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Correlation between dietary polyunsaturated fatty acids and plasma homocysteine concentration in vitamin B6-deficient rats
Nutrition, Metabolism & Cardiovascular Diseases (2005) 15, 94e99
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Correlation between dietary polyunsaturated fatty acids and plasma homocysteine concentration in vitamin B6-deficient rats L. Cabrini a,*, D. Bochicchio b, A. Bordoni a, S. Sassi c, M. Marchetti a, M. Maranesi a a
Department of Biochemistry ‘‘G. Moruzzi’’, University of Bologna, Via Irnerio 48, 40126 Bologna, Italy b Zootechnical Experimental Institute, S. Cesario s.P., Modena, Italy c Department of Angiology and Blood Coagulation, S.Orsola-Malpighi University Hospital, Bologna, Italy Received 19 May 2004; accepted 8 November 2004
KEYWORDS Vitamin B6; Homocysteine; Dietary PUFA; D6 desaturase
Summary Background and aim: Vitamin B6 as cofactor of D6 desaturase is involved in polyunsaturated fatty acid metabolism; moreover, it is a cofactor of the trans-sulfuration pathway of homocysteine. Some studies report that low concentrations of pyridoxine, by increasing homocysteine levels, are associated with coronary artery disease, and carotid and arterial lesions. The aim of this study was to verify whether different dietary amounts of polyunsaturated fatty acids associated with low content of vitamin B6 could modulate homocysteinemia. Methods and results: Thirty-two rats were divided into two groups, one fed a diet with adequate vitamin B6 content the other a diet containing low amount of the same vitamin. Within each group, rats were divided into two subgroups differing in the polyunsaturated fatty acid content of the diet (63 and 33%, respectively). The vitamin B6-deficient diet induced an increase in homocysteine concentration compared to the vitamin B6-normal diet. This increase was tenfold in the subgroup fed high polyunsaturated fatty acid levels and twofold in the other subgroup. The fatty acid composition of liver phospholipids showed a lower arachidonic acid relative molar content and a lower 20:4/18:2 ratio in vitamin B6-deficient groups compared with B6-normal groups. Conclusions: On the basis of the different biological functions of pyridoxine and considering that some factors closely related to atherosclerosis are vitamin B6 dependent, adequate pyridoxine availability could be necessary to assure
* Corresponding author. Tel.: C39 051 2091222; fax: C39 051 2091234. E-mail address: [email protected] (L. Cabrini). 0939-4753/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.numecd.2004.11.002
Dietary PUFA, vitamin B6 and homocysteinemia
95
a normal long chain fatty acid metabolism and to reduce the risk linked to hyperhomocysteinemia. Ó 2005 Elsevier Ltd. All rights reserved.
Introduction High level of homocysteine in plasma has been recognized as a further marker of cardiovascular diseases among well-known risk factors. Hyperhomocysteinemia causes endothelial damage leading to a high risk for the atherosclerotic process in coronary, cerebral and peripheral vessels [1e3] and for arterial and venous thromboembolism [4]. High plasma levels of homocysteine are attributed not only to genetic, but also to dietary factors [5,6], such as high methionine content [7] or deficiencies in B vitamins, particularly folic acid, vitamin B12 and vitamin B6 [8,9]. These vitamins are coenzymes or cofactors of the transmethylation or trans-sulfuration pathways of homocysteine. In pyridoxine deficiency, the increased plasma homocysteine concentration is due to the lower activity of B6 dependent enzyme cystathionine b-synthase, with consequent slowdown of the trans-sulfuration pathway. Since a significant proportion of the population does not meet the current RDAs for folate and pyridoxine intake, hyperhomocysteinemia could be an extensive phenomenon [10]. Many studies support the existence of an inverse correlation between plasma folate and homocysteine levels, while the relationship between pyridoxine and hyperhomocysteinemia is still controversial, although in patients with chronic renal failure, due to the vitamin B6 deficiency caused by dialysis, pyridoxine supplementation is necessary for lowering total plasma homocysteine [11,12]. Some authors report that low concentrations of vitamin B6, by increasing homocysteine levels, are related to coronary artery disease and carotid and arterial lesions [13e15]. On the contrary, Robinson et al. [16] suggest that the relationship between vitamin B6 and atherosclerosis is independent of plasma homocysteine concentration. Vitamin B6 deficiency could be related to vascular damage by altering platelet function [17], cholesterol concentration and antithrombin III activity [18]. In addition to hyperhomocysteinemia, alterations in lipid metabolism and lipid peroxidation, processes in which vitamin B6 is also involved, are other known risk factors for atherosclerosis and cardiovascular diseases. We previously demonstrated the marginal availability of pyridoxine influenced the fatty acid
composition of rat tissues, with an increase in C18:2 and a decrease in C20:4 relative molar content due to the lowest activity of vitamin B6dependent D6 desaturase [19]. Furthermore, we reported that B6 deficiency induces an increased peroxidative risk, particularly when the dietary intake of polyunsaturated fatty acids (PUFA) is high [20,21]. Since many connections appear to exist between pyridoxine, lipid metabolism, peroxidation risk and hyperhomocysteinemia, the aim of this study was to verify whether different amounts of PUFA associated with low content of vitamin B6 in the diet could modulate the vascular risk factor homocysteine.
Methods Animals and diets The trial was carried out on 32 male Wistar rats (100e110 g) randomly divided into four groups each consisting of eight animals, housed in cages in a temperature-controlled room with 12 h light/ dark cycle. Animals had free access to food and water for 8 weeks. Food consumption and animal weight were measured weekly. Each group received a diet that was different in fat quality and vitamin B6 amount (Tables 1 and 2), as follows: soybean oil, normal vitamin B6 (S-NB6): containing 8% soybean oil and 7 mg vitamin B6/kg diet soybean oil, deficient vitamin B6 (S-DB6): containing 8% soybean oil and 0.3 mg vitamin B6/kg diet soybean oil/animal fat, normal vitamin B6 (SAFNB6): containing 4% soybean oil, 4% animal fat and 7 mg vitamin B6/kg diet soybean oil/animal fat, deficient vitamin B6 (SAF-DB6): containing 4% soybean oil, 4% animal fat and 0.3 mg vitamin B6/kg diet At the end of the dietary treatment, the animals were anesthetized with ether and blood was sampled by intracardiac withdrawal. The rats were then sacrificed and the liver was excised and frozen in liquid nitrogen and stored at ÿ80 C. This study was approved by the Animal Care Committee of the University of Bologna.
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Table 1
Composition of dietsa (g/100 g)
Ingredients
Soybean oil diet
Soybean oil/ animal fat diet
Wheat starch Vitamin-free casein Dextrose Sucrose Soybean oil Animal fat Cellulose Minerals Trace element mixb Vitamin mixc
38.65 20.00 10.00 10.00 8.00 e 5.00 6.15 1.00 1.00
38.65 20.00 10.00 10.00 4.00 4.00 5.00 6.15 1.00 1.00
a
Supplied by MUCEDOLA, Settimo Milanese (Milan) Italy. Trace element mix provided in mg/kg diet: iron, 35; zinc, 40; manganese, 55; copper, 5.5; iodine, 0.2. c Vitamin mix provided in mg/kg diet: thiamin 6; riboflavin 6; pyridoxine 7 (in vitamin B6-deficient groups: 0.3); biotin 0.2; folic acid 2; niacin 30; Ca pantothenate 16; choline HCl 1000; vitamin B12 0.01; a-tocopherol 50; menadione 0.05; vitamin A 4000 IU/kg; vitamin D3 1000 IU/kg. b
Analytical Methods
Darmstadt, Germany, at 30 C; the mobile phase was n-hexane: isopropanol (99.2:0.8 v/v) with a flow rate of 1.5 ml/min, detection was fluorometric (excitation wave length 295 nm, emission 340 nm).
Vitamin B6 assay Plasma pyridoxal-5P was determined by the apotryptophanase assay method of Furth-Walker et al. [22]. Liver vitamin B6 was assayed by microbiological method: aliquots of tissues were homogenated in 0.05 N HCl and autoclaved for 1 h at 115 C. In extracts pyridoxine was evaluated using the microorganism Saccharomyces uvarum 9080 and Pyridoxine Y medium Difco [23].
Folate assay Plasma and red cell folate concentration was evaluated utilizing an Abbott IMX Folate assay based on ion capture (Abbott, Diagnostic Division, Wiesbaden, Germany).
Vitamin E assay
Homocysteine assay
a-Tocopherol (a-T) was determined by HPLC. Aliquots of liver were homogenated with 2 vols. of ethanol and then saponified in a water bath for 30 min with 50% ethanol potassium hydroxide solution in the presence of ascorbic acid under N2 reflux in the water bath. The saponified mixture was extracted with hexane, the extract evaporated to dryness and the residue diluted in an appropriate volume of mobile phase. Aliquots of this solution were injected into a chromatograph furnished with LiChrosorb Si 60 column 5 m (250!4.6 mm) E. Merck,
For homocysteine measurement, the blood samples were chilled immediately on ice and plasma was separated from blood cells within 30 min. Total homocysteine was measured utilizing the commercially available IMX Homocysteine (Hcy) immunoassay (Abbott, Diagnostic Division, Wiesbaden, Germany).
Table 2 Major fatty acid composition (%) of diet lipid content Fatty acid
Soybean oil diet
Soybean oil/animal fat diet
14:0 16:0 18:0 16:1n7 18:1n9 18:2n6 18:3n3
0.10 11.61 3.80 0.09 21.46 55.78 7.16
2.23 20.82 15.64 1.05 27.40 29.13 3.72
Total SFA Total MUFA Total PUFA
15.51 21.55 62.94
38.70 28.46 32.85
Fatty acid analysis Total lipids were extracted from the liver according to Folch et al. [24]. Phospholipids were separated by SPE C8 100 mg cartridges (Bond Elut Varian, Harbor City, CA) and methylated with methanol/hydrochloric acid (95:5 w/w) for 60 min at 70 C according to Stoffel et al. [25]. Lipid methyl esters were detected by gas chromatography (Hewlett Packard model 5890) using a capillary column (HP-FFAP, 25 m!0.32 mm!0.52 mm) at a programmed temperature (180e240 C with a 5 C/min gradient).
Statistical analysis All values are expressed as meansGstandard deviation. Statistical differences were determined by one-way analysis of variance and the Studente NewmaneKeuls test and were considered significantly different at P!0.05.
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Table 3 Final body weight, liver vitamin B6 and E contents of rats fed diets with different lipid quality and vitamin B6 amount Vitamin B6-normal SAF-NB6
S-NB6 Liver vitamin B6 (mg/g tissue) Final body weight (g) Liver vitamin E (mg/g tissue)
Vitamin B6-deficient
a
7.69G1.10 397G31a 21.10G2.10a
S-DB6 a
7.22G1.13 389G39a 21.15G2.40a
SAF-DB6 b
3.11G0.4 236G25b 15.40G1.64b
3.20G0.29b 203G29c 14.50G1.53b
Values are meansGSD of eight rats. Numbers with different superscript letters are significantly different by StudenteNewmane Keuls test (P!0.05).
Results Final body weight was significantly lower in rats fed the vitamin B6-deficient diet (Table 3) than in normal B6 fed rats. In liver, vitamin B6 and vitamin E contents appeared lower in B6-deficient rats compared to normal B6 rats (Table 3). In plasma the active form pyridoxine, PLP, was significantly decreased in vitamin B6-deficient rats; folate concentration was the same in all groups while red cell folate was lower in the SAF-DB6 group (Table 4). Plasma homocysteine level showed the most marked differences; in fact, the vitamin B6deficient diet induced an increase in homocysteine concentration compared to the normal vitamin B6 diet. This increase was tenfold in the subgroup fed high PUFA level (S-DB6) and twofold in the subgroup fed low PUFA level (SAF-DB6) (Table 4). The fatty acid composition of liver phospholipids showed a lower arachidonic acid relative molar content and a lower 20:4/18:2 ratio in vitamin B6-deficient groups, particularly in S-DB6 conditions, in comparison with B6-normal groups (Table 5).
Discussion There is increasing evidence that plasma homocysteine level is an independent risk factor for atherosclerosis, hyperhomocysteinemia possibly
interacting with conventional cardiovascular risk factors to further increase vascular diseases. Vitamin B6 deficiency is also considered a risk factor since it influences long-chain polyunsaturated fatty acids biosynthesis [26], increases lipid peroxidation [27] and affects antioxidant defenses [20,21]. Accordingly, in the present work, an increased oxidative stress in rats fed a vitamin B6-deficient diet is suggested by the observed decrease in liver vitamin E content, which can be considered as an index of oxidative condition. Since pyridoxine is a cofactor in the metabolism of methionine, its deficiency causes a substantial decrease in liver cystathionine synthase activity with consequent hyperhomocysteinemia. In our experimental conditions, in B6-deficient rats plasma homocysteine level was higher than in vitamin B6-normal rats, notwithstanding the vitamin B6 deficiency induced a lower dietary intake and consequently a lower availability of methionine. Although low availability of this amino acid had been reported to reduce homocysteinemia [7], Smolin and Benevenga [28] showed that the effect of low methionine and protein intake was insignificant compared to that due to vitamin B6 deficiency. This condition dramatically depressed the cystathionine synthase activity, with a consequent increasing effect on plasma homocysteine level. Our data confirm that the effect of vitamin B6 deficiency overcomes that due to low methionine intake.
Table 4 PLP, folate and homocysteine concentration in plasma and red cells of rats fed diets with different lipid quality and vitamin B6 amount Vitamin B6-normal Plasma PLP (pmol/ml) Plasma folate (nmol/l) Red cell folate (nmol/l) Plasma homocysteine (mmol/l)
Vitamin B6-deficient SAF-NB6
S-NB6 a
730G80 40.9G1.13a 1841G118a 4.66G1.21a
S-DB6 a
748G68 40.9G1.36a 1837G186a 6.76G1.83a
SAF-DB6 b
102G11 43.0G0.68a 1735G113a,b 47.56G8.30b
103G16b 39.6G1.29a 1601G104b 15.13G3.32c
Values are meansGSD for eight rats. Numbers with different superscript letters are significantly different by StudenteNewmane Keuls test (P!0.05).
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Table 5 Major fatty acid composition of liver phospholipids of rats fed diets with different lipid quality and vitamin B6 amount Fatty acid (mol %)
Vitamin B6-normal SAF-NB6
S-NB6 16:0 18:0 18:1n9 18:1n7 18:2 n6 20:4 n6 22:6 n3 Total SFA Total MUFA Total PUFA 20:4/18:2
Vitamin B6-deficient
a
21.2G1.2 18.6G0.9a 7.2G0.3a 2.6G0.1a 17.1G1.1a 23.5G1.3a 4.7G0.2a 39.8G0.8a 9.8G0.3a 45.3G1.3a 1.4G0.1a
S-DB6 a
SAF-DB6 b
20.6G1.0 20.3G1.2b 8.8G0.4b 2.3G0.1b 13.1G0.9b 21.7G1.3b 4.2G0.2b 40.9G1.2a 11.1G0.3b 39.0G1.0b 1.6G0.1b
22.6G1.6c 20.6G0.7b 7.8G0.2d 1.8G0.1c 17.0G0.8a 18.6G1.1c 4.7G0.2a 43.2G2.2b 9.6G0.2a 40.3G0.9c 1.1G0.2d
24.8G1.4 17.9G0.8a 5.7G0.2c 1.8G0.1c 19.2G1.3c 18.0G1.2c 3.6G0.1c 42.7G2.1b 7.5G0.2c 40.8G1.2c 0.9G0.1c
Data are meansGSD of eight rats. Numbers with different superscript letters are significantly different by StudenteNewmane Keuls test (P!0.05).
The increased plasma homocysteine concentration appeared independent of folate availability, whose normal plasma concentration was assured by dietary intake. However, in vitamin B6-deficient rats the slight decrease in red cell folate concentration could be related to a higher transmethylation activity, which was not enough to control the homocysteine level. Homocysteinemia was also influenced by PUFA dietary intake; in fact, in S-DB6 rats receiving a diet containing 63% of PUFA, the inadequate pyridoxine availability increased plasma homocysteine level ten times with respect to the S-NB6 group. In the SAF-DB6 animals, receiving a diet containing half the amount of PUFA (33%), concentration increased only twice compared to the SAF-NB6 group (Table 4). Moreover, vitamin B6-deficiency influenced arachidonate biosynthesis as demonstrated by the low 20:4/18:2 ratio. The reduction in arachidonate relative molar content in liver phospholipids was more evident in the S-DB6 group, which appeared the most prone to vitamin B6-deficiency with regard to both homocysteine and fatty acid metabolism. The possible correlations between PUFA dietary intake and some aspects of fatty acid and methionine metabolism in vitamin B6-deficiency are reported in Fig. 1. In conclusion, our data add further information about the relationship between vitamin B6 and homocysteine metabolism and underline the importance of an adequate pyridoxine intake, particularly when PUFA dietary level is high. In fact, adequate vitamin B6 intake could assure the normal long chain fatty acid biosynthesis and the
reduction of the risk linked to hyperhomocysteinemia. Clinical studies are needed before drawing final conclusions on homocysteine metabolism in man, but data reported here suggest the importance of vitamin B6 intake particularly when highly unsaturated diet are recommended, as in the case of cardiovascular patients.
+ PUFA - Vitamin B6 Lipid metabolism
Homocysteine metabolism
- Desaturase activity
- Transsulfuration pathway
- LCPUFA
+ Homocysteine
- Red cell folate Figure 1 In vitamin B6 deficiency a high PUFA dietary intake influences both lipid metabolism, decreasing the activity of PLP dependent desaturases, and homocysteine metabolism, slowing the transsulfuration process. As a consequence, a reduction in long chain PUFA biosynthesis and an increase in homocysteine plasma level, inducing a higher consumption of red cell folate for remethylation of homocysteine to methionine, are observed.
Dietary PUFA, vitamin B6 and homocysteinemia
Acknowledgements This study was supported by grants from Italian Ministry for University and Research (MIUR), Italy.
References [1] McKully KS. Homocysteine and vascular diseases. Nat Med 1996;2:386e9. [2] Refsum H, Ueland PM, Nygard O, Vollset SE. Homocysteine and cardiovascular disease. Annu Rev Med 1998;49:31e62. [3] Malinow MR, Bostom AG, Krauss RM. Homocyst(e)ine, diet, and cardiovascular diseases. Circulation 1999;99:178e82. [4] Kang SS, Wong PKW, Malinow MR. Hyperhomocysteinemia as a risk factor for occlusive vascular disease. Annu Rev Nutr 1992;12:279e98. [5] Kraus JP, Oliveriusova J, Sokolova J, Kraus E, Vlcek C, de Franchis R, et al. The human cystathionine beta-synthase (CBS) gene: complete sequence, alternative splicing, and polymorphisms. Genomics Sep 1998;1552(3):312e24. [6] Frosst P, Blom HJ, Milos R, Goyette P, Sheppard CA, Matthews RG, et al. A candidate genetic risk factor for vascular disease: a common mutation in methylenetetrahydrofolate reductase. Nat Genet 1995;10:111e3. [7] Troen AM, Lutgens E, Smith DE, Rosenberg IH, Selhub J. The atherogenic effect of excess methionine intake. Proc Natl Acad Sci U S A 2003;100(25):15089e94. [8] Lee BJ, Lin PT, Liaw YP, Chang SJ, Cheng CH, Huang YC. Homocysteine and risk of coronary artery disease: folate is the important determinant of plasma homocysteine concentration. Nutrition 2003;19:577e83. [9] Verhoef P, Stampfer MJ, Buring JE, Graziano JM, Allen RH, Stabler SP, et al. Homocysteine metabolism and risk of myocardial infarction: relation with vitamins B6, B12, and folate. Am J Epidemiol 1996;143:845e59. [10] Must A, Jacques PF, Rogers G, Rosenberg IH, Selhub J. Serum total homocysteine concentrations in children and adolescents: results from the Third National Health and Nutrition Examination Survey (NHANES III). J Nutr 2003; 133(8):2643e9. [11] De Gomez Dumm NT, Giammona AM, Touceda LA. Variations in the lipid profile of patients with chronic renal failure treated with pyridoxine. Lipids Health Dis 2003; 2(1):7. [12] Bachmann J, Tepel M, Raidt H, Riezler R, Graefe U, Langer K, et al. Hyperhomocysteinemia and the risk for vascular disease in hemodialysis patients. J Am Soc Nephrol 1995;6:121e5. [13] Robinson K, Mayer EL, Miller D, Green R, van Lente F, Gupta A, et al. Hyperhomocysteinemia and low pyridoxal phosphate: common and independent reversible risk factors for coronary artery disease. Circulation 1995;92: 2825e30.
99 [14] Kok FJ, Schrijver J, Hofman A, Witteman JC, Kruyssen DA, Remme WJ, et al. Low vitamin B6 status in patients with acute myocardial infarction. Am J Cardiol 1989;63:513e6. [15] Friso S, Girelli D, Martinelli N, Olivieri O, Lotto V, Bozzini C, et al. Low plasma vitamin B-6 concentrations and modulation of coronary artery disease risk. Am J Clin Nutr 2004;79(6):992e8. [16] Robinson K, Arheart K, Refsum H, Brattstro ¨m L, Boers G, Ueland P, et al. Low circulating folate and vitamin B6 concentrations. Circulation 1998;97:437e43. [17] Gupta A, Moustapha A, Jacobsen DW, Goormastic M, Tuzcu EM, Hobbs R, et al. High homocysteine, low folate, and low vitamin B6 concentrations: prevalent risk factors for vascular disease in heart transplant recipients. Transplantation 1998;65(4):544e50. [18] Brattstro ¨m L, Stavenow L, Galvard H, Nilsson-Ehle P, Berntorp E, Jerntorp P, et al. Pyridoxine reduces cholesterol and low-density lipoprotein and increases antithrombin III activity in 80-year-old men with low plasma pyridoxal 5-phosphate. Scand J Clin Lab Invest 1990;50: 873e7. [19] Bordoni A, Hrelia S, Lorenzini A, Bergami R, Cabrini L, Biagi PL, et al. Dual influence of aging and vitamin B6 deficiency on delta-6-desaturation of essential fatty acids in rat liver microsomes. Prostaglandins Leukotr Ess Fatty Acids 1998;58:417e20. [20] Cabrini L, Bergami R, Fiorentini D, Marchetti M, Landi L, Tolomelli B. Vitamin B6 deficiency affects antioxidant defences in rat liver and heart. Biochem Mol Biol Int 1998; 46:689e97. [21] Cabrini L, Bergami R, Maranesi M, Carloni A, Marchetti M, Tolomelli B. Effects of short-term dietary administration of marginal levels of vitamin B6 and fish oil on lipid composition and antioxidant defences in rat tissues. Prostaglandins Leukotr Ess Fatty Acids 2001;64:265e71. [22] Furth-Walker D, Leibman D, Smolen A. Changes in pyridoxal phosphate and pyridoxamine phosphate in blood, liver and brain in the pregnant mouse. J Nutr 1989;119: 750e6. [23] Cuniff P, editor. Vitamin B6 in food extracts. Microbiological method. Official Methods of Analysis. Association of official Analytical Chemist. 17th ed. vol II. Washington, DC; 1995. p. 51e3. [24] Folch J, Lees M, Sloane-Stanley GH. A simple method for the isolation and purification of total lipids from animal tissues. J Biol Chem 1957;256:497e509. [25] Stoffel W, Chu F, Ahrens Jr EH. Analysis of long chain fatty acids by gas-liquid chromatography. Anal Chem 1959;31: 307e8. [26] Cunnane SC, Manku MS, Horrobin DF. Accumulation of linoleic and gamma-linolenic acids in tissue lipids of pyridoxine-deficient rats. J Nutr 1984;114:1754e61. [27] Ravichandran V, Selvam R. Increased lipid peroxidation in kidney of vitamin B6 deficient rats. Biochem Int 1990;21: 599e605. [28] Smolin LA, Benevenga NJ. Factors affecting the accumulation of homocyst(e)ine in rats deficient in vitamin B-6. J Nutr 1984;114:103e11.
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Correlation between dietary polyunsaturated fatty acids and plasma homocysteine concentration in vitamin B6-deficient rats L. Cabrini a,*, D. Bochicchio b, A. Bordoni a, S. Sassi c, M. Marchetti a, M. Maranesi a a
Department of Biochemistry ‘‘G. Moruzzi’’, University of Bologna, Via Irnerio 48, 40126 Bologna, Italy b Zootechnical Experimental Institute, S. Cesario s.P., Modena, Italy c Department of Angiology and Blood Coagulation, S.Orsola-Malpighi University Hospital, Bologna, Italy Received 19 May 2004; accepted 8 November 2004
KEYWORDS Vitamin B6; Homocysteine; Dietary PUFA; D6 desaturase
Summary Background and aim: Vitamin B6 as cofactor of D6 desaturase is involved in polyunsaturated fatty acid metabolism; moreover, it is a cofactor of the trans-sulfuration pathway of homocysteine. Some studies report that low concentrations of pyridoxine, by increasing homocysteine levels, are associated with coronary artery disease, and carotid and arterial lesions. The aim of this study was to verify whether different dietary amounts of polyunsaturated fatty acids associated with low content of vitamin B6 could modulate homocysteinemia. Methods and results: Thirty-two rats were divided into two groups, one fed a diet with adequate vitamin B6 content the other a diet containing low amount of the same vitamin. Within each group, rats were divided into two subgroups differing in the polyunsaturated fatty acid content of the diet (63 and 33%, respectively). The vitamin B6-deficient diet induced an increase in homocysteine concentration compared to the vitamin B6-normal diet. This increase was tenfold in the subgroup fed high polyunsaturated fatty acid levels and twofold in the other subgroup. The fatty acid composition of liver phospholipids showed a lower arachidonic acid relative molar content and a lower 20:4/18:2 ratio in vitamin B6-deficient groups compared with B6-normal groups. Conclusions: On the basis of the different biological functions of pyridoxine and considering that some factors closely related to atherosclerosis are vitamin B6 dependent, adequate pyridoxine availability could be necessary to assure
* Corresponding author. Tel.: C39 051 2091222; fax: C39 051 2091234. E-mail address: [email protected] (L. Cabrini). 0939-4753/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.numecd.2004.11.002
Dietary PUFA, vitamin B6 and homocysteinemia
95
a normal long chain fatty acid metabolism and to reduce the risk linked to hyperhomocysteinemia. Ó 2005 Elsevier Ltd. All rights reserved.
Introduction High level of homocysteine in plasma has been recognized as a further marker of cardiovascular diseases among well-known risk factors. Hyperhomocysteinemia causes endothelial damage leading to a high risk for the atherosclerotic process in coronary, cerebral and peripheral vessels [1e3] and for arterial and venous thromboembolism [4]. High plasma levels of homocysteine are attributed not only to genetic, but also to dietary factors [5,6], such as high methionine content [7] or deficiencies in B vitamins, particularly folic acid, vitamin B12 and vitamin B6 [8,9]. These vitamins are coenzymes or cofactors of the transmethylation or trans-sulfuration pathways of homocysteine. In pyridoxine deficiency, the increased plasma homocysteine concentration is due to the lower activity of B6 dependent enzyme cystathionine b-synthase, with consequent slowdown of the trans-sulfuration pathway. Since a significant proportion of the population does not meet the current RDAs for folate and pyridoxine intake, hyperhomocysteinemia could be an extensive phenomenon [10]. Many studies support the existence of an inverse correlation between plasma folate and homocysteine levels, while the relationship between pyridoxine and hyperhomocysteinemia is still controversial, although in patients with chronic renal failure, due to the vitamin B6 deficiency caused by dialysis, pyridoxine supplementation is necessary for lowering total plasma homocysteine [11,12]. Some authors report that low concentrations of vitamin B6, by increasing homocysteine levels, are related to coronary artery disease and carotid and arterial lesions [13e15]. On the contrary, Robinson et al. [16] suggest that the relationship between vitamin B6 and atherosclerosis is independent of plasma homocysteine concentration. Vitamin B6 deficiency could be related to vascular damage by altering platelet function [17], cholesterol concentration and antithrombin III activity [18]. In addition to hyperhomocysteinemia, alterations in lipid metabolism and lipid peroxidation, processes in which vitamin B6 is also involved, are other known risk factors for atherosclerosis and cardiovascular diseases. We previously demonstrated the marginal availability of pyridoxine influenced the fatty acid
composition of rat tissues, with an increase in C18:2 and a decrease in C20:4 relative molar content due to the lowest activity of vitamin B6dependent D6 desaturase [19]. Furthermore, we reported that B6 deficiency induces an increased peroxidative risk, particularly when the dietary intake of polyunsaturated fatty acids (PUFA) is high [20,21]. Since many connections appear to exist between pyridoxine, lipid metabolism, peroxidation risk and hyperhomocysteinemia, the aim of this study was to verify whether different amounts of PUFA associated with low content of vitamin B6 in the diet could modulate the vascular risk factor homocysteine.
Methods Animals and diets The trial was carried out on 32 male Wistar rats (100e110 g) randomly divided into four groups each consisting of eight animals, housed in cages in a temperature-controlled room with 12 h light/ dark cycle. Animals had free access to food and water for 8 weeks. Food consumption and animal weight were measured weekly. Each group received a diet that was different in fat quality and vitamin B6 amount (Tables 1 and 2), as follows: soybean oil, normal vitamin B6 (S-NB6): containing 8% soybean oil and 7 mg vitamin B6/kg diet soybean oil, deficient vitamin B6 (S-DB6): containing 8% soybean oil and 0.3 mg vitamin B6/kg diet soybean oil/animal fat, normal vitamin B6 (SAFNB6): containing 4% soybean oil, 4% animal fat and 7 mg vitamin B6/kg diet soybean oil/animal fat, deficient vitamin B6 (SAF-DB6): containing 4% soybean oil, 4% animal fat and 0.3 mg vitamin B6/kg diet At the end of the dietary treatment, the animals were anesthetized with ether and blood was sampled by intracardiac withdrawal. The rats were then sacrificed and the liver was excised and frozen in liquid nitrogen and stored at ÿ80 C. This study was approved by the Animal Care Committee of the University of Bologna.
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Table 1
Composition of dietsa (g/100 g)
Ingredients
Soybean oil diet
Soybean oil/ animal fat diet
Wheat starch Vitamin-free casein Dextrose Sucrose Soybean oil Animal fat Cellulose Minerals Trace element mixb Vitamin mixc
38.65 20.00 10.00 10.00 8.00 e 5.00 6.15 1.00 1.00
38.65 20.00 10.00 10.00 4.00 4.00 5.00 6.15 1.00 1.00
a
Supplied by MUCEDOLA, Settimo Milanese (Milan) Italy. Trace element mix provided in mg/kg diet: iron, 35; zinc, 40; manganese, 55; copper, 5.5; iodine, 0.2. c Vitamin mix provided in mg/kg diet: thiamin 6; riboflavin 6; pyridoxine 7 (in vitamin B6-deficient groups: 0.3); biotin 0.2; folic acid 2; niacin 30; Ca pantothenate 16; choline HCl 1000; vitamin B12 0.01; a-tocopherol 50; menadione 0.05; vitamin A 4000 IU/kg; vitamin D3 1000 IU/kg. b
Analytical Methods
Darmstadt, Germany, at 30 C; the mobile phase was n-hexane: isopropanol (99.2:0.8 v/v) with a flow rate of 1.5 ml/min, detection was fluorometric (excitation wave length 295 nm, emission 340 nm).
Vitamin B6 assay Plasma pyridoxal-5P was determined by the apotryptophanase assay method of Furth-Walker et al. [22]. Liver vitamin B6 was assayed by microbiological method: aliquots of tissues were homogenated in 0.05 N HCl and autoclaved for 1 h at 115 C. In extracts pyridoxine was evaluated using the microorganism Saccharomyces uvarum 9080 and Pyridoxine Y medium Difco [23].
Folate assay Plasma and red cell folate concentration was evaluated utilizing an Abbott IMX Folate assay based on ion capture (Abbott, Diagnostic Division, Wiesbaden, Germany).
Vitamin E assay
Homocysteine assay
a-Tocopherol (a-T) was determined by HPLC. Aliquots of liver were homogenated with 2 vols. of ethanol and then saponified in a water bath for 30 min with 50% ethanol potassium hydroxide solution in the presence of ascorbic acid under N2 reflux in the water bath. The saponified mixture was extracted with hexane, the extract evaporated to dryness and the residue diluted in an appropriate volume of mobile phase. Aliquots of this solution were injected into a chromatograph furnished with LiChrosorb Si 60 column 5 m (250!4.6 mm) E. Merck,
For homocysteine measurement, the blood samples were chilled immediately on ice and plasma was separated from blood cells within 30 min. Total homocysteine was measured utilizing the commercially available IMX Homocysteine (Hcy) immunoassay (Abbott, Diagnostic Division, Wiesbaden, Germany).
Table 2 Major fatty acid composition (%) of diet lipid content Fatty acid
Soybean oil diet
Soybean oil/animal fat diet
14:0 16:0 18:0 16:1n7 18:1n9 18:2n6 18:3n3
0.10 11.61 3.80 0.09 21.46 55.78 7.16
2.23 20.82 15.64 1.05 27.40 29.13 3.72
Total SFA Total MUFA Total PUFA
15.51 21.55 62.94
38.70 28.46 32.85
Fatty acid analysis Total lipids were extracted from the liver according to Folch et al. [24]. Phospholipids were separated by SPE C8 100 mg cartridges (Bond Elut Varian, Harbor City, CA) and methylated with methanol/hydrochloric acid (95:5 w/w) for 60 min at 70 C according to Stoffel et al. [25]. Lipid methyl esters were detected by gas chromatography (Hewlett Packard model 5890) using a capillary column (HP-FFAP, 25 m!0.32 mm!0.52 mm) at a programmed temperature (180e240 C with a 5 C/min gradient).
Statistical analysis All values are expressed as meansGstandard deviation. Statistical differences were determined by one-way analysis of variance and the Studente NewmaneKeuls test and were considered significantly different at P!0.05.
Dietary PUFA, vitamin B6 and homocysteinemia
97
Table 3 Final body weight, liver vitamin B6 and E contents of rats fed diets with different lipid quality and vitamin B6 amount Vitamin B6-normal SAF-NB6
S-NB6 Liver vitamin B6 (mg/g tissue) Final body weight (g) Liver vitamin E (mg/g tissue)
Vitamin B6-deficient
a
7.69G1.10 397G31a 21.10G2.10a
S-DB6 a
7.22G1.13 389G39a 21.15G2.40a
SAF-DB6 b
3.11G0.4 236G25b 15.40G1.64b
3.20G0.29b 203G29c 14.50G1.53b
Values are meansGSD of eight rats. Numbers with different superscript letters are significantly different by StudenteNewmane Keuls test (P!0.05).
Results Final body weight was significantly lower in rats fed the vitamin B6-deficient diet (Table 3) than in normal B6 fed rats. In liver, vitamin B6 and vitamin E contents appeared lower in B6-deficient rats compared to normal B6 rats (Table 3). In plasma the active form pyridoxine, PLP, was significantly decreased in vitamin B6-deficient rats; folate concentration was the same in all groups while red cell folate was lower in the SAF-DB6 group (Table 4). Plasma homocysteine level showed the most marked differences; in fact, the vitamin B6deficient diet induced an increase in homocysteine concentration compared to the normal vitamin B6 diet. This increase was tenfold in the subgroup fed high PUFA level (S-DB6) and twofold in the subgroup fed low PUFA level (SAF-DB6) (Table 4). The fatty acid composition of liver phospholipids showed a lower arachidonic acid relative molar content and a lower 20:4/18:2 ratio in vitamin B6-deficient groups, particularly in S-DB6 conditions, in comparison with B6-normal groups (Table 5).
Discussion There is increasing evidence that plasma homocysteine level is an independent risk factor for atherosclerosis, hyperhomocysteinemia possibly
interacting with conventional cardiovascular risk factors to further increase vascular diseases. Vitamin B6 deficiency is also considered a risk factor since it influences long-chain polyunsaturated fatty acids biosynthesis [26], increases lipid peroxidation [27] and affects antioxidant defenses [20,21]. Accordingly, in the present work, an increased oxidative stress in rats fed a vitamin B6-deficient diet is suggested by the observed decrease in liver vitamin E content, which can be considered as an index of oxidative condition. Since pyridoxine is a cofactor in the metabolism of methionine, its deficiency causes a substantial decrease in liver cystathionine synthase activity with consequent hyperhomocysteinemia. In our experimental conditions, in B6-deficient rats plasma homocysteine level was higher than in vitamin B6-normal rats, notwithstanding the vitamin B6 deficiency induced a lower dietary intake and consequently a lower availability of methionine. Although low availability of this amino acid had been reported to reduce homocysteinemia [7], Smolin and Benevenga [28] showed that the effect of low methionine and protein intake was insignificant compared to that due to vitamin B6 deficiency. This condition dramatically depressed the cystathionine synthase activity, with a consequent increasing effect on plasma homocysteine level. Our data confirm that the effect of vitamin B6 deficiency overcomes that due to low methionine intake.
Table 4 PLP, folate and homocysteine concentration in plasma and red cells of rats fed diets with different lipid quality and vitamin B6 amount Vitamin B6-normal Plasma PLP (pmol/ml) Plasma folate (nmol/l) Red cell folate (nmol/l) Plasma homocysteine (mmol/l)
Vitamin B6-deficient SAF-NB6
S-NB6 a
730G80 40.9G1.13a 1841G118a 4.66G1.21a
S-DB6 a
748G68 40.9G1.36a 1837G186a 6.76G1.83a
SAF-DB6 b
102G11 43.0G0.68a 1735G113a,b 47.56G8.30b
103G16b 39.6G1.29a 1601G104b 15.13G3.32c
Values are meansGSD for eight rats. Numbers with different superscript letters are significantly different by StudenteNewmane Keuls test (P!0.05).
98
L. Cabrini et al.
Table 5 Major fatty acid composition of liver phospholipids of rats fed diets with different lipid quality and vitamin B6 amount Fatty acid (mol %)
Vitamin B6-normal SAF-NB6
S-NB6 16:0 18:0 18:1n9 18:1n7 18:2 n6 20:4 n6 22:6 n3 Total SFA Total MUFA Total PUFA 20:4/18:2
Vitamin B6-deficient
a
21.2G1.2 18.6G0.9a 7.2G0.3a 2.6G0.1a 17.1G1.1a 23.5G1.3a 4.7G0.2a 39.8G0.8a 9.8G0.3a 45.3G1.3a 1.4G0.1a
S-DB6 a
SAF-DB6 b
20.6G1.0 20.3G1.2b 8.8G0.4b 2.3G0.1b 13.1G0.9b 21.7G1.3b 4.2G0.2b 40.9G1.2a 11.1G0.3b 39.0G1.0b 1.6G0.1b
22.6G1.6c 20.6G0.7b 7.8G0.2d 1.8G0.1c 17.0G0.8a 18.6G1.1c 4.7G0.2a 43.2G2.2b 9.6G0.2a 40.3G0.9c 1.1G0.2d
24.8G1.4 17.9G0.8a 5.7G0.2c 1.8G0.1c 19.2G1.3c 18.0G1.2c 3.6G0.1c 42.7G2.1b 7.5G0.2c 40.8G1.2c 0.9G0.1c
Data are meansGSD of eight rats. Numbers with different superscript letters are significantly different by StudenteNewmane Keuls test (P!0.05).
The increased plasma homocysteine concentration appeared independent of folate availability, whose normal plasma concentration was assured by dietary intake. However, in vitamin B6-deficient rats the slight decrease in red cell folate concentration could be related to a higher transmethylation activity, which was not enough to control the homocysteine level. Homocysteinemia was also influenced by PUFA dietary intake; in fact, in S-DB6 rats receiving a diet containing 63% of PUFA, the inadequate pyridoxine availability increased plasma homocysteine level ten times with respect to the S-NB6 group. In the SAF-DB6 animals, receiving a diet containing half the amount of PUFA (33%), concentration increased only twice compared to the SAF-NB6 group (Table 4). Moreover, vitamin B6-deficiency influenced arachidonate biosynthesis as demonstrated by the low 20:4/18:2 ratio. The reduction in arachidonate relative molar content in liver phospholipids was more evident in the S-DB6 group, which appeared the most prone to vitamin B6-deficiency with regard to both homocysteine and fatty acid metabolism. The possible correlations between PUFA dietary intake and some aspects of fatty acid and methionine metabolism in vitamin B6-deficiency are reported in Fig. 1. In conclusion, our data add further information about the relationship between vitamin B6 and homocysteine metabolism and underline the importance of an adequate pyridoxine intake, particularly when PUFA dietary level is high. In fact, adequate vitamin B6 intake could assure the normal long chain fatty acid biosynthesis and the
reduction of the risk linked to hyperhomocysteinemia. Clinical studies are needed before drawing final conclusions on homocysteine metabolism in man, but data reported here suggest the importance of vitamin B6 intake particularly when highly unsaturated diet are recommended, as in the case of cardiovascular patients.
+ PUFA - Vitamin B6 Lipid metabolism
Homocysteine metabolism
- Desaturase activity
- Transsulfuration pathway
- LCPUFA
+ Homocysteine
- Red cell folate Figure 1 In vitamin B6 deficiency a high PUFA dietary intake influences both lipid metabolism, decreasing the activity of PLP dependent desaturases, and homocysteine metabolism, slowing the transsulfuration process. As a consequence, a reduction in long chain PUFA biosynthesis and an increase in homocysteine plasma level, inducing a higher consumption of red cell folate for remethylation of homocysteine to methionine, are observed.
Dietary PUFA, vitamin B6 and homocysteinemia
Acknowledgements This study was supported by grants from Italian Ministry for University and Research (MIUR), Italy.
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