1,25-Dihydroxy-24-OXO-16ene-vitamin D3, a renal metabolite of the vitamin D analog 1,25-dihydroxy-16ene-vitamin D3, exerts immunosuppressive activity equal to its parent without causing hypercalcemia in vivo.

The hormone, 1,25-(OH)2D3, is metabolized into 1,25-(OH)2-24-OXO-D3, in kidney prior to conversion to its final inactive product, calcitroic acid. Similarly, 1,25-(OH)2-24OXO-16eneD3, is produced in the kidney from the Vitamin D analog, 1,25-(OH)2-16eneD3, but resists further hydroxylation. The analog's metabolite was synthesized and its biologic activity compared to the parent compound. Naive SJL/J mice, 4 weeks old, were immunized with neuroantigen in adjuvant to induce experimental autoimmune encephalomyelitis [EAE]. Treatment with 1,25-(OH)2-24OXO-16eneD3 was given at 0.05, 0.15 and 0.3 microgram I.P., on alternate days, starting 3 days prior and for up to 5 days post immunization and compared to a similar treatment with 0.1 microgram 1,25-(OH)2D3 or 1,25-(OH)2-16eneD3. Suppression of EAE was observed with 0.15 microgram 1,25-(OH)2-24OXO-16eneD3, comparable to the suppression induced with the parent compound and more potent than 1,25-(OH)2D3. However, no hypercalcemia was seen in mice treated with 0.15 microgram of OXO-metabolite (9.7 +/- 0.6 vs 9.3 +/- 1.1 mg/dl, treated vs controls), in contrast to 1,25-(OH)2D3 and 1,25-(OH)2-16eneD3 (11.2 +/- 1.0 and 11.0 +/- 0.9 mg/dl respectively; p < 0.001). In summary, our results suggest that 1,25-(OH)2-24OXO-16eneD3, a stable intermediary metabolite of the vitamin D analog, 1,25-(OH)2-16eneD3 exerts immunosuppressive activity equal to its parent without causing hypercalcemia in vivo.

Functional block for 1 alpha,25-dihydroxyvitamin D3-mediated gene regulation in human B lymphocytes.

Elements necessary for the steroid hormone 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25-(OH)2D3) to induce a biological response include the presence of specific intracellular receptors (vitamin D3 receptors (VDR)) and modulation of gene expression via hormone-activated receptor binding to regulatory regions of target genes. These parameters were examined in normal and Epstein-Barr virus-immortalized human B cells and compared with 1 alpha,25-(OH)2D3-responsive cells of the T and monocytic lineages. Although resting tonsillar B cells did not express VDR mRNA, activation of these cells with interleukin-4 induced VDR in the absence of exogenously supplemented 1 alpha,25-(OH)2D3. As indicators of hormone-mediated gene regulation we analyzed modulation of CD23, a common B cell/monocyte surface antigen, and 24-hydroxylase. 1 alpha,25-(OH)2D3 inhibited CD23 expression in U937 cells, yet failed to modulate CD23 expression in B cells. Furthermore, 1 alpha,25-(OH)2D3 induced 24-hydroxylase mRNA expression and metabolic activity in both U937 cells and lectin-activated T cells, yet failed to induce 24-hydroxylase mRNA or its metabolic activity in B cells. These findings suggest that although human B lymphocytes can express VDR mRNA and protein, they exhibit a functional block for vitamin D-dependent gene regulation.

The orientation of substrate and reaction intermediates in the active site of ribulose-1,5-bisphosphate carboxylase.

There are four possible orientations of the substrate ribulose 1,5-bisphosphate in the active site of ribulose-1,5-bisphosphate carboxylase. Distinction between these four possible orientations has been made on the basis of 31P NMR and borohydride-trapping experiments. The orientation of the reaction-intermediate analog, 2'-carboxy-D-arabinitol 1,5-bisphosphate with respect to the divalent metal ion was determined by 31P NMR studies of the quaternary complex, enzyme.CO2.Ni2+.2'-carboxyarabinitol 1,5-bisphosphate. Assignment of the phosphorus resonances of this complex was made by labeling the phosphoryl group at either C-1 or C-5 with 17O. The phosphorus atom closer to the paramagnetic metal ion, Ni2+, to which the broader of the phosphorus resonances is attributed, has been identified as that attached to C-1. When bound to the active site of carbaminated enzyme, D-ribulose 1,5-bisphosphate was reduced by sodium borohydride with absolute stereospecificity to D-arabinitol 1,5-bisphosphate. The reduction of the enzyme-bound substrate thus occurred on the Si face of the C-2 carbonyl group. These two results together establish that ribulose 1,5-bisphosphate is oriented within the active site so that 1) the phosphoryl group at C-1 is closer to the divalent metal ion than that at C-5 and 2) the Si face of the carbonyl group points to the "outside world."

Regulation of vitamin D metabolism in normal human pregnancy.

The increasing serum concentrations of various hormones (PTH, PRL, estrogens, and human placental lactogen) are hypothesized to regulate 1,25-dihydroxyvitamin D [1,25(OH)2D] and possibly 24,25-dihydroxyvitamin D [24,25(OH)2D] production during pregnancy. We examined the correlation between the serum levels of 1,25(OH)2D and the pregnancy-related hormones in 25 normal pregnant women, followed throughout gestation and postpartum. Maternal serum levels of 1,25(OH)2D were high during the first trimester (mean +/- SE, 74 +/- 8 pg/ml), remained high until the time of delivery (95 +/- 14 pg/ml), and then fell to almost normal levels (50 +/- 9 pg/ml) on the third postpartum day. The serum levels of 1,25(OH)2D do not correlate with the serum levels of any of the aforementioned hormones. The increase in serum 1,25(OH)2D in pregnancy has been postulated to be related to the stressed calcium homeostatic mechanisms known to occur in the mother. In twin pregnancy, this maternal calcium homeostatic mechanism(s) conceivably may be stressed to a greater extent. However, serum 1,25(OH)2D levels, measured in 27 women with a twin pregnancy in both the second and third trimesters as well as at delivery, did not differ from the levels observed in women with a singleton pregnancy. There were no significant changes in the serum levels of 24,24(OH)2D or 25-hydroxyvitamin D as pregnancy progressed. However, serum 24,25(OH)2D correlated significantly with both serum 1,25(OH)2D (r - 0.51; p less than 0.001, n = 83) and serum 25-hydroxyvitamin D (r = 0.37; P less than 0.001, n = 94). In conclusion, serum levels of 1,25(OH)2D rise early in the first trimester of pregnancy, fall acutely to normal levels soon after delivery, and are similar in singleton and twin pregnancies. The changes in the serum levels of 1,25(OH)2D do not relate to the changes in the serum levels of any of the pregnancy-related hormones.