The metabolism of [3H] vitamin D3 was studied in cultured human keratinocytes (CHK). Intact CHK were incubated for 1, 6, 12, 24 and 48 h with [3H] vitamin D3 and the lipid soluble fractions from the media and cells were extracted by high‐performance liquid chromatography (HPLC). Vitamin D3 and its metabolites, 25‐OH‐D3, 24,25(OH)2D3 were added to the extracts, as markers, prior to HPLC, HPLC analysis of the lipid extracts did not reveal any monohydroxylated metabolites. CHK incubated for one hour with [3H]25‐OH‐D3 showed a 10±4% conversion to [3H]1,25,(OH)2D3 whereas no conversion to [3H]1,25(OH)2D3 was observed in control CHKs that were boiled prior to incubation with [3H]25‐OH‐D3. These findings suggest that cultured neonatal keratinocytes are incapable of metabolizing vitamin D3 to 25‐OH‐D3.

The effect of 1,25(OH)(2)D(3) on the intracellular calcium, (Ca(+2))i, in both cultured human keratinocytes and in cultured human dermal fibroblasts was investigated. When the intracellular calcium (Ca(+2))i in cultured human keratinocytes, grown in a serum-free medium containing 1.8 mM calcium, was measured by the fluorescent calcium-indicator, Furu-2, the (Ca(+2)i increased 154%, 202%, and 409% over the control value after incubation with 1,25(OH)(2)D(3) at 10(-10) m, 10(-8) m, and 10(-6) m, respectively. This response was immediate (15 seconds), specific (no effect with either 25(OH)D(3) at 10(-8) m or vitamin D(3) at 10(-8) m), and occurred with or without EGTA in the medium. In contrast, 1,25(OH)(2)D(3) did not increase the (Ca(2+))i in either cultured human keratinocytes that were grown in low calcium (0.05 mm), serum-free medium or in cultured human dermal fibroblasts that were grown in medium containing 0.05 mm calcium and 1% serum. The effect of 1,25(OH)(2)D(3) on the the turnover of phosphatidylinositol was investigated as a possible cause for the observed increase in (Ca(+2)i. Cultured human keratinocytes that were incubated with (3)H-inositol demonstrated a 50 % +/- 10% increase in the triphosphated, plasma membrane-bound metabolite of phosphatidylinositol, PIP(2), by 15 seconds, followed by a rapid decrease at 30 seconds, then a return toward basal levels by 1 minute. Lysophosphatidylinositol, which results from the sn-2 deacylation of phosphatidylinositol by phospholipase A(2), decreased 20% +/- 8% within 30 seconds, then increased to 200% +/- 10% of the control value by 5 minutes. The accumulation of IP(3) was increased 50% to 100% above the control value within 30 seconds and this increase was substained during the 5-minute incubation period. Stimulation of phosphatidylinositol turnover by 1,25(OH)(2)D(3) was not detected in either cultured human keratinocytes that were grown in serum-free, low calcium medium or in cultured human dermal fibroblasts that were grown in 1% serum.

Sunscreens block the cutaneous absorption of UV-B radiation and prevent sunburning, premature aging, and cancer of the skin. Inasmuch as UV-B radiation is also responsible for the photosynthesis of vitamin D3, we investigated the effect of sunscreens on the cutaneous formation of vitamin D3 in vivo and in vitro. Eight normal subjects, four of whom had been protected with the sunscreen para-aminobenzoic acid (sun protection factor 8), were exposed to one minimal erythema dose of UV radiation. The mean serum vitamin D3 concentration increased from 1.5 +/- 1.0 (+/- SEM) to 25.6 +/- 6.7 ng/mL in unprotected subjects, whereas it was 5.6 +/- 3.0 and 4.4 +/- 2.4 ng/mL at these times in the subjects who were protected with para-aminobenzoic acid. Para-aminobenzoic acid also prevented the photoisomerization of 7-dehydrocholesterol to previtamin D3 in human skin slices in vitro. These results indicate that the sunscreen interferred with the cutaneous production of vitamin D3.

Nutrition in Clinical PracticeVolume 1, Issue 1 p. 57-58 Current Literature Aging Decreases the Capacity of Human Skin to Produce Vitamin D3 J. MacLaughlin B.S., J. MacLaughlin B.S. Vitamin D Laboratory, Endocrine Unit, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114; and Vitamin D and Bone Metabolism Laboratory, USDA/Human Nutrition Research Center on Aging, Tufts University, Boston, Massachusetts 02111Search for more papers by this authorM. F. Holick, M. F. Holick Vitamin D Laboratory, Endocrine Unit, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114; and Vitamin D and Bone Metabolism Laboratory, USDA/Human Nutrition Research Center on Aging, Tufts University, Boston, Massachusetts 02111Search for more papers by this authorK. Kasper, K. KasperSearch for more papers by this author J. MacLaughlin B.S., J. MacLaughlin B.S. Vitamin D Laboratory, Endocrine Unit, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114; and Vitamin D and Bone Metabolism Laboratory, USDA/Human Nutrition Research Center on Aging, Tufts University, Boston, Massachusetts 02111Search for more papers by this authorM. F. Holick, M. F. Holick Vitamin D Laboratory, Endocrine Unit, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114; and Vitamin D and Bone Metabolism Laboratory, USDA/Human Nutrition Research Center on Aging, Tufts University, Boston, Massachusetts 02111Search for more papers by this authorK. Kasper, K. KasperSearch for more papers by this author First published: 01 February 1986 https://doi.org/10.1177/088453368600100118Citations: 4AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article.Citing Literature Volume1, Issue1February 1986Pages 57-58 RelatedInformation

We examined the responsiveness of cultured dermal fibroblasts from biopsies of uninvolved and involved areas of skin from six patients with psoriasis to the cell-proliferation-inhibition activity of 1,25-dihydroxyvitamin D3 (1,25-(OH)2-D3). Cultured fibroblasts from age-matched controls responded to 1,25-(OH)2-D3 (at 0.01, 1, 10, and 100 microM) in a dose-dependent fashion, whereas cultured psoriatic fibroblasts from involved or uninvolved skin showed no inhibition of proliferation when exposed to 0.01 or 1 microM of 1,25-(OH)2-D3. However, 1,25-(OH)2-D3 did inhibit proliferation of cultured psoriatic fibroblasts when the concentrations were increased to 10 and 100 microM. An analysis of the 1,25-(OH)2-D3 receptors in cultured psoriatic fibroblasts from uninvolved skin revealed that the Kd, nmax, and sedimentation coefficient were identical to the receptors found in the fibroblasts from age-matched controls. Therefore, cultured psoriatic fibroblasts from involved and uninvolved skin have a partial resistance to 1,25-(OH)2-D3, suggesting that there may be a biochemical defect that is inherent in the dermal fibroblasts of psoriatic patients. Recognition of this defect may provide a new approach for the evaluation of the cause and treatment of this disfiguring skin disorder.

The photosynthesis of previtamin D3 from 7-dehydrocholesterol in human skin was determined after exposure to narrow-band radiation or simulated solar radiation. The optimum wavelengths for the production of previtamin D3 were determined to be between 295 and 300 nanometers. When human skin was exposed to 295-nanometer radiation, up to 65 percent of the original 7-dehydrocholesterol content was converted to previtamin D3. In comparison, when adjacent skin was exposed to simulated solar radiation, the maximum formation of previtamin D3 was about 20 percent. Major differences in the formation of lumisterol3, and tachysterol3 from previtamin D3 were also observed. It is concluded that the spectral character of natural sunlight has a profound effect on the photochemistry of 7-dehydrocholesterol in human skin.

A previous study of energy-independent in vitro Ca2+ uptake by rat intestinal epithelial membrane vesicles demonstrated that uptake by Golgi membrane vesicles was greater than that by microvillus or lateral-basal membrane vesicles, was markedly decreased in vitamin D-deficient rats, and responded specifically to 1,25-(OH)2D3 repletion (R. A. Freedman, M. M. Weiser, and K. J. Isselbacher, 1977, Proc. Nat. Acad. Sci. USA74, 3612–3616; J. A. MacLaughlin, M. M. Weiser, and R. A. Freedman, 1980, Gastroenterology78, 325–332). In the present study, properties of Ca2+ uptake and release by intestinal Golgi membrane vesicles have been investigated. The initial rate of uptake was found to be saturable, suggesting carrier-mediated uptake. Uptake was markedly inhibited by Mg2+ and Sr2+, but not by Na+ or K+. Lowering the external [H+] or raising the internal [H+] resulted in enhancement of the initial rate of uptake; the intial rate was found to correlate with the internal-to-external [H+] gradient. The initial rate of uptake could be enhanced by preloading the vesicles with MgCl2 or SrCl2 but not CaCl2, NaCl, or KCl. Vesicles preloaded with K2SO4 failed to show enhanced uptake in the presence of valinomycin, suggesting that enhancement in uptake by vesicles preloaded with MgCl2 was not due to transmembrane potentials. The internal volume of the Golgi membrane vesicles was determined and found to be 9 μl/mg protein; this volume could accomodate less than 1% of the Ca2+ uptake maintained at equilibrium. Therefore, the remainder of the Ca2+ taken up was presumably bound to the Golgi membranes. A dissociation constant of 3.8 × 10−6m was found for this binding. The bound Ca2+ could be rapidly released by external Mg2+ or Sr2+, but not Ca2+, Na+, or K+. Release of bound Ca2+ could also be induced by raising the [H+] of the external medium. Failure of external Ca2+ to release bound Ca2+ suggested that the release induced by external Mg2+, Sr2+, or H+ was not due to competitive displacement of Ca2+ from its binding sites. These results indicated that Ca2+ uptake by intestinal Golgi membrane vesicles consists of carrier-mediated transport followed by binding of Ca2+ to the vesicle. The effects of H+, Mg2+, and Sr2+ on Ca2+ uptake and release suggest the existence of cation countertransport in the Golgi membrane vesicles.

When human skin was exposed to simulated solar ultraviolet radiation, epidermal 7-dehydrocholesterol was converted to previtamin D3. During prolonged exposure to simulated solar ultraviolet radiation, the synthesis of previtamin D3 reached a plateau at about 10 to 15 percent of the original 7-dehydrocholesterol content, and previtamin D3 was photoisomerized to two biologically inert isomers, lumisterol3 and tachysterol3. Increases either in skin melanin concentration or in latitude necessitated increases in the exposure time to simulated solar ultraviolet radiation required to maximize the formation, but not the total content, of previtamin D3. In order of importance, the significant determinants limiting the cutaneous production of previtamin D3 are (i) photochemical regulation, (ii) pigmentation, and (iii) latitude.

A previous study had demonstrated that rat intestinal epithelial cell Golgi, lateral-basal, and microvillus membrane vesicles translocated Ca 2+ , and that this process was markedly decreased in vitamin D-deficient rats. In the present study the kinetics of recovery of Ca 2+ uptake by intestinal Golgi vesicles was examined in vitamin D-deficient rats after a single i.v. injection of 125 ng 1,25-dihydroxycholecalciferol (1,25-(OH) 2 D 3 ). Calcium uptake was measured by determination of 45 Ca 2+ associated with Golgi membranes after collection by micropore filtration. Golgi membranes recovered rapidly (66% in 15 min, 83% in 30 min) but then quickly returned to prerepletion levels by 8 hr. This was followed by a second phase of recovery starting at 48 hr, with approximately 80% recovery by 96 hr which was maintained at least to 120 hr. By comparison, partial recovery of calcium uptake by microvillus membrane vesicles was first detected at 15 hr and full recovery did not occur until 72 hr; a biphasic pattern to microvillus membrane recovery was not apparent. The vitamin D-dependent recovery of Ca 2+ uptake by Golgi membrane vesicles was also compared to studies with gut sacs which showed 50% recovery by 3 hr and 100% recovery by 6 hr; gut sac recovery was sustained for at least 120 hr. The recovery of Ca 2+ uptake by Golgi was shown to be specific for 1,25-(OH) 2 D 3 . The first peak of recovery of Ca 2+ uptake by intestinal Golgi membrane vesicles is one of the earliest reported effects of 1,25-(OH) 2 D 3 . The biphasic nature of recovery demonstrated by Golgi membranes suggests that there may be more than one mechanism of action of 1,25-(OH) 2 D 3 on the intestine.

The simple three-step preparation of [1β-3H]1α,25-dihydroxyvitamin D3 and [1α-3H]1β,25-dihydroxyvitamin D3 from 1α,25-dihydroxyvitamin D3 is described. In the rat, 1β,25-dihydroxyvitamin D3, when compared with its α-epimer, did not stimulate intestinal calcium transport or bone calcium mobilization at doses 1000-fold higher than the doses of the natural hormone, 1α,25-dihydroxyvitamin D3.

Photosynthesis of previtamin D3 can occur throughout the epidermis in the dermis when hypopigmented Caucasian skin is exposed to solar ultraviolet radiation. Once previtamin D3 is formed in the skin, it undergoes a temperature-dependent thermal isomerization that takes at least 3 days to complete. The vitamin D-binding protein preferentially translocates the thermal product, vitamin D3, into the circulation. These processes suggest a unique mechanism for the synthesis, storage, and slow, steady release of vitamin D3 from the skin into the circulation.