Determination of trimethylpsoralen in blood, ophthalmic fluids, and skin.

Trimethylpsoralen (TMP) levels in the blood of vitiligo patients were determined through the use of a high-performance liquid chromatographic method. TMP was extracted from blood buffered at pH 9.0 with 95:5 (V/V) hexane-isopropanol mixture; evaporated to dryness, and reconstituted in 50 microliters of ethanol. A 10-microliters aliquot was injected into a Micropack MCH-10 column (Varian HPLC model #5000). The mobile phase consisted of a mixture of water and acetonitrile with a linear gradient. The retention time of TMP was 16.5 min. The calibration curve of the external standards was linear. Overall recovery of the internal stands was 75-92%, with the lower detection limit of TMP at 2 ng/ml. Peak blood levels as low as 140 ng/ml and as high as 800 ng/ml were obtained in vitiligo patients 1-2 hr following the oral administration of 30 mg of trioxsalen tablets (Paul B. Elder Co.). Blood TMP levels peaked consistently at 2 hr when patients were fasted for 8 hr prior to drug ingestion. These results are consistent with the clinical observation that maximum response due to phototherapy is obtained 1-2 hr after oral administration of the drug. Two hours after oral drug administration, TMP levels in the epidermis, dermis, and whole skin of the guinea pig (in ng per g tissue) were: epidermis, 226 +/- 15; dermis, 25 +/- 6; and whole skin 176 +/- 12. Also detected were TMP levels 244 +/- 17 ng/ml in aqueous humor and 63 +/- 6 mg/ml in vitreous humor. These results point to the fact that the eyes of patients must be protected from overexposure to sunlight after psoralen-ultraviolet treatment.

8-Methoxypsoralen levels in blood of vitiligo patients and in skin, ophthalmic fluids, and ocular tissues of the guinea pig.

8-Methoxypsoralen (8-MOP) levels in the blood of vitiligo patients were determined through the use of a reverse-phase high-performance liquid chromatographic method. The overall recovery of the internal standards was 85-94%, with the lower detection limit of 8-MOP at 2 ng. Peak blood levels as low as 130 ng/ml and as high as 3892 ng/ml were obtained in patients at 1-3 h following the oral administration of 0.6 mg/kg body weight of Oxsoralen capsules (Elder Pharmaceuticals Co.). These results are consistent with the clinical observation that maximum response in phototherapy is obtained at about 2 h after oral administration of the drug. Two hours after oral administration of 0.6 mg/kg of Oxsoralen, 8-MOP levels in the epidermis, dermis, and whole skin of the guinea pig (in ng/g) were: epidermis, 330 +/- 20; dermis, 89 +/- 16; whole skin, 379 +/- 19. Also detected were 8-MOP levels of 441 +/- 22 ng/ml in aqueous humor, 166 +/- 18 ng/ml in vitreous gel, 355 +/- 15 ng/g in lens, and 410 +/- 26 ng/g in retina. These results point to the fact that the eyes of the patient must be protected from exposure to sunlight after psoralen UV treatment, and that 8-MOP is absorbed in blood unevenly and varies from patient to patient. The fact that only 50-60% of the patients responded to psoralen photochemotherapy for vitiligo may be related to the variation of absorption of the drug in individual patients.

Autoantibodies and their clinical significance in a black vitiligo population.

The frequency of autoantibodies was determined in 70 black vitiligo patients and controls. Both groups were screened for antithyroid, antinuclear, antigastric parietal cell, anti-smooth muscle cell, and antimitochondrial autoantibodies. The significance of autoantibodies was determined in vitiligo patients by correlating their presence or absence with various clinical features of the patients. The overall frequencies of autoimmune and endocrine diseases were also assessed in vitiligo patients, controls, and their respective families. Vitiligo patients had an increased frequency of antithyroid antibodies and an increased frequency of autoimmune and/or endocrine diseases. These diseases included, especially, hyperthyroidism, hypothyroidism, and alopecia areata. Autoantibody-positive vitiligo patients had an increased frequency of first- and second-degree relatives having autoimmune and/or endocrine diseases. These findings tend to support an autoimmune cause of vitiligo in black patients.

Biochemical and morphological studies of rat submandibular gland: I. Centrifugal fractionation of granule-rich fraction.

Submandibular glands from male rats were homogenized in 0.34 M sucrose and 0.5 mM ethylenediaminetetraacetic acid in 10 mM HEPES buffer at a pH of 7.4. The extract was centrifuged and filtered through nuclepore filters to prepare a granule-rich fraction. Electron dense zymogen granules constituted approximately 85% of the particles in this fraction which also contained about a third of the total alkaline esterase activity in the gland.

Biochemical and morphological studies of rat submandibular gland: III. effects of testosterone treatment on proteins of granule-rickch fraction.

Mature adult male rats and castrated and testosterone-treated castrated adult rats were injected with pilocarpine HCl and 3-H-lysine and sacrificed sequentially over an eight-hour time period. Following homogenization and differential centrifugation, three subcellular fractions from each group of animals were analyzed by gel electrophoresis and liquid scintillation. Two proteins in the granule-rich fraction appeared in larger amounts on the densitometric scans and appeared to represent a larger proportion of newly synthesized proteins than the other proteins.

Biochemical and morphological studies of rat submandibular gland: II. Partial purification of proteins from granule-rich fraction.

Soluble proteins derived from a centrifuged and filtered granule-rich fraction of homogenized rat submandibular gland were analyzed by gel filtration, ion-exchange chromatography, and polyacrylamide gel electrophoresis. Both the granule-rich fraction and final supernatant fraction contained alkaline esterase activity. The major protein component, derived from granules of the convoluted tubules, was further resolved into a series of peptides ranging in molecular weight from 9,000 to 55,000 daltons.

Determination of 8-methoxypsoralen in plasma by scanning fluorometry after thin-layer chromatography.

A rapid and sensitive method is described for determining 8-methoxypsoralen in plasma. Plasma samples are acidified with 6 mol/liter and heated in a boiling water bath to release the plasma-bound drug nondestructively. It then is extracted into a solvent mixture consisting of benzene/ethyl acetate (9/1 by vol). The solvent phase is separated, evaporated, and an aliquot of the dissolved residue is thin-layer chromatographed, with benzene/ethyl acetete (9/1) as developing solvent. The plate is dried and the spots, made visible under ultraviolet light (320-400 nm), are scanned. The smallest amount detectable is 20 ng; the overall analytical recovery from plasma is 84%. We used the method to determine the drug in the plasma of rabbits after intravenous and oral administration of 10 mg, and in one patient after an oral dose of 30 mg.

Aberrations in T lymphocytes and natural killer cells in vitiligo: a flow cytometric study.

Twenty-five patients with vitiligo and twenty-five healthy control subjects were evaluated with the use of flow cytometry to compare percentages of peripheral T lymphocytes and natural killer cells. The percentages of total T lymphocytes, helper T cells, suppressor T cells, and natural killer cells were evaluated with the use of OKT3, OKT4, OKT8, and Leu-7 monoclonal antibodies, respectively. Mean total T lymphocytes and helper T cells were markedly depressed; mean natural killer cells were markedly elevated and mean suppressor T cells were moderately elevated in patients with vitiligo in comparison with control subjects. These results indicate that cell-mediated immunity is subject to some defect in regulation in patients with vitiligo. It remains to be determined whether these abnormalities are a direct cause or a result of vitiligo. Antibody-dependent cytotoxicity, utilizing killer cells with recently reported antimelanocyte antibodies found in patients, may be responsible for pigment cell destruction in vitiligo. Helper T cells may be reduced because of low levels or faulty production of T lymphocyte-stimulating factors in patients or because of a serum factor in patients that is toxic to helper T cells. The presence or absence of autoimmune and/or endocrine disease in patients with vitiligo had no effect on lymphocyte populations. There seemed to be a trend toward lower levels of helper T cells in patients having vitiligo for the shortest amount of time. In summary, the data indicate immunologic abnormalities in patients with vitiligo.

Ocular disturbances in vitiligo.

One hundred fifty-six patients with vitiligo were examined for ocular abnormalities. A 2:3 ratio of white to black patients allowed us to evaluate the role of race in the occurrence of ocular disturbances. A large percentage (40%) of all patients showed some degree of fundal pigment disturbance including pigment clumps, focal hypopigmented spots, and choroidal nevi. Although racial variations were found in the incidence of choroidal nevi (p = 0.001) and iris transillumination (p = 0.0012), these variations were believed to reflect normal differences found in patients without vitiligo.

Childhood vitiligo.

Eighty-two children (ages 6 months to 12 years) with clinical and/or histopathologic diagnoses of vitiligo were evaluated; 35 were male and 47, female. Fifty-six were black, 25 white, and 3 classified as "other." Children were compared with control groups of children with other skin diseases and with adults with vitiligo. Children had an increased incidence of segmental vitiligo (p less than 0.01). Children had an increased incidence of autoimmune and/or endocrine disease and also of premature graying in their immediate and extended family members (p less than 0.001). Six of 33 children with vitiligo tested had positive organ-specific serum autoantibodies, which was a higher incidence than in the control group of children (p less than 0.05). Eighteen percent of children treated with topical psoralens and long-wave ultraviolet light (PUVA) therapy had an acceptable response, which was less than an adult group similarly treated. We have found childhood vitiligo to be a distinct subset of vitiligo, showing increased segmental presentation; strong autoimmune and/or endocrine disease background and high incidence of premature graying in the families of affected children; the presence of organ-specific serum autoantibodies and a poor response to topical PUVA therapy.

Determination of optimal topical photochemotherapy for vitiligo.

The efficacy of topical 8-methoxypsoralen (8-MOP) in varying concentrations and vehicles was assessed in 73 vitiligo patients. The response rates in different anatomic sites were also assessed. Seven patients (9%) had 100% repigmentation; 26 (36%) had 50% or greater repigmentation, 29 (40%) had some degree of pigment return, but less than 50%; 11 (15%) had no repigmentation. Results suggest that neither concentration of drug nor vehicle is a crucial factor for inducing repigmentation of vitiliginous patches on the face, trunk, and extremities. Low-dose 8-MOP (0.1%) was as effective as high dose 8-MOP (0.5%, 1%), while causing fewer side effects. However, when treating recalcitrant areas (distal extremities), 1% 8-MOP may be the most efficacious preparation for topical photochemotherapy. A phototoxic response preceded repigmentation in all cases. The following responses were obtained for the various anatomic sites treated: 56% of facial lesions; 35% of trunk areas; 36% of the extremities, and 13% of the recalcitrant areas had greater than 50% repigmentation.