Uptake and bioconversion of alpha-tocopheryl acetate to alpha-tocopherol in skin of hairless mice.

The photoprotective effect of topically applied alpha-tocopheryl acetate (vitamin E acetate), a stable derivative of alpha-tocopherol (vitamin E), and its possible bioconversion to the active antioxidant species (alpha-tocopherol) was examined in skin tissue of female hairless mice (HRS/J) exposed to UV-B irradiation. Our results indicate that topically applied alpha-tocopheryl acetate is absorbed into and retained by skin tissue. Furthermore, skin tissue from UV-B-irradiated animals that received daily topical alpha-tocopheryl acetate treatments contained significantly higher levels (P < 0.001) of alpha-tocopheryl acetate than non-UV-B-irradiated mice that received identical daily topical alpha-tocopheryl acetate treatments. Finally, free alpha-tocopherol levels in skin also were significantly increased (P < 0.001) by topical applications of alpha-tocopheryl acetate and skin levels of free alpha-tocopherol were significantly greater (P < 0.001) in UV-B-irradiated animals that received daily topical alpha-tocopheryl acetate treatments than in non-UV-B-irradiated animals. These results suggest that UV-B irradiation enhances both the absorption of alpha-tocopheryl acetate and its bioconversion to free alpha-tocopherol.

Effect of topical retinoic acids on the levels of collagen mRNA during the repair of UVB-induced dermal damage in the hairless mouse and the possible role of TGF-beta as a mediator.

Topically applied retinoic acids have been found to enhance the gene expression for collagen types I and III in the skin of UVB-irradiated hairless mice. Prior damage is required because the effect is not observed in the skin of age-matched, non-irradiated control animals. Immunochemical methods have shown an increase in TGF-beta 1 and, to a lesser extent, of TGF-beta 2 in the epidermis following retinoic acid treatment. There were no changes in mRNA levels for any of the isotypes of TGF-beta induced by retinoic acid treatment. This study suggests that TGF-beta may mediate the effect of retinoic acids on dermal repair through the stimulation of collagen gene expression.

Ultrastructural effects of UVB radiation and subsequent retinoic acid treatment on the skin of hairless mice.

The ultrastructure of hairless mouse skin exposed to UVB radiation and followed by retinoic acid treatment was studied to identify alterations induced in both epidermis and dermis. Female mice were irradiated 3 times weekly for 5-6 months; a group of these mice was then treated topically 3 times weekly for 10 weeks with either 25 micrograms all-trans-retinoic acid dissolved in acetone or with acetone alone. Age-matched, unexposed, untreated mice served as controls. Cutaneous changes induced by UVB radiation included keratinocyte mitochondrial inclusions often accompanied by damaged cristae, duplication of basement membrane, increased number of dermal fibroblasts, inflammatory cells and elastic fibers, and abnormal elastic fibers. Subsequent retinoic acid treatment resulted in more prominent mitochondrial inclusions which sometimes coalesced to form irregular contoured bodies. Also observed were lipid droplets in the stratum corneum, glycogen deposits in keratinocytes and granular material in dilated keratinocyte endoplasmic reticulum. Poorly differentiated epidermis with necrotic or apoptotic cells was present in some specimens. Elastic fibers were fewer and usually morphologically normal. Skin exposed to UVB and treated with vehicle appeared similar to control except for the presence of excess basement membrane and occasional small mitochondrial inclusions. Because of the heightened concern regarding UV radiation-induced damage to the human skin and the current topical use of retinoids, the cutaneous changes described are considered worthy of attention.

Mitochondrial inclusions in keratinocytes of hairless mouse skin exposed to UVB radiation.

Mitochondrial inclusions were observed in keratinocytes during an ultrastructural investigation of the skin of hairless mice exposed to UVB radiation. Mice were irradiated 3 times a week for 5-6 months with sunlamps at individual doses seldom exceeding 0.06 J/cm2. Strips of dorsal skin were processed for electron microscopic examination; blocks were sectioned to include both epidermis and dermis. Mitochondrial inclusions were observed in keratinocytes of the basal, spinous and granular layers. They were spherical in shape and of moderate and homogeneous electron density. Mitochondria toward the upper regions of the epidermis were swollen and had fragmented cristae; mitochondria in the lower areas of the epidermis usually contained smaller and less dense inclusions and intact or partially disrupted cristae. Because mitochondria are essential in providing the energy for cellular function, keratinocyte mitochondrial damage induced by UVB radiation may have serious pathological consequences. Possible mechanisms involved in mitochondrial inclusion formation are suggested.

Retinoid effects on photodamaged skin.

The effects of retinoid treatment on wrinkling in the hairless mouse can be understood in the context of the repair of the dermal elastosis. The two isomers of retinoic acid do not differ qualitatively in their effects on the histological appearance of the tissue or on the wrinkling patterns produced. The all-trans isomer is slightly more potent in this system than the 13-cis isomer but substantially more irritating, which may limit the maximum degree of repair attainable. The "reconstructed" dermis is thickened, it contains new collagen as a result of the stimulation of gene expression, and the tangled, disorganized elastin is packed into a thin layer in the lower dermis. Thus, the framework within which a wrinkle had been established is eliminated, and the skin assumes a normal state, as observed. That the effacement is apparently permanent is additional evidence of the relationship between the integrity of the elastic fiber network and the surface appearance. The only difference in the repaired skin is the absence of filamentous surface features. The thickening effect of UVB and subsequent retinoid treatment on the epidermis does not contribute substantially to the overall thickness of the repaired skin. Nevertheless, despite having a minor role in the effacement of deep wrinkles, these epidermal changes evidently preclude the formation of fine surface features. The model is a valid one for the repair of photodamaged skin. From what is known about the role of elastin in maintaining skin integrity and from the association of wrinkling with excessive sun exposure, it is encouraging to observe the dual effect of retinoic acids.(ABSTRACT TRUNCATED AT 250 WORDS)

Retinoic acids promote the repair of the dermal damage and the effacement of wrinkles in the UVB-irradiated hairless mouse.

Chronic irradiation of hairless mice with UVB leads to elastosis as evidenced by both histologic means and an increase in skin desmosine content. Treatment with topical all-trans- or 13-cis-retinoic acid causes dose-dependent increments in the area of the dermal "repair zone"; skin desmosine content increases during irradiation but does not change significantly after irradiation is discontinued and retinoic acid treatment commenced. During the course of the irradiation the animals develop permanent wrinkles on the exposed dorsal surface, which can be recorded in plastic impressions. The extent of wrinkling can be quantitated and it has been demonstrated that topically applied retinoic acids lead to the complete effacement of these surface features and that the process appears to be permanent.

Relationship between urinary calcium and calcium intake during calcitriol administration.

The hypercalciuria that occurs when 1,25 (OH)2D3 (calcitriol) is given to humans with normal renal function depends on dietary Ca absorption and may also relate, in part, to enhanced bone resorption. To evaluate the relationship between urinary and dietary Ca during treatment with calcitriol, 12 metabolic balance studies were performed in normal volunteers ingesting a diet containing 350 mg/day of Ca, to which Ca gluconate was added. After 10 days on either 350 mg/day or 1550 mg/day of Ca, calcitriol, 0.5 microgram every 12 hr, was given. Then diet Ca was changed in successive 5-day treatment periods from 350 to 650, 950 and 1550 mg/day (group A) or from 1550 to 950, 650 and 350 mg/day (group B). On the lowest diet Ca, urinary Ca was less than Ca intake during calcitriol treatment (group A, 220 +/- 50 mg/day; group B, 247 +/- 40). As diet Ca was changed during calcitriol treatment, urinary Ca correlated with diet Ca (r = 0.60) until diet Ca reached 950 mg/day. With calcitriol, serum iPTH fell by 18 to 25% (P less than 0.01) and urinary hydroxyproline fell by 11 to 19% (P less than 0.05 to 0.01). Baseline serum levels of 1,25(OH)2D were 47 +/- 8 and 34 +/- 5 pg/ml in group A and B, respectively, and the values increased to 51 +/- 12 and 45 +/- 7.4 pg/ml during treatment with calcitriol. Serum Ca from fasted subjects was not affected by calcitriol, but the mean postabsorptive serum Ca (moon) was increased by 0.35 mg/dl.(ABSTRACT TRUNCATED AT 250 WORDS)

Pharmacokinetics and biologic effects of calcitriol in normal humans.

The dose response and pharmacokinetics of orally administered calcitriol were investigated in normal humans. In one protocol, six volunteers received calcitriol 0.25 micrograms twice a day, 0.5 micrograms daily, and 0.5 micrograms twice a day, in successive weeks. Peak plasma levels of 1,25(OH)2D occurred 4 to 8 hours after ingestion of a single dose of 0.5 micrograms, with a return to baseline within 24 hours. The 8:00 AM calcitriol plasma levels were raised only when the drug was given twice daily. Urinary calcium excretion (UCa) was significantly increased from 199 +/- 19 mg/24 hr during the control period to similar levels of 302 +/- 26 mg/24 hr after 0.25 microgram twice a day and 284 +/- 31 mg/24 hr after 0.50 microgram daily. With 0.50 microgram twice a day, UCa was 417 +/- 36 mg/24 hr, a value greater than after the lower doses (p less than 0.05). In another protocol, fourteen volunteers received calcitriol 0.25 microgram, 0.5 microgram, and 1.0 microgram twice a day each for 14 days with intervening control periods of 2 weeks. A dose-related response in urinary calcium/creatinine excretion occurred. Thus, UCa (milligrams calcium per milligram creatinine) increased with calcitriol from 0.13 +/- 0.014 mg to 0.15 +/- 0.018 mg with 0.25 microgram twice a day, from 0.13 +/- 0.010 mg to 0.22 +/- 0.022 mg with 0.5 microgram twice a day, and from 0.12 +/- 0.012 mg to 0.23 +/- 0.012 mg with 1 microgram twice a day (p less than 0.05 with 0.25 microgram, p less than 0.01 with 0.5 and 1 microgram twice a day).(ABSTRACT TRUNCATED AT 250 WORDS)

Decreased serum ionized calcium and normal vitamin D metabolite levels with anticonvulsant drug treatment.

Alterations in vitamin D metabolism are generally thought to account for the hypocalcemia and osteopenia caused by long term treatment with anticonvulsant drugs. Regional variation in the incidence and severity of anticonvulsant drug-induced bone disease has been attributed to differences in sunlight exposure, with most reports coming from areas with limited sunshine or from institutionalized patients. Serum ionized calcium levels in 109 ambulatory adult epileptic outpatients receiving chronic anticonvulsant drug therapy in Georgia were decreased [4.73 +/- 0.02 (+/-SE) vs. 4.97 +/- 0.01 mg/dl; P less than 0.001). Immunoreactive PTH concentrations were increased (5.5 +/- 0.4 vs. 4.0 +/- 0.3 microliterEq /ml; P less than 0.005), while bone mineral content was reduced, averaging only 88.8% of the predicted normal values. Hypocalcemia and osteopenia occurred in spite of normal mean levels of serum 25-hydroxyvitamin D and 1,25-dihydroxy-vitamin D. The indirect relationship between serum concentrations of antiepileptic drugs and the serum ionized calcium level, and the lack of correlation with vitamin D metabolite levels suggested that hypocalcemia was independent of the effect of the drugs on vitamin D metabolism. Bone biopsies revealed increased osteoid but normal calcification front formation, accelerated mineralization rate, and decreased mineralization lag time indicative of increased skeletal turnover, rather than osteomalacia.

Preliminary evidence of the effect of calcium supplementation on blood pressure in normal pregnant women.

In this study the hypothesis that calcium supplementation during pregnancy can modify blood pressure patterns in a population of normal pregnant women was tested. Thirty-six women with normal single pregnancies, between 20 and 35 years of age, in the second trimester of gestation (15 weeks), were randomly assigned to receive 1 gm of calcium per day (n = 11), 2 gm per day (n = 11), or a placebo (n = 14). No differences were observed at the times of admission into the study (baseline) in demographic and clinical variables or in the calcium intake of each group. Baseline blood pressure measures in several positions also were not different. After the initial blood pressure measures (fifteenth week), five follow-up blood pressure measures were obtained. The supplemented groups had significantly lower diastolic blood pressure than the control subjects between the twentieth and twenty-fourth weeks of gestation. Thereafter, an increase in the control group and the group receiving 1 gm of calcium was observed, but levels were similar at term. On the contrary, patients receiving 2 gm of calcium had blood pressure values that remained significantly lower throughout the third trimester. No differences or clear patterns were observed in the blood levels of calcium, magnesium, phosphorus, and proteins between and within groups during gestation. A possible explanation involving parathyroid hormone is attempted.

Decrease in serum levels of 1,25-dihydroxycholecalciferol in rats and chickens fed a vitamin D-deficient diet.

Twenty-one-day-old rats placed on a vitamin D-deficient diet showed no decrease in serum, 1,25-dihydroxy vitamin D levels after 13 days on this diet. Between 13 and 20 days on this D-deficient diet there was a 50% decrease in serum 1,25-dihydroxy vitamin D. After 34 days, the level of 1,25-dihydroxy vitamin D in serum had dropped to near zero. With a vitamin D-deficient diet lacking calcium, there was an apparent stimulation of 1-hydroxylase, resulting in higher 1,25-dihydroxy vitamin D serum levels after 6--20 days on the diet. After 27 days there were no differences in 1,25-dihydroxy-vitamin D levels in animals fed a calcium-replete or calcium-deficient rachitogenic diet. When 1-day-old chickens were maintained on a rachitogenic diet for 1 week, 1,25-dihydroxy vitamin D levels were higher in animals fed the calcium-deficient diet compared with the calcium-replete diet. After 2 weeks on either rachitogenic diet, 1,25-dihydroxy vitamin D levels decreased to near zero. Measurement of 1,25-dihydroxy vitamin D levels has provided a biochemical indicator of vitamin D deficiency in chicks and rats which will complement other established biological criteria for vitamin D deficiency.

Metabolic consequences of fasting in old lean and obese Zucker rats.

The effects of fasting on lipid and carbohydrate metabolism and plasma insulin and glucagon levels were compared in lean and obese Zucker rats. Sixteen-month-old female and male rats were fasted for periods of 2, 4, 6 and 12 days. Fasting produced significant decreases in hepatic rates of lipid, cholesterol, and glycogen synthesis, as well as circulating levels of triglycerides, cholesterol, phospholipids, and insulin. Significant increases in hepatic lipid levels and serum free fatty acids were noted. When compared to lean rats, obese rats had elevated rates of hepatic lipid and glycogen synthesis, hepatic lipid and glycogen stores, serum triglycerides, cholesterol, phospholipids, and plasma insulin. Lean rats had higher plasma glucagon levels. Sex differences in several parameters were observed. Females demonstrated higher levels of lipid and cholesterol synthesis and serum free fatty acids, whereas serum cholesterol levels and hepatic glycogen stores were higher in males. Following a 12-day fast, carcass fat and protein content were decreased in both lean and obese rats, but the obese animals maintained an obese body composition. It is concluded that fasting results in qualitatively similar metabolic and hormonal changes in both lean and obese rats, but that abnormalities in carbohydrate and lipid metabolism persist in obese rats even after a 12-day fast.

Paradoxical short-term effects of cyproheptadine on insulin and glucagon release in the rat.

The administration of cyproheptadine (25 mg/kg; i.p.) resulted in an increase of plasma insulin and glucagon (measured using 30 K antibody) 30, 60 and 120 min after injection to fasted rats. This dose of cyproheptadine also induced a hyperglycemia whereas a lower dose (5 mg/kg; i.p.), which did not alter plasma hormone levels, was associated with a hypoglycemia. Fed rats showed a reduction of plasma insulin with a similar elevation of blood glucose after cyproheptadine. Administration of an exogenous load of arginine resulted in increases of plasma insulin and glucagon of a greater magnitude than induced by cyproheptadine, however, cyproheptadine pretreatment (25 mg/kg) completely suppressed the pancreatic response to the amino acid, resulting in blood hormone levels similar to values seen after cyproheptadine administered alone. Cyproheptadine pretreatment also prevented the hyperinsulinemia and hypoglucagonemia resulting from glucose loading. alpha-Adrenergic receptor blockade (with phentolamine), beta adrenergic receptor blockade (with propranolol) and adrenodemedullation did not alter pancreatic responsiveness to the drug.

The acute effects of 5HTP, fluoxetine and quipazine on insulin and glucagon release in the intact rat.

5-hydroxytryptophan (5HTP), the immediate precursor of serotonin, induces a release of insulin and glucagon in the intact rat. These effects of 5HTP, which have previously been shown to be blocked by L-aromatic amino acid decarboxylase inhibition, were also prevented by methysergide (a serotonin receptor antagonist). Quipazine (a serotonin receptor agonist) did not alter pancreatic hormone release. Fluoxetine, a serotonin neuronal reuptake blocker did not effect insulin secretion and had a slight glucagon stimulatory effect, however the effects of 5HTP on insulin and glucagon release were not potentiated by fluoxetine pretreatment. Alpha and beta-adrenergic receptor blockade did not alter the pancreatic effects of 5HTP.

The acute pharmacologic effects of serotonin on the release of insulin and glucagon in the intact rat.

Serotonin (5HT) (5 mg/kg-25 mg/kg; i.p.) induced a dose-related increase of plasma glucagon (IRG) (using 30K antibody) 3 to 60 min after administration to overnight fasted rats. Blood glucose (BS) also increased as early as 10 min post-injection whereas plasma insulin (IRI) increased in a non dose-related (30 min to onset) manner. Adreno-demedullation prevented the rise of BS and IRI, but not IRG. Pretreatment with reserpine (5 mg/kg; i.p.; 24 hr earlier) did not prevent the actions of 5HT. Pretreatment with the alpha-adrenergic antagonist phentolamine (3 mg/kg-6 mg/kg; i.p.) reduced but did not prevent the subsequent rise of IRG, whereas beta-adrenergic blockade with propranolol (5 mg/kg-10 mg/kg; i.p.) was without effect. Phentolamine and the lower dose of propranolol (5 mg/kg) reduced the 5HT-induced hyperglycemia; whereas the higher dose (10 mg/kg) prevented the hyperglycemia. Phentolamine potentiated and propranolol prevented (5 mg/kg) or reversed (10 mg/kg) the 5HT-induced IRI rise. Pretreatment with the 5HT-antagonist, methysergide, prevented all the effects of 5HT. Precursor loading with 5HTP (5 mg/kg-50 mg/kg; i.p.) also resulted in a dose-related increase of IRG and a slight increase of IRI. Blockade of the conversion of 5HTP to 5HT with Ro-4-4602 (an L-aromatic acid decarboxylase inhibitor) blocked the subsequent rise of IRG. These results suggest that the 5HT-induced changes in BS and IRI may be secondary to a release of epinephrine and/or norepinephrine, but that the effects of 5HT on the release of IRG cannot be explained solely by this mechanism.

Insulin and glucagon: plasma levels and pancreatic release in the genetically obese Zucker rat.

Circulating levels of insulin and glucagon, as well as their release from isolated pancreatic islets, have been measured in Zucker rats to examine the effect of genotype, sex and diet. The obese animals had higher plasma insulin levels and enhanced release from islets when compared to lean controls. Conversely, obese animals, despite no significant differences in fed plasma levels of glucagon, showed substantially reduced release from islets. Diet had no main effect on any of these parameters.