A Protein Extract from Chicken Reduces Plasma Homocysteine in Rats

The present study aimed to evaluate effects of a water-soluble protein fraction of chicken (CP), with a low methionine/glycine ratio, on plasma homocysteine and metabolites related to homocysteine metabolism. Male Wistar rats were fed either a control diet with 20% w/w casein as the protein source, or an experimental diet where 6, 14 or 20% w/w of the casein was replaced with the same amount of CP for four weeks. Rats fed CP had reduced plasma total homocysteine level and markedly increased levels of the choline pathway metabolites betaine, dimethylglycine, sarcosine, glycine and serine, as well as the transsulfuration pathway metabolites cystathionine and cysteine. Hepatic mRNA level of enzymes involved in homocysteine remethylation, methionine synthase and betaine-homocysteine S-methyltransferase, were unchanged, whereas cystathionine gamma-lyase of the transsulfuration pathway was increased in the CP treated rats. Plasma concentrations of vitamin B2, folate, cobalamin, and the B-6 catabolite pyridoxic acid were increased in the 20% CP-treated rats. In conclusion, the CP diet was associated with lower plasma homocysteine concentration and higher levels of serine, choline oxidation and transsulfuration metabolites compared to a casein diet. The status of related B-vitamins was also affected by CP.


Introduction
Hypolipidemic effects of processed proteins or peptides have been reported in numerous different animal models [1][2][3][4][5][6][7]. These effects are most likely due to the amino acid composition or specific peptides in the different protein sources [1,[8][9][10]. Recently, we have shown that a water-soluble protein extract of chicken (CP) had both hypotriglyceridemic and hypocholesterolemic effects in male Wistar rats [11]. The triglyceride lowering effect of the CP extract was associated with reduced hepatic triglyceride synthesis and increased mitochondrial oxidation of fatty acids, perhaps mediated by peroxisome proliferator-activated receptor (PPAR) α, of which the gene expression was increased. We also observed different plasma concentrations of several amino acids between the groups, with lower methionine and higher glycine and serine in animals fed CP.
The aim of the present study was to provide further insight into effects of a water-soluble protein extract of chicken with particular emphasis on diet related changes in Hcy metabolism. Thus, we measured the effects of the protein source on one-carbon metabolites and vitamin B status in plasma, as well as changes in hepatic gene expression. The results show substantial protein-induced alterations of plasma tHcy, as well as of B-vitamins, metabolites and gene expression related to homocysteine metabolism.

Animals and Diets
Male Wistar rats (Tactonic Europe A/S, Ry, Denmark) 12 weeks old, were housed in Makrolon type II cages, 3 animals per cage, in an open system. They were kept under standard laboratory conditions with temperature 22 ± 1 °C, dark/light cycles of 12/12 h, relative humidity 55% ± 5% and 20 air changes per hour. The animal study was conducted according to the Guidelines for the Care and Use of Experimental Animals, and the Norwegian State Board of Biological Experiments with Living Animals approved the protocol.
Animals were divided at random into 4 groups of 6 rats, and after 7 days of acclimatization, the control group were fed a 20% casein diet with 7% fat (5% lard and 2% soy oil) while in the intervention diets, casein was replaced with 6%, 14% or 20% CP (group 2-4), respectively. The composition of the diets, the procedures of making the water soluble CP extract and the amino acid profile of casein and CP are previously described [11], and the amino acid composition is given in Table 1. Notably, the 20% CP diet contains only half the amount of methionine, and has a 6-fold higher content of glycine compared to the casein diet, resulting in a 14-fold lower ratio of methionine to glycine. This resulted in a 57-fold lower ratio in plasma [11]. All groups had free access to tap water and feed during the 28 days experiment. Rat feed intake and weight gain were determined twice a week. Rats in the 20% CP group had a higher feed intake as compared to control, but gained less weight [11]. Ala, alanine; Arg, arginine; Asx, aspartic acid + aspargine; Glx, glutamic acid + glutamine; Gly, glycine; His, histidine; Hyp, hydroxyproline; Ile, isoleucine; Leu, leucine; Lys, lysine; Met, methionine; Phe, phenylalanine; Pro, proline; Ser, serine; Tau, taurine; Thr, threonine.
All rats were killed on day 28 after an overnight fast. They were anesthetized by inhalation of 2% Isofluorane (Forane, from Abbott Laboratories Ltd, Chicago, IL, USA) in an anesthesia chamber and thoracotomy, cardiac puncture, and exsanguination was performed. Plasma and liver samples were stored at −80 °C.
Three different reference genes were included: 18s (Kit-FAM-TAMRA (Reference RT-CKFT-18s)) from Eurogentec, Belgium, glyceraldehyde-3-phosphate dehydrogenase (Gapdh, Taqman Rodent GAPDH, No. 4308313) and ribosomal protein, large, P0 (Rplp0, Rn00821065_g1) from Applied Biosystems. cDNA was generated from universal rat reference RNA (Agilent Technologies) and used in 5-6 serial dilutions as a standard curve in all gene runs. The NormFinder software was used to evaluate the reference genes [25], and data were normalized to 18 s. Finally, expression values relative to the control group were calculated by dividing all individual values with the control group mean, and the results are presented as relative values ± SDs.

Statistical Analysis
Data sets were analyzed using Prism Software (Graph-Pad Software, San Diego, CA, USA) to determine statistical significance. The results are shown as means of 6 animals per group with their standard deviations. For the metabolite analyses, between-group differences were tested by one way ANOVA, and Dunnet's post hoc test was performed to evaluate statistical differences between the individual intervention groups toward the control group. For the gene expression analyses, normal distribution was determined by the Kolmogorov-Smirnov test (with Dallal-Wilkinson-Lilliefor p value), and p-values < 0.05 were considered not normally distributed. Unpaired t-test was performed to evaluate statistical differences between groups, or Mann Whitney test when values were not normally distributed. p-Values < 0.05 were considered significant.

Metabolites
The plasma levels of methionine ( Figure 2A) was reduced, and tHcy ( Figure 2B) was lowered by CP. Metabolites of the transsulfuration pathway were also affected by CP treatment as the plasma concentrations of both cystathionine ( Figure 2C) and cysteine ( Figure 2D) were increased by 14% and 20% CP. In addition, CP increased the creatinine level in a dose-dependent manner ( Figure 2E).  Values shown are means ± standard deviation (n = 6). One-way ANOVA with Dunnet's post hoc test was used to determine values significantly different from control (* p < 0.05, ** p < 0.01, *** p < 0.001).

Hepatic Gene Expression
The hepatic mRNA level of Mtr was unchanged, while Mtrr showed a small but significant increase in expression. The hepatic gene expression of Cth, the final enzyme in the transsulfuration pathway, was significantly increased 1.4 fold after CP administration. However, the mRNA level of the first enzyme in this pathway, Cbs, was unchanged.
The gene expression of Pemt was unchanged. The hepatic mRNA level of the gene encoding Chka, Chkb and of genes involved in the choline oxidation pathway, Chdh, Bhmt, Dmgdh, Sardh and Gnmt, were all unchanged in the CP-treated animals. The expression of genes involved in serine and glycine interconversion, Shmt1 and Shmt2, however, was significantly increased (Table 2).

Vitamin B Status
The plasma concentration of flavin mononucleotide (FMN) and riboflavin was significantly increased in rats fed the 20% CP diet compared to control ( Figure 4A,B). The plasma concentration of PLP and pyridoxal was unchanged in CP-fed rats compared to control ( Figure 4C,D). However, an increased plasma level of pyridoxic acid was observed in the 20% CP group ( Figure 4E). Plasma levels of folate were increased in all CP-fed rats ( Figure 4F) while cobalamin was increased only by the 20% CP ( Figure 4G). In contrast, the functional marker of vitamin B12, MMA, was unchanged ( Figure 4H). Rats were fed a 20% (w/w) casein control diet, or a diet containing 6%, 14% or 20% chicken protein (CP) for 4 weeks. Flavin mononucleotide (FMN, A), riboflavin (B), pyridoxal phosphate (PLP) (C), pyridoxal (D), pyridoxic acid (E), folate (F), cobalamin (G), and methylmalonic acid (MMA, H), were measured in fasting plasma samples. Values shown are means ± standard deviation (n = 6). One-way ANOVA with Dunnet's post hoc test was used to determine values significantly different from control (* p < 0.05, ** p < 0.01, *** p < 0.001).

Discussion
In this study in rats fed a normal fat diet (7%, w/w) we demonstrated that a water-soluble protein extract of chicken (CP) with a low methionine/glycine ratio reduced plasma tHcy and increased levels of serine and metabolites of choline oxidation and transsulfuration compared to a casein diet. The CP diet also affected the status of several B vitamins.
Hcy is produced from methionine during a myriad of methylation reactions, and early experiments demonstrated that curtailed dietary supply of labile methyl groups like methionine and choline, increases the rate of Hcy remethylation back to methionine [26]. Additionally, excess methionine intake has been shown to decrease remethylation of Hcy [27]. Finally, a low methionine-glycine ratio, as in CP compared to casein (0.11 vs. 1.57, respectively [11]), has previously been suggested to lower tHcy in rats [1]. Together, this suggests the substantially lower content of methionine in the CP diet to be an important determinant of the lower tHcy observed among the CP fed rats. A probable increase in BHMT activity, a known effect following reduced methionine intake [28], would also help explain the higher concentrations of DMG and sarcosine after CP treatment. Additionally, according to the USDA food composition database (http://ndb.nal.usda.gov), the betaine content of chicken meat from legs and wings is about twice as high as compared to dairy products. This could explain the higher plasma concentration of betaine, and also supports increased BHMT-mediated remethylation of Hcy, leading to increased production of the downstream metabolites DMG and sarcosine. However, the gene expression of BHMT was similar between groups, indicating that a potential altered activity must be related to post-transcriptional regulation.
Although gene expression of MS was unchanged, an increased remethylation by this enzyme cannot be excluded. The CP fed rats showed higher plasma folate compared to control which is associated with lower tHcy levels in humans [29,30], as well as in mouse models [31], generally believed to be mainly related to mTHF being the methyl donor for MS-mediated remethylation of homocysteine. However, reduced Hcy production due to mTHF being an inhibitor of methyltransferases like GNMT [18] seems to be the most important mechanism for this association, as demonstrated in subjects with marginal folate status [32]. We also observed higher levels of vitamin B2, which could increase enzyme activity of important enzymes in the folate cycle, including methylene tetrahydrofolate reductase, responsible for mTHF production, and MS reductase, important for maintaining MS function [33,34]. This would support increased production of mTHF, thus contributing to the Hcy-lowering.
Importantly, the tHcy decrease could also be the consequence of increased Hcy catabolism through the transsulfuration pathway, as the gene expression of Cth was significantly increased. This was accompanied by increased plasma concentrations of cystathionine and cysteine. However, when dietary supply of methionine is low, previous studies have shown that transsulfuration flux decreases [35]. Additionally, CTH mRNA changes were small and may not alter its protein expression.
The concentrations of other metabolites along the choline oxidation pathway, namely DMG, sarcosine, glycine and serine were increased after CP administration. The gene expression of Bhmt, Dmgdh and Sardh was unchanged after CP treatment. Notably, the 20% CP diet contained six-fold higher levels of glycine, while the content of serine was only half compared with the control diet [11]. Thus, we cannot rule out the possibility that the increased level of plasma DMG and sarcosine could be a direct consequence of product inhibition of SARDH and DMGDH, respectively, due to the higher intake of glycine in the CP fed animals. Glycine is also a product of the catabolism of threonine [36], but as the threonine content of CP was much lower than in casein, this was most probably not a significant contributor to the elevated plasma glycine. Serine is used as a substrate for the first enzyme in the transsulfuration pathway, where serine and Hcy condenses to form cystathionine. If transsulfuration flux is reduced to spare Hcy for methionine synthesis, this could contribute to the accumulation of serine. However, an increased expression of Shmt1 and Shmt2 mRNA was observed, which suggest that the higher concentrations of serine observed among the animals receiving the CP diet may also be due to increased conversion from glycine. We observed increased catabolism of vitamin B6, which is used for degradation of glycine through the glycine cleavage system in the mitochondria [37].
Thus, further studies should evaluate if increased B6 catabolism is a mechanism induced to spare glycine for serine production.

Conclusions
In summary, a water-soluble protein extract of chicken (CP) reduced plasma tHcy and interfered with circulating markers of B vitamin status. The low ratio of methionine to glycine in CP may play an important role due to the importance of regulating methionine availability for methylation reactions, and the higher folate concentrations may have contributed to decreased Hcy synthesis due to its ability to inhibit methyltransferases like GNMT. Further studies are needed to further clarify the specific effects of individual dietary amino acids or different amino acid composition on the metabolic pathways related to Hcy metabolism.