J. Nutr. Sci. Vitaminol., 29, 271-281, 1983 Variation of 25-Hydroxyvitamin D3 and 25-Hydroxy vitamin D2 Levels in Human Plasma Obtained from 758 Japanese Healthy Subjects Tadashi KOBAYASHI,1 Toshio OKAN0,1 Sumiko SHIDA,1 Kaoru OKADA,1 Toshie SUGINOHARA,1 Hideto NAKAO,2 Eizo KURODA,2 Soichi KODAMA,2 and Tamotsu MATSUO2 1Department of Hygienic Sciences, Kobe Women's College of Pharmacy, Higashinada-ku, Kobe 658, Japan 2Department of Pediatrics, Kobe University School of Medicine, Chuo-ku, Kobe 650, Japan (Received January 6, 1983) Summary Plasma levels of 25-hydroxyvitamin D3 (25-OH-D3) and 25 - hydroxyvitamin D2 (25-OH-D2) in 758 Japanese healthy subjects (most of them adults) were determined by a high-performance liquid chromato graphic method previously reported (6) and the following results were obtained: 1) The mean and standard deviation (M }SD) of the assayed values of 25-OH-D (sum of 25-OH-D3 and 25-OH-D2) was 23.8 }10.1ng/ml. 2) 25-OH-D3 was detected in all the samples and the M }SD was 23.0 }10.1ng/ml. The plasma levels clearly showed the seasonal variation that the levels in summer were significantly higher than those in winter. Moreover, the plasma levels were significantly correlated with the amounts of UV light in solar radiation. These results strongly suggested that 25-OH-D3 in plasma mainly originated from endogenous vitamin D3 formed by photo-conversion of 7-dehydrocholesterol in skin. 3) 25-OH-D2 was detected only in 18.3% of the plasma samples and the M }SD in the detected samples was 4.4 }2.9ng/ml which was much lower than those of 25-OH-D3. The results suggested that few healthy Japanese are taking daily exogenous vitamin D2 from multivitamin preparations or others. 4) The M }SD values of 25-OH-D3 plasma levels in men and women were 26.2 }10.4 and 19.3 }8.0ng/ml, respectively. The formers were significantly higher than the latters. The results were thought to be due to Abbreviations: 25-OH-D3/D2, 25-hydroxyvitamin D3/D2 (25-hydroxy-cholecalciferol/ -ergocalciferol); 1ƒ, 25-(OH)2-D, 1ƒ, 25-dihydroxyvitamin D; HPLC, high-performance liq uid chromatography; UV, ultraviolet. 271
272 T. KOBAYASHI et al. the reason that men might be outdoors for longer periods than women. 5) When age variation of plasma 25-OH-D3 levels was examined, the levels in the twenties were significantly lower than the other generations. This was confirmed to be due to the low values observed in the female twenties group, but the detailed reason is unclear at the present time. b) When 4 healthy volunteers were orally administered 400I.U./day of vitamin D2 every day for 8 weeks, maximum levels (average: 11.5ng/ml) were observed at the 8 weeks and the levels gradually decreased after stopping the administration. The results suggested that the half life of 25 OH-D2 in plasma might be 4-5 weeks. Key Words 25-hydroxyvitamin D3, 25-hydroxyvitamin D2, 25-hydrox ycholecalciferol, 25-hydroxyergocalciferol, high-performance liquid chro matography, plasma level, seasonal variation, vitamin D3, vitamin D2 It has been documented that the major circulating metabolite of vitamin D in plasma is 25-OH-D of which concentration directly reflects the repletion status of vitamin D (1, 2). There are two forms of vitamin D with different side chain structures, vitamin D3 and D2, which are similarly metabolized into 1ƒ, 25-(OH)2-D via 25-OH-D to show the same biological activity for mammals including hu mans (2). Although some quantities of vitamin D3 are naturally found in foods (e.g., fishes, egg yolk, cow's milk and others), the vitamin is mainly derived from the photo-biogenesis of 7-dehydrocholesterol in skin (3-5). On the other hand, since the synthesis of vitamin D2 is easily performed by the photochemical reaction of ergosterol and the oral administration of vitamin D2 is convenient, the vitamin has been predominantly used in commercial drugs (e.g., multivitamin preparations) and enriched foods (e.g., fortified dried milk) in Japan. Therefore, when humans exposed to sunlight receive such drugs or enriched foods, a mixture of 25-OH-D3 and 25-OH-D2 circulates in their plasma. In a previous paper (6), we established a simple and precise method for simultaneous determination of the two metabolites in human plasma. The method makes it possible to evaluate the repletion status of vitamin D, because the plasma levels of 25-OH-D3 mainly derived from endogenous vitamin. D3 formed in skin reflect the status of sunlight exposure while the levels of 25-OH-D2 derived from exogenous vitamin D2 reflect the status of supplemen tation. We previously investigated the levels of the metabolites in the plasma of mothers, cords and neonates, and very reasonable results were obtained (7). In this paper, the proposed method applied to determine the levels of 25-OH-D3 and 25 - OH-D2 in human plasma obtained from 758 healthy subjects and their seasonal, sex and age variations are described. EXPERIMENTAL 1. Materials and reagents. The materials and reagents described in the previous paper (6) were used. J. Nutr. Sci. Vitaminol.
PLASMA LEVELS OF 25-OH-D3 AND 25-OH-D2 273 2. Plasma samples. Samples of human plasma were collected from 758 Japanese healthy subjects. Seventeen samples were collected from healthy volunteers of laboratory workers, while the other 741 samples of plasma used had been collected for testing hepatic functions and judged as healthy at the Hanshin Army Hospital (Jieitai Hanshin Hospital). Among them, 411 were from men and 347 were from women while their age was distributed between 6 and 73. The plasma samples collected from December in 1979 to November in 1980 were investigated. 3. Determination of 25-OH-D3 and 25-OH-D2 in plasma. Exactly 0.5ml of plasma was placed in a test tube with a stopper, and then 25-OH-D3 and 25-OH-D2 were simultaneously determined according to the procedure described in the previous paper (6). 4. Determination of amounts of ultraviolet light in solar radiation. Amounts of UV light in solar radiation were estimated by an integrating photometer of PH-11 type from Suga Analytical Instrumental Co. (Japan). The phototube was set to the direction of south with the wave angle of 45 against the horizon on the roof of Kobe Women's College of Pharmacy. The amounts of UV light between 300 and 330nm in solar radiation were automatically recorded from 11 to 14 o'clock and the average per hour was calculated. RESULTS AND DISCUSSION 1. Assayed values of 25-OH-D3 and 25-OH-D2 in human plasma The assayed values of 25-OH-D3 and 25-OH-D2 in human plasma obtained from 758 Japanese healthy subjects are shown by month in Fig. 1. The closed and opened circles represent values of 25-OH-D3 and 25-OH-D2, respectively. 25 Fig. 1. Assayed values of 25-OH-D3 and 25-OH-D2 in human plasma obtained from 758 healthy Japanese subjects. Vol. 29, No. 3, 1983
274 T. KOBAYASHI et al. Table 1. Assayed values of 25-OH-D levels in human plasma of healthy subjects. a 25 -OH-D means the sum of 25-OH-D3 and 25-OH-D2 levels. b The values in the parentheses represent the percentages of subjects in which the metabolite existed as compared to the total. c The value is shown as M }SD in the subjects which showed the existence of 25-OH-D2 in their plasma. Fig. 2. Histograms of 25-OH-D (25-OH-D3+25-OH-D2) and 25-OH-D3 plasma levels. Hydroxyvitamin D3 was detected in all the samples and the values in summer were generally higher than those in winter. On the other hand, 25-OH-D2 was detected only in 18.3% of the samples (men, 13.1%; women, 24.2%) and the assayed values were generally much lower than those of 25-OH-D3. The results strongly suggested that 25-OH-D3 in plasma is mainly derived from the photo-biogenesis of vitamin D3 in skin while few healthy Japanese are taking daily exogenous vitamin D2 from multivitamin preparations or others. Table 1 shows the mean values and standard deviations (M }SD) and ranges of 25-OH-D (25-OH-D3+25-OH-D2), 25-OH-D3 and 25-OH-D2, respectively. Since both the histograms of 25-OH-D (25-OH-D3+25-OH-D2) and 25-OH-D3 plasma levels show nearly normal distribution curves as shown in Fig. 2 but the values of standard deviations (SD) were rather large, M+1.65 ~SD (90.1%) instead of M }2 ~SD (95.4%) was adopted as the normal range. According to the direction, the normal ranges of 25-OH-D and 25-OH-D3 in healthy Japanese plasma were calculated as 7.1-40.5 and 6.3-39.7ng/ml, respectively. J. Nutr. Sci. Vitaminol.
PLASMA LEVELS OF 25-OH-D3 AND 25-OH-D2 275 Table 2. Comparison of serum or plasma 25-OH-D3 levels reported in the literature. 1) The value of 25-OH-D3 shown as M }SD except for the data of Eisman et al. (14) shown as M }SE.2) HPLC and CPBA stand for high-performance liquid chroma tography and competitive protein binding method, respectively.3) Winter, spring, summer and autumn in our data represent Dec.-Feb., Mar.-May, June-Aug. and Sept.-Nov., respectively. Table 2 shows the comparison of our data in this study with the plasma or serum levels reported in the literature. Our data of 25-OH-D3 levels per year (M }SD: 23.0 }10.8ng/ml) were higher than the respective data of UK (10) (16.4 }6.2ng/ml) and Finland (12) (10.2 }6.1ng/ml) as shown in Table 2. This might be due to the difference of amounts of UV light in solar radiation among the areas. On the other hand, when our data for season were compared with the respective data, the values were rather similar to each other except for data of USA (15) (27.6 }9.2ng/ml) in winter which gave somewhat higher values. Since there are methodological and dietary factors giving variations on data, evaluation of the results was very difficult. However, we thought that the people living in the north area of the world are making efforts to receive solar radiation and to get vitamin D3 from supplemented foods or drugs although they have little sunshine (Although vitamin D2 is predominantly supplied to drugs or enriched foods in Japan, this is not the case in most of the other countries and vitamin D3 is predominant in them). Therefore, when the plasma levels were compared to each other at a short term, there might be little difference among them. Vol. 29, No. 3, 1983
276 T. KOBAYASHI et al. 2. Seasonal variation of plasma 25-OH-D2 levels Since seasonal variation on the plasma 25-OH-D3 levels was observed as shown in Fig. 1, monthly variation of plasma 25-OH-D3 levels and amounts of UV light in solar radiation were examined. The results are shown in Fig. 3. The highest and lowest values were observed in August and February, respectively, which showed good agreement with the amounts of UV light effective for conversion of pro vitamin D to vitamin D (300-330nm). The plasma 25-OH-D3 levels were signi ficantly correlated with the amounts of UV light in solar radiation (r=0.48, p<0.05). Table 3 shows the comparison of plasma 25-OH-D3 levels among four seasons. The value in winter was significantly lower than the other three seasons while the values in summer and autumn were significantly higher than those in Fig. 3. Monthly variation of plasma 25-OH-D3 levels and amounts of UV light in solar radiation. Table 3. Comparison of plasma 25-OH-D3 levels among seasons. The values of season (B) are compared with those of season (A). J. Nutr. Sci. Vitaminol.
PLASMA LEVELS OF 25-OH-D3 AND 25-OH-D2 277 spring and winter. The value in autumn was nearly the same as that in summer. This was due to the high value in September which has strong sunshine as mid-summer in Japan. All of the results mentioned above strongly suggested that 25-OH-D3 in plasma mainly originated from endogenous vitamin D3 formed by the photo conversion of 7-dehydrocholesterol in skin while exogenous vitamin D3 mainly existing in animal origin foods, i.e., cow's milk, egg yolk and fishes, might give little effect on the plasma levels of 25-OH-D3. The plasma levels of 25-OH-D2 giving much lower values than 25-OH-D3 were not correlated with the amounts of UV light in solar radiation. The results were reasonable, because the metabolite was not derived from photosynthesized vitamin D3 in skin but from exogenous vitamin D2. 3. Variation of plasma 25-OH-D3 levels in men and women Our data on the plasma 25-OH-D3 levels were divided into men (411 subjects) and women (347 subjects). The M }SD values were 26.2 }10.4 and 19.3 }8.0 ng/ml, respectively. The former was significantly higher than the latter (p<0.01). Figure 4 shows the seasonal and monthly variation of plasma 25-OH-D3 levels in men and women. The values of men were always higher than the respective values of women and the former were significantly higher than the latter (p<0.01) except for the values of January and February. The results were thought to be due to the Fig. 4. Seasonal and monthly variation of plasma 25-OH-D3 levels in men and women. Vol. 29, No. 3, 1983
278 T. KOBAYASHI et al. reason that men might be outdoors for longer periods than women. 4. Age variation of plasma 25-OH-D3 levels Our data on the plasma 25-OH-D3 levels were divided into ages of the subjects. Table 4 shows the M }SD values in each generation. The value in the twenties (men+women) was significantly lower than those in the thirties, fourties and older than fifty (p<0.01). As shown in Table 4, the phenomenon was mainly derived from the results that the women's values in the twenties were very low (14.1 }5.4ng/ml) although the men's values in the same generation were high (26.2 }8.6ng/ml) and there was no significant difference among the men's data. Since the number of the subjects was sufficiently large (n=207), the results observed in the female twenties group might be normal in Japan. The detailed reason in unclear at the present time, Table 4. Comparison of plasma 25-OH-D3 levels among age groups. The value of a was significantly lower than those of b, c and d (p<0.01). Fig. 5. Seasonal and age variation of plasma 25-OH-D3 levels. J. Nutr. Sci. Vitaminol.
PLASMA LEVELS OF 25-OH-D3 AND 25-OH-D2 279 but one could speculate that women in that generation were making efforts to protect themselves from strong sun burning in Japan. Figure 5 shows the seasonal variation on each generation. In all the generation, the values in summer and autumn were significantly higher than the respective data in winter (p<0.01). 5. Variation of plasma 25-OH-D2 and 25-OH-D3 levels in the subjects after daily intake of exogenous vitamin D2 Four healthy volunteers of laboratory workers were orally administered 400 I. U./day of vitamin D2 from multivitamin tablets every day for 8 weeks and the variation of plasma 25-OH-D2 and 25-OH-D3 levels was examined. The adminis tration was stopped after 8 weeks, but the investigations on assaying the plasma Fig. 6. Variation of plasma 25-OH-D2 and 25-OH-D3 levels in the subjects after daily intake of exogenous vitamin D2. Vol. 29, No. 3, 1983
280 T. KOBAYASHI et al. levels were continued for the following 23 weeks. The investigations was started at July in 1979 and ended at February in 1980. The results are shown in Fig. 6. The plasma levels of 25-OH-D3 (shown as opened columns) gradually decreased, but the phenomenon might be due to seasonal variation because the investigation was carried out from summer to winter. After starting a daily intake of vitamin D2, the levels (shown as closed columns) gradually increased and reached a maximum after 8 weeks of intake. The plasma levels of 25-OH-D2 of 4 subjects at the maximum were 13.4, 10.6, 12.5 and 9.6ng/ml (average, 11.5ng/ml), respectively. The values were 28.5%, 35.9%, 35.5% and 39.0% (average, 34.7%) for those of 25-OH-D (25 OH-D2+25-OH-D3), respectively. After stopping the administration, the levels gradually decreased and two of them disappeared after 23 or 11 weeks. It was deduced from the results that the half life of 25-OH-D2 in plasma might be 4-5 weeks. samples. The authors wish to thank the Hanshin Army Hospital for donating so many plasma REFERENCES 1) DeLuca, H. F. (1976): Recent advances in our understanding of the vitamin D endocrine system. J. Lab. Clin. Med., 87, 7-26. 2) Holick, M. F., and DeLuca, H. F. (1978): Metabolism of vitamin D, in Vitamin D, ed. by Lawson, D. E. M., Academic Press, New York, pp. 51-91. 3) Okano, T., Mizuno, K., and Kobayashi, T. (1978): Identification and determination of 25-hydroxyvitamin D3 in the blood and liver of vitamin D-deficient rats irradiated with ultraviolet light. J. Nutr. Sci. Vitaminol., 24, 511-518. 4) Okano, T., Mizuno, K., Matsuyama, N., Nobuhara, N., and Kobayashi, T. (1979): Gas-liquid chromatography/mass spectrometric identification of previtamin D3 and vitamin D3 in the skin of vitamin D-deficient rats irradiated with ultraviolet light. Reel. Trav. Chim. Pays-Bas, 98, 253-257. 5) Takada, K., Okano, T., Tamura, Y., Matsui, S., and Kobayashi, T. (1979): A rapid and precise method for the determination of vitamin D3 in rat skin by high-performance liquid chromatography. J. Nutr. Sci. Vitaminol., 25, 385-395. 6) Okano, T., Mizuno, N., Shida, S., Takahashi, N., Kobayashi, T., Kuroda, E., Kodama, S., and Matsuo, T. (1981): A method for simultaneous determination of 25-hy droxyvitamin D2 and 25-hydroxyvitamin D3 in human plasma by using two steps of high-performance liquid chromatography. J. Nutr. Sci. Vitaminol., 27, 43-54. 7) Kuroda, E., Okano, T., Mizuno, N., Kobayashi, T., Kodama, S., Ninomiya, M., Morishita, Y., and Matsuo, T. (1981): Plasma levels of 25-hydroxyvitamin D2 and 25 - hydroxyvitamin D3 in maternal, cord and neonatal blood. J. Nutr. Sci. Vitaminol., 27, 55-65. 8) Jones, G. (1978): Assay of vitamin D2 and D3, and 25-hydroxyvitamin D2 and D3 in human plasma by high-performance liquid chromatography. Clin. Chem., 24, 287-298. 9) Delvin, E. E., Glorieux, F. H., Dussault, M., Bourbonnais, R., and Watters, G. (1979): Simultaneous measurement of serum 25-hydroxycholecalciferol and 25-hydroxyergo calciferol. Med. Biol., 57, 165-170. 10) Poskitt, E. M. E., Cole, T. J., and Lawson, D. E. M. (1979): Diet, sunlight, and 25 J. Nutr. Sci. Vitaminol.
PLASMA LEVELS OF 25-OH-D3 AND 25-OH-D 2 281 hydroxy vitamin D in healthy children and adults. Br. Med. J., 1979, 221-223. 11) Savolainen, K., Maenpaa, P. H., Alhava, E. M., and Kettunen, K. (1980): A seasonal difference in serum 25-hydroxyvitamin D3 in a Finnish population. Med. Biol., 58, 49-52. 12) Dabek, J. T. (1980): Plasma profile of hydroxylated vitamin D metabolites: Methods and results in normals for spring-winter in southern Finland. Ann. Clin. Res., 12, 17-24. 13) Lambert, P. W., Syverson, B. J., Arnaud, C. D., and Spelsberg, T. C. (1977): Isolation and quantitation of endogenous vitamin D and its physiologically important meta bolites in human plasma by high pressure liquid chromatography. J. Seroid Bio chem., 8, 929-937. 14) Eisman, J. A., Shepard, R. M., and DeLuca, H. F. (1977): Determination of 25 - hydroxyvitamin D2 and 25-hydroxyvitamin D3 in human plasma using high-pressure liquid chromatography. Anal. Biochem., 80, 298-305. 15) Shepard, R. M., Horst, R. L., Hamstra, A. J., and DeLuca, H. F. (1979): Determination of vitamin D and its metabolites in plasma from normal and anephric man. Biochem. J., 182, 55-69. 16) Stryd, R. P., Gilbertson, T. J., and Brunden, M. N. (1979): A seasonal variation study of 25-hydroxyvitamin D3 serum levels in normal humans. J. Clin. Endocrinol. Metab., 48, 771-775. 17) Kano, K., Yoshida, H., Yata, J., and Suda, T. (1980): Age and seasonal variations in the serum levels of 25-hydroxyvitamin D and 24, 25-dihydroxyvitamin D in normal humans. Endocrinol. Jpn., 27, 215-221. Vol. 29, No. 3, 1983