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Vitamin D is not technically a vitamin, since it is not an essential dietary factor. It is rather a prohormone produced photochemically in the skin from 7-dehydrocholesterol. Vitamin D and its metabolites may be categorized as either cholecalciferols or ergocalciferols. Cholecalciferol (vitamin D3) is the parent compound of the naturally occurring family and is produced in the skin from 7-dehydrocholesterol on exposure to the ultraviolet B portion of sunlight. Vitamin D2 (ergocalciferol), the parent compound of the other family, is manufactured by irradiation of ergosterol produced by yeasts and its potency is less than one-third of vitamin D3's potency. The steps in the vitamin D endocrine system include the following: 1) the photoconversion of 7-dehydrocholesterol to vitamin D3 in the skin or dietary intake of vitamin D3; 2) metabolism of vitamin D3 by the liver to 25-hydroxyvitamin-D3 [25(OH)D3], the major form of vitamin D circulating in the blood compartment; 3) conversion of 25(OH)D3 by the kidney (functioning as an endocrine gland) to the hormone 1,25-dihydroxyvitamin D3 [1,25(OH)2D3 ]; 4) systemic transport of the dihydroxylated metabolite 1,25(OH)2D3 to distal target organs; and 5) binding of 1,25(OH)2D3 to a nuclear receptor (VDR) at target organs, followed by generation of appropriate biological responses. The activation of vitamin D to its hormonal form is mediated by cytochrome P450 enzymes. Six cytochrome P450 (CYP) isoforms have been shown to hydroxylate vitamin D. Four of these, CYP27A1, CYP2R1, CYP3A4 and CYP2J3, are candidates for the enzyme vitamin D 25-hydroxylase that is involved in the first step of activation. The highly regulated, renal enzyme 25-hydroxyvitamin D-1a-hydro xylase contains the component CYP27B1, which completes the activation pathway to the hormonal form 1,25(OH)2D3. A five-step inactivation pathway from 1,25(OH)2D3 to calcitroic acid is attributed to a single multifunctional CYP, CYP24A1, which is transcriptionally induced in vitamin D target cells by the action of 1,25(OH)2D3. An additional key component in the operation of the vitamin D endocrine system is the plasma vitamin D binding protein (DBP), which carries vitamin D3 and its metabolites to their metabolism and target organs. DBP is a specific, high-affinity transport protein. It is synthesized by the liver and circulates in great excess, with fewer than 5% of the binding sites normally occupied. 1,25(OH)2D3, acts as a ligand for a nuclear transcription factor, vitamin D receptor - VDR, which like all other nuclear receptors, regulates gene transcription and cell function. The widespread presence of VDR, and the key activating (1a-hydroxylase, CYP27B1) and inactivating (24-hydroxylase, CYP24A1) enzymes in most mammalian cells means that the cells in these tissues have the potential to produce biological responses, depending on the availability of appropriate amounts of vitamin D3. Thanks to this widespread presence of elements of vitamin D endocrine system, its biological features are being recognized outside bone tissue, i.e. calcium and phosphate metabolism.

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Determination of 25-hydroxyvitamin D [25(OH)D] represents a unique challenge, considering its lipophilic nature. Considering the widespread prevalence of vitamin D deficiency, which leads to increasing number of requests for 25(OH)D determination, immunoassay measurements adjusted to automated analyzers are being developed. Because of the variability among assays, it is often difficult to monitor vitamin D status and supplementation. The aim of this study was to compare the results of two immunoassays with high performance liquid chromatography with ultraviolet detection (HPLC-UV). Also, the aim was to estimate vitamin D status, since up to date the prevalence of vitamin D deficiency in Serbia was not examined. We have evaluated analytical characteristics of two automated immunoassays for 25(OH)D determination, from Roche (Cobas® e601) and Abbott (Architect). For comparison studies we used HPLC analysis of 25-(OH)-Vitamin D3/D2 from Chromsystems (Waters isocratic system). In order to estimate vitamin D status in general population, we have searched the database of the laboratory information system and analyzed the data from 533 patients whose 25(OH)D was determined together with intact parathyroid hormone (iPTH). For imprecision assessment, four serum pools were prepared with following 25(OH)D concentrations: 35 nmol/L, ~50 nmol/L, ~75 nmol/L and ~125 nmol/L. Obtai ned CVs for Roche method were 1.5-2.8% for within-run and 4.0-6.7% for between-run imprecision. For Abbott method, CVs were 0.7-4.4% for within-run and 3.8-7.2% for between-run imprecision. Inaccuracy was analyzed with commercial control sera. Obtained deviations from target value were 2.1% for Roche assay and 1.3-1.5% for Abbott method, and were not statistically significant (P>0.05). Comparison of Roche and HPLC-UV methods using Passing-Bablok regression analysis gave the following equation for the regression line y=0.937x+9.518 (r=0.739; n=97) and the regression line equation from the comparison of Abbott and HPLC-UV methods was y=0.745x+10.343 (r=0.793; n=97). Mean difference and SD for Bland-Altman plot were -4.5 nmol/L and 21.75 nmo/L, respectively for Roche method and 6.4 nmol/L and 18.8 nmol/L, respectively for Abbott. Statistical analysis (Chi-square test) of frequency distribution among different vitamin D status categories (<25 nmol/L severe deficiency, 25-50 nmol/L deficiency, 50-75 nmol/L insufficiency and >75 nmol/L sufficiency) showed that the frequency distribution obtained with Abbott method was significantly different from the distribution of the HPLC results, in contrast to Roche results frequency distribution which did not differ significantly. Also, statistical analysis of the agreement between the three methods for each vitamin D status category showed that results of both Roche and Abbott methods were significantly higher than HPLC in the two deficiency categories (P=0.005 for Roche, P=0.0407 for Abbott), and in the sufficiency category Abbott method significantly underestimated concentration of 25(OH)D compared to HPLC results (P<0.0001). Median population values of 25(OH)D and iPTH were 41.8 nmol/L and 76.6 ng/L, respectively. ANOVA analyses showed significant (P<0.05) decrease in iPTH and Ca2+ concentrations across the 25(OH)D concentration categories. Stepwise multiple linear regression analysis indicated independent correlation of iPTH with 25(OH)D concentration (β=-0.290, P=0.0008). Also, one-way ANOVA with Student-Newman-Keuls test demonstrated that 25(OH)D concentrations measured in summer and autumn were significantly (P<0.001) higher compared to those determined in winter and spring. Despite acceptable imprecision and inaccuracy of both examined methods, results obtained with them did not correlate well with HPLC-UV (r<0.9), which was used as a reference. However, methods showed satisfactory ability to classify patients into vitamin D status categories, which is important for diagnosis of vitamin D deficiency and therapy follow-up. About two thirds (68.5%) of the examined po pulation had vitamin D deficiency (25(OH)D<50 nmol/L) and only 8% had sufficient 25(OH)D concentration (>75 nmol/L). [ABSTRACT FROM AUTHOR]