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© 2006 American Society for Nutrition J. Nutr. 136:745S-749S, March 2006

Supplement: Significance of Garlic and Its Constituents in Cancer and Cardiovascular Disease

Homocysteine-Lowering Action Is Another Potential Cardiovascular Protective Factor of Aged Garlic Extract1–3,

Yu-Yan Yeh4 and Shaw-mei Yeh

Department of Nutritional Sciences, The Pennsylvania State University, University Park, PA 16803, USA

4 To whom correspondence should be addressed. E-mail: haru-amagase{at}wakunaga.com.


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
We investigated hypohomocysteinemic action as a cardiovascular protective property of aged garlic extract (AGE). Hyperhomocysteinemia was induced in rats by feeding folate-depleted diets. Plasma folate concentrations of 5, 24, and 202 nmol/L were detected in rats fed a folate-deficient L-amino acid diet containing succinyl sulfathiazole, an AIN-93G folate-deficient diet, and an AIN-93G folate-sufficient diet, respectively. Plasma concentrations of total homocysteine were elevated to the highest level (32 µmol/L) by severe folate deficiency and to a moderate level (9 µmol/L) by mild folate deficiency, compared with the lowest level of (5 µmol/L), noted for the folate-sufficient group. The addition of AGE to the severely folate-deficient diet decreased plasma total homocysteine concentration by 30%. Hyperhomocysteinemia caused by mild folate deficiency remained unaltered by AGE supplementation. The reduction in total homocysteine of the severely folate-deficient rats was accompanied by a proportional decrease in protein-bound and free homocysteine, resulting in an unchanged protein-bound:free homocysteine ratio. AGE added to the diet did not alter plasma concentrations of other aminothiol compounds: cysteine, glutathione, and cysteinylglycine. These data, together with increased S-adenosylmethionine and decreased S-adenosylhomocysteine concentrations in the liver, suggest that the hypohomocysteinemic effect of AGE most likely stems from impaired remethylation of homocysteine to methionine and enhanced transsulfuration of homocysteine to cystathionine. More importantly, in addition to its cholesterol-lowering potential, blood pressure–lowering effect, and antioxidant property, a hypohomocysteinemic action may be another important cardiovascular protective factor of AGE.

KEY WORDS: • folate deficiency • garlic • hyperhomocysteinemia • total homocysteine

Despite extensive intervention efforts over the past decades, heart disease remains the leading cause of death, followed by cancer and stroke, in the United States (1) and other countries (2). Environmental risk factors for cardiovascular diseases include hyperlipidemia, hypertension, diabetes, obesity, cigarette smoking, and physical inactivity (3,4). Among these factors, elevated plasma levels of total and low-density lipoprotein cholesterol and suboptimal high-density lipoprotein cholesterol are most closely associated with cardiovascular disease (5). Aside from these, elevated plasma concentration of homocysteine has recently been identified as a risk factor for cardiovascular diseases independent of hypercholesterolemia (6–11). Cardiovascular risk is further increased by a combination of hyperhomocysteinemia, hypertension, and smoking (12). It has been documented that plasma total-homocysteine levels in patients with vascular disease are significantly higher than those of normal subjects (13).

Similarly, patients with myocardial infarction had increased levels of homocysteine compared with men free of infarction (14). The risk for cardiovascular diseases caused by hypercholesterolemia is associated with atherosclerosis. However, the mechanism underlying homocysteine-induced cardiovascular diseases is still controversial. It has been suggested that homocysteine may impair production of endothelium-derived relaxing factor, stimulate proliferation of smooth cells, retard endothelial nitric oxide activity, and induce cardiovascular fibrosis (15–18). Because of the direct relation between plasma homocysteine level and risk of cardiovascular diseases, any attempt to reduce plasma homocysteine is warranted (19).

Mild hyperhomocysteinemia occurs commonly amid deficiencies of folic acid, vitamin B-6, and vitamin B-12, alone or in combination (20–22). Severe hyperhomocysteinemia has been reported in individuals with genetic defects in enzymes such as cystathionine ß-synthase (7,23) and N5,N10-methylenetetrahydrofolate reductase (23,24). Conversely, folic acid supplementation is effective in reversing elevated homocysteine level (9,12,19). Garlic contains a variety of aminothiol compounds that may interact with free and protein-bound homocysteine. However, our previous report indicated that a reduction in plasma level of homocysteine could not be attributed to disulfide-disulfide exchange and thiol-disulfide exchange among aminothiol compounds and homocysteine (25). The objectives of this article are to review hypohomocysteinemic effects of aged garlic extract (AGE)5 and to integrate the new finding into currently documented mechanisms underlying cardiovascular protective properties of AGE (26–28).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 LITERATURE CITED
 
    Animals and diets. Growing male rats of the Sprague-Dawley strain purchased from Harlan Sprague-Dawley Co. (Indianapolis, IN) were used throughout the study. A folic acid–deficient L-amino acid diet containing 1% succinyl sulfathiazole (Dyets, Inc.) was used to produce severe folic-acid deficiency (–SFA) (29). Two groups of 8 rats each were fed the diet either supplemented with AGE or without it. AGE was supplemented at 4% of diet. Rats were fed for 4 wk because they began to show signs of slow growth after this time period. Animals had access to diet and water at all times. Body weight was measured every 4 d during the feeding. In another series of experiments, rats were made mildly deficient in folate. They were divided into 4 groups of 10 each and fed for 6 wk with 1 of 4 diets: AIN-93G folic-acid sufficient (2 mg/kg of diet); AIN-93G folic-acid deficient; AIN-93G folic-acid sufficient and supplemented with AGE (4% of diet, wt:wt); AIN-93G folic-acid deficient and supplemented with AGE. All animals were decapitated and used for hematological and biochemical analysis. The animal protocol was reviewed and approved by The Pennsylvania State University Institutional Animal Care and Use Committee.

    Blood and tissue preparation. After overnight fasting prior to the termination of feeding period, blood samples were obtained and centrifuged at 200 x g for 5 min at 4°C within 1 h. Plasma was obtained and stored at –80°C until analysis. Liver samples were excised and stored in glass tubes with caps at –80°C. For determination of S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH), 1 g of fresh liver was homogenized in 5 mL of 0.4 mol/L perchloric acid (30). The homogenate was centrifuged at 25,700 x g for 10 min. The resulting supernatant was collected and filtered through a 0.4-µm Gelman syringe filter (VWR Scientific).

    Folate assays. Folic acid in plasma was analyzed by microbiological assay (31) using Lactobacillus casei as the test organism. 5-Formyltetrahydrofolate (5-HCO-H4OteGlu, calcium salt) served as the standard. For determination of tissue folic acid, frozen liver samples (~1 g) were added to 15 volumes of fresh folate extraction buffer consisting of sodium ascorbic acid (2%, wt:v), 0.2 mol/L 2-mercaptoethanol, 50 mol/L Hepes, 50 mol/L Ches, pH 7.85 (32). The resulting supernatant was used for assay. Folate was determined by the microbiological assay after treatment of the supernatant with rat serum folate conjugase to convert polyglutamates to their corresponding monoglutamate derivatives (33). For determination of folate in AGE the same microbiological assay was used.

    Homocysteine assays. Plasma total homocysteine was determined by the HPLC method of Araki and Sako (34), as modified by Vester and Rasmussen (35), using a fluorescence detector. A Waters HPLC system (Waters Instruments) was equipped with a column (Waters Nova-Pak C18, 4-µm particle size, 60 x 150 mm column) protected by a guard column (Waters Sentry Guard Column), pumps (Waters 501), injector (Waters U6K), scanning fluorescence detector (Waters 470), and chromatography workstation (Waters Baseline 810). The assays were performed isocratically with acetic-acid buffer (0.1 mol/L acetic acid–0.1 mol/L sodium acetate, pH 4.8) at a flow rate of 0.6 mL/min. The fluorescence intensities were detected with excitation at 385 nm and emission at 515 nm. Homocysteine, cysteinylglycine, cysteine, and glutathione were quantified by comparing their fluorescence intensity peaks with those of corresponding standards. For analysis of free homocysteine, plasma samples were first treated with 0.6 mol/L perchloric acid containing 1 mmol/L EDTA.

    SAH and SAM assays. The protein-free liver supernatant prepared above was used for measurement of hepatic SAM and SAH according to the method described by Miller et al. (30). A buffer containing 4% acetonitrile in 10 mmol/L ammonium formate/4 mmol/L heptanesulfonic acid (pH 4.0) was used in an isocratic system at flow rate of 0.7 mL/min. The peaks of compounds that appeared on the chromatograph were detected at 254 nm by a UV-VIS detector (Waters 486).

Materials. Folate-deficient L-amino acid rodent diets were prepared by Dyets, Inc. All ingredients for AIN-93G diet were purchased from ICN Biochemicals. AGE was donated by Wakunaga of America Co., Ltd. Chemicals. Standards used for HPLC analyses of homocysteine and aminothiol compounds were obtained from Sigma Chemicals. For analysis of folate, buffer ingredients, enzymes, and folate conjugase were products of Sigma Chemicals. Rat serum was obtained from Harlan Bioproducts for Science. Lactobacillus casei was provided by the American Type Culture Collection. Acetonitrile, ammonium formate, and heptanesulfonic acid were provided by Fisher Scientific.

Statistics. Data are presented as means ± SD for 8 or 10 rats. For determination of statistically significant differences, Student's t-test was used when 2 diet groups were compared, and ANOVA was employed for comparison of 4 dietary groups. When statistical significance was indicated by ANOVA, Fisher's protected least significant difference multiple-comparison test was applied to identify significant difference among means at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 LITERATURE CITED
 
Rats fed a folate-deficient L-amino acid diet in the presence of 1% succinyl sulfathiazole had 5.2 ± 0.5 nmol of folic acid per milliliter of plasma and 5.4 ± 0.9 nmol/g of folic acid in the liver (Fig. 1). The rats clearly developed a severe folate deficiency compared with 202.1 ± 29.1 nmol/L of folate seen in plasma of rats fed an AIN-93G diet containing a sufficient amount of folic acid. The markedly low plasma and liver folate levels were unchanged by supplementation with AGE (Fig. 1). Despite severe folate deficiency, rats supplemented with or without AGE grew equally well as indicated by the comparable initial and final body weights and body weight gains over the 4 wk of feeding (Fig. 2). Growth retardation became apparent when animals were fed beyond 4 wk (data not shown). Plasma level of total homocyteine was 31.9 ± 12.7 µmol/L detected in rats fed the folate-deficient L-amino acid diet containing no AGE (Fig. 3) as compared with 9.3 ± 3.2 µmol/L for rats fed a folate-deficient AIN-93G diet. AGE added to the folate-deficient L-amino acid diet reduced plasma total homocysteine by 30%. Plasma levels of protein-bound and free homocysteine were decreased by AGE in proportion to that of total homocysteine (Fig. 3). Adding AGE to the folate-deficient L-amino acid diet had no effect on plasma levels of the aminothiol compounds cysteine, glutathione, and cysteinylglycine (Fig. 4). Liver concentration of SAM of the rats fed the nonsupplemented diet was 22.5 ± 5.2 nmol/g (Fig. 5). AGE supplementation increased SAM concentration by 26%. Conversely, AGE supplementation lowered liver concentration of SAH by 15%.


Figure 1
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  		FIGURE 1  Plasma and liver concentrations of folate in severely folate-deficient rats supplemented with and without AGE. –SFA = severe folate-deficiency, –SFA+AGE = severe folate deficiency supplemented with AGE. Values are means ± SD for 8 rats.

 

Figure 2
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  		FIGURE 2  Body weights and weight gains in severely folate-deficient rats supplemented with and without AGE. –SFA = severe folate deficiency, –SFA+AGE = severe folate deficiency supplemented with AGE. Values are means ± SD for 8 rats.

 

Figure 3
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  		FIGURE 3  Plasma concentrations of total, protein-bound, and free homocysteine in severely folate-deficient rats supplemented with and without AGE. –SFA = severe folate deficiency, –SFA+AGE = severe folate deficiency supplemented with AGE . Values are mean ± SD for 8 rats. Asterisk indicates significant difference from –SFA at *P < 0.05.

 

Figure 4
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  		FIGURE 4  Relative concentrations of aminothiol compounds in severely folate-deficient rats supplemented with and without AGE. –SFA = severe folate deficiency, –SFA+AGE = severe folate deficiency supplemented with AGE. Concentrations of cysteine, glutathione, and cysteinylglycine of 75.3 ± 5.6, 5.0 ± 0.6, and 0.12 ± 0.02 µmol/L, respectively, in control group were set as 100%. Values are means ± SD for 8 rats.

 

Figure 5
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  		FIGURE 5  Liver concentrations of S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) in severely folate-deficient rats supplemented with and without AGE. –SFA = severe folate deficiency, –SFA + AGE = severe folate-deficiency supplemented with AGE. Values are mean ± SD for 8 rats. Asterisk indicates significant difference from –SFA at *P < 0.05.

 
In a series of experiments, rats were made mildly deficient in folate by being fed the AIN-93G diet. Four groups of animals fed diets supplemented with or without folate and/or AGE had similar growth rates as indicated by the comparable initial and final body weights (Table 1). Plasma folate levels in 2 groups of animals that received folate-deficient diets ranged from 24 to 35 nmol/L, which was only 11–16% of that of rats fed folate-sufficient diets (Table 1). Addition of AGE to a folate-sufficient diet slightly increased folate levels in plasma and the liver. Liver concentrations of folate were ~50% lower in rats fed a folate-deficient diet than in those fed folate-sufficient diets. As a result of the changes in plasma and liver folate, plasma level of homocysteine in the folate-deficient group was moderately increased to 9.4 µmol/L, 85% higher than that of the folate-sufficient group. AGE supplementation of diets deficient or sufficient in folate had no effect on plasma levels of homocysteine (Table 1).


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  		TABLE 1 Change of body weight, folate, and homocysteine in rats fed AIN-93G diets supplemented with or without folate and AGE1

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
LITERATURE CITED
 
Severe folate deficiency was produced by a folate-deficient L-amino acid diet in the presence of succinyl sulfathiazole, whereas an AIN-93G folate-depleted diet caused relatively mild folate deficiency. These were clearly demonstrated by plasma folate concentrations of 5, 24, and 202 nmol/L observed for rats fed the folate-deficient L-amino acid diet, the AIN-93G folate-depleted diet, and the AIN-93G folate-sufficient diet, respectively. The reduction of plasma folate levels was accompanied by increased plasma homocysteine concentrations, with the highest noted for the folate-deficient L-amino acid diet group (32 µmol/L), followed in decreasing order by those for the AIN-93G folate-depleted diet (9 µmol/L) and for the AIN-93G folate-sufficient diet (5 µmol/L). Interestingly, AGE supplementation significantly attenuated hyperhomocysteinemia caused by severe folate deficiency but not that caused by mild folate deficiency. Although there was a strong inverse relation between plasma concentration of folate and homocysteine, the homocysteine-lowering effect of AGE could not be attributed to folate content of AGE. It was estimated in our study that AGE contained 7.1 µg of folate/g. AGE added to diets at a 4% level did not increase plasma and liver folate levels in rats fed the folate-deficient L-amino acid diet (data not shown). However, addition of AGE to the AIN-93G folate-depleted diet slightly increased (6–11%) plasma and liver folate but did not lower plasma homocysteine level (Table 1).

Homocysteine homeostasis is modulated by synthesis of homocysteine, remethylation of homocysteine, and transsulfuration of homocysteine (19,23). Homocysteine may be remethylated to methionine by cobalamin (B-12)-dependent methionine synthase requiring methyltetrahydrofolate. Methyltetrahydrofolate, on the other hand, is the reaction product of methylenetetrahydofolate catalyzed by N5,N10-methylenetetrahydrofolate reductase (MTHFR) (19,23,36). Transsulfuration for converting homocysteine to cystathionine is facilitated by cystathionine ß-synthase (19,23). SAH is a feed-forward activator of S-adenosylhomocysteine hydrolase (37). A decrease in the activity of MTHFR, an increase in the activity of cystathionine ß-synthase and/or inhibition of S-adenosylhomocysteine hydrolase could lead to reduced level of homocysteine. SAM is an inhibitor of MTHFR, and an activator of cystathionine ß-synthase (19,36). Our study demonstrated that AGE supplementation resulted in higher hepatic SAM but lower SAH concentrations than those of the nonsupplemented group. The results led us to speculate that the hypohomocysteinemic action of AGE stems in part from inhibition of MTHFR and stimulation of cystathionine ß-synthase. A reduction of SAH concentration resulting in relieving the activation of S-adenosylhomocysteine hydrolase could further decrease plasma homocysteine.

The possibility of roles of disulfide–disulfide exchange and thiol–disulfide exchange (25,38) leading to alteration in homocysteine distribution and hence concentration of total homocysteine was also explored. AGE supplementation, however, did not affect the exchange reactions as indicated by unchanged concentrations of cysteine, glutathione, cysteinylglycine, and unaltered ratio of protein-bound to free homocysteine.

Finally, the cardiovascular protective effects of AGE have been attributed to its cholesterol-lowering potential (26,27), blood pressure–lowering effect (26), and antioxidant property (28). The current study revealed that hypohomocysteinemic action may be another important factor to protect against cardiovascular disease.


   ACKNOWLEDGMENTS
 
The authors are grateful for technical assistance provided by Dr. Xi-Bin Wang.


   FOOTNOTES
 
1 Published in a supplement to The Journal of Nutrition. Presented at the symposium "Significance of Garlic and Its Constituents in Cancer and Cardiovascular Disease" held April 9–11, 2005 at Georgetown University, Washington, DC. The symposium was sponsored by Strang Cancer Prevention Center, affiliated with Weill Medical College of Cornell University, and Harbor-UCLA Medical Center, and co-sponsored by American Botanical Council, American Institute for Cancer Research, American Society for Nutrition, Life Extension Foundation, General Nutrition Centers, National Nutritional Foods Association, Society of Atherosclerosis Imaging, Susan Samueli Center for Integrative Medicine at the University of California, Irvine. The symposium was supported by Alan James Group, LLC, Agencias Motta, S.A., Antistress AG, Armal, Birger Ledin AB, Ecolandia Internacional, Essential Sterolin Products (PTY) Ltd., Grand Quality LLC, IC Vietnam, Intervec Ltd., Jenn Health, Kernpharm BV, Laboratori Mizar SAS, Magna Trade, Manavita B.V.B.A., MaxiPharm A/S, Nature's Farm, Naturkost S. Rui a.s., Nichea Company Limited, Nutra-Life Health & Fitness Ltd., Oy Valioravinto Ab, Panax, PT. Nutriprima Jayasakti, Purity Life Health Products Limited, Quest Vitamins, Ltd., Sabinco S.A., The AIM Companies, Valosun Ltd., Wakunaga of America Co. Ltd., and Wakunaga Pharmaceutical Co., Ltd. Guest editors for the supplement publication were Richard Rivlin, Matthew Budoff, and Harunobu Amagase. Guest Editor Disclosure: R. Rivlin has been awarded research grants from Wakunaga of America, Ltd. and received an honorarium for serving as co-chair of the conference; M. Budoff has been awarded research grants from Wakunaga of America, Ltd. and received an honorarium for serving as co-chair of the conference; and Harunobu Amagase is employed by Wakunaga of America, Ltd. Back

2 Supported in part by Wakunaga of American Company, Ltd. and Elmore Funds. Back

3 Author disclosure: No relationships to disclose. Back

5 Abbreviations used: AGE, aged garlic extract; MTHFR, N5,N10-methylenetetrahydrofolate reductase; SAH, S-adenosylhomocysteine; SAM, S-adenosylmethionine. Back


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