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Introduction There is a balance between oxidative stress, antioxidant capacity and immune response. Their roles in physiological and behavioural mechanisms are important for the maintenance of the organism’s internal equilibrium. This study aimed to evaluate the antioxidant effects of the exogenous alga Spirulina platensis (Arthrospira platensis) in a stress-induced rat model, and to describe its possible mechanism of action. Material and Methods Thirty-six adult male Sprague Dawley rats were separated into four groups: control (C), stress (S), S. platensis (Sp), and S. platensis + stress (SpS). The rats in groups Sp and SpS were fed with 1,500 mg/kg b.w./day Spirulina platensis for 28 days. All rats were exposed to prolonged light phase conditions (18 h light : 6 h dark) for 14 days. The SpS and S groups were exposed to stress by being kept isolated and in a crowded environment. Blood samples were obtained by puncturing the heart on the 28th day. The effect of stress on serum corticosterone, oxidative stress markers (TOS, TAC, PON1, OSI) and immunological parameters (IL-2, IL-4, IFN-ɣ) were tested. Also, the brain, heart, intestines (duodenum, ileum, and colon), kidney, liver, spleen, and stomach of the rats were weighed. Results Serum corticosterone levels were higher in the S group than in the C group, and significantly lower in the SpS group than in the S group. Mean total antioxidant capacity were lower in the S group than in the C group, and Spirulina reversed this change. Although not significantly different, IL-2 was lower in the S group than in the C group. However, in the SpS group, IL-2 increased due to Spirulina platensis mitigating effects of stress. Conclusion Male rats fed a diet with Spirulina platensis could experience significantly milder physiological changes during stress, although stress patterns may be different. Exogenous antioxidant supplements merit further investigation in animals and humans where the endogenous defence mechanism against stress may not be sufficient.

Background The enzymatic method of caeruloplasmin measurement is based on copper-dependent oxidase activity. The advantage of the oxidase determination is that it has a much lower detection limit compared with immunoassay-based methods. It has found its application in both the diagnosis of Wilson’s disease and also in the monitoring of patients’ response to treatment. Methods The method previously described in literature was adapted for use on a 96-well plate. Caeruloplasmin oxidase activity results were derived from the equation: caeruloplasmin oxidase activity = (A15−A5) × 185 U/L. Results Repeatability (intra-batch) imprecision ranged from 6 to 15% and intermediate (inter-batch) imprecision varied from 7 to 16% for caeruloplasmin oxidative activities of 14, 29, 45 and 99 U/L. Between 3 and 92 U/L, the assay appeared linear with a regression coefficient R2 = 0.9958. The lower limit of quantification was 4 U/L. Samples were stable over a five-week period at 4℃ and for at least four freeze–thaw cycles. There was a statistically significant difference between the areas under ROC curve for copper-to-caeruloplasmin ratios between caeruloplasmin oxidative activity and immunoassay-based methods ( P Conclusions Using the oxidative assay provides a cost-effective means of estimating caeruloplasmin concentrations. The method is easily adaptable to a 96-well plate format that facilitates high throughput of samples in a busy laboratory. The enzymatic method is more sensitive and specific for differentiating between Wilson’s and non-Wilson’s when compared with immunoassay-based methods.

Studies on the oxidation products of organic pollutants and their toxicity in textile dyeing sludge after the sludge was treated by the advance oxidation processes were limited, since textile dyeing sludge was a complicated mixture. For the first time, simulated sludge was used to study the degradation mechanism of 3,3′-dimethoxybenzidine (DMB) during the combined ultrasound-Mn(VII) treatment. The toxicity of DMB and its products was also evaluated. The results indicated that the compositions and microstructures of polyaluminium chloride (PAC)- and polyferric sulphate (PFS)-based simulated sludge were similar to those of real textile dyeing sludge. The optimum conditions of ultrasound-Mn(VII) treatment were: a KMnO4 dosage of 40 μM, an ultrasound power density of 0.36 W cm−3, and a reaction time of 20 min. 98.24% of DMB and 63.04% of total organic carbon (TOC) in the simulated sludge were removed. Six products, that is, 2-nitroanisole, 3-methoxy-4-nitrophenol, vanillylmandelic acid, vanillyl alcohol, m-anisic acid, and benzoic acid, were identified by GC-MS and LC-MS-MS. It was noted that all of these identified products were also detected in the real textile dyeing sludge after the ultrasound-Mn(VII) treatment. All of them were less toxic than DMB. Moreover, 53.30% and 54.80% of toxicity toward the alga Desmodesmus subspicatus and the bacterium Vibrio fischeri were removed in simulated sludge, respectively. Therefore, simulated sludge was helpful for studying a pollutant's degradation mechanism in the complex sludge mixtures. The results would also provide some useful suggestions for the sludge disposal after the sludge was treated by the advance oxidation processes.

Fruit ripening associated full length cDNA of a peroxidase from papaya was cloned and heterologously expressed. The expressed peroxidase was activated by in-vitro re-folding in the presence of hemin and calcium. The purified recombinant peroxidase exhibited broad substrate affinity in the order of o-dianisidine > pyrogallol > guaiacol and was found to be a homotetramer of 155 kDa with each subunit having a size of 38 kDa. The basis of the distinctive preferences for various substrates was investigated through in-silico molecular modeling approaches. Thus, when the modeled papaya peroxidase–heme complex was docked with these substrates, the in-silico binding efficiency was found to be in agreement with those of wet lab results with the involvement of Arg37, Phe40, His41, Pro137, Asn138, His139, His167, and Phe239 as the common interacting residues in all the cases. However, the binding of the different substrates were found to be associated with conformational changes in the peroxidase. Thus, in the case of o-dianisidine (the most efficient substrate), the protein was folded in the most compact fashion when compared to guaiacol (the least efficient substrate). Protein function annotation analyses revealed that the papaya peroxidase may have biological roles in oxidation-reduction processes, stresses, defense responses etc. In order to further validate its role in lignifications, the papaya peroxidase was compared with a lignin biosynthetic peroxidase from Leucaena leucocephala, a tree legume. Thus, based on 3D structure superimposition and docking, both peroxidases exhibited a great extent of similarity suggesting the papaya peroxidase having a role in lignification (defense response) too. The predicted functions of papaya peroxidase in defense response and lignification were further validated experimentally using qRT-PCR analyses and measurement of oxidation of coniferyl alcohol.

Herein we report a label-free microRNA (miRNA) biosensor in which the formation of a thin insulating film is used to amplify the analytical signal. Briefly, the biosensor is made of an oligonucleotide-coated gold electrode. After hybridizing with a target miRNA, free capture probe (CP) strands on the biosensor are removed by a nuclease digestion. A second hybridization with an oligonucleotide-tailed DNAzyme is performed to introduce the DNAzyme to the biosensor. The DNAzyme triggers the polymerization of 3,3′-dimethoxybenzidine (DB) in the presence of H2O2 and the hybridized miRNA-CP duplexes serve as templates to guide the deposition of poly (3,3′-dimethoxybenzidine) (PDB). The formation of the insulating PDB film alters the impedance of the biosensor, rendering it readily distinguishable by electrochemical impedance measurements. The accumulative nature of the PDB deposition drastically improves the detectability of the biosensor. A proof-of-concept study is conducted on the detection of miRNAs in total RNA extracted from cultured cells.

Aminopropyl-functionalized SBA-15 mesoporous silica was used as a support to adsorb myoglobin. Then, in order to avoid the leakage of adsorbed myoglobin, lysozyme was covalently tethered to the internal and external surface of the mesoporous silica with glutaraldehyde as the coupling agent. The property of amino-functionalized mesoporous silica was characterized by N(2) adsorption-desorption and thermogravimetric (TG) analysis. The feature of the silica-based matrix before and after myoglobin adsorption was identified by fourier transform infrared (FTIR) and UV/VIS measurement. With o-dianisidine and H(2)O(2) as the substrate, the peroxidase activity of adsorbed myoglobin was determined. With Micrococus lysodeilicus as the substrate, the antibacterial activity of covalently tethered lysozyme was measured. Results demonstrated that the final product not only presented peroxidase activity of the myoglobin but yielded antibacterial activity of the lysozyme.

Directed evolution methods were developed for Cu-containing nitrite reductase (NiR) from Alcaligenes faecalis S-6. The PCR cloning strategy allows for the efficient production of libraries of 100 000 clones by a modification of a megaprimer-based whole-plasmid synthesis reaction. The high-throughput screen includes colony lift onto a nylon membrane and subsequent lysis of NiR-expressing colonies in the presence of Cu(2+) ions for copper incorporation into intracellularly expressed NiR. Addition of a chromogenic substrate, 3, 3'-diaminobenzidine (DAB), results in deposition of red, insoluble color at the site of oxidation by functional NiR. Twenty-thousand random variants of NiR were screened for improved function with DAB as a reductant, and five variants were identified. These variants were shuffled and screened, yielding two double variants. An analog of the DAB substrate, o-dianisidine, which is oxidized to a water-soluble product was used for functional characterization. The double variant M150L/F312C was most proficient at o-dianisidine oxidation with dioxygen as the electron acceptor (5.5X wt), and the M150L single variant was most proficient at o-dianisidine oxidation with nitrite as the electron acceptor (8.5X wt). The library generation and screening method can be employed for evolving new reductase functions in NiR and for screening of efficient folding of engineered NiRs.

This paper describes the applicability of in vitro digestion models as a tool for consumer products in (ad hoc) risk assessment. In current risk assessment, oral bioavailability from a specific product is considered to be equal to bioavailability found in toxicity studies in which contaminants are usually ingested via liquids or food matrices. To become bioavailable, contaminants must first be released from the product during the digestion process (i.e. become bioaccessible). Contaminants in consumer products may be less bioaccessible than contaminants in liquid or food. Therefore, the actual risk after oral exposure could be overestimated. This paper describes the applicability of a simple, reliable, fast and relatively inexpensive in vitro method for determining the bioaccessibility of a contaminant from a consumer product. Different models, representing sucking and/or swallowing were developed. The experimental design of each model can be adjusted to the appropriate exposure scenarios as determined by the risk assessor. Several contaminated consumer products were tested in the various models. Although relevant in vivo data are scare, we succeeded to preliminary validate the model for one case. This case showed good correlation and never underestimated the bioavailability. However, validation check needs to be continued.

Hollow nanoshells of gold entrapping an enzyme, horseradish peroxidase (HRP), in the cavity of the nanoshell have been prepared in the reverse micelles by leaching out silver chloride (AgCl) from Au(shell)AgCl(core) nanoparticles with dilute ammonia solution. The particles have been characterised by dynamic laser light scattering (DLS), transmission electron microscopy (TEM), X-ray diffraction (XRD), and electron diffraction. The particle size is below 100 nm diameter, depending upon the size of the aqueous core of reverse micelles in which these particles have been prepared. This soft-chemical method for the preparation of such particles allows the entrapped enzyme to remain active inside the hollow gold nanoparticles. Small substrate molecules such as o-dianisidine can easily enter through the pores of the nanoshell and can undergo enzymatic oxidation by H2O2. The enzyme kinetics follows Michaelis-Menten mechanism. When the substrate is chemically conjugated with dextran molecule (10 kDa), the enzymatic reaction is practically completely prevented perhaps by the inability of dextran-o-dianisidine conjugate to penetrate the pores of the nanoshells. However, HRP did not show any activity when trapped inside solid gold nanoparticles.

Due to limitations of the existing preparative methods of hollow nanoparticles by either heating at high temperature (>600 °C) or by using strong acid, alkali, or an organic solvent, it was not possible up till now to encapsulate any sensitive organic molecule like enzyme or others inside the cavity of hollow nanoparticles. We have demonstrated a much softer method of preparing hollow silica nanoparticles with horseradish peroxidase (HRP) inside the cavity by synthesizing HRP-doped core-shell silica-coated silver chloride nanoparticles and finally leaching out silver chloride with dilute ammonia at low temperatures. TEM pictures showed the hollow cavity inside the nanoparticles. The enzyme entrapped in these particles was active. The turnover number of HRP entrapped into these hollow particles and dispersed in aqueous buffer (pH 7.2) ( k cat = 2.56 × 10 6 s −1 ) was found to be less than that of free enzyme in aqueous buffer ( k cat = 6.133 × 10 7 s −1 ) but higher than that of HRP entrapped in solid-core silica nanoparticles and dispersed in aqueous buffer ( k cat = 1.05 × 10 5 s −1 ). The result showed that hollow nanoparticles could be prepared using soft chemical methods and sensitive chemicals like active enzyme could be entrapped in the cavities and it retains its activity.

A spectrophotometric method based on myeloperoxidase activity for the determination of leukocytes in urine is described. Red cells that may be found in urine samples were lysed by an ammonium chloride method. Leukocytes were then sedimented by centrifugation and lysed using Triton X‐100 (Sigma Chemicals Co., St. Louis, MO). Myeloperoxidase‐catalyzed oxidation of o‐dianisidine was carried out at 37°C, pH 7. The reaction was stopped with the addition of 2 M H(2)SO(4), and a stable form of oxidized o‐dianisidine in acidic solution was obtained. Solid particles that may be found in urine samples were removed by centrifugation to avoid turbidity, and absorbance values of the supernatants were recorded at 400 nm. An Average number of leukocytes were noted per number of fields by microscopic examination and were related with the absorbance values of the supernatants at 400 nm. Pearson correlation (r) between our presented spectrophotometric analysis results and visual microscopic analysis was 0.877. Roche Combur 10‐test M strips (Roche, Mannheim, Germany) and Multistix 10 SG Bayer test strips (Bayer Diagnostics, UK) were 0.645 and 0.648, respectively (P

The model compounds for horseradish peroxidase (HRP) is reported, based on the association of H 2 O 2 with iron and manganese porphyrins immobilized onto a functionalized silica gel. The models were developed in an attempt to find a biomimetical compound for systems containing heme groups. The ortho -dianisidine is a useful substrate model compound for checking the ability of degradation promoted by delignificant natural enzymes. The heterogeneous catalysts were obtained by grafting of three metalloporphyrins: (Fe(TFPP)—iron porphyrin from the 5,10,15,20-tetrakis (pentafluorophenyl) porphyrin—and Mn(TCPP) and Fe(TCPP), iron and manganese porphyrins from the 5,10,15,20-tetrakis (4-carboxyphenyl) porphyrin), onto the modified surface of silica gel. The oxidation of ortho -dianisidine was monitored by UV-Vis spectroscopy at room temperature using different ratios of catalyst, oxidant and substrate. At high H 2 O 2 concentration, the perhalogenated iron porphyrin Fe(TFPP) produced best results, showing a turnover number of about 1100. This value was higher than those obtained for Fe(TCPP) and Mn(TCPP) systems.

The determination of leukocyte concentration in human blood depending on the detection of oxidized o‐dianisidine in acidic solution is studied. The oxidation of o‐dianisidine was carried out by peroxidase enzymes found in leukocytes. The reaction was stopped by the addition of 4N H(2)SO(4) to the solution, and a very stable, colored o‐dianisidine derivative was obtained. The calibration graph was plotted with the recorded absorbance values at 400 nm assigned to the y‐axis, and leukocyte counts in 1‐mL blood samples to the x‐axis. The equation of the calibration graph was y=0.0025x+0.0904, with a correlation coefficient of R=0.994. The coefficient of variation and P‐value of the method were 4.00% and 0.05%, respectively. J. Clin. Lab. Anal. 16:233–236, 2002. © 2002 Wiley‐Liss, Inc.

A total of 11 manganese peroxidase isoenzymes (MnP 1 -MnP 11 ) with isoelectric points (pIs) in the range of 4.58–3.20 were isolated from liquid- and solid-state cultures of the basidiomycete Ceriporiopsis subvermispora . In the presence of hydrogen peroxide, these isoenzymes showed different requirements for Mn(II) in the oxidation of vanillylacetone, o-dianisidine , p-anisidine and ABTS, whereas oxidation of guaiacol by any isoenzyme did not take place when this metal was omitted. K m values for o-dianisidine and p-anisidine in the absence of Mn(II) are in the range of 0.5–1.0 mM and 4.5–42.0 mM, respectively. Oxalate and citrate, but not tartrate, accelerate the oxidation of o-dianisidine , both in the presence and in the absence of Mn(II). MnPs from this fungus are able to oxidize kojic acid without externally added hydrogen peroxide, indicating that they can also act as oxidases. In this reaction, however, the requirement for Mn(II) is absolute.

The unique antitubercular activity of isoniazid requires that the drug be oxidized by the katG-encoded mycobacterial catalase-peroxidase to an activated drug form. In order to quantitatively assess the catalytic capabilities of the enzyme, the native catalase-peroxidase from Mycobacterium smegmatis was purified over 200-fold to homogeneity. The enzyme was shown to exhibit both catalase and peroxidase activities, and in the presence of either hydrogen peroxide or t-butyl peroxide, was found to catalyze the oxidation of the reduced pyridine nucleotides, NADH and NADPH, as well as artificial peroxidase substrates, at rates between 2.7 and 20 s-1. The homogeneous enzyme exhibited a visible absorbance spectrum typical of ferric heme-containing catalase-peroxidases, with a Soret maximum at 406 nm. Low temperature (10 K) electron paramagnetic resonance spectra in the presence of ethylene glycol revealed a high spin Fe(III) signal with g values of 5.9 and 5.6. The enzyme was very slowly (t1/2 = approximately 20 min) reduced by dithionite, and the reduced form showed typical spectral changes when either KCN or CO were subsequently added. The M. smegmatis catalase-peroxidase was found to contain 2 heme molecules per tetramer, which were identified as iron protoporphyrin IX by the pyridine hemochromogen assay. The peroxidatic activity was inhibited by KCN, NaN3, isoniazid (isonicotinic acid hydrazide), and its isomer, nicotinic acid hydrazide, but not by 3-amino-1,2,4-triazole. The role of mycobacterial catalase-peroxidases in the oxidative activation of the antitubercular prodrug isoniazid is discussed.

Made available in DSpace on 2013-08-28T14:09:25Z (GMT). No. of bitstreams: 1 WOS000306752700025.pdf: 1046807 bytes, checksum: cd877a54c25980a5175ee0db7f82685f (MD5) Made available in DSpace on 2013-09-30T19:09:11Z (GMT). No. of bitstreams: 2 WOS000306752700025.pdf: 1046807 bytes, checksum: cd877a54c25980a5175ee0db7f82685f (MD5) WOS000306752700025.pdf.txt: 45557 bytes, checksum: 744d1bc7b9755cb558b4b0abb89c3d2d (MD5) Previous issue date: 2012-07-01 Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-20T14:19:33Z No. of bitstreams: 2 WOS000306752700025.pdf: 1046807 bytes, checksum: cd877a54c25980a5175ee0db7f82685f (MD5) WOS000306752700025.pdf.txt: 45557 bytes, checksum: 744d1bc7b9755cb558b4b0abb89c3d2d (MD5) Made available in DSpace on 2014-05-20T14:19:33Z (GMT). No. of bitstreams: 2 WOS000306752700025.pdf: 1046807 bytes, checksum: cd877a54c25980a5175ee0db7f82685f (MD5) WOS000306752700025.pdf.txt: 45557 bytes, checksum: 744d1bc7b9755cb558b4b0abb89c3d2d (MD5) Previous issue date: 2012-07-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) The room temperature ionic liquid (IL) 1-butyl-3-methylimidazolium bis-(trifluorometanesulfonyl) imide BMIm[NTf2] was used as a novel medium for improvement of separation and quantization of 16 aromatic amines typically present as contaminants in consumer products and detected by HPLC coupled to an electrochemical detector. The aromatic amines, namely 4,4'-diaminodiphenylmethane, 4-chloroaniline, 2-methoxy-5-methyl-aniline, 3,3'-dimethylbenzidine, 2,4-diaminotoluidine, 2-chloro-4-nitroaniline, 4,4'-oxydianiline, aniline, 3,3'-dichlorobenzidine, benzidine, 4-aminobiphenyl, o-dianisidine, o-anisidine, o-toluidine, 4,4'-methylene-bis-2-chloroaniline and 2-naphthylamine are oxidized in methanol/BMIm[NTf2] at a potential around +0.68V to +0.93V vs. Ag/AgCl at a glassy carbon electrode, which is the base for their determination by HPLC/ED. Using the optimized conditions of methanol/BMIm[NTf2] 70: 30 (v/v) as mobile phase, flow-rate of 0.8 mL.min(-1), column CLC-ODS, E-ap = +1.0 V and T = 40 C analytical curves were constructed for each of the tested amines. Good linearity was obtained in the concentration range of 1.09 mg.L-1 to 217 mg.L-1, with excellent correlation coefficients. The limits of detection reached 0.021 mg.L-1 to 0.246 mg.L-1 and good relative standard deviations (RSD, n = 3) were obtained from the measurements. Satisfactory recovery for each aromatic amine was achieved, ranging from 95 to 103%. The developed method was successfully applied to determine six aromatic amines present as contaminants in commercial hair dye samples. State Univ Julio de Mesquita Filho UNESP, Inst Chem, BR-14800900 Araraquara, SP, Brazil State Univ Julio de Mesquita Filho UNESP, Inst Chem, BR-14800900 Araraquara, SP, Brazil

With an assay that generates free radicals (FR) through photooxidation of dianisidine sensitized by riboflavin, 4 x 10(-5) M captopril, epicaptopril (SQ 14,534, captopril's stereoisomer), zofenopril, and fentiapril [all sulfhydryl (-SH)-containing angiotensin-converting enzyme (ACE) inhibitors] were shown effective scavengers of nonsuperoxide free radicals whereas non-SH ACE inhibitors were not. Captopril was a more effective FR scavenger at pH 5.0 than at pH 7.5. Captopril (2 x 10(-5) M) also scavenged the other toxic oxygen species hydrogen peroxide and singlet oxygen and inhibited microsomal lipid peroxidation. Finally, captopril reduced the amount of superoxide anion-radical detected after neutrophils in whole blood were activated with zymosan, probably by inhibiting leukocyte superoxide production.

The insulin-like growth factor (IGF) family is essential for normal embryonic growth and development and it is highly conserved through vertebrate evolution. However, the roles that the individual members of the IGF family play in embryonic development have not been fully elucidated. This study focuses on the role of IGF-2 in zebrafish embryonic development. Two igf-2 genes, igf-2a and igf-2b, are present in the zebrafish genome. Antisense morpholinos were designed to knock down both igf-2 genes. The neural and cardiovascular defects in IGF-2 morphant embryos were then examined further using wholemount in situ hybridisation, TUNEL analysis and O-dianisidine staining. Knockdown of igf-2a or igf-2b resulted in ventralised embryos with reduced growth, reduced eyes, disrupted brain structures and a disrupted cardiovascular system, with igf-2b playing a more significant role in development. During gastrulation, igf-2a and igf-2b are required for development of anterior neural structures and for regulation of genes critical to dorsal-ventral patterning. As development proceeds, igf-2a and igf-2b play anti-apoptotic roles. Gene expression analysis demonstrates that igf-2a and igf-2b play overlapping roles in angiogenesis and cardiac outflow tract development. Igf-2b is specifically required for cardiac valve development and cardiac looping. Injection of a dominant negative IGF-1 receptor led to similar defects in angiogenesis and cardiac valve development, indicating IGF-2 signals through this receptor to regulate cardiovascular development. This is the first study describing two functional igf-2 genes in zebrafish. This work demonstrates that igf-2a and igf-2b are critical to neural and cardiovascular development in zebrafish embryos. The finding that igf-2a and igf-2b do not act exclusively in a redundant manner may explain why both genes have been stably maintained in the genome.

A method was devised for detecting Lactobacillus mutants that produced amounts of dicarbonyl compounds different from those of their parents. The mutants were recognized by contacting colonies with glass filter paper, which later was sprayed with a solution of o -dianisidine and heated. The amount of dicarbonyl could be estimated by the intensity of the resulting brown spots. The amounts of glyoxal, methylglyoxal, and diacetyl produced by mutants and parents were determined by a high performance chromatography method after growth on two media. Mutants that differed in the production of all three dicarbonyls were noted. The expression of the mutation varied with the medium used.

We recently published in this journal an article entitled ‘‘Plasma oxidative stress biomarkers, nitric oxide and heat shock protein 70 in trained elite soccer players’’ (Banfi et al. 2005) but our results with the d-ROMs test (Diacron International, Grosseto, Italy) were believed invalid by Harma et al. (2006), who reported the findings of a previous paper of Erel (2005) comparing the above test with the new iron-o-dianisidine/xylenol orange assay. In this article we provide the following evidence strongly indicating that the above authors made some relevant errors in the assessment of the d-ROMs test validity. Firstly, the relationships between d-ROMs test and ferroxidase activity are neither new nor an original finding. Indeed, Alberti et al. (2000), just in the study leading to the definitive validation of the d-ROMs test by electron paramagnetic resonance (EPR) spectrometry, reported that the contribution of ceruloplasmin ferroxidase activity to the typical change of absorbance at 505 nm (DA505/min) in such a test (see below), although not negligible, appeared relatively small. This is because in the d-ROMs test the serum sample is 100-fold diluted. Moreover, Alberti et al. (2000) demonstrated that an amount of sodium azide equivalent to the maximum expected concentration of ceruloplasmin in the serum (i.e. 4.0 lM) causes only an approximately 7% decrease of the DA505/min value. This may explain the reported correlation between d-ROMs test results and ferroxidase activity (Erel 2005). Of course, the above correlation does not mean that d-ROMs test measures only and exactly the serum ferroxidase activity. Indeed, a 70% of the DA505/min value is still detectable even with a five-to-ten fold excess of the azide in the test (Alberti et al. 2000). Furthermore, the found correlation between ferroxidase activity and d-ROMs test (Erel 2005) is not a general rule. For instance, in hemodyalised patients d-ROMs test value was shown to be increased (Gerardi et al. 2002) while the ferroxidase activity of ceruloplasmin was reduced (Roxborough et al. 2000). In this respect, if right that d-ROMs test measures only the ferroxidase activity, Erel (2005) failed to demonstrate and/or explain how an increased ceruloplasmin activity may justify the experimental/clinical findings, which have been reported to be associated to an increased result of d-ROMs test. On the other hand, even granting, for the sake of an argument, that the above relation is the rule, this is not consistent with the recently recognised controversial role of ferroxidase (Shukla et al. 2006). About the lack of response of d-ROMs test during copper-induced lipoprotein autoxidation, apart from the questionable significance of this experimental approach, this finding may be explained by the fact that d-ROMs test is significantly inhibited by the adding of chelants (Alberti et al. 2000) which sequester the iron thus, inhibiting the Fenton’s reaction. Indeed, Erel (2005), in the autoxidation tests used a citrate buffer to obtain lowdensity lipoprotein and ethylendiamine tetra acetate (EDTA) to prevent further oxidation during the eating. This reply refers to the Letter to the Editors, at http://dx.doi.org/ 10.007/s00421-006-0202.

A multicopper oxidase gene from Staphylococcus aureus was cloned and overexpressed. Purified recombinant multicopper oxidase oxidized the substrate 3,3′-dimethoxybenzidine in the presence of copper. Disruption of mco showed copper sensitivity and H 2 O 2 resistance, suggesting roles for mco in copper homeostasis and oxidative stress response. Northern blot analysis showed copper-induced mco transcription.

Cardiac thrombosis, one of the causes of sudden death throughout the world, plays a principal role in several cardiovascular diseases, such as myocardial infarction and stroke in humans. Data from studies of induction of chemical thrombosis in rodents help to identify substances in our environment that may contribute to cardiac thrombosis. Results for more than 500 chemicals tested in rodents in 2-year bioassays have been published as Technical Reports of the National Toxicology Program (NTP) 〈 http://ntp-server.niehs.nih.gov/index 〉. We evaluated atrial thrombosis induced by these chemical exposures and compared it to similarly induced lesions reported in the literature. Spontaneous rates of cardiac thrombosis were determined for control Fischer 344 rats and B6C3F1 mice: 0% in rats and mice in 90-day studies and, in 2-year studies, 0.7% in both genders of mice, 4% in male rats, and 1% in female rats. Incidences of atrial thrombosis were increased in high-dosed groups involving 13 compounds (incidence rate: 20–100%): 2-butoxyethanol, C.I. Direct Blue 15, bis(2-chloroethoxy)methane, diazoaminobenzene, diethanolamine, 3,3′-dimethoxybenzidine dihydrochloride, hexachloroethane, isobutene, methyleugenol, oxazepam, C.I. Pigment Red 23, C.I. Acid Red 114, and 4,4′-thiobis(6- t-butyl- m-cresol). The main localization of spontaneously occurring and chemically induced thromboses occurred in the left atrium. The literature survey suggested that chemical-induced atrial thrombosis might be closely related to myocardial injury, endothelial injury, circulatory stasis, hypercoagulability, and impaired atrial mechanical activity, such as atrial fibrillation, which could cause stasis of blood within the left atrial appendage, contributing to left atrial thrombosis. Supplementary data referenced in this paper are not printed in this issue of Toxicologic Pathology. They are available as downloadable files at http:taylorandfrancis.metapress.com/openurl.asp?genre=journal&issn=0192-6233 . To access them, click on the issue link for 33(5), then select this article. A download option appears at the bottom of this abstract. In order to access the full article online, you must either have an individual subscription or a member subscription accessed through www.toxpath.org .

Although benzidine (Bz), 4-aminobiphenyl (ABP), 3,3'-dichlorobenzidine HCl (DCBz), 3,3'-dimethylbenzidine (DMBz), 3,3'-dimethoxybenzidine (DMOBz) and the benzidine congener-based dye trypan blue (TB) produce primarily frameshift mutations in Salmonella typhimurium, the base-substitution strain TA100 also responds to these compounds when S9 is present. Performing DNA sequence analysis, other investigators have shown that ABP induces frameshift, base-pair and complex mutations. Also, it was found that an uninduced hamster liver S9 preparation with glucose-6-phosphate dehydrogenase, FMN, NADH and four times glucose 6-phosphate gave a stronger mutagenic response than the conventional plate incorporation with rat S9 activation mixture for all the compounds tested. Using the base-specific tester strains of S. typhimurium (TA7001-TA7006) with the above reductive metabolic activation system, we surveyed these compounds for the ability to produce specific base-pair substitutions after reductive metabolism. Bz was weakly mutagenic in TA7005 (0.04 revertants/microg). ABP was mutagenic in TA7002 (1.4 revertants/microg), TA7004 (0.6 revertants/microg), TA7005 (2.98 revertants/microg) and TA7006 (0.4 revertants/microg). DCBz was weakly mutagenic in TA7004 (0.01 revertants/microg). It was concluded that benzidine induced some CG-AT transversions in addition to frameshift mutations. ABP induced TA-AT, CG-AT, and CG-GC transversions as well as GC-AT transitions. DCBz induced only GC-AT transitions. Because DMBz, DMOBz and TB were not mutagenic in this base-substitution mutagen detection system, their mutagenic activity was attributed strictly to frameshift mechanisms.

A sensitive and convenient method is described for estimating superoxide dismutase activity using a photochemical augmentation procedure. This method is applicable to both liquid assays and polyacrylamide gel electropherograms. The flux of superoxide is generated by illuminating a reaction mixture containing dianisidine and riboflavin by either a laser source or light from a fluorescent lamp. The oxidation of dianisidine, as sensitized by riboflavin, is enhanced by superoxide dismutase. The increase is linearly dependent on superoxide dismutase concentration. The photochemical reaction is allowed to proceed uninterrupted for a standardized optimum time and intensity of illumination and then terminated by addition of a buffer, 'finibuf', which stabilizes the chromophoric complex formed. This permits the spectrophotometric absorbance measurements of a number of samples collectively and also eliminates the interruption of illumination with the concomitant requirement of a spectrophotometer for constant recording of the absorbance. This method is of utility to both biochemists and clinicians.

This method is suitable for the determination of bromate residues in a variety of baked goods. The peer-verified method trial was performed on white bread, multigrain bread, and coffee cake spiked with known levels of potassium bromate. The analytical portion is extracted with deionized water to remove bromate from the bulk of the baked product. The aqueous extract is carried through a series of steps to remove co-extractives that would interfere with the liquid chromatography (LC) in the determinative step or hasten the deterioration of the LC column. The extract is filtered before passing it through a reversed-phase solid-phase extraction (SPE) column and a cation-exchange column in the silver form to remove lipids and chloride, respectively. Ultrafiltration is then used to remove proteins with molecular weights of >30 000 daltons. Finally, a cation-exchange column in the sodium form is used to remove silver ions from the extract. The determinative step uses LC with a reversed-phase column and an ion-pairing agent in the mobile phase. Detection is based on the post-column reaction of bromate with o-dianisidine to form an oxidation product that is quantitated spectrophotometrically at 450 nm. Overall agreement between the submitting and peer laboratories was quite good. For bromate levels of 10–52 ppb, overall mean recoveries were 76.9 and 78.8% for the submitting and peer laboratories, respectively. The standard deviations were higher for the results of the peer laboratory, probably because of the generally higher level of baseline noise present in the chromatograms. The results demonstrate that the method provides adequate accuracy with low-fat as well as high-fat foods. Bromate at levels as low as 5 ppb (ng/g) can be detected with the method.

Benzidine and its 3,3′-diamino, 3,3′-dimethyl, 3,3′-dimethoxy, 3,3′-difluoro, 3,3′-dichloro, 3,3′-dibromo, 3,3′-dicarbomethoxy and 3,3′-dinitro derivatives together with 2-nitrobenzidine and 3-nitrobenzidine were compared for their in vitro and in vivo genotoxicity. Relative mutagenicity was established with Salmonella strains TA98, TA98/1,8-DNP 6 and TA100 with and without S9 activation. All the derivatives in the presence of S9 were more mutagenic than benzidine with 3,3′-dinitro- and 3-nitro-benzidine having the greatest mutagenicity. Mutagenicity in all 3 strains with S9 activation could be correlated to electron-withdrawing ability of substituent groups, as measured by the basicity of the amines. This correlation was explained on the basis that electron-withdrawing groups could favor the stability of the mutagenic intermediate N -hydroxylamine and also enhance the reactivity of the ultimate mutagenic species, the nitrenium ion. Mutagenicity was also correlated to the energy of the lowest unoccupied molecular orbitals ( E LUMO ). Hydrophobicity was found to have very limited effect on the relative mutagenicity of our benzidine derivatives. The in vivo endpoint was chromosomal aberrations in the bone-marrow cells of mice following intraperitoneal administration of benzidine and its derivatives. In contrast to the in vitro results, while all the amines were genotoxic in vivo, only the 3-nitro derivative had a significant increase in toxicity over benzidine.

Dimethoxybenzidine (DMO) and dimethylbenzidine (DM) are used to synthesize dyes such as C.I. Direct Blue 15 and C.I. Acid Red 114, respectively. These commercially used dyes are metabolically degraded to DMO or DM in the intestinal tract of rodents and subsequently DMO and DM are absorbed into the blood stream. Animals were exposed to DMO, DM, or the dyes in the drinking water. Tumors obtained from control and chemical-treated animals were examined for the presence of activated oncogenes by the NIH 3T3 DNA transfection assay. Activated oncogenes were detected in less than 3% (1/38) of the tumors from control animals whereas 68% (34/50) of the tumors from chemical-treated animals contained detectable oncogenes. Activated oncogenes were detected in both malignant (25/36) and benign (9/14) tumors from the chemically treated animals but only in one of 13 malignant tumors from the control animals. The presence of oncogenes in the chemically induced benign tumors suggests that oncogene activation was an early event in those tumors. Southern blot analysis of transfectant DNA showed that the transforming properties of the chemically induced rat tumor DNAs were due to the transfer of an activated H-ras (31/34) or N-ras (3/34) gene. One spontaneous rat tumor DNA was found to contain an activated H-ras gene. Oligonucleotide hybridization analysis indicated that the H-ras oncogenes from chemical-associated tumors contained mutations at codons 12, 13, or 61 whereas the spontaneously activated H-ras gene contained a point mutation at codon 61. These data suggest that activation of cellular ras genes by point mutation is an important step in the induction of tumors, at least in rats, by this class of benzidine-derived dyes. Moreover, in light of common histogenesis of the normal counterparts of many of the chemically induced neoplasms and histological evidence of varied tissue differentiation in some basal cell neoplasms, it is possible that most or all of the chemically induced neoplasms were derived from a common epidermal progenitor stem cell population.

A new method of horseradish peroxidase (HRP) neurohistochemistry using o-dianisidine (OD) as the chromogen was described. In labelling neurones of the hypoglossal nucleus in rats and cats, the new OD method was as sensitive as Mesulam's method using tetramethyl benzidine (TMB) as the chromogen.

Four methods used for the neurohistochemical demonstration of horseradish peroxidase (HRP) were quantitatively compared by counting retrogradely labeled neurons found after each method was used. HRP used as a retrograde marker is an important neuroanatomical tracing method, and maximum sensitivity in its demonstration of retrogradely, labeled neurons is important if these neuroanatomical studies are to completely demonstrate afferent neurons. The four methods compared were a diaminobenzidine (DAB) procedure, a Hanker-Yates procedure using P-phenylenediamine and pyrocatechol, an o-dianisidine procedure, and a tetramethyl benzidine (TMB) procedure. The TMB procedure resulted in a more complete topography of neurons afferent to the HRP application site, and demonstrated many more neurons in all afferent cell groups that either of the three other procedures. Use of the TMB method was especially critical in the cases of small HRP applications, a size useful for neuroanatomical studies, where the other methods demonstrated very few or no retrogradely labeled neurons. Neurons were judged to be retrogradely HRP labeled if they had small granules of the reaction product (the color varying with the chromogen) describing the somal shape, usually extending into the processes, and a clear nucleus. In addition, after the o-dianisidine or the TMB reaction a small number of retrogradely labeled neurons had soma and processes especially well filled with reaction product, giving the appearance of neurons from Golgi preparations. For a sensitive TMB reaction giving good results, exact H2O2 concentration, freshly prepared solutions, minimal postreaction exposure to alcohol, counterstaining, and clean glassware were each found to be important.

Enteric bacterial and hepatic azoreductase enzymes are capable of reducing azo dyes to yield the constituent aromatic amines. Azo dyes based on benzidine and benzidine congeners have received particular attention because of their widespread use and the known carcinogenicity of benzidine to humans. Azo dyes based on β-diketone coupling components exist preferentially as the tautomeric hydrazones. A series of hydrazone dyes based on benzidine and benzidine congeners was prepared and characterized by NMR and UV-visible spectroscopy. These dyes were tested for mutagenicity using a modified Ames assay and, unlike the true azo dyes, showed no significant mutagenic activity. The hydrazone dyes were resistant to enzymatic reduction by FMN-supplemented hamster-liver post-mitochondrial supernatant (S-9); under identical conditions, azo dyes such as trypan blue were rapidly reduced.

Iodide fixation by murine polymorphonuclear leukocytes (PMN) incubated with viable Candida albicans blastoconidia increases directly with yeast cell concentration up to about 3 x 10(6) cells per ml, but above this concentration bound activity declines dramatically. To understand the basis for this decline, we examined the oxidative metabolism of fungi and stimulated PMN and found some remarkable similarities between these cell types. Both produced 14CO2 when incubated with [1-14C]glucose, both reduced cytochrome c, and both fixed radiolabeled iodide, although the fungi required exogenous lactoperoxidase. In dose-response experiments, iodination by fungi with lactoperoxidase was identical to that with PMN, i.e., the maximum bound activity occurred in cultures with 10(6) to 3 x 10(6) blastoconidia per ml. Iodination by fungi with lactoperoxidase was reduced when blastoconidia were incubated at 25 degrees C or in the presence of catalase and the metabolic inhibitors rotenone, antimycin A, and 2-deoxyglucose. Results from assays for oxidation of scopoletin and o-dianisidine showed that 10(6) blastoconidia in 1.0 ml of medium released 0.5 to 0.7 nmol of H2O2 after 1 h, but 3 X 10(6) and 10(7) cells released significantly less H2O2. These results suggest that iodide fixation by PMN and low numbers of fungal cells may reflect a cooperative effort, with fungi generating some H2O2 that reacts with the myeloperoxidase released from the PMN. With high concentrations of blastoconidia, H2O2 activity appeared to be specifically inhibited, possibly to protect fungal cells from damage.

Extracts of aerobically grown Escherichia coli B exhibit both catalase and dianisidine peroxidase activities. Polyacrylamide gel electrophoresis demonstrates two distinct catalases which have been designated hydroperoxidases I and II (HP-I and HP-II) in order of increasing anodic mobility. HP-I has been purified to essential homogeneity and found to be composed of four subunits of equal size. Its molecular weight is 337,000, and it contains two molecules of protoheme IX per tetramer. Its amino acid composition is unusual, for so large a protein, in lacking half-cystine. HP-I is a very efficient catalase with an activity optimum at pH 7.5, a Km for H2O2 of 3.9 mM, and a turnover number of 9.8 x 10(5) per min. It is also a broad specificity peroxidase capable of acting upon dianisidine, guaiacol, p-phenylenediamine, and pyrogallol. Dianisidine acted as a powerful reversible inhibitor of the catalatic activity of HP-I and as a suicide substrate when HP-I functioned in its peroxidatic mode.

Separation of blood serum on Sephadex G-100 reveals three fractions that interfere with the glucose oxidase— peroxidase method for serum glucose when o-dianisidine is used as the chromogen. A low-molecular-weight fraction containing primarily uric acid, a fraction containing protein with a molecular weight of about 40 000, and a fraction of even higher molecular weight (∼ 500 000) each interfered with glucose recovery when glucose was measured by this procedure. The uric acid fraction and the isolated 40 000 molecular weight fraction interfere by competing with o-dianisidine for hydrogen peroxide in the peroxidase-catalyzed color-formation step. The high-molecular-weight fraction not only interferes with the peroxidase reaction, but also with the glucose oxidase reaction itself. These agents cause values to be low by as much as 20% in the manual determination of glucose in normal serum if their interference is not recognized.

The sensitivity of the hemolysis-in-gel (HIG) test with rubella antigen is not improved by chemical linkage of the virus to the erythrocytes, and after such modification, IgM specific antibodies are not detectable. In the influenza HIG test with tetraazotized o-dianisidine (TOD), chromic chloride and potassium periodate as coupling reagents, increased sensitivity was observed with allantoic fluid of infected eggs as antigen. If Tween-ether treated hemagglutinin is used in the HIG test, zones of hemolysis are detectable only after treatment of the erythrocytes with TOD, chromic chloride and potassium periodate.

o-Phenylenediamine, 2,2'-azino-di(3-ethylbenzthiazoline sulphonic acid-6) (ABTS), o-dianisidine and 4-aminoantipyrine were compared as chromogens for the determination of horseradish peroxidase. Highest sensitivity in the determination of horseradish peroxidase-IgG conjugates in dissolved form was obtained with o-phenylenediamine. When these conjugates were used in a two-site binding enzyme immunoassay for hepatitis B surface antigen (HBsAg), the steepest calibration curve and the lowest detection limit were obtained when ABTS was used to determine the immune complexes bound to the solid phase. Non-ionic detergents, such as polyoxyethylene-sorbitol ester, retarded horseradish peroxidase inactivation, resulting in a chromogen-dependent activity rise of horseradish peroxidase. An optimised determination of horseradish peroxidase is reported, in which the sensitivity of the solid phase enzyme immunoassay is doubled by the use of o-dianisidine.

We describe a method for calculating the absorptivity (in terms of substrate consumed) of the colored solution obtained when o-dianisidine dihydrochloride is oxidized by ceruloplasmin. By oxidizing o-dianisidine dihydrochloride with known amounts of hydrogen peroxide we could determine that the molar reacting ratio of o-dianisidine to hydrogen peroxide is 2:1. So calculated, absorptivity is 9.6 ml µmol-1 cm-1, the figure used to estimate ceruloplasmin oxidase activity in terms of International Units.

A variety of nitrogenous compounds broaden the activity versus pH profile for the peroxidation of dianisidine catalyzed by horseradish peroxidase (HRP), but not by myeloperoxidase, chloroperoxidase, Escherichia coli hydroperoxidase I, methemoglobin, or microperoxidases. The peroxidation of dianisidine catalyzed by cytochrome c peroxidase was affected by the nitrogenous compounds, but to a lesser extent than was the action of HRP. The peroxidations of a variety of phenols by HRP exhibited broad activity versus pH profiles and were unaffected by the nitrogenous compounds. The energy of activation for the peroxidation of dianisidine by HRP was unaffected by changes of pH in the range 6.5-8.5 and was unchanged by the presence of the nitrogenous compounds. The nitrogenous compounds markedly increased Vm for the peroxidation of dianisidine by HRP, but did not change the slope of Lineweaver-Burk plots of kinetic data. These results are accommodated by a mechanism in which nitrogenous compounds hydrogen-bond to the distal histidine of HRP and in so doing raise its pK alpha. Since the acid form of the distal histidine is thought to facilitate peroxidations catalyzed by HRP by hydrogen bonding to the ferryl oxygen of compound II, raising its pK alpha broadens the activity versus pH profile for the peroxidation of anilino substrates, such as dianisidine. We propose that phenolic substrates hydrogen-bond directly to the ferryl oxygen, thus displacing the distal histidine and eliminating the possibility of being influenced by nitrogenous compounds.

The condensation of 2-picoline with various aromatic primary amines (or nitro compounds) in the presence of sulfur was carried out at elevated temperature. This reaction was tentatively divided into three types : The first type is a simple thiopicolinoyl ; the second, a mixed type, yielding thiopicolinoyl and thiazolyl cyclization products ; and the last, unchanged (or resinous). It is noteworthy that protomeric amino group, such as that of N1-position of sulfanilamide and 2-position of pyridine, was active to some extent for this reaction. Oxidative cyclization of five thiopicolinoyl intermediates (XXI, XXIII, and XXIV) from dianisidine, sulfanilamide, homosulfanilamide, 2-aminopyridine, and 5-nitro-2-aminopyridine with potassium ferricyanide was unsuccessful, but not for (XXII) and (XXV). The chemical structure of these cyclization products was discussed on the basis of their ultraviolet spectral observation and electronic theory. Microbiological activity of these compounds will be reported elsewhere.

The dichroic spectra in the stretched polyvinyl alcohol (PVA) sheet of benzidine, 3,3′-dichlorobenzidine, o -tolidine, and dianisidine were analyzed in the region above 220 mμ. The first electronic band appearing at around 310 mμ in the neutral state corresponds to the first band of biphenyl (ca. 250 mμ), and its transition moment is directed along the long axis of the molecule. At the shorter wavelengths of the first band a weak absorption (ca. 270 mμ) is hidden, the transition moment of which is directed along the short axis of the molecule and is attributed to the 1 L b transition of benzene. But in the case of dianisidine, the corresponding band appears at about 290 mμ as a shoulder. The intense band below 220 mμ, of which only the tail was observable, has the same polarization as that of the hidden band and is assigned to the 1 B b transition of benzene. All samples in the acid state have the first band with the longitudinal polarization at about 250 mμ and the second band with the latitudinal below 220 mμ. Besides, dianisidine in the acid state has another band at 292 mμ with the polarization slanting against the molecular axis. This may have originated from the 1 L b transition of benzene which has become intensified and has changed its polarization due to the perturbation of the methoxy groups.

1. The absorption spectra of the dyes prepared by coupling disazotized 2S-, H-, chromotropic, NW-, γ- and J- acid with dianisidine, and of their copper derivatives were observed in aqueous solutions of the binary mixture with Chrysophenine G, and were compared with the sum curves of the component absorption spectra. 2. The order of the equilibrium constants for a 1:1 complex formed between the dyes not containing copper and Chrysophenine G was parallel to the respective values of the difference in wave number (Δν cm−1) between maximum positions in the region of 390∼420 mμ of the curve simply added and that observed with the equimolar binary mixture of about 1×10−5mol./1.; it was also parallel to those of the decreasing ratio in optical density (ΔD⁄D) of the mixed system at the first maximum position of the sum curve. 3. Under the same conditions as above, Δν and ΔD⁄D were also determined for the mixture of the copper derivatives and Chrysophenine G. In this case, the copper derivatives of 2S-, H-, and chr...

Experimental data for absorption on cotton at 90°C with three dyes, Aizen Direct Blue BBH (C. I. No.406), Diamine Blue 3B (C. I. No.477) and Nippon Sky Blue (C. I. No.520) (which differ only slightly in structure, being derived from benzidine, tolidine and dianisidine respectively coupled with H acid) was analysed by the Donnan treatment of Neale et al.. The value of the absorption constant k and the volume of the surface phase of the fibre was determined from the results of the experiment. It was found that the volume of the surface phase increases with increased concentration of the dye. The data of low dye concentrations are analysed by the Donnan treatment using the volume term of 22g/100g cotton, whereas at high dye concentrations it is necessary to use a much larger value of 42g/100g cotton. This value of 42g/100g cotton is in close agreement with the date Boulton et al. for the amount of water imbibed by American cotton.

A procedure is presented for the automated determination of acid phosphatase activity in biological materials using the Robot Chemist. Although either phenylphosphate and α-naphthylphosphate may be used as substrate in this analysis, the procedure is described in detail for serum acid phosphatase using α-naphthylphosphate in 0.1 M citrate, pH 5.2, since this substrate is more selective for prostatic acid phosphatase in human serum. Enzymically generated aα- naphthol is determined by the Emerson reaction (alkaline aminoantipyrine and ferricyanide), modified for use with this automated system. Correlations are presented between the results obtained on the Robot Chemist and the identical procedure developed for the AutoAnalyzer.

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Urocanic acid (UCA) was first demonstrated in human sweat (1), later on in aqueous extracts of superficial (2) and deep human horny layer (barrier” (3)) and in the whole epidermis of guinea pigs (4). The epidermal nature of UCA in guinea pig epidermis, normally exempt from sweat glands, was proven by its existence after X-ray-induced atrophy of sebaceous glands (5). The known origin of UCA from histidine could also be demonstrated in mammalian epidermis by radio-carbon tracers (6). The identity of water soluble ultraviolet-absorbing constituent of normal human horny layer with UCA was confirmed by means of several quite different analyses: as there were characteristic ultraviolet spectra at pH 1 and pH 13 (1, 2), their loss after hydrogenation depending on a transformation to imidazolyl propionic acid (1), ionophoretic mobility (1, 2), Rf-values by paper chromatography in three different solvents (1, 2), coupling reactions with diazosulfanilic acid (Pauly's reagent) (1, 2), and with dianisidine (2), further coloring with eosine-Hg (2), and finally, growing pale after spraying with KMn04 (2).

An automated method for the enzymatic determination of ceruloplasmin with o-dianisidine dihydrochioride as substrate is described. The method enables the measurement of 30 samples per hour with a coefficient of variation (day-to-day) of 2.8%. Results correlate well (r = 0.99) with those obtained by the corresponding manual method.