Stability of the mercaptobenzothiazole compounds.

An analytical quantitative high-pressure liquid chromatography (HPLC) method was developed for simultaneous determination of all mercaptobenzothiazole derivatives in the mercapto mix patch testing standard. The stability of the mercaptobenzothiazoles constituting the mercapto mix was studied both in petrolatum and in buffer solution at pH 6.5, with and without glutathione. In petrolatum vehicle, dibenzothiazyl disulfide was the dominant compound found in stored mercapto mix. In buffer solution at pH 6.5, 2-mercaptobenzothiazole and the sulfenamide derivatives morpholinyl mercaptobenzothiazole and N-cyclohexyl-2-benzothiazyl sulfenamide were converted into dibenzothiazyl disulfide. In the presence of glutathione, both the sulfenamide derivatives and the dibenzothiazyl disulfide were rapidly converted into 2-mercaptobenzothiazole. The findings explain the "cross-sensitivities" reported for the mercaptobenzothiazole group as a result of chemical reactions resulting in one main hapten. The use of a single substance for patch testing for mercaptobenzothiazole hypersensitivity is proposed.

High performance concentration and gel filtration of rat urinary protein allergens.

Allergies to laboratory animals, notably rats, have become an increasingly recognized occupational problem. Identification and isolation of the individual proteins causing allergic reactions, could form the basis for early recognition of sensitivity, diagnosis, control of degree of pollution of the environment and desensibilization treatments. Frequently, allergens originate from dried rat urine. Because earlier published methods were found unsatisfactory we have developed a new strategy for isolation of rat urinary proteins including a high performance technique for their mild concentration on hydroxyapatite. The concentrated allergens have been fractionated according to molecular size by high performance gel filtration and according to carbohydrate content by wheat germ lectin-Sepharose 6 MB affinity chromatography. The obtained fractions have been examined by denaturing and non-denaturing polyacrylamide gradient gel electrophoresis followed by sensitive staining procedures, and tested with respect to allergenicity by skin tests on allergic patients.

Sensitization studies in the guinea pig with the active ingredients of Euxyl K 400.

The preservative Euxyl K 400 consists of the 2 active ingredients, 2-phenoxyethanol and 1,2-dibromo-2,4-dicyanobutane. Sensitization studies with the guinea pig maximization test were performed with these substances, but no sensitizing capacity was demonstrated in the case of either compound.

Allergy to laboratory animals in laboratory technicians and animal keepers.

The prevalence of allergy to laboratory animals (LAA) was investigated in laboratory technicians and animal keepers. In a questionnaire 41 of 101 technicians reported symptoms provoked by work with laboratory animals. On clinical investigation 30 were found to have symptoms and signs related to contact with animals, and allergy was confirmed by radioallergosorbent tests (RAST) and skin tests in 19. All had rhinitis and 10 also had bronchial asthma. Forty seven other technicians who had stopped working with laboratory animals showed the same relative numbers of respiratory tract symptoms and of confirmed allergy to laboratory animals as did those currently handling animals. Seven of 23 animal keepers had work related symptoms. LAA symptoms were found in four and confirmed animal allergy in two. All four animal keepers with animal related symptoms had rhinitis, none had bronchial asthma. Positive animal RAST and skin tests were found only among people with animal related symptoms. A history of atopic disease was commoner among those with positive animal test results than among those with negative test results. No relation between smoking and the development of allergy to laboratory animals emerged. Simple prophylactic measures often sufficed to help technicians with animal related symptoms to remain at work.

Contact urticaria in laboratory technicians working with animals.

The occurrence of contact urticaria was studied in 101 laboratory technicians investigated for allergy to laboratory animals. Fourteen cases of contact urticaria caused by rat were found. Other animals causing contact urticarial reactions were mouse (7), guinea-pig (4), and cat (2). Hand eczema was not found in laboratory technicians with contact urticaria and IgE-antibodies to laboratory animals.

Pompholyx--a one year series.

During one year 68 patients with pompholyx attending the outpatient clinic at the Department of Dermatology, Lund, were examined with special regard to occupational disability. Owing to pompholyx 56 patients had sick-leave, and in 48 sick-leave lasted more than 1 month. No patient needed change of occupation because of pompholyx, and no disability pension was granted because of the disease.

Methaemoglobin-catalysed formation of dopa and 6-OH-dopa from tyrosine.

An extract of bovine retina and choroid with tyrosine hydroxylating and dopa-oxidizing capacity also showed marked formation of 6-OH-dopa on incubation with tyrosine and ascorbic acid. The extract contained appreciable amounts of methaemoglobin as determined spectrophotometrically, and boiled extracts also showed catalytic activity. The effect of methaemoglobin on the oxidation of tyrosine and dopa was therefore investigated. Methaemoglobin catalysed the formation of dopa and 6-OH-dopa in the presence of tyrosine and ascorbic acid. Hydrogen peroxide plays an important role in this reaction: the rates of formation of both dopa and 6-OH-dopa were increased by addition of hydrogen peroxide but diminished by addition of catalase. Methaemoglobin also catalysed the formation of cysteinyldopa from dopa and cysteine.

Diastereomers of 5-S-cysteinyldopa.

Diastereomers of 5-S-cysteinyldopa formed from D-dopa and L-cysteine or from L-dopa and D-cysteine can be separated from 5-S-cysteinyldopa formed from L-dopa and L-cysteine by liquid chromatography. The diastereomers have a great potential as internal standards in the analysis of 5-S-cysteinyldopa. They can be used as reference substances in the differentiation of stereospecific enzymatic oxidation of dopa from non-specific oxidation.

5-OH-dopa, product of and substrate for tyrosinase.

The formation of 5-OH-dopa on incubation of tyrosine or dopa with mushroom tyrosinase was studied. Dopa oxidase activity was defined by measuring the formation of 5-S- and 2-S-cysteinyldopa from dopa in the presence of excess amounts of cysteine. This procedure quantitating the immediate nucleophilic reaction products of dopaquinone constitutes a new method for assessing tyrosinase activity. The rate of 5-OH-dopa formation from dopa was similar to that of the formation of dopa from tyrosine. The rate of dopaquinone formation was one order of magnitude higher. When tyrosine was oxidized in the presence of ascorbic acid, 5-OH-dopa formation represented 14% of the original amount of tyrosine. At low concentration of dopa, formation of 5-OH-dopa was proportional to the dopa concentration. At high concentrations the relative quantity of 5-OH-dopa formed decreased. Tyrosine in high concentrations inhibited the formation of 5-OH-dopa from dopa. 5-OH-dopa proved to be a substrate for tyrosinase. The rate of oxidation of 5-OH-dopa was similar to that of dopa. The oxidation products of 5-OH-dopa were transformed into relatively stable fluorophores.

The effect of cysteine on oxidation of tyrosine, dopa, and cysteinyldopas.

The influence of cysteine on the oxidation of tyrosine, dopa, and monocysteinyldopas by mushroom tyrosinase was reexamined. During oxidation of tyrosine in the presence of cysteine the concentration of dopa increased slowly, whereas the concentration of cysteinyldopas increased more rapidly. When the concentration of cysteine decreased the cysteinyldopas were rapidly consumed and dopa concentrations increased sharply. Experiments on the oxidation of dopa by tyrosinase in the presence of cysteine showed that this thiol does not inhibit the oxidation. Dopa concentrations decreased more rapidly in the presence of cysteine because cysteine addition to dopaquinone prevented reformation of dopa from dopaquinone. Both 2-S-cysteinyldopa and 5-S-cysteinyldopa are substrates for tyrosinase. The oxidation of cysteinyldopas was inhibited at high cysteine concentrations. The greater part of 2,5-S,S-dicysteinyldopa formed during the oxidation of monocysteinyldopas in the presence of cysteine is derived from 5-S-cysteinyldopa, which is a better substrate for tyrosinase than 2-S-cysteinyldopa. The fact that cysteine binds more rapidly to 5-S-cysteinyldopaquinone than to 2-S-cysteinyldopaquinone further stresses the importance of 5-S-cysteinyldopa in the formation of 2,5-S,S-dicysteinyldopa. Oxidation of dopa in the presence of cysteine and glutathione or methionine showed that glutathione is added to dopaquinone but less rapidly than cysteine. Methionine showed insignificant addition to dopaquinone. When dopa or 5-OH-dopa is added to an incubate of cysteinyldopa and tyrosinase the oxidation of cysteinyldopa is accelerated owing to oxidation of cysteinyldopa by dopaquinone or 5-OH-dopaquinone.

Clinical trial of a potent non-halogenated topical steroid, Budesonide.

In a double-blind, randomized multi-centre study, 116 patients with psoriasis have been treated for 1-3 weeks with budesonide ointment (Preferid, Draco/Tika; a subsidiary of AB ASTRA), a new non-halogenated topical steroid. In a series of 11 patients a 0.025% budesonide ointment was significantly superior to placebo. In a second series, of 54 patients, a 0.025% fluocinolone acetonide ointment (Synalar, ICI). In a third series, of 51 patients, a 0.010% budesonide ointment was compared with 0.025% fluocinolone acetonide ointment. No statistically significant difference between these two preparations was found to exist. No adverse reactions were observed.

Dopa and 5-S-cysteinyldopa in the serum of albino, black, and red guinea pigs.

Dopa and 5-S-cysteinyldopa levels in serum of albino, red, and black guinea-pigs were quantified. The levels of both amino acids were lower in the albino than in the pigmented animals. It is suggested that the serum levels of dopa and 5-S-cysteinyldopa in pigmented animals reflect tyrosine and dopa oxidation activity in the melanocytes.

5-S-cysteinyldopa, dopa, and dopamine in the kidney and some other tissues.

5-S-Cysteinyldopa, dopa, and dopamine levels were determined by HPLC and electrochemical detection in serum, urine, kidney, spleen, heart, and sympathetic ganglion of guinea-pig. The kidney showed high levels of 5-S-cysteinyldopa, but also of dopa and dopamine, thus demonstrating the role of the renal ti-sue in the excretion of these substances. The concentrations found in spleen and heart may indicate specific catechol-containing structures in these organs.

Treatment of malignant melanoma with dacarbazin (DTIC-DOME) with special reference to urinary excretion of 5-S-cysteinyldopa.

Seventeen patients were given DTIC, 200 mg/m2/day in five-day courses every four to six weeks. In four patients (stage II) treated on an adjuvant basis, tumor recurrence has been verified in three. Four of the palliatively treated patients were also given DTIC by regional intra-arterial infusion with minimal positive tumor effect and minimal toxicity. 5-S-cysteinyldopa excretion in urine was checked continuously in all patients. Tumor recurrence was revealed in two patients given DTIC on an adjuvant basis three and four months before clinical signs of tumor. In the palliatively treated patients, 5-S-cysteinyldopa excretion increased in 5/6 patients judged to have stable disease, before tumor progression was clinically detectable. The use of 5-S-cysteinyldopa examination is a valuable adjunct to the follow-up of the effect of DTIC therapy in melanoma patients.

Two-step leukocyte migration in agarose technique.

A two-step modification of Clausen's technique of leukocyte migration under agarose is described. Blood leukocytes from a subject are incubated for approximately 4 days with or without antigen. The cultures, after reconstitution with antigen followed by repeated washing of the cultured cells, are added to fresh, dextran-separated blood leukocytes, and migration tests are performed in the absence of the antigen. Cells preincubated with antigen give a very marked reduction of the migration of the target leukocytes which must not necessarily be autologous. An inhibition of target cell migration can be seen with cells cultured without antigen for up to 3 days; with longer culture time, this effect is reduced or disappears. The effect of cells cultured with antigen, however, is equally strong irrespective of the culture time up to 1 week, and the most clearcut results will, therefore, be obtained after approximately 4 days culture. Also migration enhancement has been seen. Treatment of cells during the incubation with antigen and cycloheximide gives a dose-dependent blocking of the inhibiting effect. 5 patients were studied both before and after sensitization with DNCB: low concentrations of DNCB gave an inhibition and high concentrations an enhancement of the migration when the patients were sensitized. The technique is more sensitive and reproducible than the original Clausen technique. It is also suitable in studies where shortage of cells is a problem.

Intracellular distribution of dopa and 5-S-cysteinyldopa in pigment cells with minimal pigment formation.

The hypothesis that only melanosomal catecholic amino acids contribute to melanin formation was tested by studying adult bovine eyes in which pigment synthesis is considered to be low or absent. Dopa and 5-S-cysteinyldopa were investigated in different cell fractions of the choroid and retinal pigment epithelium of cattle. Most of the dopa and 5-S-cysteinyldopa was found in the cytoplasm and very little in the large granule fraction. The presence of cysteinyldopa in the adult eye is evidence of tyrosinase activity, but the catechol amino acids in the cytoplasm probably do not give rise to melanin formation. It is assumed that they instead are excreted from the cells.

Analysis of cysteinyldopas, dopa, dopamine, noradrenaline and adrenaline in serum and urine using high-performance liquid chromatography and electrochemical detection.

The catecholic amino acids, dopa, 2-S- and 5-S-cysteinyldopa, and 2,5-S,S-dicysteinyldopa were determined qualitatively in serum from patients with malignant melanoma by reversed-phase high-performance liquid chromatography, using electrochemical detection. In urine the catecholamines dopamine, noradrenaline and adrenaline were also determined qualitatively, as well as the above-mentioned compounds, in a single chromatographic run. The conditions were optimized by changing the pH of the mobile phase and by the addition of methanesulphonic acid. A comparison was made between the performance of four commercial reversed-phase packing materials containing chemically bonded octadecyl groups, using a standard mixture of catecholic amino acids. The influence of ionic strength, pH and amount of methanesulphonic acid on retention was investigated.