japan and * Dainippon Ink and Chemicals, Chiba 290-8585, Japan
1To whom correspondence should be addressed. E-mail: nagaoka{at}cc.gifu-u.ac.jp.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • algae • C-phycocyanin • cholesterol • rat • spirulina
Spirulina platensis and Spirulina maxima have long histories of use as foods for humans. Although spirulina is used extensively as a food supplement in many countries, regulatory authorities have not yet approved its use as a "food for specified health use" in Japan. This type of approval is generally withheld until active components beneficial to health are fully identified. Dietary proteins were shown to influence serum cholesterol concentrations in many studies (1–4). Most animal and human studies on the effects of dietary proteins on serum cholesterol concentrations to date have focused on a comparison of soybean protein and milk casein. As a result, limited data are available on the effects of algal protein on cholesterol metabolism.
Among the few studies that were conducted on algal proteins, 2 suggested that Spirulina platensis concentrate (SPC)2 imparts a hypocholesterolemic effect in rats (5) and humans (6). If this is so, SPC must contain one or more factors that attenuate diet-induced hypercholesterolemia. SPC consists of protein, lipid, chlorophyll, carotenoids, vitamins, minerals, and phycobiliproteins such as C-phycocyanin (PHY). The mechanism by which SPC induces hypocholesterolemia and the active factor responsible for the hypocholesterolemic action have yet to be identified. Moreover, no studies to date have examined the effect of the major phycobiliprotein, PHY.
Until now, no researchers have identified any algal proteins or proteins in SPC that exert hypocholesterolemic action. As an alternative, we hypothesized that a protein derived from SPC might induce hypocholesterolemic action. To pursue this hypothesis, we attempted to clarify the mechanism of SPC-induced hypocholesterolemia by in vivo studies and in vitro assays related to the taurocholate binding capacity, micellar solubility of cholesterol, and cholesterol absorption in Caco-2 cells. We also attempted to identify a novel hypocholesterolemic protein derived from SPC.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Preparation of SPC acetone extract (SPC lipid-rich fraction). SPC was dispersed in 85% (v:v) acetone, stirred with a stirrer at 4°C for 16 h, filtered, concentrated by an evaporator, and mixed with an equal portion of dextrin (dextrin:concentrate = 1:1). The concentrate with dextrin was used as the SPC acetone extract.
Preparation of PHY and PHY residue. PHY was prepared by a previously described procedure (7) with slight modifications. SPC was dispersed in 0.1 mol/L Na-phosphate buffer (pH 6.0) 5% (wt:v), stirred with a stirrer at 4°C for 16 h, and centrifuged at 10,000 x g for 30 min. The supernatant obtained was freeze-dried and used as crude PHY. Next, a portion of the precipitate was washed 3 times with 2 volumes of deionized water, freeze-dried, and used as the PHY residue. The crude PHY was dissolved in 30 mmol/L potassium-phosphate buffer (pH 6.0) at a concentration of 4% (wt/v), precipitated in 20% (NH4)2 SO4, and recovered by centrifugation at 10,000 x g for 30 min.
The resulting supernatant was precipitated in 60% (NH4)2 SO4 and recovered by centrifugation at 10,000 x g for 30 min. The precipitate was then dissolved in 50 mmol/L potassium-phosphate buffer (pH 6.0) and dialyzed against the same buffer. The dialyzed preparation was placed in a DEAE-Cellulose (DE-52) (Whatman International) column and purified by a stepwise elution with 50 mmol/L potassium-phosphate buffer (pH 6.0) of increasing ionic strengths as follows. The first fraction was eluted by 0.1 mol/L KCl containing 50 mmol/L potassium-phosphate buffer (pH 6.0) and discarded. The second fraction (the one with the highest 618 nm:280 nm ratio) was eluted by 0.2 mol/L KCl containing 50 mmol/L potassium-phosphate buffer (pH 6.0), precipitated in 65% (NH4)2 SO4, and recovered by centrifugation at 10,000 x g for 30 min. The precipitate was dissolved in deionized water and dialyzed against deionized water. The dialyzed preparation was freeze-dried and used as PHY.
Chemical analyses. The protein content was determined by the Kjeldahl method (8) with an N:protein conversion factor of 6.25. Lipids were extracted using chloroform:methanol (2:1, v:v) and weighed. The sugar content was determined by the phenol-sulfonic acid method (9). The dietary fiber content was determined by previously described methods (8). Moisture was determined as the loss in weight after drying at 105°C for 24 h. The ash content was determined by the direct ignition method (550°C overnight).
The PHY content was determined by a method based on the absorption spectrum of PHY as described previously (7,10): 500 mg of sample was weighed into a 50-mL centrifuge tube; 25 mL of 0.1 mol/L Na-phosphate buffer (pH 6.0) was pipetted into this tube and mixed. This tube was kept at 30°C for 16 h and then centrifuged at 2000 x g for 15 min. The supernatant was filtered, and distilled water was added to 2 mL of the filtrate to a volume of 50 mL; absorbance was measured at 560, 618, and 650 nm. The PHY concentration (g/100 g) was calculated by:
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where AS 618 is the absorbance at 618 nm, AS 560 is the absorbance at 560 nm, AS 650 is the absorbance at 650 nm, W is the weight of the sample (500 mg), and D is the dilution ratio (625). Casein was generously supplied by the Central Research Institute of Meiji Dairy Products. The chemical compositions of casein, SPC, SPC acetone extract, PHY, and PHY residue are given in Table 1. The amino acid composition (Table 2) and tryptophan content were determined by a previously described method (11).
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Animals and diets. Male Wistar rats (Japan SLC) were used in these studies. The room was maintained at 22 ± 2°C with a 12-h light cycle (800–2000 h). The Gifu University Animal Care and Use Committee approved all of our animal experiments. All rats were housed individually in metal cages and allowed free access to food and water.
Casein/SPC and taurocholate binding capacity in vitro. The binding capacity of cholestyramine, casein, or SPC with taurocholate was measured as previously described (11) with slight modifications. The mixture contained 1.85 kBq of tauro [carbonyl-14C] cholic acid (sodium salt) (1.89 Gbq/mmol, Amersham International), 0.1 mol/L sodium taurocholate in 2 mL of 0.1 mol/L Tris-HCl buffer (pH 7.4), and 1–200 mg binding substances [cholestyramine, casein: casein sodium (Wako Pure Chemical), SPC].
Casein/SPC and micellar solubility of cholesterol in vitro. The micellar solubility of cholesterol with casein or SPC in vitro was measured by a previously described method (11) with some modifications. The [14C]-labeled micellar solutions (1.0 mL) were prepared at the following concentrations and mixed by sonication (Ultrasonic Homogenizer, Model VP-5, Taitec): 0.74 kBq [4-14C]-cholesterol (2.1 Gbq/mmol, NEN), 0.1 mmol/L cholesterol (Sigma), 6.6 mmol/L sodium taurocholate (Sigma), 1 mmol/L oleic acid (Sigma), 0.6 mmol/L phosphatidylcholine (Sigma), 0.5 mmol/L monoolein (Sigma), 132 mmol/L NaCl, and 15 mmol/L sodium phosphate (pH 7.4). After incubation at 37°C for 24 h, casein (casein sodium, Wako Pure Chemical) or SPC (5 g/L, respectively) was added to the micellar solution, solubilized by sonication, incubated at 37°C for 1 h, and centrifuged at 100,000 x g for 60 min at 37°C. The supernatant was collected for the determination of [14C]-cholesterol by a liquid scintillation counter.
Casein/SPC and cholesterol absorption in Caco-2 cells in vitro. Caco-2 cells were acquired from the American Type Culture Collection. The cells were maintained as described previously (11,12). Monolayers were grown in 48-well plastic dishes containing 0.5 mL fetal bovine serum supplemented with DMEM. [14C]-Labeled micellar cholesterol uptake in Caco-2 cells was measured as described previously (11,12).
The [14C]-labeled micellar solutions (0.2 mL) contained casein (casein sodium, Wako Pure Chemical) or SPC (5 g/L, respectively). The cellular protein was determined by a commercially available kit (Bio-Rad Protein Assay, Bio-Rad). The amount of cholesterol absorbed into the cells was expressed as pmol/mg protein.
Dietary casein/SPC and cholesterol metabolism in rats in vivo (Expt. 1). After acclimation to a commercial nonpurified MF diet (Oriental Yeast) for 3 d, 5-wk-old rats weighing 90–110 g were divided into 2 groups of 6 rats each based on body weight. Each group had free access to one of the test diets containing casein or SPC as the protein source for 10 d. The basal diet composition followed that of our previous study (11). The diet composition (13) was (g/100 g): casein, 20; corn oil, 1.0; lard, 5; AIN93G mineral mixture, 3.5; AIN93G vitamin mixture, 1.0; choline chloride, 0.2; cholesterol, 0.5; sodium cholate, 0.25; sucrose, 24.02; and starch, 48.03.
SPC (10% protein level) was added to the diet. Adjustments in the amounts of carbohydrate (1 part sucrose and 2 parts gelatinized cornstarch) were used to compensate for the changes in the dietary levels of SPC. After 24 h of food deprivation, the rats were anesthetized with diethyl ether and killed. Blood was collected by cardiac puncture and the liver was removed. Fecal collections (7–9 d) for the determination of fecal steroid were completed before the 24-h food deprivation and blood sampling. Serum, liver, and fecal lipids were determined.
Dietary casein/SPC acetone extract and cholesterol metabolism in rats in vivo (Expt. 2). Acclimation and treatment of rats were as in Expt. 1. Casein or casein containing SPC acetone extract w as the protein source for 10 d. Adjustments in the amounts of carbohydrate were as in Expt. 1. Food deprivation, blood sampling, and killing were as in Expt. 1. Serum and liver lipids were determined.
Dietary casein/SPC, PHY/PHY residue and cholesterol metabolism in rats in vivo (Expt. 3). Acclimation and treatment were as in the earlier experiments. Rats were divided into 4 groups of 6 rats each based on the body weight. Each group had free access to one of the test diets containing casein, casein containing SPC, casein containing PHY, or casein containing PHY residue as the protein source for 5 d. The basal diet included the same components used in in vivo Expt. 1. SPC, PHY, or PHY residue (as 3% protein level) was added to the diets. Adjustments in the amounts of carbohydrate were as in Expt. 1. Fecal collections were for 2–4 d; food deprivation, killing, blood collection, and liver removal were as in Expt. 1. Serum, liver, and fecal lipids were determined.
Statistical analyses. Results are expressed as means and SEM. Data were analyzed by Student’s t test (14) or one-way ANOVA and Duncan’s multiple range test (15). Differences were considered significant at P < 0.05.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Cholesterol absorption in Caco-2 cells in vitro. The cholesterol uptake from micelles containing SPC (53.8 ± 2.4 pmol/mg protein) was lower than that of cholesterol micelles containing casein (102.3 ± 11.2 pmol/mg protein, P < 0.01).
Dietary casein/SPC and cholesterol metabolism in rats in vivo (Expt. 1). Body weight gains, liver weight, and food intake were all unaffected by dietary treatment (Table 3). The serum total cholesterol and atherogenic index were significantly lower in the SPC group than in the casein group. The serum HDL cholesterol concentration was significantly higher in the group fed SPC than in the casein group. The total lipid and cholesterol concentrations in the liver were significantly lower in the SPC group than in the casein group. The fecal dry weight was significantly higher in the groups fed SPC than in the casein group. The fecal outputs of total steroids were significantly higher in the SPC groups than in the casein group.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Our first step in this study was to clarify experimentally the mechanism underlying the hypocholesterolemic effects of SPC. The serum cholesterol concentration and atherogenic index were significantly lower in rats fed a diet containing 10% SPC than in rats fed a diet containing casein. The serum HDL cholesterol concentration was significantly higher in the group fed 10% SPC than in the group fed casein. It was postulated that the degree of serum cholesterol–lowering activity depends on the degree of fecal steroid excretion (acidic steroids+ neutral steroids) (21). We found a significant negative correlation between fecal total steroid excretion and serum total cholesterol in rats fed diets containing soy protein (11), soy protein peptic hydrolysate (SPH) (11), and ß-lactoglobulin tryptic hydrolysate (LTH) (22). The present study demonstrated a higher fecal excretion of total steroids (acidic steroids + neutral steroids) in rats fed SPC, indicating that the effect is due at least in part to an enhancement of fecal steroid excretion.
Cholesterol is solubilized in bile salt-mixed micelles and then absorbed (23). The present study indicated that the micellar solubility of cholesterol was significantly lower in the presence of SPC than in the presence of casein. We were also very interested to discover that the micellar solubility of cholesterol was significantly suppressed in the presence of SPH (11), LTH (22), and egg ovomucin (24) in vitro. Sitosterol (25), sesamin (26), and catechin (27) also lowered the micellar solubility of cholesterol in conjunction with serum cholesterol–lowering effects in rats. When coupled with our results for SPC, these findings suggest that the suppressed micellar solubility of cholesterol inhibits the cholesterol absorption in the jejunum, a process that may be closely related to the action of these agents in lowering serum cholesterol. As shown in the cases of SPC, LTH (22), SPH (11), and egg ovomucin (24), other dietary proteins and peptides may also affect the micellar solubility of cholesterol. Several recent studies used monolayers of Caco-2 cell cultures as a model system to examine the process of lipid metabolism (28,29). For example, Field et al. (28) reported that Caco-2 cells possess features also found in the small intestine and small intestinal cells, namely, the abilities to absorb micellar cholesterol and express marker enzymes such as alkaline phosphatase, respectively. Earlier studies by our group demonstrated for the first time that cholesterol micelles containing SPH (11), LTH (22), and egg ovomucin (24) significantly suppressed cholesterol absorption by Caco-2 cells in vitro. To make up for the dearth of information on the effects of SPC on cholesterol absorption, we used Caco-2 cells or rats to investigate the mechanisms of the serum cholesterol–lowering action of SPC. Our experimental system to evaluate cholesterol absorption with Caco-2 cells proved very useful in clarifying the molecular mechanism underlying the inhibitory effect of SPC on cholesterol absorption from the small intestine, a mechanism heretofore unknown. Few experimental studies to date have used cultured intestinal cells to evaluate the effects of proteins and peptides on cholesterol-absorbing cells (11,12,22,24). In this study, SPC lowered serum cholesterol concentrations in rats and inhibited cholesterol absorption in Caco-2 cells. We hypothesize that SPC or its hydrolysate binds to bile acids in the jejunum; this binding affects the micellar solubility of cholesterol and then suppresses cholesterol absorption. Furthermore, our preliminary observations suggested that SPC-derived lipid (SPC acetone extract, SPC derived lipid mainly consists of palmitic acid) or carbohydrates did not induce a significant effect on either the suppression of micellar solubility of cholesterol or the inhibition of cholesterol absorption in Caco-2 cells under our experimental conditions as shown in this study. These results suggest that the suppressed cholesterol absorption via the direct interaction between cholesterol-mixed micelles and SPC in the jejunal epithelia constitutes at least part of the mechanism of SPC-induced hypocholesterolemia. SPC increases fecal cholesterol and bile acid excretion (Table 3). SPC may also induce the inhibition of the reabsorption of bile acids in the ileum, thus attenuating hypercholesterolemia in rats. The SPC-induced increase in bile acid excretion was enhanced by PHY feeding. Because the PHY residue exhibits a weaker hypocholesterolemic activity than PHY itself, the latter can be presumed to play a crucial role in SPC-induced hypocholesterolemia. Although an earlier paper (30) reported that SPC-derived lipids exert hypocholesterolemic action, our present study using lipids derived from an SPC acetone extract could not corroborate this result.
Thus, our findings indicated that SPC-derived lipids cannot play the principal role in SPC-induced hypocholesterolemia. Another previous report identified algal carbohydrates as the most likely hypocholesterolemic factor in red microalga, yet the dietary concentration of algal carbohydrates that elicited hypocholesterolemia in rats was as high as 14% (31). In contrast, our diet in the present study contained only 1.8% dietary carbohydrates derived from SPC. Because SPC contains lower amounts of carbohydrates than red microalga, the carbohydrates in SPC cannot play the key role in the SPC-induced hypocholesterolemia. Many previous studies suggested that dietary fiber induces a hypocholesterolemic action (32). In our study, however, we were very interested to find that our PHY preparation containing no dietary fiber still elicited a hypocholesterolemic action. In conclusion, the present study clearly suggests that SPC-derived phycobiliprotein (PHY) itself plays a crucial role in SPC-induced hypocholesterolemia. The novel hypocholesterolemic protein C-phycocyanin will play a pivotal role in both the study of the effect of algal proteins on cholesterol metabolism and the use of spirulina in "food for specified health use" in Japan or worldwide.
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FOOTNOTES |
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Manuscript received 6 May 2005. Initial review completed 2 June 2005. Revision accepted 20 July 2005.
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LITERATURE CITED |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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