This is cross-sectional study investigating status vimain D in children new diagnosed CeD, who were refered to the Gastroenterology department of the Republican Specialized Scientific and Practical Medical Center of Pediatrics of the Ministry of Health of the Republic of Uzbekistan during the period from Septeber 2016 till Desember 2017 year. None of the children had a history of vitamin D supplementation or any comorbid conditions that could have contributed to vitamin D deficiency or insufficiency (such as bone deformations or parathyroid gland diseases).

Children presenting with gastrointestinal symptoms indicative of celiac disease, who were referred to the Department of Pediatric Gastroenterology from community clinics or peripheral hospitals, were assessed based on the 2012 diagnostic guidelines of the European Society for Pediatric Gastroenterology, Hepatology, and Nutrition (ESPGHAN) (15). The study excluded participants who: (i) had a confirmed diagnosis of CeD prior to referral; (ii) were following a gluten-free diet at the time of enrollment; or (iii) did not provide informed consent to participate.

The study was carried out in accordance with international ethical standards and received approval from the Ethics Committee of the Republican Specialized Scientific-Practical Medical Center of Pediatrics (RSSPMCP) (approval number IP-2016, dated May 17, 2016). Written informed consent was obtained from the legal guardians of all participants, and the study complied with the principles of the Declaration of Helsinki. The physical development of children was assessed using reference tables of anthropometric indicators proposed by experts from the World Health Organization, using the WHO Anthro, WHO AnthroPlus programs (16).

For symptomatic children, the no-biopsy diagnostic pathway—applicable when anti-tissue transglutaminase IgA (anti-tTG IgA) levels exceeded 10 times the upper limit of normal and endomysial antibodies (EMA IgA) were positive in a second serum sample—was considered (11). However, due to the unavailability of EMA IgA testing in our country, all children with positive anti-tTG IgA results were advised to undergo esophagogastroduodenoscopy (EGDS) with histological examination of the duodenal mucosa.

Consequently, CD diagnosis was based on positive serologic findings (anti-tTG IgA) in combination with Marsh grade 2 or higher histopathological changes. In IgA-deficient individuals, anti-tTG IgG was measured, and a diagnosis was confirmed if histologic findings were consistent with CD. All patients diagnosed through this diagnostic process were also genotyped for HLA-DQ2 and DQ8.

Total serum IgA was quantified using a two-step sandwich ELISA with monoclonal anti-IgA antibodies (Cat. No. A-8666, Vector-BEST, Novosibirsk, Russia). In cases of reduced IgA levels, total IgG and anti-tTG IgG were also measured. Total IgG was assessed with a similar sandwich ELISA kit using monoclonal antibodies (Cat. No. A-8662, Vector-BEST, Novosibirsk, Russia). Quantitative determination of anti-tTG IgA (or anti-tTG IgG, when applicable) was performed using ELISA kits from Orgentec Diagnostika GmbH (Cat. No. 416-5400A, ORG 540G, Mainz, Germany).

HLA genotyping was conducted using sequence-specific primer polymerase chain reaction (SSP-PCR) with DQ kits targeting DQA105, DQB102, DQA10301, DQB10302, DQA10505, and DQB10202 alleles to detect DQ2.5, DQ2.2, and DQ8 haplotypes (Celiacstrip HLA DQ2DQ8, OPERON, Inmuno and Molecular Diagnostics, Caparoca, Spain).

25(OH)D and parathyroid hormone (PTH) in the serum were determined by the ELISA method on the Elecsys apparatus (Switzerland). Total and ionized calcium, inorganic phosphorus and alkaline phosphatase (ALP) were determined biochemically. The results were assessed in accordance with the recommendations of the International Society of Endocrinologists (2011): vitamin D deficiency—less than 20 ng/ml (less than 50 nmol/L); vitamin D insufficiency—21–29 ng/ml (51–75 nmol/L); normal vitamin D content—30–100 ng/ml (76–250 nmol/L). A concentration below 10 ng/ml (less than 25 nmol/L) is interpreted as severe vitamin D deficiency. A level above 100 ng/ml (more than 250 nmol/L) is considered as excess vitamin D (17). The control group consisted of 31 children aged 3–15 years (mean age: 7 ± 2.1 years) who had not received vitamin D supplementation. The group included 14 boys and 17 girls.

Histological evaluation of duodenal mucosal changes was conducted by experienced histopathologists using the Marsh–Oberhuber classification system. According to this grading, Marsh–Oberhuber grade 2 is characterized by an increased number of intraepithelial lymphocytes along with crypt hyperplasia. Marsh–Oberhuber grade 3 (subtypes a, b, and c) indicates partial, subtotal, or total villous atrophy, respectively, in combination with intraepithelial lymphocyte infiltration and crypt hyperplasia.

Statistical analysis was carried out using Microsoft Excel with built-in statistical tools and STATISTICA 10.0 software [StatSoft, Inc. (2011)]. Methods of descriptive and inferential statistics were employed. This included calculation of relative frequencies (percentage, %), measures of central tendency and dispersion (arithmetic mean [M], standard deviation [σ], and standard error [m), and comparison of means using Student's t-test (t). A p-value of ≤ 0.05 was considered statistically significant. Correlations between variables were analyzed using Pearson's correlation coefficient. Descriptive statistics are presented as absolute numbers and percentages (%).

The sample size was not calculated preliminarily, a continuous study of children who came to our center with the first established CeD was carried out. Studies were carried out with the written consent of their parents.

Missing data were handled using the Complete Case Analysis method, in which rows/columns containing gaps were excluded from the data set. Statistical analysis was performed using GraphPad Prism (version 9.3.1, 2021). Descriptive statistics included the calculation of the mean (M), standard deviation (SD), median (Me), and interquartile range (Q1; Q3). The Kolmogorov–Smirnov test was used to evaluate the normality of data distribution. Group comparisons were conducted using either Student's t-test for normally distributed variables or the Mann–Whitney U-test for non-normally distributed data.

For measuring the strength and direction of the relationship between two variables Pearson's correlation coefficient was used. Categorical variables were expressed as absolute and relative values. 95% CI for the proportion was calculated using the Wald normal approximation method. The differences were considered statistically significant at p < 0.05, the calculation was made by the two-sided p-value.

During the study period, celiac disease was diagnosed in 69 children who came to our centre, of whom 9 refused the study, therefore, the study population consisted of 60 children (age range: 1–16 years, 60 children with celiac disease aged from 1 year to 16 years were examined, the average age was 6 ± 2.3 y including 36 boys and 24 girls. According to the distribution of the decrease in vitamin D: deficiency was detected in 48 (80%) patients with CeD, and in every fourth patient (15/25%) extremely low levels were established (below 10 ng/ml). Insufficient vitamin D content was detected in 12 patients with CeD (20%) (p < 0.05) (Table 1).

Comparison of clinical manifestations in children with CeD depending on the deficiency and insufficiency of vitamin D demonstrated the severity of bone deformations and metabolic disorders in group with vitamin D deficiency compared to insufficiency: bone pain, dull hair, dry skin, hair loss (Table 2).

When studying anthropometric data depending on vitamin D deficiency and insufficiency, we found that with its deficiency, every third patient with CeD suffered from severe weight deficiency, growth retardation of more than 3 SD was established in 41.7 ± 7.1% of cases. Whereas with insufficiency, these indicators were 8.3 ± 10.8% and 16.7 ± 10.8%, respectively (Table 3).

The results of studies of calcium-phosphorus metabolism and its regulators demonstrated a relationship between the level of vitamin D deficiency and a deficiency of total and ionized calcium, phosphorus in patients with CeD, as well as an increase in alkaline phosphatase and parathyroid hormone in patients with CeD. An increased level of parathyroid hormone (PTH) was observed among patients with CeD, exceeding the values of the control group by 3.2 times (p < 0.001). Thus, the alkaline phosphatase (ALP) indicators were increased by 2.3 times with vitamin D deficiency and by 1.2 times with insufficiency; parathyroid hormone—by 3.2 times with vitamin D deficiency and by 2.6 times with vitamin D insufficiency (p < 0.05, p < 0.001) (Table 4).

A reliable significant difference in the levels of total, ionized calcium and phosphorus was also established in vitamin D deficiency compared to its insufficiency. Analysis of the correlation relationship between vitamin D concentration and biochemical parameters characterizing phosphorus-calcium metabolism and the level of parathyroid hormone showed that in CeD, an inverse correlation was found between vitamin D content and the level of alkaline phosphatase (respectively r = −0.710 and r = −0.623, r = −0.589) and parathyroid hormone (respectively r = −0.610 and r = −0.659, r = −0.623) (Table 5).