Impact of Compliance to a Gluten-Free Diet on Vitamin and Trace Element Deficiencies in Celiac Patients

Objective: Celiac disease (CD) is a chronic immune-mediated disorder characterized by growth retardation and malabsorbtion related to mucosal damage and inflammation of small intestine in genetically predisposed people as a result of gluten exposure. In CD treatment, clinical, histological and serological improvement is possible with gluten-free diet. Thus, we aimed to assess the vitamin and trace element levels of CD patients in regard to their compliance with gluten-free diet. Methods: In our study, 77 patients diagnosed with CD were evaluated retrospectively. All cases were assessed with Marsh classification histopathologically and surveyed when they follow a gluten-free diet. Demographic features, age of disease onset, physical examination findings, anthropometric measurements and laboratory findings along with clinical and laboratory outcomes of patients after a gluten-free diet were compared between compliant to diet and non-compliant to diet groups. Results: A total of 77 cases consisting of 48 females and 29 males with a diagnosis of CD and mean age of 9,81 ± 4,73 years on admission were reqruited in our study. Patients were mostly found to be Marsh type 3a (n = 22) and Marsh type 3b (n = 20) histopathologically. The results of serological screening revealed that 40,3% of cases (n = 31) were compliant with diet whereas 59,7% (n = 46) non-compliant. Non-compliant group had significantly lower mean vitamin B12, vitamin D, folate, zinc and selenium levels compared to compliant group (p values = 0.000, 0.000, 0.000, 0.000 and 0.031, respectively). In addition, a significantly higher mean serum total IgA level was detected in non-compliant group in comparison to compliant group (p = 0,027). Conclusion: In our study, high efficacy of gluten-free diet (GFD) in correcting nutritional insufficiencies and deficiencies was shown. Thus, there is no doubt that informing patients and their families about lifelong GFD in detail is beneficial although GFD treatment contains many social and practical difficulties.


Introduction
Celiac disease (CD) is a chronic immune-mediated disorder characterized by malabsorbtion and growth retardation in children as a result of inflammation, infiltration and villous atrophy in small intestine [1]. There is a lifelong intolerance to gluten of wheat in particular, together with grain proteins of rye, barley and sometimes contaminated oat products in genetically predisposed people [2].
Even if the disease presents often with chronic diarrhea, weight loss and bloating, asymptomatic course is also likely. Moreover, a broad spectrum of clinical symptoms including stomach ache and chronic fatigue are found along with disorders related to vitamin and mineral uptake such as iron deficiency anemia, vitamin D deficiency and calcium deficiency. Although the prevalence of CD is reported to be almost 1 % in Europe, actual prevalence is considered as high as 7-10 fold due to its insidious presentation and globally increased incidence in recent years [2][3][4].
Though histopathological assessment is regarded as the gold standard of diagnosis, tissue transglutaminase antibody, anti-endomysial antibody and human leukocyte antigene tests are among first line diagnostic tools [5]. In fact, there is a well known pathophysiological interaction between environmental and genetic risk factors in CD. Accordingly, CD does not develop in absence of allelle coding HLA-DQ2 or HLA-DQ8 proteins while presence of that allelle does not always suffice for the disease to emerge. In addition, a mild gluten exposure of celiac patients may lead to mucosal damage, however clinical, histological or even serological (TGA/G) improvement is likely after a gluten-free diet [1,2]. Thus, we purposed to analyze the vitamin and trace element levels of patients we followed up with a diagnosis of CD, in regard to dietary compliance after a gluten free diet GFD) therapy. A total of 77 patients being between 1 to 18 years of age diagnosed with CD who have no other chronic illness leading to additional malabsorbtion were investigated retrospectively. Demographic features of patients, age of disease onset, physical examination findings, anthropometric measurements and laboratory findings along with clinical and laboratory outcomes of patients after a GFD were compared between compliant to diet and non-compliant to diet groups. Biochemical analysis of serum derived from patients was performed with the Abbott Architect c8000 autoanalyzer (Abbott Diagnostics, Illinois, USA) by using electrochemiluminescence method whereas serum 25-hydroxyvitamin D 3 , vitamin B 12 and folate levels were measured and reported by using test kits compatible with chemiluminescence method in Roche Cobas 6000 (Tokyo, Japan) immunological autoanalyzer device.

Materials and methods
Serological screening for CD was carried out with enzyme-linked immunosorbent assay by using anti tissue transglutaminase antibodies (TGAb). All patients were also screened with immunoflorescence method ((INOVA, San Diego, Calif., USA) for anti-endomysial antibodies, umbilical cord and fluorescein isothiocyanate conjugated anti-human IgA. To detect serum IgA deficiency, total serum IgA levels were assessed with nephelometric method. In addition, presence of HLA antigenes (DQ2 and DQ8) were investigated by using polimerase chain reaction (PCR) technique on genomic DNA isolated from peripheral blood of all patients. Patients with positive TGA and EMA antibodies got a definite diagnosis after the endoscopic biopsy taken from distal duodenum and cases were then evaluated histopathologically in regard to Marsh classification. All CD cases were followed up with gluten-free diet.
Statistical analysis. For the statistical analysis of our study, the 21.0 version of SPSS software ((Statistical Package of the Social Sciences, IBM, Armonk, NY, USA) was utilized. Descriptive statistics were expressed as mean ± standard deviation or median (minimum-maximum) for discrete and continuous quantitative variables whereas categorical variables were given as number of cases and percentage (%). For the comparison of categorical variables, cross table statistics were used (Chi square, Fisher). While normally distributed parametric data sets were compared by using Student t-test and ANOVA, non-parametric data with abnormal distribu-tion were compared via Mann Whitney U and Kruskal Wallis tests. Comparisons between multiple groups were carried out with post hoc Tukey analysis. In results, p < 0.05 was defined as statistically significant.

Results
A total of 77 cases consisting of 48 females (62.3 %) and 29 males (37.7 %) followed up with the diagnosis of CD were reqruited in our study. Female to male ratio of patients was found to be 1.65 and mean age of all patients on admission was 9.81 ± 4.73 (range of distribution; 2-19) years. In particular, mean age of males was 9.25 ± 4.23 years whereas mean age of females was found to be 10.16 ± 5.02 years (p = 0.381). Additionally, overall mean age of cases at diagnosis was 2.28 ± 2.37 years (range of distribution: 0-11years) (  15.03 ± 5.08 mg/l, zinc 82.32 ± 9.84 µg/dl and selenium 49.91 ± 5.77 µg/dl in the group compliant to diet. Hence significantly higher mean serum total IgA levels were observed in non-compliant group compared to compliant group (p = 0.027). In addition, significantly lower mean serum vitamin B 12 , vitamin D, folate, zinc and selenium levels were noted in non-compliant group compared to compliant group (p va lues; 0.000; 0.000; 0.000; 0.000 and 0.031, respectively).

Discussion
CD developed into a crucial global health issue as its prevalence increases in recent years and its atypical and asym ptomatic presentations suggest presence of many undiag nosed cases. Accordingly, current data show that children and adolescents with a higher mean age compared to that of past reports are diagnosed with CD [6]. Predominance of female gender in CD was also reported many times [7]. N.R. Reilly et al. reported a mean age of 8.3 years based on 318 pediatric patients of whom 57 % were female and diag nosed with CD histopathologically [8]. E. Lurz et al. also reported a group of 94 pediatric patients at a mean age of 6.8 years diag nosed with CD, which displayed a female predominance of 62 % [9]. Consistent with the published data, female/male ratio in our study was calculated as 1.65 and the mean age on admission was found to be 9.81 ± 4.73 years.
Celiac patients frequently face nutritional deficiencies resulting from malabsorbtion of macro-and micronutrients induced by basal enteropathy. Micro-nutritional insufficien-Mìžnarodnij endokrinologìčnij žurnal, ISSN 2224-0721 (print), ISSN 2307-1427 (online) Оригінальні дослідження /Original Researches/ cies and deficiencies mostly comprise iron, zinc, copper, calcium and selenium elements accompanied by vitamin E, D, B 12 and B 6 [10]. Iron deficiency is so widespread in adult patients without a diagnosis of CD yet that it is worth to perform serological screening [11].
Systemic and neurological disorders may also develop in celiac patients without treatment, leading a decline in quality of life [12]. At the present day, most effective treatment for celiac patients is GFD whereby clinical symptoms and morbidity lessen along with a quick improvement in body weight, bone density and nutritional parameters [13]. For the efficacy of GFD, one should strictly avoid all products of wheat, rye and barley [14]. Since enteropathy may worsen even with as low as 50 mg of gluten, patients also have to abstain from products such as oat which may be contaminated with gluten although it is essentially gluten-free [15]. With an appropriate GFD, iron deficiency is corrected in 6-12 weeks and zinc deficiency in a few weeks [16].
S. Terlemez et al. investigated the influence of a 6 months GFD followed by 66 pediatric CD patients on their laboratory parameters. Researchers detected iron deficiency anemia in 36.6 % of patients, folate deficiency in 3 % and vitamin B 12 deficiency in 1.5 %. By means of GFD, however, vitamin B 12 and folate deficiencies were completely corrected and iron deficiency anemia significantly improved [17]. P. Rawal et al. randomized 134 patients with CD into two groups and treated them with GFD or GFD accompanied by zinc supplementation. While final plasma zinc levels in both groups were significantly increased compared to initial values, researchers concluded that this rise in zinc levels occured solely due to GFD independent of supplementation [18]. Additionally, several neurological and thyroid disorders related to copper and selenium deficiency were reported in patients with CD from previous studies. After the GFD, serum copper and selenium levels were observed approaching the normal ranges despite a minimum improvement in neurological disturbance [19,20] E. Topal et al. reported the rate of vitamin D and zinc deficiency in 52 newly-diagnosed pediatric celiac patients as 51.9 % and 67.3 %, respectively [21]. Moreover, K. Öhlund et al. notified in their study that vitamin D absorbtion was significantly lower than required amount in 17 of 25 patients diagnosed with CD whereas none of the children being 12 years old and younger could achieve normal serum levels of vitamin D in this group [22]. In addition, M. Unubol et al. showed that vitamin D deficiency of celiac patients is corrected by taking vitamin D supplementation along with GFD therapy [23]. A. Dahele et al., on the other hand, detected vitamin B 12 deficiency in 41 % of 39 patients with CD and folate deficiency in 31 %. After an 11-months GFD therapy, normal serum vitamin B 12 and folate levels were achieved in all patients except one whose data were unavailable [24]. Correspondingly, our study has also demonstrated a significantly lower mean serum vitamin B 12 , vitamin D, folate, zinc and selenium levels in non-compliant to diet group compared to compliant ones, supporting all these data.

Conclusions
High efficacy of GFD on CD was also shown in our results even if GFD treatment contains lots of social and practical difficulties due to low satisfaction and high costs for the patient and his family. Thus, it is undoubted that informing patients and their families in depth about lifelong GFD may contribute both to abate CD-related morbidity for patients' safety and to use health resources productively.

Conflicts of interests.
Author declares the absence of any conflicts of interests and their own financial interest that might be construed to influence the results or interpretation of their manuscript.