Effects of sample matrix in the measurement of antithrombin by LC-MS: A role for immunocapture.

Introduction: The sample matrix composition, which is greatly affected by the type of blood collection tube used during phlebotomy, is of major importance in laboratory testing as it can influence test results. We developed an LC-MRM-MS test to molecularly characterize antithrombin in citrate plasma. The test principle differs greatly from traditional laboratory tests and the influence of varying plasma sample matrices is largely unknown. Objectives: To identify whether variations in sample matrix affect the LC-MRM-MS test for antithrombin and assess whether sample pre-processing by immunocapture reduces matrix-specific effects. Methods: Samples (n = 45) originating from four different blood collection tubes (sodium citrate, lithium heparin, K2-EDTA and K2-EDTA with protease inhibitors) were processed directly or after immunocapture. Antithrombin was digested into proteotypic peptides, which were monitored by LC-MRM-MS. Results from lithium heparin and the K2-EDTA matrices were compared to the standard sample matrix, sodium citrate, using Deming regression analysis and repeated measures one-way ANOVA. Results: Deming regression analysis of directly processed samples revealed slopes deviating >5% from the line of identity for at least six out of 22 peptides in all matrices. Significant differences between all matrices were found upon analysis by ANOVA for at least 10 peptides. Pre-processing by immunocapture led to slopes within 5% of the line of identity for nearly all peptides of the matrices. Furthermore, significant differences between matrices after immunocapture were only observed for four peptides. Conclusion: Variations in the sample matrix affect the measurement of antithrombin by LC-MRM-MS, but observed effects are greatly reduced upon pre-processing by immunocapture.

The effect of RBC transfusions on cytokine gene expression after cardiac surgery in patients developing post-operative multiple organ failure.

AIM: To determine the effect of red blood cell (RBC) transfusions during cardiac surgery on cytokine gene expression (GE) in relation to multiple organ failure (MOF) development after systemic inflammatory response syndrome (SIRS). BACKGROUND: RBC transfusion in cardiac surgery patients is dose-dependently associated with post-operative MOF, possibly acting as a second hit after cardiopulmonary bypass. METHODS: For this observational study, 29 patients divided into four groups of cardiac surgery patients were selected from a randomised controlled trial (RCT). Group 1: no-RBC, no-MOF (N = 8); group 2: MOF, no-RBC (N = 7); group 3: RBC, no-MOF (N = 6); group 4: RBC and MOF (N = 8). Selection was based on age, gender, number of (leukocyte-depleted) RBC transfusions, type and duration of surgery. A 114 cytokine GE array was applied to blood samples withdrawn before and 24 h after surgery. Expression of selected genes was confirmed with reverse transcriptase real time-polymerase chain reaction (RT-PCR). RESULTS: Nineteen of the 39 detectable genes showed a significant change in GE after surgery. Confirmed by RT-PCR, transfused MOF patients exhibit significantly less downregulation of CD40 ligand than control patients. Patients who would develop MOF show significantly larger increases in GE of transforming growth factor-α (TGF-α), tumour necrosis factor (TNF)-superfamily members 10 and 13B (TNFsf10/13B). CONCLUSIONS: When tested at 24 h after surgery, cytokine GE in peripheral blood leucocytes showed no significant differences between those transfused and those not transfused. Some alterations were seen in those developing MOF compared to those who did not, but the findings offer no role of leukocyte depleted (LD) RBC transfusion in the development of MOF.

Regulatory mechanisms of calcineurin phosphatase activity.

Calcineurin (protein phosphatase 3, Cn) is best known for its central position in Ca(2+)-dependent T-cell signaling. Interest in calcineurin has, however, conserved its momentum as new Ca(2+)-dependent pathways have been steadily surfacing in several other cell types, such as brain, heart, skin cells and beta pancreatic cells, and Cn appears to serve as a central controller of stress, immune response, and cellular proliferation and differentiation. Calcineurin is the principal target of the immunosuppressive drugs cyclosporin A (CsA) and tacrolimus (TRL). Therapy based on these immunosuppressants has markedly reduced the incidence of transplant rejection in allograft recipients. In addition, these drugs have proven very useful for patients suffering from chronic inflammatory skin conditions. Unfortunately, their application is somewhat limited by a broad spectrum of toxic side-effects, affecting several organ systems. This calls for enhancements in the design of this class of immunosuppressants. An intricate constellation of regulatory systems allows for precise modulation and adaptation of calcineurin activity in vivo. The last few years have been very fruitful in elucidating several long-standing issues regarding the binding patterns of substrates and inhibitors to Cn. This new knowledge may enable more precise manipulation of the Ca(2+)-calcineurin pathway in the near future, preferably targeted towards one specific substrate or cell system. In this review, we will discuss the factors and mechanisms underlying calcineurin activity regulation and their exploitation in recent approaches towards better immunosuppressants.

Non-transferrin bound iron measurement is influenced by chelator concentration.

Release of non-protein bound iron plays an important role in the toxicity inflicted by chemotherapy in cancer patients. Since large variations have been described for different methods measuring non-transferrin bound iron (NTBI), we aimed to obtain more accurate values. After binding to the chelator nitrilotriacetic acid disodium salt (NTA) and ultrafiltration, the NTBI can be measured spectrophotometrically by the addition of thioglycolic acid (TGA) and baptophenanthroline disulfonic acid (BPT). Results demonstrated that NTBI values increased with NTA concentration. In samples incubated with 80 mM NTA, >5-fold higher NTBI values were found compared to using 10 mM NTA. Optimal concentration of NTA was established by additions of iron to serum with known latent iron-binding capacity (LIBC). Iron addition curves showed that NTBI could be measured starting from the LIBC of the serum with optimal yield after incubation with 4 mM NTA in 5 mM Tris-HCl pH 6.5, with 3mM TGA and 6.2 mM BPT for the colour reaction. The results showed excellent correlation with 195 samples measured also by HPLC. For the spectrophotometric method, significantly higher NTBI values were measured in patient samples with maximal iron saturation compared to patients with lower iron saturation.

Calcineurin activity and inhibition in skin and (epi)dermal cell cultures.

Calcineurin (Cn) is the target of the immunosuppressive drugs cyclosporine A (CsA), tacrolimus (Trl), and pimecrolimus (Prl). Trl and Prl are often used topically for treatment of various skin diseases. The Cn inhibitors CsA and Trl are mostly used for maintenance therapy of transplant patients. Their long-term use, however, causes a dramatic increase in skin cancer risk. By using a newly developed assay for Cn measurement in blood, we were able to demonstrate Cn activity in total skin homogenates. A significantly higher activity was found in epidermis compared to dermis. In skin cell cultures, fibroblasts showed the highest activity as compared to keratinocytes and melanocytes. Of the Cn inhibitors, Trl showed stronger inhibition than CsA and Prl (57 and 55% in fibroblast and keratinocyte cultures, respectively). Also, the lowest IC(50) (the half maximal inhibitory concentration) values were found for Trl (0.5 and 1.3 nM in two different fibroblast cultures). Cn activity and its inhibition can thus be studied in dermatological samples. The effects of Cn inhibition in fibroblasts and keratinocytes may be of influence on the overall functioning of the skin immune system.

Ultraviolet-A (UVA-1) radiation suppresses immunoglobulin production of activated B lymphocytes in vitro.

Previous studies have shown that low-dose ultraviolet-A (UVA-1) total body irradiations were capable of improving disease activity in patients with systemic lupus erythematosus (SLE). We hypothesized that UVA-1-induced suppression of immunoglobulin production by activated B cells in the dermal capillaries could be (partly) responsible for this effect. Our experiments with donor skin demonstrated that approximately 40% of UVA-1 could penetrate through the epidermis. Irradiation of peripheral blood mononuclear cells (PBMCs) with 2 J/cm(2) of UVA-1 resulted in 20% cell death. This toxic effect could be prevented totally by preincubation of the cell cultures with catalase. This indicates that the generation of hydrogen peroxide plays a role in UVA-1 cytotoxicity. T cells and B cells appeared to be less susceptible to UVA-1 cytotoxicity than monocytes. With the use of a CD40-CD40L B cell activation method we measured immunoglobulin production after various doses of UVA-1 irradiation (0-2 J/cm(2)). The doses of 2 J/cm(2) caused a significant decrease of IgM, IgG, IgA and IgE production under the conditions of interleukin (IL)-10 or IL-4 (IgE) stimulation. Although UVA-1 can cause apoptosis of B lymphocytes, we show that relatively low doses of UVA-1 radiation also affect the function of these cells. Both effects may be responsible for the observed improvement of disease activity in SLE patients.

Glutamate receptors on human melanocytes regulate the expression of MiTF.

Glutamate is the major excitatory neurotransmitter in the central nervous system but has also important functions in the epidermis. It is involved in keratinocyte barrier function and in re-epithelialization processes after wounding. Recently, glutamate signalling has been suggested to be implicated in the development of melanoma. The present study examined the expression and functionality of metabotropic and ionotropic glutamate receptors on normal human melanocytes. We found that cultured melanocytes expressed the ionotropic glutamate receptors GluR2 and 4 [alpha-amino-3-hydroxy-5-methyl-4-isoxsazolepropionic acid (AMPA) receptors] and N-methyl-d-aspartate (NMDA) receptors 2A and 2C and possibly the metabotropic glutamate receptor 1. Melanocytes were also found to express specific glutamate transporters and decarboxylases, but appeared neither to produce nor to release l-glutamate. Stimulation with 10 or 100 microM AMPA or NMDA elevated intracellular calcium concentrations in melanocytes, and thus demonstrated the functionality of the glutamate receptors. Millimolar concentrations of l-glutamate did not induce melanocyte toxicity and had no stimulating effect on melanin production. However, blockage of AMPA and NMDA receptors with CFM-2, memantine or MK801 caused a rapid and reversible change in melanocyte morphology, which was associated with disorganisation of actin and tubulin microfilaments. After 24 h of treatment with the AMPA receptor inhibitor CFM-2, there was a sharp reduction in the expression of the crucial melanocyte differentiation and proliferation factor MiTF. The results of this study demonstrate a role for glutamate in melanocyte regulation that may have implications in melanocyte associated disorders.

[Risk factors for skin melanoma: genetic factors probably more important than exposure to sunlight]. / Risicofactoren voor huidmelanoom: genetische factoren waarschijnlijk belangrijker dan expositie aan zonlicht.

Pigmented naevi (moles) are increasingly regarded as risk factors for the development of melanoma. The probability of melanoma developing from congenital naevi is proportional to the volume of the naevi. The risk of melanoma development from large naevi (diameter > 20 cm) is already present in the early years of childhood. The most important risk factor is the higher number of acquired naevi. This applies particularly to dysplastic (also called clinically atypical) naevi that not only represent the highest risk group but are also considered potential melanoma precursors. The development of acquired naevi (including dysplastic naevi) is dependant on the degree of skin pigmentation. The role of sunlight (ultraviolet radiation) in the development of melanoma is less significant than is generally assumed. The indirect effect of sunlight on melanoma development is to stimulate naevogenesis. One of the risk-modifying genes is the gene coding for melanocortin-1-receptor (MC1R). The presence of some gene variants has been found to lead to changes in melanin synthesis and is associated with a higher risk of melanoma. Recent research has shown that dysplastic naevi synthesise more phaeomelanin. There are also strong indications that dysplastic naevus cells suffer from chronic oxidative stress. This situation can lead to hypermutability and genetical instability.

Melanin has a role in Ca2+ homeostasis in human melanocytes.

We have examined whether melanin affects Ca2+ homeostasis in cultured normal human melanocytes. Intracellular Ca2+ concentrations ([Ca2+]i), were measured in four Caucasian and in three Negroid melanocyte cultures. Under resting conditions [Ca2+]i was around 100 nM in all cultures, but differences between cells within cultures were observed. All cultures responded to endothelin-1 (ET-1) with increases in [Ca2+]i and there were no differences between Caucasian and Negroid cultures. However, large differences in responses between cells within cultures were observed, indicating that melanocyte cultures are very heterogeneous. The addition of 2.5 mM CaCl2 to melanocytes kept in Ca2+-free medium resulted in rapid and transient increases in [Ca2+]i of up to 1500 nM. These increases were on average more than two times smaller in melanocyte cultures established from Negroid donors compared with Caucasian cultures. In addition, well melanized Caucasian melanocytes, cultured in the presence of 400 microM tyrosine and 10 mM NH4Cl, showed a reduced increase in cytoplasmic Ca2+ concentration upon the addition of extracellular Ca2+. The difference in maintaining Ca2+ homeostasis between poorly and well melanized melanocytes may be the result of the clearance of cytoplasmic Ca2+ into melanosomes and the greater capacity for this in the more pigmented melanocytes.

Ligand-dependent activation of the melanocortin 5 receptor: cAMP production and ryanodine receptor-dependent elevations of [Ca(2+)](I).

The melanocortins are involved in the regulation of various cognitive and physiological processes such as learning, feeding, immune suppression, pigmentation, and sebum production. Five melanocortin receptors have been identified, of which the melanocortin 5 receptor (MC5R) has the most widespread distribution. This subtype is found in the brain, and at numerous peripheral sites including the skin where it is expressed in the sebaceous glands. The purpose of this study was to identify the peptide that functions as a natural ligand at the MC5R in the skin. alpha-MSH, ACTH1-39, ACTH1-17, ACTH1-10, and ACTH4-10 all increased the production of cAMP in HEK293 cells transfected with the mouse MC5R. alpha-MSH and ACTH1-17 were the most potent in this respect. In addition, all peptides stimulated a rapid and transient increase in [Ca(2+)](i), and, ACTH1-10 was the most potent. The increases in [Ca(2+)](i) were of intracellular origin, but not associated with inositol phosphate production. The elevations in [Ca(2+)](i) were reduced by ruthenium red and procaine and it is therefore possible that they were mediated via ryanodine receptors.

Melanin offers protection against induction of cyclobutane pyrimidine dimers and 6-4 photoproducts by UVB in cultured human melanocytes.

The goal of this investigation was to correlate the melanin content in human pigmentary cells with the generation of UVB-induced photoproducts and to examine the relationship between the melanin content and the removal of the photoproducts. Cultured melanocytes from light-skinned individuals synthesized less melanin and produced more cyclobutane pyrimidine dimers and 6-4 photoproducts upon UVB exposure than did melanocytes from black skin. Tyrosine-stimulated melanogenesis provided protection against DNA damage in both cell types. In another set of pigmented cell lines a ratio between eumelanin and pheomelanin was determined. The assessment of association between DNA damage induction and the quantity and quality of melanin revealed that eumelanin concentration correlated better with DNA protection than pheomelanin. Skin type-I and skin type-VI melanocytes, congenital nevus (CN)-derived cells and skin type-II melanocytes from a multiple-melanoma patient were grown in media with low or high L-tyrosine concentration. The cells were irradiated with 200 J/m2 UVB, and the levels of the photoproducts were determined immediately and after 6 and 24 h. Once again the induction of the photoproducts was mitigated by increased melanogenesis, and it was inversely correlated with the skin type. No significant differences were found for the removal of photoproducts in the cultures of skin types I and VI and CN cells. No indications of a delay in the removal of photoproducts in the melanocytes from the multiple-melanoma patient were found either.

Melanin content of cultured human melanocytes and UV-induced cytotoxicity.

Cultured melanocytes originating from persons with different skin phototypes were utilized for measurement of endonuclease sensitive sites induced by UVB and the determination of cell survival after UVA or UVB irradiation. During culture, the melanocytes largely maintained their phenotypic characteristics according to their original skin phototype. Total melanin concentrations were 4.9 times higher in the darker skin phototype (IV-VI) melanocytes when compared to the cells from lighter skin phototypes (I-III). Also phaeomelanin contents were higher (2.5 times) in the skin phototype (IV-VI) melanocytes which implies that the cells from light skin types contain less melanin, but a relatively high proportion of phaeomelanin. After UVB irradiation a stronger induction of endonuclease sensitive sites was found for melanocytes with a lower level of total melanin and a high content of pheomelanin. By measuring the clone forming ability in different melanocyte cultures after UVB irradiation, significant better survival was found in case of the cells with the higher melanin content. Despite the large variations in melanin content, no significant difference in survival after UVA irradiation could be demonstrated in this way. Our results suggest a protective effect of melanin for UVB and indicate the importance of the measurements of melanin content and composition when different parameters of UV-induced damage are studied in melanin producing cells.

Melanosomal pH controls rate of melanogenesis, eumelanin/phaeomelanin ratio and melanosome maturation in melanocytes and melanoma cells.

The skin pigment melanin is produced in melanocytes in highly specialized organelles known as melanosomes. Melanosomes are related to the organelles of the endosomal/lysosomal pathway and can have a low internal pH. In the present study we have shown that melanin synthesis in human pigment cell lysates is maximal at pH 6.8. We therefore investigated the role of intramelanosomal pH as a possible control mechanism for melanogenesis. To do this we examined the effect of neutralizing melanosomal pH on tyrosinase activity and melanogenesis in 11 human melanocyte cultures and in 3 melanoma lines. All melanocyte cultures (9 of 9) from Caucasian skin as well as two melanoma cell lines with comparable melanogenic activity showed rapid (within 24 h) increases in melanogenesis in response to neutralization of melanosomal pH. Chemical analysis of total melanin indicated a preferential increase in eumelanin production. Electron microscopy revealed an accumulation of melanin and increased maturation of melanosomes in response to pH neutralization. In summary, our findings show that: (i) near neutral melanosomal pH is optimal for human tyrosinase activity and melanogenesis; (ii) melanin production in Caucasian melanocytes is suppressed by low melanosomal pH; (iii) the ratio of eumelanin/phaeomelanin production and maturation rate of melanosomes can be regulated by melanosomal pH. We conclude that melanosomal pH is an essential factor which regulates multiple stages of melanin production. Furthermore, since we have recently identified that pink locus product (P protein) mediates neutralization of melanosomal pH, we propose that P protein is a key control point for skin pigmentation. We would further propose that the wide variations in both constitutive and facultative skin pigmentation seen in the human population could be associated with the high degree of P-locus polymorphism.

Study of DT-diaphorase in pigment-producing cells.

DT-diaphorase is an FAD-containing enzyme capable of a two-electron reduction of ortho- and paraquinones. Nicotinamide coenzymes (NADH + H+ and NADPH + H+) serve as hydrogen sources in these reactions. The role of DT-diaphorase has been thoroughly investigated in situations when the enzyme is able to reduce exogenous and endogenous quinones, hence protecting the cells against these reactive intermediates. The enzyme has also been studied in connection with its ability to activate some quinoid cytostatics. It is surprising that DT-diaphorase has never been investigated in pigment-producing cells that are known to generate considerable amounts of ortho-quinones. Using a spectrophotometric method we could readily measure the activity of DT-diaphorase in epidermis and various cultured pigment cells. The melanocytes isolated from dark skin showed generally higher DT-diaphorase activity than those from fair skin samples. Also, darkly pigmented congenital naevus cells exhibited higher activity of this enzyme. The most striking was the high DT-diaphorase activity in melanoma cell cultures. In these cells DT-diaphorase activity could be induced by incubation of the cells with 4-hydroxyanisole. A similar effect was seen when a catechol-O-methyltransferase (COMT) inhibitor (3-(3,4-dihydroxy-5-nitrobenzylidene)-2,4-pentanedione (OR-462) was utilised. The induction was inhibited by cyclohexidine.

(Pheo)melanin photosensitizes UVA-induced DNA damage in cultured human melanocytes.

The question of whether melanins are photoprotecting and/or photosensitizing in human skin cells continues to be debated. To evaluate the role of melanin upon UVA irradiation, DNA single-strand breaks (ssb) were measured in human melanocytes differing only in the amount of pigment produced by culturing at two different concentrations, basic (0.01 mM) or high (0.2 mM), of L-tyrosine, the main precursor of melanin. In parallel, pheo- and total melanin contents of the cells were determined. Identical experiments were performed with two melanocyte cultures derived from a skin type I and a skin type VI individual. For the first time the correlation between UVA-induced genotoxicity and pheo-/total melanin content has been investigated. We observed that cultured in basic medium, the skin type VI melanocytes contained 10 times more total melanin and about seven times more pheomelanin than the skin type I melanocytes. Elevation of tyrosine level in the culture medium resulted in an increase of both pheo- and total melanin levels in both melanocyte cultures; however, the melanin composition of skin type I melanocytes became more pheomelanogenic, whereas that of skin type VI melanocytes remained the same. The skin type VI melanocytes cultured in basic medium demonstrated a very high sensitivity (1.18 ssb per 10(10) Da per kJ per m2) toward UVA that is probably related to their high pheo- and total melanin content. Their UVA sensitivity, however, did not change after increasing their melanin content by culturing at high tyrosine concentration. In contrast, the skin type I melanocytes demonstrated a low sensitivity (0.04 ssb per 10(10) Da per kJ per m2) toward UVA when cultured in basic medium, but increasing their melanin content resulted in a 3-fold increase in their UVA sensitivity (0.13 ssb per 10(10) Da per kJ per m2). These results demonstrate that UVA-irradiated cultured human melanocytes are photosensitized by their own synthesized chromophores, most likely pheomelanin and/or melanin intermediates.

Variations in melanin formation by cultured melanocytes from different skin types.

In many laboratories, culturing skin melanocytes has become a routine research activity. However, recent investigations have revealed that the quality and quantity of the pigment formed in the cultured cells may differ significantly from those of the original skin pigment cells. To shed more light on this issue, we examined the influence of different culture media on pigment production. We showed that there were notable passage-to-passage variations in the synthesis of melanin. This was particularly true for phaeomelanin. It is therefore advisable to analyse the melanin in the cells before the start of experiments. In spite of the variations, basic differences in the pigmentation pattern between melanocytes isolated from light-skinned and dark-skinned individuals remained preserved in the corresponding cultures as observed by electron microscopy. Also, the total melanin content was higher in a skin type VI melanocyte culture than in skin type I and II melanocyte cultures. In contrast to total melanin, the phaeomelanin concentration of skin type VI cells was similar to that of the skin type I melanocytes. With higher L-tyrosine concentrations in the medium, as well as increased eumelanin synthesis, phaeomelanogenesis was also stimulated in all cultures tested. This stimulation was particularly prominent in skin type I melanocytes. Our preliminary experiments also showed that a melanocyte culture from atypical naevus cells exhibited a similar preference for phaeomelanogenesis when pigmentation was stimulated.

Melanogenesis in cultured melanocytes can be substantially influenced by L-tyrosine and L-cysteine.

We investigated the effect of varying concentration of 1-tyrosine and 1-cysteine in culture medium on melanin production by human skin melanocytes (skin phototype II/III). In addition to the analyses of dopa oxidase activity and total melanin, pheomelanin production in the cells was assessed by high-performance liquid chromatography determinations of pheomelanin degradation products, 3-aminotyrosine and 4-amino-3-hydroxyphenylalanine. As another marker for pheomelanin, melanosomal sulfur was determined by the use of X-ray microanalysis. With varying concentration of both amino acids, profound changes in the pigmentation patterns of the melanocytes were observed. A high concentration of 1-tyrosine (0.2 mM) was always connected with increased pigmentation. In combination with a low 1-cysteine content we saw an increase in tyrosinase activity and the highest melanin content. At high concentrations of both 1-tyrosine and 1-cysteine, the melanocytes showed reduced tyrosinase activity and they produced notably more pheomelanin. In case of the pheomelanin measurements by high-performance liquid chromatography and the sulfur detection with X-ray microanalysis, strongly increased concentrations were found when cells were maintained in high 1-tyrosine medium as compared with those grown with low 1-tyrosine. This was especially true for the combination with low 1-cysteine showing that the 1-tyrosine content of the medium strongly influences not only the eumelanin but also the pheomelanin production in the cultured melanocyte. It can be concluded that variations in the concentrations of 1-tyrosine and 1-cysteine in culture medium can be used to regulate the melanogenetic phenotype under in vitro conditions.

Determination of pheomelanin by measurement of aminohydroxyphenylalanine isomers with high-performance liquid chromatography.

We describe an improved method for the analysis of pheomelanin in biological samples. The method is based on a chemical degradation of the melanin polymer and HPLC analysis of specific degradation products. Hydriodic hydrolysis provides 4-amino-3-hydroxyphenylalanine (AHP) and 3-amino-l-tyrosine (AT) which are detected with an electrochemical detector. We have examined each step of the analysis and the results are presented in this paper. First the samples are hydrolyzed for 16 h. AT and AHP are then isolated from the hydrolysates by ion-exchange chromatography and then separated and quantitated by HPLC and electrochemical detection. The method shows good reproducibility with a total imprecision below 5.6%. The linearity of the method was shown from 0 to 490 ng AT and 0 to 850 ng AHP per sample, using a melanoma cell suspension (27 mg protein/ml) with up to 24-fold dilutions of the original sample. For cultured "normal" human melanocytes a minimal amount of 0.1 mg protein is sufficient for analysis of pheomelanin in the samples. This method provides the opportunity to study the composition of the formed melanin in cell lines, cultured in different growth media.

Evidence of the indirect formation of the catecholic intermediate substrate responsible for the autoactivation kinetics of tyrosinase.

Tyrosinase (EC 1.14.18.1) exhibits unusual kinetic properties in the oxidation of monohydric phenol substrates consisting of a lag period that increases with increasing substrate concentration. The cause of this is an autocatalytic process dependent on the generation of a dihydric phenol substrate, which acts as an activator of the enzyme. Experiments with N-substituted dihydric phenol substrates (N-methyldopamine, N-acetyldopamine) demonstrate that oxygen consumption is retarded in the N-acetyl substituted material due to a diminished rate of cyclization. The oxygen uptake exhibited a similar pattern when N-acetyltyramine was oxidized, and this was reflected by a prolongation of the lag period. N,N-Dipropyldopamine was oxidized with normal kinetics but with an oxygen stoichiometry of 0.5 mol of oxygen/mol of substrate. We show that this is the result of the formation of a stable indoliumolate product with oxidation-reduction properties that prevent the formation of dopaminochrome, thus blocking further stages in the tyrosinase-catalyzed oxidation. Evidence that the indoliumolate product is formed by cyclization of the ortho-quinone is presented by pulse radiolysis studies, which demonstrate the formation of the ortho-quinone (by disproportionation of the corresponding semiquinones), which cyclizes to give the indoliumolate. The rate constant for cyclization was shown to be 48 s-1 (at pH 6.0). Tyrosinase-catalyzed oxidation of the monohydric phenol analogue, N, N-dimethyltyramine, was shown to require the addition of a dihydric phenol. Oxygen utilization then exhibited a stoichiometry of 1.0, indicating that the reactions proceed only as far as the cyclization. The analogous stable cyclic indoliumolate product was shown to be formed, with UV absorption and NMR spectra closely similar to the indoliumolate derived from N,N-dipropyldopamine. This material was methylated by catechol O-methyltransferase but was unreactive to redox reagents. The formation of the cyclic product accounts for the indefinite lag when N,N-dimethyltyramine is used as the substrate for tyrosinase in the absence of a dihydric phenol cofactor.