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The antigenecity and nutritional adequacy of N-253 formula, a partly hydrolyzed formula, were evaluated in high risk infants with a family history of atopy, on immunological and hematological parameters, weight gain, clinical conditions and stool characteristics; during the first four months of life. Its effects were compared with those observed in infants taking a conventional infant formula and breast-fed infants, There were no changes in serum IgE, cow's milk specific IgG, cow's milk-specific IgG4 concentrations and the number of eosinophils in the N-253 formula-fed infants during the study period. Whereas, those parameters were increased in infants fed with the conventional infant formula and in breast-fed infants. Positive RAST against cow's milk was not detected in the N-253 formula-fed infants (0/33), while it was detected in conventional infant formula-fed infants (2/13). Green and watery stools were observed more frequently in the N-253 formula-fed infants than in the conventional infant formula-fed infants. Weight gain and hematological values were within normal ranges in all three groups during the study period. These results suggest that the antigenecity of cow's milk is reduced in N-253 formula and that N-253 formula has nutritional efficacy for infants.

A high-performance liquid chromatographic method for simultaneous assay of 25-hydroxyvitamin D2 (25-OH-D2) and 25-hydroxyvitamin D3 (25-OH-D3) proposed in a previous paper (1) was applied to determine the plasma levels of the metabolites in perinatal and postnatal periods. The plasma samples of maternal, cord and neonatal blood were collected in summer and winter seasons. 25-Hydroxyvitamin D3 was detected in all the samples. The plasma levels of the metabolite in mothers, cords and newborn infants (at life within 24 hr) in summer were 33.9 +/- 12.5, 18.9 +/- 8.4 and 16.6 +/- 6.4 (mean +/- S.D.) ng/ml, respectively, while those in winter were 15.8 +/- 6.6, 8.8 +/- 3.4 and 7.7 +/- 3.2 ng/ml, respectively. The data in summer were significantly higher than the respective data in winter and there were high significant correlations between the mothers and cords and between mothers and newborns. In both seasons, the plasma levels of mothers were about two times higher than the respective data of cords and newborns which were nearly identical with one another. The similar tendency was always observed in the individual data of mothers-cords-newborns pair samples. In this study, many plasma samples from mothers, cords and newborns were examined, but 25-OH-D2 was detected in only few samples (6/41 for mothers, 3/36 for cords and 2/34 for newborns). However, the metabolite began to appear in all the samples during nursing with vitamin D2-fortified dry milk to show 4.6 +/- 1.3 and 4.8 +/- 1.2 ng/ml in the summer and winter samples of neonates at life of 5-6 days, respectively. When the variation of plasma 25-OH-D2 and 25-OH-D3 levels was examined in postnatal periods until one month, the levels of exogenous 25-OH-D2 was increased while those of endogenous 25-OH-D3 was decreased. The sum of vitamin D2 intake from fortified dry milk was highly significantly correlated with the plasma levels of 25-OH-D2, which indicates that daily uptake of exogenous vitamin D2 is very important in nutrition during postnatal periods of bottle-fed infants.

The presence of water-soluble vitamin D and 25-OH-D sulfates in human breast and cow's milk was studied. We first confirmed that synthetic vitamin D2 and D3 sulfates could not be hydrolyzed by alkali but by acid. Breast or cow's milk was separated into milk whey containing water-soluble components and milk curd containing crude proteins and lipophilic components. The separated milk whey and curd were hydrolyzed by acid or alkali and each lipid extract was subjected to HPLC analysis. Neither peak due to vitamin D and 25-OH-D was observed in the chromatograms of acid- and alkali-hydrolyzed milk whey, whereas the peaks due to vitamin D3 and 25-OH-D3 were found in the chromatograms of both acid- and alkali-hydrolyzed milk curd and there was no significant difference between the respective peak heights. The eluates corresponding to the respective peaks observed on the latter's chromatograms were collected and subjected to UV, HPLC, GC-MS and GLC to identify the existence of vitamin D3 and 25-OH-D3, respectively. We concluded from these results that neither breast nor cow's milk contained water-soluble vitamin D and 25-OH-D sulfates, whereas they contained fat-soluble vitamin D3 and 25-OH-D3. The concentrations of vitamin D3 and 25-OH-D3 in breast milk were about 125 and 350 ng/liter, while those in cow's milk were about 420 and 270 ng/liter, respectively. The experiments on the transfer of 3H-D3 and 3H-25-OH-D3 perorally dosed to lactating rats into suckling pups through their milk also supported the above conclusion.

A method for simultaneous determination of 25-hydroxyvitamin D2 (25-OH-D2) and 25-hydroxyvitamin D3 (25-OH-D3) in human plasma has been developed by using two steps of high-performance liquid chromatography (HPLC). Lipids extracted from 0.51 ml of human plasma were first subjected to the preparative HPLC using a Nucleosil 5C18 column (reversed-phase type) and a 25-OH-D fraction containing 25-OH-D2 and 25-OH-D3 was separated. The separated fraction was subsequently subjected to the analytical HPLC using a Zorbax SIL column (straight-phase type). Since the peaks corresponding to 25-OH-D2 and 25-OH-D3 were clearly separated from one another on the chromatogram of the analytical HPLC, the metabolites could be simultaneously determined by estimating the respective peak heights. When the fractions corresponding to the respective peaks were separately collected by repeatedly applying rather large quantities of human plasma and were subjected to gas chromatography-mass spectrometry (GC-MS), they were identified as containing 25-OH-D2 and 25-OH-D3, respectively. The proposed method was applied to plasma samples of human adults taking 400 I.U./day of vitamin D2 for 8 weeks and the values were 22.5 +/- 8.1 ng/ml for 25-OH-D3 and 11.5 +/- 1.8 ng/ml for 25-OH-D2 (mean +/- S.D.), respectively.

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 chromatographic method previously reported (6) and the following results were obtained: 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 +/- 10.1 ng/ml. 25-OH-D3 was detected in all the samples and the M +/- SD was 23.0 +/- 10.1 ng/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. 25-OH-D2 was detected only in 18.3% of the plasma samples and the M +/- SD in the detected samples was 4.4 +/- 2.9 ng/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. The M +/- SD values of 25-OH-D3 plasma levels in men and women were 26.2 +/- 10.4 and 19.3 +/- 8.0 ng/ml, respectively. The formers were significantly higher than the latters. The results were thought to be due to the reason that men might be outdoors for longer periods than women. 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. When 4 healthy volunteers were orally administered 400 I.U./day of vitamin D2 every day for 8 weeks, maximum levels (average: 11.5 ng/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.

In order to obtain a standard compound of 25-hy-droxyvitamin D2 (25-OH-D2), a method for isolating in vivo-generated 25-OH-D2 from the blood of rats or rabbits was established by using several steps of preparative high-performance liquid chromatography (HPLC). When the unsaponifiable matter of the plasma obtained from rats or rabbits receiving a large dose of vitamin D2 was applied to the preparative HPLC using a Zorbax SIL column, a peak denoted as peak X was observed on the chromatogram. Since the peak X was thought to be due to 25-OH-D2 from the experiments of time course and dose-response, it was purified by subjecting it to successive preparative HPLC using several kinds of columns. From the results of ultraviolet (UV) absorption spectrum, gas chromatography-mass spectrometry (GC-MS) and mass chromatography, the purified peak X compound was confirmed to be 25-OH-D2. The proposed method for isolating in vivo-generated 25-OH-D2 is very convenient, because the time to perform each HPLC is very short though several steps of HPLC are used.