Age, gender and season dependent 25(OH)D levels in children and adults living in Istanbul

Introduction

Vitamin D (Vit D) is a fat-soluble steroid hormone that is bound tightly to a special carrier vitamin D-binding protein (DBP) in the circulation. Smaller amounts are bound to albumin and lipoproteins. Only very little amounts are free and biologically active.

25(OH)D plays an important role in calcium and phosphate homeostasis. People with Vit D deficiency show mineralization defects in forms of osteoporosis, osteomalacia, rickets and fractures. In recent studies, its deficiency is associated with autoimmune, infectious, cardiovascular diseases, several types of cancer and depression [1], [2]. Based on several research articles and society recommendations [3], [4], we used the following groups: >75 nmol/L sufficient, 50–75 nmol/L moderate deficiency, 25–50 nmol/L deficiency, <25 nmol/L severe deficiency.

According to the Institute of Medicine; Vit D deficiency occurs when the serum 25(OH)D concentration is below 30 nmol/L individuals with serum 25(OH)D levels between 30 and 50 nmol/L are considered at risk of developing bone diseases [5], [6], [7], [8], [9], [10].

25(OH)D deficiency has emerged as a common worldwide problem. Besides age, gender and ethnicity; availability of direct sunlight as a result of latitude, duration and area of exposure, season, time of day and skin pigmentation are the variables associated with sufficient synthesis. Low 25(OH)D levels are mostly seen with older age, pregnancy and non-Western ethnicity. Levels change depending on the season and peak during summer [11], [12]. The most critical factor affecting seasonal changes in 25(OH)D levels is the latitudinal position of a country. For useful synthesis the angle at which the sun’s incident light hits the surface of the earth is of primary importance. Turkey is located at 39°57′N latitude and it is considered to have abundant sunlight. However Turkey is one of the countries where 25(OH)D deficiency is endemic [13]. Covering clothing style, and a diet low in fish and dairy products may be responsible for deficient levels. In cases where synthesis via sunlight is insufficient, specific nutrition and supplementation becomes more important.

The aim of this study was to define current 25(OH)D status of Turkish people living in Anatolian region of Istanbul both for children and adults, assessing 25(OH)D levels concerning age, sex and seasons and may be to guide national healthcare actions.

Materials and methods

Laboratory 25(OH)D records of 103,509 adults (78,909 females and 24,600 males) aged between 18 and 80 years and 19,186 children (10,362 girls and 8824 boys) aged between 0 and 17.9 years attending Dr. Lütfi Kırdar Kartal Education and Training Hospital outpatient clinics for any reason, between 31.07.16 and 31.07.17 were retrospectively evaluated. 25(OH)D values >413 nmol/L and recurrent admissions were excluded.

Median (2.5–97.5 percentiles) values for ages were 47.6 (20.3–76.6) years for adults and 7.8 (0.51–17.6) years for the children. Both adults and children were divided into subgroups regarding age. Groups were; <40 years, 40–50 years, 50–60 years and >60 years for the adults [8] and 0–1 month, 1–12 months,1–3 years, 4–6 years, 7–9 years, 10–12 years, 13–15 years and 16–18 years for the children [5]. Effects of gender and age on 25(OH)D levels were evaluated for each group. Both adults and children data were also evaluated regarding seasons. Patients were defined according to 25(OH)D levels as; (sufficient) >75 nmol/L, (moderate deficiency) 50–75 nmol/L, (deficiency) 25–50 nmol/L, (severe deficiency) <25 nmol/L [3], [4] patient rates were calculated for each interval. Data did not include any information about Vit D supplementation, sunlight exposure or diet.

Measurements were done by Architect i1000 SR (Abbott Diagnostics, Lake Forest, IL, USA) analyzer using competitive chemiluminescent microparticle immunoassay (CMIA) without any method change throughout the study. Abbott 25(OH)D reagent uses microparticles coated with a polyclonal sheep anti-vitamin D IgG antibody, and a biotinylated vitamin D anti-biotin IgG acridinium-labelled conjugate complex for the quantitative determination of 25(OH)D2 and D3 in human serum and plasma. The limit of quantitation of this method is 6 nmol/L, and it is linear up to 413 nmol/L. The total coefficient of variations provided by the manufacturer was 3% for 50 nmol/L and 2.4% for 188 nmol/L. Samples provided by Randox International Quality Assessment Scheme external quality assessment monthly immunoassay program (code RQ128524/B) were used for evaluating accuracy. The Architect assay was standardized against NIST SRM 2972 (National Institute of Standards and Technology Standard Reference Material 2972).

Results

One hundred three thousand five hundred and nine adults (female 76.2%, male 23.8%) and 19,186 children (girls 54%, boys 46%) participated in the study. Characteristics of the study population and median serum 25(OH)D levels were shown in Table 1. 25(OH)D levels of adult females and males were found as 37.5 (9–162.5) and 41.25 (12.75–139.8) nmol/L, and children girls and boys were found as 39 (10–124) and 48 (14.5–127.5) nmol/L, respectively (p’s<0.001). In adults group, males had higher levels than females till 50 years (p’s<0.001), females had higher levels than males after 50 years (p’s<0.001). Age subgroups had significantly different serum 25(OH)D levels from each other and levels increased with increasing age for both females and males (p’s<0.0001) Table 2. Comparison of serum 25(OH)D levels according to gender and ages for adults were shown in Figure 1. Approximately 70% of the females and males younger than 40 years had 25(OH)D levels below 50 nmol/L, the highest rate of deficiency and severe deficiency. In contrast the lowest rate (52%) was observed in females over 60 years of age Table 3. There were significant differences among seasonal values of 25(OH)D; highest in summer, followed by autumn, spring and winter (p’s<0.001) Table 4.

Figure 1:

Comparison of serum 25(OH)D levels according to gender and ages for adults.

Children seems to be born with insufficiency. After 1 month, levels increased, having the highest 25(OH)D concentrations between 1 and 12 months interval; 79.2 (16.2–174.5) nmol/L for girls and 78.8 (12.5–197.3) nmol/L for boys. At the end of first year levels began to decrease and insufficiency begins after the age of 4. Age subgroups had significantly different levels from each other (p<0.001). In children group gender difference was observed after 4 years of age (p’s<0.0001) Table 2. Figure 2 represented comparison of serum 25(OH)D levels according to gender and age groups for children. Children, 65% of the girls and 53% of the boys had 25(OH)D levels below 50 nmol/L. Especially 1–12 months children having the highest 25(OH)D levels in the group, only showed a 17.6% rate of deficiency Table 3.

Figure 2:

Comparison of serum 25(OH)D levels according to gender and ages for children.

Seasonal variations among children were similar to adults; highest in summer, lowest in winter (p’s<0.0001) except for 1–12 months’ girls (p=0.855) Table 5. Figures 3 and 4 represented comparison of serum 25(OH)D levels according to seasons for adults and children, respectively.

Figure 3:

Comparison of serum 25(OH)D levels according to season for adults.

Figure 4:

Comparison of serum 25(OH)D levels according to season for children.

Discussion

Inadequate Vit D levels is primarily associated with disorders of calcium and phosphate metabolism affecting skeletal health. Recently there have been many studies relating this compound with important life functions such as cell proliferation and differentiation, immune system, reproduction, cardiovascular system and neurological system. The subject has drawn more and more attention recently; many screening studies defined Vit D status of populations almost all over the world and this enabled application of disease control and prevention protocols by healthcare professionals and providers.

In North America the largest study was NHANES (National Health and Nutrition Examination Survey). This study included both children and adults and measured both 25(OH)D3 and 25(OH)D2. The prevalence of both deficiency and insufficiency was found higher in 20–39 years of age group than >60 years group. This result was attributed to widespread prescription of Vit D supplements in the elderly group [14].

In Europe, ODIN study (Food-based solutions for optimal Vit D nutrition and health through the life cycle) was carried after standardization with a NIST standard and LC-MS/MS technology. Data from countries such as United Kingdom, Ireland, Germany, Greece, Norway was presented. In spite of their lower latitude and less sunshine Vit D levels of Nordic people were better than Mediterranean countries. This result was attributed to highly consuming cod and cod liver products in Norway and Sweden. Besides Nordic people had a light skin and possible sunbathing [15]. In French SUVİMAX (Supplementation en Vitamine et Mineraux Antioxydants) study [16] Vit D concentrations were lower in Northern Region (43 nmol/L) than in Southwest Regions (94 nmol/L) of France as expected. In Italy and Greece also Vit D concentrations were found quite low despite their abundant sunshine. This was attributed to a more pigmented skin and long duration of home rests in too hot climate conditions [17].

In Middle-East countries such as Jordan and Saudii Arabia with plenty of sunshine, low levels of Vit D was attributed to clothing style of women [18], [19].

In Asian countries, Vit D status in Russian people was insufficient in common [20] as well as Mongolia [17] where Rickets is very common. Indian people, though localized between 13 and 27° latitudes, had very low levels also, probably because of pigmented skin, skin covering clothes and sun avoiding creams [21], [22].

In “Turkish Diabetes, Hypertension, Obesity and Endocrinological Diseases Prevalence Study II” 9560 adults living in rural and urban areas were examined and 93% had serum 25(OH)D levels less than 75 nmol/L [23] Turkish Ministry of Health initiated ‘Vit D prophylaxis augmentation programme’ in 2005, which included free distribution of Vit D drops to all newborns and infants [13]. Özkan et al conducted a study on children in 2009 and found the prevalence of (25(OH)D) deficient rickets as <1%. Prevalence in children was significantly reduced from 6% to a level of 1% within 3 years since the beginning of the campaign [24].

In a Turkish study held by Öğüş et al. with 4168 patients, 50% of women and 38% of men had values below 20 ng/mL (50 nmol/L); women had lower concentrations than men and seasonal changes were significant [25]. In another study with 440 subjects [26] although Vit D values were higher in summer than in winter it was insufficient all through the year. Alagöl and colleagues studied 48 premenopausal women with three different clothing styles, women with whole body covering clothing style had the lowest values of Vit D because of no exposure to sunshine [27].

In this retrospective study, we evaluated 25(OH)D levels of 103,509 adults and 19,186 children who attended our hospital’s outpatient clinics in a year. Our study includes the most substantial dataset assessing 25(OH)D levels concerning age, sex and seasons in the literature. Age interval is from newborn to 80 years of age. Study was carried out in Istanbul (latitude: 41°–29°), a city known for the abundance of its sunny days, especially in August, and the climate can be described as the transitional Mediterranean, with relatively cold winters, and hot sunny summers [27]. Approximately 4000 patients a day attend to our laboratory and 13,000 samples are analyzed per day. So the study population is the habitants of a large area in the east of Istanbul.

Results of our study were very similar with those of literature. Age subgroups had significantly different serum 25(OH)D levels from each other. Mainly children had the highest Vit D concentrations between 1 and 12 months interval probably because of using Vit D drops as the Turkish Ministry of Health policy. After 4 years of old levels began to decrease, lowest in puberty, adolescence and young adults. Adolescence is a period of rapid growth which necessitates the requirement of Vit D for skeletal growth and development which caused lower 25(OH)D levels for both genders [5]. We did not observe sufficient 25(OH)D levels in any other age, either in adults or children, even in summer periods. Gender difference was observed after 4 years of age; females having lower levels of Vit D than males till 50 years. An association which could be explained by the wearing of skin-covering conservative clothing, spending less time outdoors, less physical activity and obesity for females [28]. After 50 years females had higher 25(OH)D levels. This may be due to the use of calcium and Vit D for osteoporosis in this age groups. Levels increased with increasing age for both females and males. Seasonal variations were similar both in children and adults; highest in summer, lowest in winter, except for 1–12 months. In the general study population; 63.1% of females, 62.9% of males, 65% of girls and 63% of boys had 25(OH)D levels below 50 nmol/L. In the summer period, deficiency rates dropped from 53.1% to 40.5% for females and from 70.2% to 26.2% for males compared with the winter period. However, Vit D treatment is not without risks, as Vit D toxicity has potentially serious effects. Vit D toxicity occurs from exposure to extremely high doses of Vit D supplementation, which can be the result of manufacturing errors, accidental or intentional incorrect dosing, incorrect interpretation of Vit D status by the clinician or self supplement administration [29].

There were potential limitations in majority of studies including ours: Many of them were retrospective or cross-sectional. In retrospective studies exclusion and inclusion criteria were not well defined. Additionally, the relation between serum 25(OH)D levels and alkaline phosphatase, PTH, calcium, and phosphorus were not evaluated probably these tests are not ordered together with vit D at first visit. Also studies were held in different seasons of the year.

Total Vit D concentration is affected from the diseases or conditions that affect the synthesis of DBP or albumin. Over-synthesis of DBP is physiological because of high sex steroids, particularly estrogens in pregnancy. Acute and chronic kidney diseases may also cause variations of both free and complex vit D concentrations [30].

People of African descent have DBP isoforms with a lower binding affinity to Vit D as compared to Caucasians. Thus, total Vit D serum levels for a given concentration of free Vit D in African people is always lower as compared to Caucasians [31], [32].

Though many of them were standardized with a NIST standard, many different techniques with different CV values were used in the assays. Many of the assays did not distinguish between the DBP-bound Vit D, albumin-bound Vit D and free Vit D like ours. There are different recommendations about the optimal serum 25(OH)D cut off levels as 50 nmol/L according to the Institute of Medicine or 75 nmol/L according to the Endocrine Society [33].

In spite of their heterogeneity, almost all studies point out at least insufficiency of Vit D in almost all populations and age groups. In each study results are attributed to any of the factors, whichever suitable. Are all these people all over the world, at any season, at every age and gender insufficient/deficient? May be present cut off’s should be reevaluated, at least for different genders, age groups and seasons. Clinical presentation of insufficient Vit D values must be followed by longitudinal studies before rearrangement of present cut-offs. In several studies, most of the subjects defined as hypovitaminosis remained asymptomatic during years, and these authors expressed uncertainty about the treatment of asymptomatic patients [34], [35], [36], [37]. Thus some authors questioned it whether a significant increase in serum tests lead to a potential increase in unnecessary healthcare costs [38], [39].

Conclusion

The prevalence of deficiency was very high in our population for all ages. Although Turkey was a Mediterranean country and had adequate sunlight this wasn’t enough to maintain optimal 25(OH)D levels in any season. We can conclude that for better clinical and economic outcomes, measurement of Vit D should be indicated in cases with clinical signs of deficiency; in case of uncertainty other metabolic parameters such as PTH, calcium and phosphorus should be considered to support the diagnosis.