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© 2005 The American Society for Nutritional Sciences J. Nutr. 135:544-548, March 2005

Nutritional Neurosciences

Folic Acid Supplementation Can Reduce the Endothelial Damage in Rat Brain Microvasculature Due to Hyperhomocysteinemia1,2

Hwayoung Lee*,{dagger}, Ji-myung Kim**, Ho Jin Kim*, Insun Lee** and Namsoo Chang**,{ddagger},3

* Department of Anatomy, College of Medicine, ** Department of Food and Nutritional Sciences, and {dagger} Medical Research Center and {ddagger} Asia Food and Nutrition Research Institute, Ewha Womans University, Seoul, Republic of Korea

3To whom correspondence should be addressed. E-mail: nschang{at}ewha.ac.kr.


   ABSTRACT
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 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
To evaluate the effects of dietary folic acid supplementation on the cerebral vascular damage induced by hyperhomocysteinemia, rats were fed a diet containing 3.0 g/kg homocystine for 2 wk and then either 3.0 g/kg homocystine or 3.0 g/kg homocystine plus 0.008 g/kg folic acid for 8 wk. Control rats consumed the AIN-93 Maintenance diet throughout the experiment. The cerebral expression of glucose transporter-1 was measured by Western blot analysis and cerebrovascular structural alterations were evaluated by electron microscopy. The homocystine diet significantly increased the plasma levels of homocysteine and TBARS and decreased the cerebral expression of glucose transporter-1 (GLUT-1) with a concomitant increase in the percentage of damaged cerebral vessels. The inclusion of dietary folic acid for 8 wk caused plasma homocysteine levels to be the same as in control rats and it significantly upregulated the cerebral expression of GLUT-1 that was significantly reduced by hyperhomocysteinemia. Folic acid supplementation also significantly decreased the incidence of damaged vessels due to hyperhomocysteinemia. These results and the electron microscopy findings suggested that folic acid supplementation might reduce the detrimental effects on the endothelium caused by experimentally induced hyperhomocysteinemia.

KEY WORDS: • brain • hyperhomocysteinemia • electron microscopy • folic acid • TBARS

Hyperhomocysteinemia, defined as an elevation of plasma total homocysteine, is a prevalent risk factor for a variety of vascular-based diseases (1). Although associations between hyperhomocysteinemia and adverse vascular outcomes have been observed in many retrospective and some prospective studies, the mechanisms responsible for vascular dysfunction in hyperhomocysteinemia remain poorly understood. It has been suggested that increased oxidative stress plays a key role in the reduction of endothelial nitric oxide bioactivity observed in hyperhomocysteinemia (2). The endothelial dysfunction induced by hyperhomocysteinemia may be due to oxidative inactivation of endothelium-derived nitric oxide, a major mediator of endothelium-dependent relaxation, in addition to an increase in the generation of hydrogen peroxide (3,4). Homocysteine treatment increases the rate of endothelial senescence (5) and potentially enhances the cytotoxic effects of agents or conditions known to cause oxidative stress (6). Hyperhomocysteinemia has been reported to impair endothelium-dependent dilatation in the aorta (7,8) and mesenteric vessels (9) in mice, as well as in the middle cerebral artery in humans (10).

Homocysteine concentrations depend on a series of intracellular metabolic reactions in which folic acid acts as a substrate and vitamin B-12 serves as a coenzyme, and it is thought that dietary supplementation with these 2 vitamins can lower the levels of homocysteine and thus avoid its adverse effects. Dietary supplementation with folic acid is currently recommended to hyperhomocysteinemic patients, because it has been shown to significantly lower plasma homocysteine concentrations (11) and, therefore, to decrease cardiovascular risk in such patients, in part through amelioration of endothelial function (12). Further, because some of the damage induced by elevated plasma concentrations of homocysteine is mediated by oxidative stress (13), strengthening the antioxidant capacity of the organism may provide benefits in addition to lowering plasma homocysteine.

Endothelial injury induced by hyperhomocysteinemia may alter the vasculature. Although there are several lines of evidence that hyperhomocysteinemia alters vascular structure (14–16) and hyperhomocysteinemia and stroke are clinically related (17), little is known about the effects of hyperhomocysteinemia on cerebral vascular structures. We previously found, using Western blot analysis and immunohistochemistry, that the probable functional impairment in cerebral endothelium resulting from diet-induced hyperhomocysteinemia and the ameliorative effects of a 2-wk supplementation of folic acid (18). However, the structural evidence for the effects of plasma homocysteine on cerebral vasculature has not been investigated.

In the present study, we evaluated the effects of 8 wk of dietary folic acid supplementation on cerebral vascular damage induced by hyperhomocysteinemia in vivo, in particular investigating the structural features of the cerebral vasculature by electron microscopy. The changes therein were found to be related to the cerebral expression level of a marker for endothelial dysfunction, the glucose transporter protein-1 (GLUT-1).4


   MATERIALS AND METHODS
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 MATERIALS AND METHODS
RESULTS
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 LITERATURE CITED
 
    Animal preparation. The experiments followed an animal protocol approved by the NIH Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80–23, revised 1996). Male Sprague-Dawley rats (8 wk old) (Samtaco) were housed individually in stainless-steel cages under controlled conditions (25°C, 12:12 h light:dark cycle). Their diets consisted of a nutritionally complete formula [AIN-93 Maintenance diet (19)] plus homocystine at 3.0 g/kg of diet (Table 1). A total of 48 rats (initial weight, 280.0 ± 10.5 g; mean ± SD) were used, of which 8 were killed for baseline value. The control rats (group C, n = 24) were fed a normal diet for 10 wk, and those in the treatment group were fed a diet containing 3.0 g/kg homocystine for the first 2 wk and then, after 8 rats were killed, they were divided into 2 treatment subgroups, H and HF. For the next 8 wk, group H continued to receive 3.0 g/kg homocystine, and group HF received 3.0 g/kg homocystine plus 0.008 g/kg folic acid. All of the experimental rats were allowed free access to food and water.


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  		TABLE 1 Composition of diets1

 
    Analysis of plasma homocysteine, vitamin B-12, and folic acid. Rats were anesthetized intraperitoneally with pentobarbital sodium. Blood samples were drawn from a heart puncture and promptly centrifuged at 1750 x g for 15 min at 4°C. Plasma homocysteine was analyzed by the HPLC (Waters 474) fluorescence detection method developed by Araki and Sako (20). Plasma vitamin B-12 and folic acid were measured with the 57Co vitamin B-12 and 125I folic acid Dualcount solid-phase-no-boil radioassay (Diagnostic Products).

    Analysis of plasma TBARS. The presence of TBARS in the plasma was tested using the method described by Yagi (21). Fluorescence was measured with a luminescence spectrophotometer (Perkin-Elmer LS50) at 553 nm (excitation at 515 nm).

    Western blot analysis. Samples of whole brains from each of the 3 groups were freshly frozen in liquid nitrogen and subsequently homogenized in a lysis buffer using a tissue homogenizer as previously described (18). Equal amounts of protein were loaded for 8% SDS-PAGE. Immunoblotting was performed using monoclonal antibodies for GLUT-1 (Santa Cruz Biotechnology). The membranes were subsequently probed with a secondary antimouse antibody conjugated to horseradish peroxidase (Southern Biotechnology) and developed by chemiluminescence. Densitometry was performed with gel documentation (Gel Doc 2000, Quantity One, Bio-Rad) and densitometric units were measured.

    Electron microscopy. Electron microscopy investigation was carried out as described previously (22). The brain sections were examined and quantified using a transmission electron microscope (Hitachi H-600). The number of vessels displaying alterations of damaged capillaries was counted directly on the screen of the electron microscope in region CA1 of the hippocampus. A person who was unaware of the dietary treatments of the brain specimens counted the proportion of damaged vessels in at least 100 vessels in each section. Five randomly selected hippocampal sections in each experimental group were used for quantification of the damaged vessels.

    Statistical analysis. Statistical analysis was performed using the Statistical Package for Social Science (Microsoft Windows version 11.0). Weight gain and food intake were analyzed by one-way ANOVA. The plasma homocysteine, folic acid, vitamin B-12, and TBARS levels at 2 wk were analyzed by Student’s t test, and these levels at 10 wk were analyzed by one-way ANOVA, followed by Duncan’s multiple range test. For densitometric data, Wilcoxon’s Signed Rank test was performed to determine whether differences were significant. Results for plasma homocysteine, folate, vitamin B-12, and TBARS are presented as means ± SEM; data for Western blotting analysis and percentage of damaged vessels are presented as means ± SD. Significance was accepted at probability values of P < 0.05.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 LITERATURE CITED
 
Weight gain, food intake, and plasma homocysteine, folate, and vitamin B-12

Weight gain and food intake did not differ between the control and treated groups throughout the experiment. The homocystine diet increased plasma homocysteine by up to 400% at 10 wk compared to the controls (Fig. 1A). Dietary folic acid supplementation significantly decreased the plasma homocysteine levels that had been increased by the homocystine diet. The plasma homocysteine levels were decreased by dietary folic acid (0.008 g/kg) supplementation for 2 wk, but not to control levels. However, 8 wk of folic acid supplementation caused the plasma homocysteine levels to return to control levels (Fig. 1B). The plasma vitamin B-12 level did not differ among the groups (Fig. 1C).



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  		FIGURE 1 Levels of plasma homocysteine (A), folate (B), and vitamin B-12 (C) after folic acid supplementation in rats fed a control diet (C), 3.0 g/kg homocystine (H), or 3.0 g/kg homocystine for 2 wk followed by 3.0 g/kg homocystine plus 0.008 g/kg folic acid (HF). Values are means ± SEM, n = 8. *Different from the control at wk 2. At wk 10, means without a common letter differ, P < 0.05.

 
Plasma TBARS

The homocystine diet increased the plasma TBARS levels significantly both at 2 and at 10 wk. At 10 wk, the plasma TBARS level in rats fed a homocysteine diet was still significantly higher than that of the control rats. Dietary folic acid supplementation for 8 wk (group HF) decreased the plasma levels of TBARS that had been increased by a homocystine diet for 2 wk (Fig. 2).



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  		FIGURE 2 Plasma concentration of TBARS in rats fed a control diet (C), 3.0 g/kg homocystine (H), or 3.0 g/kg homocystine for 2 wk followed by 3.0 g/kg homocystine plus 0.008 g/kg folic acid (HF). Values are means ± SEM, n = 8. Means without a common letter differ, P < 0.05.

 
Western blotting

The expression of cerebral GLUT-1 protein was lower in rats with induced hyperhomocysteinemia (group H) than in control rats (group C) both at 2 wk (C, 30.9 ± 4.4 INT/mm2 vs. H, 64.0 ± 2.4 INT/mm2, P < 0.05) and 10 wk (C, 41.2 ± 1.7 INT/mm2 vs. H, 65.7 ± 1.9 INT/mm2, P < 0.05). Eight weeks of folic acid supplementation significantly increased the cerebral contents of GLUT-1, which had been decreased by the homocystine diet for 2 wk, to that of control rats (Fig. 3).



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  		FIGURE 3 Effects of folic acid on the expression level of GLUT-1 protein in the brain of a hyperhomocysteinemic rat. The data were normalized by reblotting with an antibody against ß-actin and analyzed by densitometry. Data are means ± SD, n = 4. *P < 0.05.

 
Electron microscopy

    Ultrastructural alterations in cerebral vasculature. The cerebral capillary wall of a control rat had a normal appearance (Fig. 4A). It has a relatively smooth luminal surface and a regular thin layer of basement membrane around the endothelium, with the endothelial cells forming a thin, regular sheet on the inner side of the basement membrane. We categorized capillary alterations induced by a homocystine diet as follows: microvascular fibrosis, local thickening of the basement membrane, perivascular detachment, and pericytic degeneration. Damaged capillaries appear in region CA1 of the hippocampus in experimental rats (Fig. 4B–F). An abnormal electron-lucent structure indicating perivascular amorphous fibrosis was observed within the basement membrane and in the perivascular area (Fig. 4B). A locally and irregularly thickened basement membrane was also observed in cerebral microvessels (Fig. 4C). The capillaries with perivascular detachment are more evident (Fig. 4D) and degenerative pericytes that include swollen mitochondrial and cytoplasmic profiles with amorphous fibrosis are shown (Fig 4E–F). In the folic acid–supplemented group damaged vessels such as annihilation of cell organelles (Fig. 4G), degeneration of mitochondrial bilayer (Fig. 4H), and perivascular detachment (Fig. 4I) were also observed.



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  		FIGURE 4 Electron microscopic features in cerebral vessels of control and diet-induced hyperhomocysteinemic rats. (A) Control diet group; (B–F) H, 3.0 g/kg homocystine diet group; (G–I) HF, 3.0 g/kg homocystine for 2 wk followed by 3.0 g/kg homocystine plus 0.008 g/kg folic acid diet groups. (A) A cerebral capillary with a normal appearance. (B) A capillary with perivascular fibrosis. Electron-lucent structures (arrow) are present in the basement membrane and perivascular area. (C) A capillary with local basement membrane thickening. (D) Perivascular detachments (stars) appear frequently. (E,F) Membranous inclusion bodies and swollen pericytic profiles indicating pericytic degeneration (arrows) are present between the basement membrane. Electrolucent fibrosis is also evident. (G) Annihilation of cell organelles. (H) Degeneration of mitochondrial bilayer. (I) Perivascular detachments (stars). Scale bars = 1 µm.

 
    Percentage of damaged vessels. Approximately 5% of the vessels in the CA1 region of the hippocampus were altered ultrastructurally in the rats killed at baseline (Fig. 5). The incidence of damaged capillaries increased to 14.9% in rats fed a homocystine diet for 10 wk (group H) and differed significantly from both control rats (group C) and rats on a homocystine diet for only 2 wk (group H). Dietary supplementation with folic acid for 8 wk (group HF) reduced the percentage of damaged vessels compared to a homocystine diet for 10 wk (group H), but the percentage was still higher than in controls (group C).



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  		FIGURE 5 The percentage of damaged vessels in the hippocampus in control rats and rats fed a homocystine diet. Data are means ± SD, n = 4. *P < 0.05.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
LITERATURE CITED
 
We investigated the in vivo effects of dietary folic acid supplementation on structural alterations of cerebral microvasculature due to hyperhomocysteinemia. We demonstrated that diet-induced hyperhomocysteinemia decreased the cerebral content of GLUT-1 and increased the plasma concentration of TBARS and the percentage of damaged cerebral vessels in rats. Moreover, our results suggest that dietary folic acid supplementation may reduce the detrimental effects on the endothelium due to experimentally induced hyperhomocysteinemia.

Homocysteine is emerging as a vascular risk factor that leads to a cascade of vascular damage, which likely begins with endothelial dysfunction followed by progressive atherosclerosis that eventually can lead to increased cardiovascular or cerebrovascular ischemic risk (23–26). The recent experimental data reported by Nappo et al. (27) show that hyperhomocysteinemia impairs endothelial function, activating coagulation and modifying the adhesive properties of endothelia and the vascular responses to L-arginine. Pretreatment with the antioxidants vitamin E and ascorbic acid blocks these effects, which suggests the involvement of an oxidative mechanism.

The incidence of vascular disorders increases dramatically with age, but no mechanistic link has been found between aging and vascular disease. Hernanz et al. (28) showed that homocysteine synthesis increases with aging, which in turn could produce an augmented oxidant effect on endothelia and an impaired intracellular antioxidant capacity, leading to enhanced lipid peroxidation and decreased total intracellular glutathione content. Recently it was shown that homocysteine treatment increases the rate of endothelial senescence (5).

We have shown that morphological changes in the vascular ultrastructure in region CA1 of the hippocampus of hyperhomocysteinemic rats are similar to the cerebrovascular degeneration typically found in cerebral diseases such as Alzheimer’s and Parkinson’s diseases, as well as with age (29–31). We also demonstrated that the percentage of microvessels with deposits, basement membrane thickening or fibrosis, pericytic degeneration, or perivascular detachment significantly increases in rats fed a 10-wk homocystine diet.

The brain has an absolute dependence on glucose from the peripheral circulation for its normal metabolism, because its glycogen stores are negligible compared to the metabolic demand (32,33). For this, the brain capillary endothelium has a high density of GLUT-1, which facilitates the transport of glucose (34,35). Therefore, the brain endothelium, which constitutes < 0.1% of the brain weight, must transport glucose to the much larger mass of surrounding neurons and glia. Brain GLUT-1 content was significantly lower after 2 wk of diet-induced hyperhomocysteinemia. Studies employing positron-emission tomography revealed that the rates of glucose metabolism are reduced in the cerebral cortex of patients with Alzheimer’s disease (36). Reduced concentrations of glucose transporters (37) and reduced transport of glucose across the blood-brain barrier (33,35) may underlie this change. Therefore, morphological alterations in their blood-brain barriers might have influenced the brain glucose metabolism detrimentally.

Folic acid administration has decreased homocysteine levels (38), lowered the cardiovascular risk in homocystinuric patients (39), and improved endothelial function in patients with mild to moderate hyperhomocysteinemia (40). The present study found that folic acid, which is the precursor to the coenzyme that is critical in metabolizing homocysteine, reduces hyperhomocysteinemia-induced changes in brain GLUT-1 levels, and 8 wk of folic acid supplementation resulted in a full recovery in the cerebral contents of GLUT-1 to the control level. Also, dietary folic acid supplementation for 8 wk showed a similar appearance in microvascular structural damage with higher magnification, but the percentage of damaged vessels, which had been increased by hyperhomocysteinemia, was reduced with dietary folic acid supplementation. It is likely that folic acid supplementation cannot repair the structural damage done by hyperhomocysteinemia completely. However, it may prevent the vascular damage from progressing. This could be due to a direct antioxidant effect and also to an indirect effect of folic acid by improvement of the cellular antioxidant defense system (41), reduction of the pro-oxidant homocysteine (4), or an increase in the availability of tetrahydrobiopterin, a mediator of the eNOS regulation (42). Our data on the plasma TBARS levels seem to support the hypothesis that folic acid might improve cellular antioxidant defense systems.

Taken together, it is likely that folic acid supplementation may reduce cerebrovascular damage induced in hyperhomocysteinemia by affecting cellular oxidative metabolism. Further studies will be needed to warrant the potential of dietary folic acid supplementation as a novel, safe, and inexpensive tool to reduce cerebrovascular risk in hyperhomocysteinemia or in other risk factors for vascular disease associated with oxidative stress.


   FOOTNOTES
 
1 Presented in part at Experimental Biology 04, April 2004, Washington, DC [Kim, J. M., Lee, H., Kim, H. J. & Chang, N. (2004) Dietary folic acid may halt endothelial damage caused by hyperhomocysteinemia in the rat brain microvasculature. FASEB J. 18: A173 (abs.)]. Back

2 Supported in part by a grant from the Korea Health 21 R&D Project, Ministry of Health & Welfare, Republic of Korea (02-PJ1-PG10-22003-0002). Back

4 Abbreviations used: C, control diet group; GLUT-1, glucose transporter-1; H, 3.0 g/kg homocystine diet group; HF, 3.0 g/kg homocystine for 2 wk followed by 3.0 g/kg homocystine plus 0.008 g/kg folic acid diet group. Back

Manuscript received 28 October 2004. Initial review completed 12 November 2004. Revision accepted 31 December 2004.


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 MATERIALS AND METHODS
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 DISCUSSION
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J. Durga, P. Verhoef, L. J.C. Anteunis, E. Schouten, and F. J. Kok
Effects of Folic Acid Supplementation on Hearing in Older Adults: A Randomized, Controlled Trial
Ann Intern Med, January 2, 2007; 146(1): 1 - 9.
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