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Similar to HM diet, a B-vitamin deficient diet to induce HHcy in animal models has been studied too. Caudill et al. administered a diet low in methionine and deficient in folate and choline to C57BL/6J mice. After 24 weeks, Hcy was higher, compared to the control diet mice. SAM concentrations and SAM:SAH ratio were lowered in all the analyzed tissues; liver, kidney, brain and testes. SAH was higher in the liver, but not in other tissues. However, global DNA methylation measured by cytosine extension assay did not change in any of the tissues [22].

In the context of vascular pathologies, effects of HHcy induced by a B-vitamin deficient diet were not as clear as compared to the effects by the HM diet. In a study by Liu et al., B-vitamin deficient diet with or without HM was given to a group of ApoE- deficient mice. The aortic sinus plaque areas were shown to be larger as a result of HM diets, but not with B-vitamin deficient diet alone. Plasma Hcy was elevated, but there was no change in the plasma SAH or aortic global methylation levels [18]. Another study investigated the effects of folate and/or vitamin B12 deficiency on endothelium- dependent relaxation in rats. Serum Hcy was higher in the folate deficient (FD) and folate-vitamin B12 deficient diet groups, compared to controls. Liver SAM and SAM: SAH ratio were lower, and SAH showed no difference. However no association of folate, vitamin B12, Hcy, SAM, SAH or SAM:SAH ratio was found with vascular reactivity [23]. In yet another study, moderate HHcy resulted when ApoE-deficient mice were treated with either a FD or folate, vitamin B6 and B12 deficient diet for 16 weeks. The hepatic SAM:SAH ratio for both the diets were reduced up to 80 and 90% respectively. However, they showed no association with atherosclerosis and global DNA methylation of the vascular or liver tissue, even after an increased atherosclerotic lesion formation in the aortic arch [24,25].

In relation to pregnancy outcomes, maternal folate status affects the homeostasis of Hcy pathway in their offsprings. In a study by Blaise et al., female Wistar rats were fed a diet deficient in vitamin B12, B2, folate and choline from one month before pregnancy until weaning at day 21. Hcy concentrations were moderately elevated in mothers. The pups, who were fed on dams with such a deficient diet, also developed HHcy and had a decline in hepatic SAM:SAH ratio due to the decrease in SAM. Cbs, Mthfr and Ms activities in the liver were extremely lower in deficient pups in comparison to normal fed pups [26]. In another similar study of a FD diet given to female pregnant rats starting 2 weeks before mating until gestation at day 21, 4.5-fold increased maternal plasma Hcy concentrations were seen. However in this case, no change in the global DNA methylation levels was observed in the maternal or fetal rat livers [27]. Similarly, Mejos et al. randomly administered a FD or a folate supplemented (FS) diet to male and female rats for 4 weeks until mating. Hcy concentrations were

elevated in the postnatal rats of either or both of the deficient parents, in comparison to the postnatal rats of both FS parents. There was a marked reduction of hepatic folate and global DNA methylation levels as identified by detection antibodies and quantified using an ELISA [28]. In another study, pregnant Mthfr (+/+) mice were fed a FD diet to induce HHcy. Hepatic SAM concentrations were decreased and SAH concentrations were increased, which was accompanied by a reduction of SAM:SAH ratio. Placental SAH also increased with decrease in SAM:SAH ratio [29].

Two other studies measured the levels of DNA methylation and its metabolites in the brain of FD diet induced HH rats. The treatment periods for the studies were 30 days and 36 weeks. SAM, SAH and SAM: SAH ratio remained unaltered in both cases. In the first case, global DNA methylation measured by in vitro methyl acceptance capacity assay, was lowered in the deficient group compared to controls [30]. Interestingly, in the second case, at 18 and 36 weeks, the global DNA methylation levels quantified by high-performance liquid chromatography–(electrospray)-mass spectrometry, were higher in the FD diet group, as compared to controls, although the same mice group had also shown global hypomethylation in the liver, when quantified by the cytosine extension assay [31,32]. The trend in the alteration of DNA methylation differed between studies, or might suggest a different mechanism with prolonged treatment period. Furthermore in an additional study, such a similar HH model of Cbs (+/+) mice showed elevated SAH with lower SAM:SAH ratio in the brain [33].

Folate deficiency, which is also implicated in relation to colon cancer, was experimented in the colon of HH rats by two groups. In the first study, SAM concentrations of the colonic mucosa and methylation of Trp53 promoter region remained unchanged in the FD diet group. A 3–3.5 fold higher SAH and 64–71% lower SAM:SAH ratio was observed at 5 weeks, in comparison to controls. A 30% higher colonic global DNA methylation, measured by an in vitro methyl acceptance capacity assay was observed only at 3 weeks, which directly correlated with plasma Hcy [34]. The second study group conducted a FD treatment of 24 weeks. An ~11% lower hepatic SAM:SAH ratio was observed, in comparison to controls. But no change was found in colonic SAM and SAH concentrations or their ratio. Global DNA methylation in the liver and colon did not lower significantly, even after this longer treatment period of 24 weeks [35].

In general, different diets may have variable effects on Hcy, DNA methylation and its markers. Devlin and associates conducted a study, where they assigned one of the 3 diets for 7–15 weeks to random groups of both Mthfr (+/+) and Mthfr (+/−) mice; 1) HM 2) LF 3) HM-LF. Plasma Hcy showed no difference between control and the HM diet group. But higher Hcy was observed in the LF and HM-LF group, as compared to controls. Hepatic and brain SAM:SAH ratio were lower in all experimental diet groups, being the most in the LF and HM-LF groups. In the liver, the lower SAM:SAH ratio in LF and HM-LF groups was due to higher SAM, whereas, in the HM group, it was due to

higher SAH. No effect on global hepatic or brain DNA methylation was observed in any of the three cases [36].