Serum homocysteine , vitamin B 12 and folate levels in healthy Turkish Van Cats

Objective: The present study was conducted for the purpose of setting forth the normal serum Hcy, vitamin B12 and folate levels in Van cats of varying ages and genders, and the age-dependent variations of these parameters. Materials and methods: The material of the study consisted of a total of 60 healthy Van cats including 30 female and 30 male cats. Cats from both genders were separated into 3 groups on the basis of their ages. While the cats of 6 12 months of age were included in the first group, cats of 12-24 months of age were included in the second and those of more than 24 months of age were included in the third group. Results: From the blood samples collected; serum normal homocysteine, vitamin B12 and folate levels were determined as 7.1±2.2 nmol/mL, 850.7±231.8 pg/mL and 16.7±0.8 ng/mL, respectively. In the statistical comparison of the determined serum homocysteine, vitamin B12 and folate levels; some variations among different groups of age and genders were determined. However, none of these differences were determined to be statistically significant. Conclusion: The normal levels of serum Hcy, vitamin B12 and folate of healthy Van cats were set forth for the first time by the present study. It is believed that the normal values of these parameters in Van cats can be used in the diagnosis and prognosis of various diseases and particularly cardiovascular diseases, that they will be helpful for researchers and will serve as a guideline to the studies to be conducted in the future.


INTRODUCTION
Turkish Van cat is a significant endemic cat species living in the area of Van province of Turkey and attracting great attention all around the world.With qualities of having odd colored eyes, being affectionate, the white and silky fur and being quite fond of water, Van cats have built a well-earned reputation worldwide.Due to their diminishing numbers in the recent years, they have been taken into protection and subjected to many scientific studies (Sonmez and Agaoglu, 2010;Ozkan et al., 2013;Kayar et al., 2014).
Homocysteine (Hcy) is a sulfuric amino acid generated in consequence of the metabolism of the essential amino acid methionine and is not taken in the primary structure of proteins.It is known that there are close relations between Hcy and folic acid, B6 and B12 vitamins.In the body it is metabolized through remethylation where folic acid and vitamin B12 serves as cofactors and transsulfuration where vitamin B6 is the cofactor (Aksoy et al., 2006;Nursalim et al., 2013;Ganguly and Alam, 2015).Generated through transsulfuration, Hcy is disintegrated and excreted via urine and during these reactions the cofactor vitamin B6 is needed.Therefore, it is reported that Hcy levels increase in cases of folic acid, vitamin B6 and vitamin B12 deficiencies, and that these vitamins are essential for Hcy metabolism (

Animals:
The animal material of the present study consisted of a total of 60 (30 female and 30 male) clinically healthy Van  Blood sampling and biochemical analyses: For biochemical analysis, blood samples were collected from vena radialis (V.cephalica) to vials without anticoagulant.Blood samples collected in vials without anticoagulant were centrifuged for 10 min at 3000 rev/min (Rotofix 32 ® -Hettich) and serums were prepared.Until the time of measurements, obtained serums were kept at -20 0 C. Serum vitamin B12 and folate levels were examined by means of Cobas-6000 e601 (Roche-Hitachi Japan) hormone analyzer.Homocysteine levels, on the other hand, were determined by means of an ELISA equipment (ELISA reader ® -DAS) and through the use of a commercial homocysteine test kit (Human Homocysteine ELISA Kit-CUSABIO, Catalog Number; CSB-E13814h), at 450 nm wavelength as advised in the user manual of the test kit.
Statistical analysis: Kalmagrov Simirnov was performed on the data of the study as a normality test.Parametric methods were used for statistical analyses because the data showed normal distribution.Definitive statistics were performed for analysing different gender and age groups.While independent t-test was used for comparing the data of female and male animals, One WAY ANOVA test was used for comparing data of different ages belonging to female and male animals.Tukey test was used as multiple comparison test between groups.For this purpose SPSS 20.0 software was used.For making the calculations, P<0.05 values were accepted as statistically significant.

RESULTS
While the arithmetic means and standard deviations of the serum Hcy, vitamin B12 and folate levels of different genders of Van cats and the results of the statistical    comparisons between the groups are presented in Table 1.The arithmetic means, standard deviations of the same values for age-based groups and the statistical comparisons between these groups are presented in Table 2, and Figure 1, 2 and 3.
Examining Table 1 shows that while the serum Hcy and vitamin B12 levels of male cats are higher than those of female cats, folate levels are higher in female cats.However, the differences in these parameters were not found out to be statistically significant in terms of the cats' genders.While the average Hcy level was determined to be 7.1±2.2nmol/mL, the average levels of vitamin B12 and folate were determined to be 850.7±231.8pg/mL and 16.7±0.8ng/mL respectively.
Although statistically insignificant, these parameters were determined to be different among the groups of different ages, and the values are presented in Table 2.While the highest Hcy levels were observed in the 1 st female group, the highest levels of vitamin B12 were found out to be in the 1st male group, and the highest folate levels were determined to belong the 2 nd male group (Figure 1, 2 and 3).In another study (Kocabalkan et al., 2000), tHcy levels were reported to be 6.7±1.7 nmol/mL in female and 7.5±2.6nmol/mL in male test subjects, and that serum Hcy levels may be about 10% higher in males when compared to females.In a study conducted by Aksoy et al. (2006), levels of Hcy, vitamin B12 and folate were reported to be 10.3 µmol/L, 357±163 pmol/L and 8.8±4.1 ng/mL respectively.They reported plasma Hcy levels of males (11.8 µmol/L) to be statistically higher than those of females (10.6 µmol/L).

DISCUSSION
Also in our present study serum Hcy levels of male animals (7.5±2.6 nmol/mL) were determined to be higher than those of female animals (6.7±1.7 nmol/mL), yet the difference was not found out to be statistically significant.This finding is in line with several studies reporting that the Hcy levels of males are higher than the Hcy levels of females (Kocabalkan et al., 2000;Aksoy et al., 2006).In addition, it was reported in studies conducted on humans that serum or plasma Hcy levels may increase in line with the increasing age (Kocabalkan et al., 2000;Aksoy et al., 2006).Examining the Hcy levels determined in our study also revealed that there are some differences among the Hcy levels of different groups of age, while none of these differences were determined to be statistically significant (Figure 1, 2, 3).

CONCLUSION
In conclusion, the normal levels of serum Hcy, vitamin B12 and folate of healthy Van cats were set forth for the first time by the present study.It is believed that the normal values of these parameters in Van cats can be used in the diagnosis and prognosis of various diseases and particularly cardiovascular diseases, that they will be helpful for researchers and will serve as a guideline to the studies to be conducted in the future.

Figure 1 .
Figure 1.Serum homocysteine levels of male and female Van cats of different groups of age.

Figure 2 .
Figure 2. Serum vitamin B12 levels of male and female Van cats of different groups of age.

Figure 3 .
Figure 3. Serum folate levels of male and female Van cats of different groups of age.
It is reported in recent studies that Hcy levels may increase in the early periods of several diseases and particularly cardiovascular diseases(Uren etal., 2009; Lippi and Plebani, 2012; Amin et al., 2016; Smith and Refsum, 2016).Due to this reason blood Hcy levels has become a significant parameter in the early diagnosis of many diseases.Although there are many studies conducted on Hcy levels within the scope of human medicine, the same topic is not studied in details in veterinary medicine (McMichael et al., 2000; Ozden et al., 2005; Geesaman et al., 2016).

Table 1 .
Serum Hcy, vitamin B12 and folate levels of healthy male and female Van Cats.

Table 2 .
Serum Hcy, vitamin B12 and folate levels of male and female Van cats of different groups of age.
(Aksoy et al., 2000;t al., 2010;Nursalim et al., 2013;Geesaman et al., 2016)d Agaoglu, 2010), their serum Hcy, vitamin B12 and folate levels were never taken as the topic of a study.Due to this reason, in the present study it was aimed to determine the normal values serum Hcy, vitamin B12 and folate levels of healthy Van cats of varying ages and different genders, and to set forth the variations in these values based on cats' age and gender.Homocysteine is a sulfuric amino acid generated in consequence of the metabolism of methionine and is not taken in the primary structure of proteins (Gauthier et al., 2003; Lippi and Plebani, 2012; Nursalim et al., 2013; Çayır and Kozat, 2016).Several studies conducted in the recent years report that the increases in homocysteine levels cause several negative effects in the organism (Bostom and Lathrop, 1997; Lippi and Plebani, 2012; Geesaman et al., 2016).Conducted studies report that there are close relations between Hcy and the vitamins that are essential for Hcy metabolism, namely folic acid, vitamin B6 and vitamin B12 and that in the case of the deficiency of these, Hcy levels may increase(Klee, 2000;Collin et al., 2010;Nursalim et al., 2013;Geesaman et al., 2016).The serum tHcy (7.1±2.2 nmol/mL), vitamin B12 (850.7±231.8pg/mL)and folate (16.7±0.8 ng/mL) levels determined in healthy Van cats within the scope of our study are considered to be close to the normal values reported for cats and humans in various studies(Aksoy et al., 2000;