Our study was planned as a case-control study in a single center with a Training and Research Hospital Pediatrics Clinic in order to examine the relationship between vitamin B12 deficiency and thiol/disulfide hemostasis in adolescence.
The study was planned by selecting 45 of 141 adolescent patients who applied to Aksaray Training and Research Hospital Pediatrics Outpatient Clinic and the control group, was formed by selecting 45 healthy adolescents who did not have any symptoms and applied to the polyclinic to obtain a sports license.
Patients between the ages of 11-18 were selected among the patients who applied to the outpatient clinic for the first time with one of the complaints of headache, decreased school performance, dizziness, and fatigue. Except for vitamin B12 deficiency, patients without iron deficiency anemia and vitamin D deficiency, without any comorbite disease, and with normal cranial magnetic resonance imaging  were selected.
Of the 96 patients who were not included in the study because, 43 patients had upper respiratory tract infections such as sinusitis and flu, 33 patients had vitamin D deficiency and iron anemia, 14 patients had comorbidit disease and 2 patients use of antidepressant medication. Four patients were excluded as they were diagnosed with migraine.
Hemogram, vitamin B12, native and total thiol, disulfide and homocysteine levels were measured from the patients. Serum samples were collected into vacutainer tubes containing EDTA, during routine blood sample taking for the measurement of vitamin B12 and other biochemical parameters, and were centrifuged at 1500 g × 10 min after sampling. Then, serum samples were portioned and stored -80°C in the eppendorf tube until the time of assay.
Measurement of Thiol/ Disulfide Homeostasis
In this study, native and total thiol levels were measured with a new spectrophotometric method developed by Erel and Neselioglu . Disulfide levels were calculated the half of the difference between the total and native thiols.
Measurement of Homocysteine Levels
Homocysteine levels (6-14 μmol/L) were studied with the colorimetric method (automatic analyzer MINDRAY-BS400® device). First, oxidized homocysteine (HCY) is reduced to free HCY. Free HCY then reacts with a co-substrate, catalysed by cycling enzymes, and was significantly amplified. Then through dehydrogenation, NADH converts to NAD+.The concentration of HCY in the sample is indirectly proportional to the amount of NADH converted to NAD+.
In this study, the data were analyzed by using the SPSS 22.0 (IBM, USA) statistical package program. Shapiro-Wilk test was used to determine the distribution patterns of the variables. Numerical values were expressed as mean ± standard deviation, and categorical values were expressed as n (%). The mean differences between two independent groups were compared using the Student's t-test; The Mann-Whitney U test was used to compare the median values that did not fit the parametric distribution. The Kruskal-Wallis test was used to evaluate oxidative stress in the age category divided into three groups. One-way ANOVA was used when differences between the groups were evaluated because the variables showed normal distribution. Linear regression analysis was applied to evaluate the relationship between dependent and independent variables. P <0.05 were considered statistically significant.
Written informed consent was obtained from all parents after the procedures regarding the study were fully explained to the families. This study was approved by ethical committee of Hatay Mustafa Kemal University (protocol number: 2022/10).