A comprehensive review on tyrosinase inhibitors.

Tyrosinase is a multi-copper enzyme which is widely distributed in different organisms and plays an important role in the melanogenesis and enzymatic browning. Therefore, its inhibitors can be attractive in cosmetics and medicinal industries as depigmentation agents and also in food and agriculture industries as antibrowning compounds. For this purpose, many natural, semi-synthetic and synthetic inhibitors have been developed by different screening methods to date. This review has focused on the tyrosinase inhibitors discovered from all sources and biochemically characterised in the last four decades.

Tyrosinase inactivation in its action on dopa.

Under aerobic or anaerobic conditions, tyrosinase undergoes a process of irreversible inactivation induced by its physiological substrate L-dopa. Under aerobic conditions, this inactivation occurs through a process of suicide inactivation involving the form oxy-tyrosinase. Under anaerobic conditions, both the met- and deoxy-tyrosinase forms undergo irreversible inactivation. Suicide inactivation in aerobic conditions is slower than the irreversible inactivation under anaerobic conditions. The enzyme has less affinity for the isomer D-dopa than for L-dopa but the velocity of inactivation is the same. We propose mechanisms to explain these processes.

Melanogenesis inhibition by tetrahydropterines.

There is controversy in the literature concerning the action of tetrahydropterines on the enzyme tyrosinase and on melanogenesis in general. In this study, we demonstrate that tetrahydropterines can inhibit melanogenesis in several ways: i) by non-enzymatic inhibition involving purely chemical reactions reducing o-dopaquinone to L-dopa, ii) by acting as substrates which compete with L-tyr and L-dopa, since they are substrates of tyrosinase; and iii) by irreversibly inhibiting the enzymatic forms met-tyrosinase and deoxy-tyrosinase in anaerobic conditions. Three tetrahydropterines have been kinetically characterised as tyrosinase substrates: 6-R-L-erythro-5,6,7,8-tetrahydrobiopterin, 6-methyl-5,6,7,8-tetrahydropterine and 6,7-(R,S)-dimethyl-5,6,7,8-tetrahydropterine. A kinetic reaction mechanism is proposed to explain the oxidation of these compounds by tyrosinase.

COLOR II. A randomized clinical trial comparing laparoscopic and open surgery for rectal cancer.

INTRODUCTION: Laparoscopic resection of rectal cancer has been proven efficacious but morbidity and oncological outcome need to be investigated in a randomized clinical trial. TRIAL DESIGN: Non-inferiority randomized clinical trial. METHODS: The COLOR II trial is an ongoing international randomized clinical trial. Currently 27 hospitals from Europe, South Korea and Canada are including patients. The primary endpoint is loco-regional recurrence rate three years post-operatively. Secondary endpoints cover quality of life, overall and disease free survival, post-operative morbidity and health economy analysis. RESULTS: By July 2008, 27 hospitals from the Netherlands, Belgium, Germany, Sweden, Spain, Denmark, South Korea and Canada had included 739 patients. The intra-operative conversion rate in the laparoscopic group was 17%. Distribution of age, location of the tumor and radiotherapy were equal in both treatment groups. Most tumors are located in the mid-rectum (41%). CONCLUSION: Laparoscopic surgery in the treatment of rectal cancer is feasible. The results and safety of laparoscopic surgery in the treatment of rectal cancer remain unknown, but are subject of interim analysis within the COLOR II trial. Completion of inclusion is expected by the end of 2009. TRIAL REGISTRATION: Clinicaltrials.gov, identifier: NCT00297791 (www.clinicaltrials.gov).

Mean lifetime and first-passage time of the enzyme species involved in an enzyme reaction. Application to unstable enzyme systems.

Taking as starting point the complete analysis of mean residence times in linear compartmental systems performed by Garcia-Meseguer et al. (Bull. Math. Biol. 65:279-308, 2003) as well as the fact that enzyme systems, in which the interconversions between the different enzyme species involved are of first or pseudofirst order, act as linear compartmental systems, we hereby carry out a complete analysis of the mean lifetime that the enzyme molecules spend as part of the enzyme species, forms, or groups involved in an enzyme reaction mechanism. The formulas to evaluate these times are given as a function of the individual rate constants and the initial concentrations of the involved species at the onset of the reaction. We apply the results to unstable enzyme systems and support the results by using a concrete example of such systems. The practicality of obtaining the mean times and their possible application in a kinetic data analysis is discussed.

A review on spectrophotometric methods for measuring the monophenolase and diphenolase activities of tyrosinase.

Tyrosinase is a copper enzyme with broad substrate specifity toward a lot of phenols with different biotechnological applications. The availability of quick and reliable measurement methods of the enzymatic activity of tyrosinase is of outstanding interest. A series of spectrophotometric methods for determining the monophenolase and diphenolase activities of tyrosinase are discussed. The product of both reactions is the o-quinone of the corresponding monophenol/diphenol. According to the stability and properties of the o-quinone, the substrate is classified as four substrate types. For each of these substrate types, we indicate the best method for measuring diphenolase activity (among eight methods) and, when applicable, for measuring monophenolase activity (among four methods). The analytical and numerical solutions to the system of differential equations corresponding to the reaction mechanism of each case confirm the underlying validity of the different spectrophotometric methods proposed for the kinetic characterization of tyrosinase in its action on different substrates.

Kinetic characterization of the oxidation of chlorogenic acid by polyphenol oxidase and peroxidase. Characteristics of the o-quinone.

Chlorogenic acid is the major diphenol of many fruits, where it is oxidized enzymatically by polyphenol oxidase (PPO) or peroxidase (POD) to its o-quinone. In spectrophotometric studies of chlorogenic acid oxidation with a periodate ratio of [CGA]0/[IO4-]0 < 1 and [CGA]0/[IO4-]0 > 1, the o-quinone was characterized as follows: lambda(max) at 400 nm and epsilon = 2000 and 2200 M-1 cm-1 at pH 4.5 and 7.0, respectively. In studies of o-quinone generated by the oxidation of chlorogenic acid using a periodate at ratio of [CGA]0/[IO4-]0 > 1, a reaction with the remaining substrate was detected, showing rate constants of k = 2.73 +/- 0.17 M-1 s-1 and k' = 0.05 +/- 0.01 M-1 s-1 at the above pH values. A chronometric spectrophotometric method is proposed to kinetically characterize the action of the PPO or POD on the basis of measuring the time it takes for a given amount of ascorbic acid to be consumed in the reaction with the o-quinone. The kinetic constants of mushroom PPO and horseradish POD are determined.

Enzymatic method with polyphenol oxidase for the determination of cysteine and N-acetylcysteine.

Thiols, such as cysteine and N-acetylcysteine, are included in many pharmaceutical products for their mucolytic properties. The method described here uses mushroom polyphenol oxidase (PPO) to determine two thiols and consists of measuring the lag period in the formation of the product generated as PPO acts on o-diphenol in the presence of a thiol. In the experimental conditions, o-quinone is formed enzymatically and then reacts stoichiometrically with the thiol, originating the corresponding thiol-diphenol adduct, which does not absorb visible light. Once the thiol has been used up, the o-quinone can be observed in the medium. It must be borne in mind that the inhibition of PPO is practically null at low concentrations of thiol, and the only effect observed is the formation of the thiol-diphenol adduct. In the following, an exact kinetic method capable of rapidly and accurately assaying thiols with PPO and o-diphenol is optimized and is shown to be a straightforward way of calculating thiol concentration. The method has been successfully applied to the determination of cysteine in model solutions and of N-acetylcysteine in pharmaceutical products.

Retroperitoneal pelvic hemangiopericytoma. Combined abdominoperineal resection.

We report a case of large-size pelvic hemangiopericytoma (HPC) incidentally discovered in a 32-year-old woman. Prior to surgery the patient was studied using a CT scan and magnetic resonance imaging (MRI). Because of the size and scarce extent of the tumour (it expanded downwards, into the right ischiorectal fossa) a combined abdomino-perineal approach was needed for complete resection. Seventy-two months after initial surgery the patient remains asymptomatic, with no signs of local recurrence or distant metastasis.

Computerized tomographic assessment of the composition of gallstones.

We present the results of an in vitro study of biliary lithiasis (n = 106) with computerized tomography (CT). We analyzed the correlation between composition of gallstones (crystallographic analysis of thin sections and atomic absorption spectrophotometric analysis of total calcium content) with CT patterns (densitometry) and their corresponding attenuations. Six main CT patterns were distinguished: hypodense (homogeneous and heterogeneous), dense, homogeneous hyperdense, ringed hyperdense and irregular hyperdense. Statistically significant differences (p < 0.05) were found between attenuations for cholesterol (mixed and pure) and pigmentary stones. We found a good inverse correlation between attenuations and cholesterol content; the contents of pigment, inorganic calcic salts and total calcium content also showed significant direct correlations (p < or = 0.01). Of the samples classified as pure cholesterol type stones, 86.4% showed hypodense patterns, and the remaining 13.6% showed irregular hyperdense patterns. Of the pigmentary stones, 80% showed homogeneous hyperdense images. All stones that contained more than 3% calcium produced hyperdense patterns, whereas 72.4% of the stones that contained less calcium produced hypodense images. Tomodensitometric measurements provided more information than simple radiography, and made it possible to distinguish a greater variety of gallstone types based on structural complexity.

Primary colorectal lymphoma.

OBJECT: To analyze our experience in the treatment of primary colorectal lymphomas. PATIENTS: During the recent period from 1989 to 1995, a total number of 406 cases of malignant neoplasms of large intestine were treated at Department of Surgery, Hospital del S.A.S de Jerez de la Frontera. There were 5 cases of primary colorectal Non-Hodgkin's lymphomas, constituting 1.2% of all colorectal malignancies. METHODS: Medical records of all five patients were carefully reviewed. Surgical pathology specimens were retrospectively reexamined to confirm the diagnosis, and immunohistochemistry staining methods were performed. Dawson, Cornes and Morson criteria were used to distinguish primary colonic lymphoma. For tumor staging, a modification of the Ann-Arbor system for gastrointestinal lymphoma proposed by Musshoff and Schmidt-Vollmer was used. RESULTS: All cases were Non-Hodgkin's lymphomas. There were no cases of lymphoma of the rectum. There were 2 males and 3 females. Their ages ranged from 12 to 73 years, with an average age of 31 years. The cecum was the site of the lesions in 4 patients and the other was located in the sigmoid colon. Acute abdomen in three cases, undiagnosed abdominal mass in one and intussusception in the other one were the clinical presentation. Inmunohistochemical studies showed B-cell phenotype in 4 and non-B non-T Ki-1-positive phenotype in 1 patient. Chemotherapy was administered following resection in 100% of patients. CONCLUSIONS: Intraoperative diagnosis of lymphoma was not suspected in all cases but the malignancy was always present. The cecum is the commonest site of involvement. Both of our two patients presented with perforated lymphoma were associated with a poor short term prognosis. It is necessary to unify the staging system and the histologic classifications enabling a comparison of several studies.

Determination and applications of the molar absorptivity of phenolic adducts with captopril and mesna.

Captopril and mesna are molecules with a free thiol group, used as active ingredients due to their hypotensor and mucolytic properties, respectively. These compounds cross the hematoencephalic barrier and, due to the reactivity of their thiol group, can form adducts with the o-quinones formed during the oxidation of mono- and o-diphenols. Polyphenol oxidase from plants and fungi can be used as a tool for generating o-quinones in their action on o-diphenols and facilitate the formation of adducts in the presence of captopril or mesna. The spectrophotometric characterization of these adducts is useful from several points of view. Here, using the end-point method, which involves the exhaustion of oxygen in the medium, we determined the molar absorptivity of the adducts of different o-diphenols with captopril and mesna. Besides the analytical interest of this approach, we also use it to make a kinetic characterization of polyphenol oxidase as it acts on o-diphenolic substrates that produce unstable o-quinones.

Kinetic characterization of the enzymatic and chemical oxidation of the catechins in green tea.

The oxidation of green tea catechins by polyphenol oxidase/O2 and peroxidase/H2O2 gives rise to o-quinones and semiquinones, respectively, which inestability, until now, have hindered the kinetic characterization of enzymatic oxidation of the catechins. To overcome this problem, ascorbic acid (AH2) was used as a coupled reagent, either measuring the disappearance of AH2 or using a chronometric method in which the time necessary for a fixed quantity of AH2 to be consumed was measured. In this way, it was possible to determine the kinetic constants characterizing the action of polyphenol oxidase and peroxidase toward these substrates. From the results obtained, (-) epicatechin was seen to be the best substrate for both enzymes with the OH group of the C ring in the cis position with respect to the B ring. The next best was (+) catechin with the OH group of the C ring in the trans position with respect to the B ring. Epigallocatechin, which should be in first place because of the presence of three vecinal hydroxyls in its structure (B ring), is not because of the steric hindrance resulting from the hydroxyl in the cis position in the C ring. The epicatechin gallate and epigallocatechin gallate are very poor substrates due to the presence of sterified gallic acid in the OH group of the C ring. In addition, the production of H2O2 in the auto-oxidation of the catechins by O2 was seen to be very low for (-) epicatechin and (+) catechin. However, its production from the o-quinones generated by oxidation with periodate was greater, underlining the importance of the evolution of the o-quinones in this process. When the [substrate] 0/[IO4 (-)] 0 ratio = 1 or >>1, H2O2 formation increases in cases of (-) epicatechin and (+) catechin and practically is not affected in cases involving epicatechin gallate, epigallocatechin, or epigallocatechin gallate. Moreover, the antioxidant power is greater for the gallates of green tea, probably because of the greater number of hydroxyl groups in its structure capable of sequestering and neutralizing free radicals. Therefore, we kinetically characterized the action of polyphenol oxidase and peroxidase on green tea catechins. Furthermore, the formation of H2O2 during the auto-oxidation of these compounds and during the evolution of their o-quinones is studied.

Kinetic analysis of a general model of activation of aspartic proteinase zymogens involving a reversible inhibitor. I. Kinetic analysis.

Starting from a simple general reaction mechanism of activation of aspartic proteinases zymogens involving a uni- and a bimolecular simultaneous activation route and a reversible inhibition step, the time course equation of the zymogen, inhibitor and activated enzyme concentrations have been derived. Likewise, expressions for the time required for any reaction progress and the corresponding mean activation rates as well as the half-life of the global zymogen activation have been derived. An experimental design and kinetic data analysis is suggested to estimate the kinetic parameters involved in the reaction mechanism proposed.

Effect of tetrahydropteridines on the monophenolase and diphenolase activities of tyrosinase.

This study explains the action of compounds such as 6-tetrahydrobiopterin, (6BH4) and 6,7-dimethyltetrahydrobiopterin (6,7-di-CH3BH4) on the monophenolase and diphenolase activities of tyrosinase. These reductants basically act by reducing the o-quinones, the reaction products, to o-diphenol. In the case of the diphenolase activity a lag period is observed until the reductant is depleted; then the system reaches the steady-state. In the action of the enzyme on monophenol substrates, when the reductant concentration is less than that of the o-diphenol necessary for the steady-state to be reached, the system undergoes an apparent activation since, in this way, the necessary concentration of o-diphenol will be reached more rapidly. However, when the reductant concentration is greater than that of the o-diphenol necessary for the steady-state to be reached, the lag period lengthens and is followed by a burst, by means of which the excess o-diphenol is consumed, the steady-state thus taking longer to be reached. Moreover, in the present kinetic study, we show that tyrosinase is not inhibited by an excess of monophenol, although, to confirm this, the system must be allowed to pass from the transition state and enter the steady-state, which is attained when a given amount of o-diphenol has accumulated in the medium.

Expressions for the fractional modification in different monocyclic enzyme cascade systems: analysis of their validity tested by numerical integration.

This paper presents the derivation, under a minimal set of assumptions, of a general expression for the steady-state fractional modification of an interconvertible protein involved in four different schemes of monocyclic enzyme cascade systems. From this general expression we derive, as particular cases, other, simpler expressions by applying additional assumptions and which have, therefore, a smaller range of validity. Some of these particular expressions coincide with those already obtained in previous contributions on individualised analyses. We discuss the relationships between the kinetic parameters and the concentrations needed for the fulfilment of the additional assumptions. The goodness of the analysis was tested by reference to the shape in the steady-state of the simulated time progress curves obtained by numerical integration.