Vitamin D status in adults and children in Transcarpathia, Ukrainein 2019

Background: Vitamin D deciency is a global health problem, it is assessed by measuring serum 25-hydroxivitamin D (25(OH) D), nevertheless epidemiological data for many countries remains underreported. Objectives:To study the prevalence of vitamin D deciency throughout the calendar year in a large cohort recruited ina multiethnic Transcarpathian region of Ukraine. Methods: In this retrospective study 25(OH)D serum concentration was measured during all 12 months of the year 2019 by electrochemoluminescent assay on the automatic analyzer Cobas e411 in 1,823 subjects, including both children and adults (1551 females (85.03%) and 273 males (14.97%)). The mean 25(OH) D concentration in adults demonstrates signicantly lower levels compared to children (22.67±8.63 ng/ml vs. 26.00±10.72 ng/ml respectively, p<0.001). Adult women expressed signicantly lower mean annual serum 25 (OH) D concentrations in comparison to men (22.29±8.46 ng/ml vs. 25.75±9.38 ng/ml respectively, p< 0.001). In contrast, children did not show a signicant difference between sexes (girls 24.98±10.38 ng/ml vs. boys 27.01±11.01 ng/ml, p=0.2003). In the winter months, 25(OH)D levels fell below 20 ng/ml in 51,74 % of adult population of Thranscarpathia, and in 12.91%, below 12 ng/ml. The results of this study contradict the previously reported evaluations of the vitamin D levels in Ukraine which were assessed by measuring serum 25(OH) D. Specically, only approximately half of the studied population is vitamin D decient during winter season. This study features the most representative sample size in Ukraine to date.


Introduction
Vitamin D is an important fat-soluble vitamin that acts as a steroid prohormone playing a key role in bone mineralization. It is synthesized in skin under UV light exposure and is ingested in food [1]. To date, the effects of vitamin D have been associated not only with musculoskeletal disorders, but also with such frequently encountered pathologies as cancer, autoimmune, in ammatory, infectious and cardiovascular diseases, as well as diabetes [2]. Levels of vitamin D in the population depend on UV light exposure, consequently geographic location, skin type, clothing culture, testing methods, nutritional habits including supplements consumption, as well as genetic factors [3].
Vitamin D status is generally evaluated by measuring serum 25-hydroxyvitamin D (25(OH)D), the gold standard method for this test is being liquid chromatography-tandem mass spectrometry (LC-MS/MS) [4]. However, despite the vast knowledge amassed on the topic, the concentration of 25 (OH) D that indicates vitamin D de ciency in the human body still remains controversial. The National Academy of Medicine (USA) [5] considers levels of 25(OH)D below 12ng/ml to be a sign of vitamin D de ciency, while The Endocrine Society (www.endocrine.org) [6] contends that the threshold level is at 20 ng/ml. Also, a concentration below 10ng/ml is commonly de ned as "severe de ciency" and indicates the risk of rickets and symptomatic osteomalacia development [7,8].
Indisputably, vitamin D de ciency is a global health problem, which has been reported in many countries, particularly among institutionalized elderly, postmenopausal women, and immigrants. Reportedly, from 2 to 30% of Europeans may have levels of 25(OH)D below 10 ng/ml [3]. Interestingly, there is a sizable regional variation, as population of Nordic countries seem to have higher vitamin D levels, that may be attributed to the traditionally high consumption of sh liver oil [9].
Ukraine is the largest European territory that spreads over various climate zones, from temporal to subtropical. Previous studies have reported that as much as 37,3 % of the Ukrainian population could bevitamin D de cient (<10 ng/ml), which is a very high proportion, speci cally when compared to other European countries [8], [10]. In this study we attempted to validate the previous studies, and to reevaluate the prevalence of vitamin D de ciency using a large and the most representative sample to date collected in a multiethnic Transcarpathian region (Transcarpathia) of Ukraine, bordering four other European countries (Poland, Slovakia, Hungary and Romania).

Methods
This retrospective study included 1,823 randomly selected subjects among those, whose serum concentration of 25(OH)D was recorded in 2019 at the medical laboratory center "Astra Dia"(The recorded data was depersonalized and the following information was included: sex, age, and month of blood collection. The 25(OH)D serum concentration was measured on the automatic analyzer Cobas e411using the "Elecsys Vitamin D total" electrochemoluminescent assay (Roche®, Germany).To the best of our knowledge, as self-declared by individuals studied, no dietary supplements were consumed at the time of blood draw. The data was analyzed taking into account two established 25(OH)D concentration cut-offs indicating de ciency: <12 ng/ml by National Academy of Medicine, USA and <20 ng/ml by Endocrine Society, USA. All the statistical data analysis, including normality of the distribution (Shapiro-Wilk test), the differences between groups (2-sample independent T-test) and ANOVA (Analysis of variance) were performed using STATISTICA® software (StatSoftInc., Tulsa, OK, USA). P values less than 0.05 were regarded as statistically signi cant.
We analyzed crude 25(ОН)D levels every month of the calendar 2019 year among adults and children, and observed signi cant seasonal variability in 25-hydroxyvitamin D expression levels (ANOVA test, p= <0.001). Among the adults the lowest levels were observed in February (19.44 ng/ml), and the highest -in September and July (26.97 ng/ml and 26.83 ng/ml, respectively). Similarly, children and the adolescentsexpressedthe lowest levelsof 25(ОН)D in February (16.60 ng/ml), and the highest in August (32.53 ng/ml). Monthly 25(OH)D concentrations in different age categories are illustrated in Table 1. The concentration of 25(OH)D negatively correlated with age both among adult males (r= -0.16, p<0.05) and females(r=-0.12, p<0.05). Strikingly, even stronger negative correlation of 25(OH)D with age was observed in children: among males (r= -0.42, p<0.05) and females(r=-0.43, p<0.05).
Since many studies calculate and report the prevalence of vitamin D de ciency in winter months [9] we also analyzed and utilized the winter data for comparison. In our sample, 51,74 % of adult population of Thranscarpathia have 25(OH)D levels below 20 ng/ml and 12,91% -below 12 ng/ml in the winter (December through February of 2019). The prevalence of vitamin D de ciency in different sex and age groups in our sample is shown in Table 2.
The prevalence of vitamin D de ciency in different age groups throughout the year is shown in Table 3. Across the age groups, the highest level of vitamin D de ciency is observed in adults over 60 years (58.7% of people with levels below 20 ng/ml, and 9.9% of people with levels below12 ng/ml).Among the children and adolescents, the highest prevalence of vitamin D de ciency is observed in the age range of 8-12 years old (41.86% below 20 ng/ml and 4.65% below 12 ng/ml). Vitamin D de ciency was completely absent in the infants and toddlers (0-3 years age group).

Discussion
To the best of our knowledge, this is the largest study of the vitamin D de ciency in Ukraine to date and the rst study of this condition in the Transcarpathian region. Previously, the only countrywide study of vitamin D levels in Ukraine conducted and published in 2010 by Povoroznyuk et al. [10] reported that 81.8% of the Ukrainian population was de cient with levels of 25(OH)D below 20 ng/ml, while 37.3% had levels below 10 ng/ml. In addition, Povoroznyuk et al. [10] reported that the average level of 25(OH)D in Ukraine was 13.87 ng/ml, and the lowest level of 12.61 ng/ml was in western Ukraine, which geographically includes Transcarpathia as the westernmost region of the country. Unfortunately, the times of the year when the blood samples were drawn have not been reported for this study [10].
Overall, our data signi cantly differs from the results obtained by the other study discussed here; our results suggest a lesser prevalence of vitamin D de ciency than was thought previously. In contrast to the earlier study [10], our results indicate that only 51.74% of the studied population have a de ciency of vitamin D (below 20 ng /ml) in the winter months, and only 12.91% of individuals havelevels below 12 ng/ml in Transcarpathia. Moreover, in our sample the mean 25(OH)D concentrations during the winter months was at 20.84 ng/ml, while the annual average was at 22.67 ng/ml, almost twice the level previously reported [10].
There may be several reasons for the stark discrepancies in resultsbetween the two studies. First, Povoroznyuk et al. [10] reported using the electrochemoluminescent method, polyclonal Vitamin D assay (RocheDiagnostics ® , Germany) on Elecsys 2010 analyzer. There were reports in 2011 that Roche Diagnostics ® withdrew several lots of this particular assay from use referring to a deterioration of conformity to the reference method for the results reported (liquid chromatography -tandem mass spectrometry; LCMSMS), the same studies reported those polyclonal assays tend to lower the actual serum 25(OH)D levels [11]. In contrast, in the current study the samples were tested using the Elecsys assay with a newer generation kit containing a different analyzer (Cobas® e411, RocheDiagnostics ® , Germany). Second, the season of sample collection can signi cantly contribute to the difference in our results. The exact difference between seasons cannot be established, due to the fact that the earlier study did not specify the timing of samples collection [10]. It is unlikely that the climate could account for a large difference, as Transcarpathia has similar average solar activity compared to other regions of Ukraine. The total annual amount of sun exposure in Transcarpathia lowlands is around 2,000 hours, while in mountainous areas it is approximately 1,700 hours, which is very similar to the total sun exposure in Ukraine overall (1,700 to 2,400 hours annually) [12].
Furthermore, there could be a difference in the levels of 25 (OH) D in different studies due to different amount of sun exposure and dietary supplements intake by study participants. Some studies suggest that clothing practices (wearing a veil) or tendency to avoid sun may highly contribute to the vitamin D levels [13]. We did not access sun exposure by any means, but the patients have declared to have not taken any dietary supplements.[с1] Given the close geographic proximity of Transcarpathia to other European countries it seems reasonable to compare our ndings to those from neighboring countries.The similar values reported for these countries give additional credence to our results. For instance, a recent study in Poland that involved 5,775 adults with a mean age of 54.0±15.9 years reportedthe mean level of 25(OH)D at 18.0 ± 9.6 ng/ml [14]. This study also indicates that 65.8% of the population had a level of 25(OH)D below 20 ng/ml, which is closer to our results (51.74%) than to the earlier study of the Ukrainian population overall (81.8%). In addition, while our results showed lower levels in females in all age groups of adults in almost all months of the year, the study conducted in Poland showed lower levels of vitamin D in men compared to women. Although the authors believe the geographic location plays a minor role in vitamin D status in Poland, this may in fact account for the relatively higher prevalence of the de ciency in Poland (14.06% higher compared to Transcarpathia) because the territory of Poland is located more to the north (52.13°N ,21.02° E ) compared to Transcarpathia (48.41°N, 23.29°E). It should be noted that yet a different method of measurement of 25(OH)D was used in this Polish study(Liaison XL system (DiaSorin; CLIA method). The collection of material in this study was conducted from February 14 to March, and from April 28 to May 2, which is considered to be a "low" solar activity season. In the neighboring country Slovakia, a study of healthy women aged 25-40 years showed an average level of 25(OH)D at 32.6 ng/ml [15],which is 10.3 ng/ml above 22.3 ng/ml 25(OH)D reported for women in Transcarpathia during the year (mean age 39.87 ± 13.22 years). Unfortunately, this study did not account for the season. Similarly, the prevalence of severe vitamin D de ciency in Transcarpathia (12.91% below 12 ng/ml in the winter months) is lower than in Germany [16] and Great Britain [17], but higher than that reported in Spain [18], Italy [19], and France, where a study of 2,007 individuals, in the age ranges 30-54 reported only 5,2 %prevalence of vitamin D de ciency below 10 ng/ml [20]. The average level of vitamin 25(OH)D in adult population of Transcarpathia (20.84 ng/ml in the winter months) is closest to the levels reported in Austria (20.88 ng/ml, adults, age range 21-76 years) and Belgium (19.28 ng/ml, adults, age range 21-69) [21,22].

Limitations
While our study had several limitations that prevented the direct comparisons to others (the method of measurement we used in our study is not the gold standard method for this type of test though it shows the correlation with tandem mass-spectrometry of r=0,89 according to the manufacturer), and can be considered a rough population-based study on vitamin D status in the region, it highlights an important geographical trend that should be further explored and could be useful to aid the development of health care strategies in the region. Unfortunately, while we collected the seasonality data, we did not obtain data on the exact place of residence, and the origin of the people who were included in the study. Furthermore, the information on supplements consumption was self-reported. Also, our sample cannot be considered as a completely accurate representation of the general population, as we sampled a highly disproportional number of men and women, unfortunately this is due to the lack of testing of 25 (OH)D amongst males in general, in the region and in the country as a whole, and to a lesser extent, a lower number of males comparatively to females living permanently and temporarily in the country due to socio-economic reasons.
[с2] All these could be useful covariates in the future investigations.

Conclusions
This study showed a noticeable prevalence of de ciency of vitamin D in the Transcarpathian population in Ukraine, documented by a moderate decreasein 25(OH)D levels (<20 ng/ml) in approximately half of the tested individuals in winter months. Only a limited part of the studied population (12.91%) expressed severely decreased 25(OH)D levels (<12 ng/ml). Taking together, our ndings contradict the previous study conducted in Ukraine, which showed much higher prevalence of vitamin D de ciency, but are consistent with the majority of the regional European reports on vitamin D status, thus tting in the hypothetical south to north trend in de ciency across Europe. Adult women have signi cantly lower vitamin D levels than men, and children have higher levels than adults in our cohort (p<0,001). Serum 25(OH)D concentration negatively correlates with age both in children and adults Declarations Ethics approval and consent to participate This study was approved by IRB of SU "Uzhhorod National University" with the waiver of consent as it is a retrospective anonymous study with the minimal possible risk to participants Consent for publication Waiver of consent by IRB decision. Availability of data and material The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests
The author declares that there are no competing interests.

Funding
This research did not receive any speci c grant from funding agencies in the public, commercial or notfor-pro t sectors. The author was paid graduate assistantship during the Ph.D. program.

Authors' contributions
The author solely designed the study, performed the analysis, data interpretation, and preparation of the manuscript.   Figure 1 Monthly distribution of 25(OH)D concentration among adults presented by sex