Involvement of oxidoreductive reactions of intracellular haemoglobin in the metabolism of 3-hydroxyanthranilic acid in human erythrocytes.

3-Hydroxyanthranilic acid, a metabolite of tryptophan, was rapidly metabolized by human erythrocytes. The final product was determined to be cinnabarinic acid as detected by spectrophotometry, paper chromatography and t.l.c. The formation of cinnabarinic acid from 3-hydroxyanthranilic acid in the cells was markedly inhibited by CO when intracellular haemoglobin was in a ferrous state, and by cyanide when it was in a ferric state. Ferrous haemoglobin in erythrocytes was oxidized to (alpha 3+ beta 2+)2, (alpha 2+ beta 3+)2 and (alpha 3+ beta 3+)2 by 3-hydroxyanthranilic acid, and the oxidation rates were very high, like those of cinnabarinic acid formation, suggesting that the metabolism of 3-hydroxyanthranilic acid is coupled with oxidoreductive reactions of intracellular haemoglobin. This view was further confirmed by the findings that 3-hydroxyanthranilic acid was metabolized by ferrous or ferric haemoglobin and that ferrous and ferric haemoglobins were oxidized and reduced by the compound respectively. The significance of the metabolism of 3-hydroxyanthranilic acid and the oxidoreductive reactions of haemoglobin with this compound may be associated with the pathological conditions with increased 3-hydroxyanthranilic acid levels in the blood of diabetic subjects.     

Mechanism of reaction of 3-hydroxyanthranilic acid with molecular oxygen

The autoxidation of the tryptophan metabolite, 3-hydroxyanthranilic acid, at pH 7 gives rise to a p-quinone dimer and cinnabarinic acid. A novel dimer formed by radical-radical coupling of 3-hydroxyanthranilic acid is also produced. Labelling studies have shown that the C-2 oxygen in the p-quinone dimer is derived from molecular oxygen. A product versus time study of this reaction has revealed that, in the absence of catalase, cinnabarinic acid is formed but undergoes decomposition by hydrogen peroxide. At pH 7, in the presence of catalase, both the p-quinone dimer and cinnabarinic acid are formed at approximately the same rate and this rate of formation increases with increasing pH. Inclusion of superoxide dismutase was found to increase the rate of formation of cinnabarinic acid, suggesting that superoxide ions may also cause decomposition of cinnabarinic acid. This was confirmed by treating cinnabarinic acid with superoxide. A mechanism involving a common anthranilyl radical intermediate is proposed to account for the formation of the different oxidation products.     

Novel Interaction between Laccase and Cellobiose Dehydrogenase during Pigment Synthesis in the White Rot Fungus Pycnoporus cinnabarinus

When glucose is the carbon source, the white rot fungus Pycnoporus cinnabarinus produces a characteristic red pigment, cinnabarinic acid, which is formed by laccase-catalyzed oxidation of the precursor 3-hydroxyanthranilic acid. When P. cinnabarinus was grown on media containing cellobiose or cellulose as the carbon source, the amount of cinnabarinic acid that accumulated was reduced or, in the case of cellulose, no cinnabarinic acid accumulated. Cellobiose-dependent quinone reducing enzymes, the cellobiose dehydrogenases (CDHs), inhibited the redox interaction between laccase and 3-hydroxyanthranilic acid. Two distinct proteins were purified from cellulose-grown cultures of P. cinnabarinus; these proteins were designated CDH I and CDH II. CDH I and CDH II were both monomeric proteins and had apparent molecular weights of about 81,000 and 101,000, respectively, as determined by both gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The pI values were approximately 5.9 for CDH I and 3.8 for CDH II. Both CDHs used several known CDH substrates as electron acceptors and specifically adsorbed to cellulose. Only CDH II could reduce cytochrome c. The optimum pH values for CDH I and CDH II were 5.5 and 4.5, respectively. In in vitro experiments, both enzymes inhibited laccase-mediated formation of cinnabarinic acid. Oxidation intermediates of 3-hydroxyanthranilic acid served as endogenous electron acceptors for the two CDHs from P. cinnabarinus. These results demonstrated that in the presence of a suitable cellulose-derived electron donor, CDHs can regenerate fungal metabolites oxidized by laccase, and they also supported the hypothesis that CDHs act as links between cellulolytic and ligninolytic pathways.     

Cinnabarinic acid formation in Malpighian tubules of the silkworm, Bombyx mori. Participation of catalase in cinnabarinic acid formation in the presence of manganese ion

Cinnabarinic acid was formed from 3-hydroxyanthranilic acid during incubation with a soluble fraction from Malpighian tubules of the silkworm, Bombyx mori, in the presence of manganese ion. The enzyme having this activity was purified to homogeneity by ammonium sulfate fractionation, gel filtration and ion exchange chromatography. Enzyme activity was accompanied by parallel catalase activity at all steps of purification; the two activities could not be separated from each other. The purified protein was concluded to be catalase. Manganese was shown to be present in 0.1 mM concentration in Malpighian tubules of Bombyx mori. These findings suggest that in Malpighian tubules catalase participates in the formation of cinnabarinic acid. A possible mechanism for the formation of cinnabarinic acid from 3-hydroxyanthranilic acid by catalase in the presence of manganese ion is proposed.     

Redox reactions related to indoleamine 2,3-dioxygenase and tryptophan metabolism along the kynurenine pathway

The heme enzyme indoleamine 2,3-dioxygenase (IDO) oxidizes the pyrrole moiety of L-tryptophan (Trp) and other indoleamines and represents the initial and rate-limiting enzyme of the kynurenine (Kyn) pathway. IDO is a unique enzyme in that it can utilize superoxide anion radical (O2*- ) as both a substrate and a co-factor. The latter role is due to the ability of O2*- to reduce inactive ferric-IDO to the active ferrous form. Nitrogen monoxide (*NO) and H2O2 inhibit the dioxygenase and various inter-relationships between the nitric oxide synthase- and IDO-initiated amino acid degradative pathways exist. Induction of IDO and metabolism of Trp along the Kyn pathway is implicated in a variety of physiological and pathophysiological processes, including anti-microbial and anti-tumor defense, neuropathology, immunoregulation and antioxidant activity. Antioxidant activity may arise from O2*- scavenging by IDO and formation of the potent radical scavengers and Kyn pathway metabolites, 3-hydroxyanthranilic acid and 3-hydroxykynurenine. Under certain conditions, these aminophenols and other Kyn pathway metabolites may exhibit pro-oxidant activities. This article reviews findings indicating that redox reactions are involved in the regulation of IDO and Trp metabolism along the Kyn pathway and also participate in the biological activities exhibited by Kyn pathway metabolites.     

Determination of tryptophan and its kynurenine pathway metabolites in human serum by high-performance liquid chromatography with simultaneous ultraviolet and fluorimetric detection

An isocratic reversed-phase high-performance liquid chromatographic method for the simultaneous determination of tryptophan and four metabolites of the kynurenine pathway (kynurenine, 3-hydroxykynurenine, kynurenic acid and 3-hydroxyanthranilic acid) in human serum is described. This new method, which uses both isocratic elution and two on-line connected programmable ultraviolet and spectrofluorimetric detectors, allows the determination of these metabolites, in the physiological ranges, with satisfying specificity and sensitivity within 30 min.     

Synthesis of cinnabarinic acid by metabolically engineered Pseudomonas chlororaphis GP72

Cinnabarinic acid is a valuable phenoxazinone that has broad applications in the pharmaceutical, chemical, and dyeing industries. However, few studies have investigated the production of cinnabarinic acid or its derivatives using genetically engineered microorganisms. Herein, an efficient synthetic pathway of cinnabarinic acid was designed and constructed in Pseudomonas chlororaphis GP72 for the first tim, which was more straightforward and robust than the known eukaryotic biosynthetic pathways. First, we screened and identified trans-2,3-dihydro-3-hydroxyanthranilic acid (DHHA) dehydrogenases from Escherichia coli MG1655 (encoded by entA), Streptomyces sp. NRRL12068 (encoded by bomO) and Streptomyces chartreusis NRRL3882 (encoded by calB₃) based on the structural similarity of the substrate and product, and the DHHA dehydrogenase encoded by calB₃ was selected for the synthesis of cinnabarinic acid due to its high DHHA conversion rate. Subsequently, cinnabarinic acid was synthesized by the expression of the DHHA dehydrogenase CalB₃ and the phenoxazinone synthase CotA in the DHHA-producing strain P. chlororaphis GP72, resulting in a cinnabarinic acid titer of 20.3 mg/L at 48 hr. Further fermentation optimization by the addition of Cu²⁺, H₂O₂, and with adding glycerol increased cinnabarinic acid titer to 136.2 mg/L in shake flasks. The results indicate that P. chlororaphis GP72 may be engineered as a microbial cell factory to produce cinnabarinic acid or its derivatives from renewable bioresources.     

Utilization of free fatty acids by starved and pregnant sheep

Rat-liver cinnabarinate synthase (3-hydroxyanthranilic acid-oxygen oxido-reductase) was partially purified. Stoicheiometric studies indicated the consumption of 3 atoms of oxygen/molecule of cinnabarinic acid formed. There was an initial lag in enzyme activity. The reaction had an optimum pH about 7.2 and an optimum temperature of 37 degrees . The enzyme was highly specific for 3-hydroxyanthranilic acid. The system showed an absolute requirement for Mn(2+) ions. Several bivalent metal ions and metal-chelating agents inhibited the reaction. Thiol inhibitors had no effect on enzyme activity, but reducing agents such as ascorbic acid were potent inhibitors. There was no requirement for any cofactor other than Mn(2+) ions. The probable significance of the reaction in mammals is discussed.     

Evidence for a close link between the thyroid hormone transport system and the aromatic amino acid transport system T in erythrocytes

The transport of [125I]triiodothyronine ([125I]T3) and [3H]tryptophan ([3H]Trp) by washed rat erythrocytes was studied at 25 degrees C in the presence of leucine in order to block the neutral amino acid transport system L. Eadie-Hofstee plots of initial velocity data gave the following values of Km (micromolar) and Vmax (nanomole/min/10(8) cells): 0.122 +/- 0.007 and 0.140 +/- 0.021 for T3, and 558 +/- 28 and 17.4 +/- 2.3 for Trp (n = 5). The Trp transport system in rat erythrocytes is similar to the human erythrocyte aromatic amino acid-specific system T described by Rosenberg et al. (Rosenberg, R., Young, J. D., and Ellory, J. C. (1980) Biochim. Biophys. Acta 598, 375-384). Unlabeled aromatic amino acids (e.g. Trp, phenylalanine, tyrosine) competitively inhibited [125I]T3 uptake and unlabeled iodothyronine analogues (e.g. T3, D-T3, thyroxine, thyronine) competitively inhibited [3H]Trp uptake. The inhibition constants of these competitors measured with each labeled substrate were highly correlated. N-Ethylmaleimide irreversibly inhibited T3 and Trp transport and each substrate protected the transport system of the other from inactivation by N-ethylmaleimide. The Vmax of T3 and Trp transport by human erythrocytes were 500 and 120 times lower, respectively, than those of rat erythrocytes (0.30 and 126 pmol/min/10(8) cells, respectively). The T3 and Trp transport activities of sheep erythrocytes were undetectable. These results indicate that T3 and Trp either share a common multi-specific transport system or are transported by closely linked systems which interact in the erythrocyte membrane.     

A new biological assay, utilizing parasitic protozoa, for screening carcinogenic substances which induce bladder cancer in man and other mammals

Injections of aromatic amines (β-naphthylamine, benzidine, O-dianisidine or N-2-fluorenyl acetamide), tryptophan metabolites (3-hydroxyanthranilic acid, xanthurenic acid or LD-kynurenine sulphate), oestrone, and nicotine, which are known bladder carcinogens in man and some other mammals induced sexual reproduction (encystation) in Opalina sudafricana when injected into its host Bufo regularis. This may be used as a new biological assay for screening substances which induce bladder cancer in man and some other mammals. It is speculated that the metabolites of the injected carcinogenic substances used in this work are excreted in the urine of the host, hydrolysed by the hydrolytic enzymes and become carcinogenic. These carcinogenic metabolites reach the parasites in the rectum of the toads and induce them to divide mitotically to form small forms which eventually encyst. It is speculated that the presence of cysts in the rectum of the injected toads is indicative that a carcinogenic effect took place in the parasites. Oestrone is the only carcinogenic substance which induced encystation in the opalinids in vitro. Urine of toads injected with β-naphthylamine, benzidine, O-dianisidine, N-2-fluorenyl acetamide, 3-hydroxyanthranilic acid, xanthurenic acid, DL-kynurenine sulphate, oestrone and nicotine induced cyst formation in the parasites in vitro.     

Synthesis of Quinolinic Acid by 3-Hydroxyanthranilic Acid Oxygenase in Rat Brain Tissue In Vitro

In mammalian peripheral organs, 3-hydroxyanthranilic acid oxygenase (3HAO), catalyzing the conversion of 3-hydroxyanthranilic acid to quinolinic acid, constitutes a link in the catabolic pathway of tryptophan to NAD. Because of the possible involvement of quinolinic acid in the initiation of neurodegenerative phenomena, we examined the presence and characteristics of 3HAO in rat brain tissue. A simple and sensitive assay method, based on the use of [carboxy-14C]3-hydroxyanthranilic acid as a substrate, was developed and the enzymatic product, [14C]quinolinic acid, identified by chromatographic and biochemical means. Kinetic analysis of rat forebrain 3HAO revealed a Km of 3.6 +/- 0.5 microM for 3-hydroxyanthranilic acid and a Vmax of 73.7 +/- 9.5 pmol quinolinic acid/h/mg tissue. The enzyme showed pronounced selectivity for its substrate, since several substances structurally and metabolically related to 3-hydroxyanthranilic acid caused less than 25% inhibition of activity at 500 microM. Both the Fe2+ dependency and the distinct subcellular distribution (soluble fraction) of brain 3HAO indicated a close resemblance to 3HAO from peripheral tissues. Examination of the regional distribution in the brain demonstrated a 10-fold variation between the region of highest (olfactory bulb) and lowest (retina) 3HAO activity. The brain enzyme was present at the earliest age tested (7 days postnatum) and increased to 167% at 15 days before reaching adult levels. Enzyme activity was stable over extended periods of storage at -80 degrees C. Taken together, these data indicate that measurements of brain 3HAO may yield significant information concerning a possible role of quinolinic acid in brain function and/or dysfunction.     

Kynurenines and the respiratory parameters on rat heart mitochondria

It has been shown recently that the L-kynurenine metabolite kynurenic acid lowers the efficacy of mitochondria ATP synthesis by significantly increasing state IV, and reducing respiratory control index and ADP/oxygen ratio of glutamate/malate-consuming heart mitochondria. In the present study we investigated the effect of L-tryptophan (1.25 microM to 5 mM) and other metabolites of L-kynurenine as 3-hydroxykynurenine (1.25 microM to 2.5 mM), anthranilic acid (1.25 microM to 5 mM) and 3-hydroxyanthranilic acid (1.25 microM to 5 mM) on the heart mitochondria function. Mitochondria were incubated with saturating concentrations of respiratory substrates glutamate/malate (5 mM), succinate (10 mM) or NADH (1 mM) in the presence or absence of L-tryptophan metabolites. Among tested substances, 3-hydroxykynurenine, 3-hydroxyanthranilic acid and anthranilic acid but not tryptophan affected the respiratory parameters dose-dependently, however at a high concentration, of a micro molar range. 3-Hydroxykynurenine and 3-hydroxyanthranilic acid lowered respiratory control index and ADP/oxygen ratio in the presence of glutamate/malate and succinate but not with NADH. While, anthranilic acid reduced state III oxygen consumption rate and lowered the respiratory control index only of glutamate/malate-consuming heart mitochondria. Co-application of anthranilic acid and kynurenic acid (125 or 625 microM each) to glutamate/malate-consuming heart mitochondria caused a non-additive deterioration of the respiratory parameters determined predominantly by kynurenic acid. Accumulated data indicate that within L-tryptophan metabolites kynurenic acid is the most effective, followed by anthranilic acid, 3-hydroxykynurenine, 3-hydroxyanthranilic acid to influence the respiratory parameters of heart mitochondria. Present data allow to speculate that changes of kynurenic acid and/or anthranilic acid formation in heart tissue mitochondria due to fluctuation of L-kynurenine metabolism may be of functional importance for cardiovascular processes. On the other hand, beside the effect of 3-hydroxyanthranilic acid and 3-hydroxykynurenine on respiratory parameters, their oxidative reactivity may contribute to impairment of mitochondria function, too.     

Human plasma lipofuscin melanins formed from tryptophan metabolites

Incubation of human plasma with the tryptophan metabolites 3-hydroxy-DL-kynurenine (3HK) or 3-hydroxyanthranilic acid (3HAA) yields soluble brown pigments with the fluorescence spectrophotometric and paper chromatographic properties of plasma lipofuscin (PL). An alternative to the recognized tyrosine pathway of melanogenesis is therefore demonstrated in human plasma.     

Oxidation of 3-hydroxyanthranilic acid to the phenoxazinone cinnabarinic acid by peroxyl radicals and by compound I of peroxidases or catalase.

Since 3-hydroxyanthranilic acid (3HAA), an oxidation product of tryptophan metabolism, is a powerful radical scavenger [Christen, S., Peterhans, E., & Stocker, R. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 2506], its reaction with peroxyl radicals was investigated further. Exposure to aqueous peroxyl radicals generated at constant rate under air from the thermolabile radical initiator 2,2'-azobis[2-amid-inopropane] hydrochloride (AAPH) resulted in rapid consumption of 3HAA with initial accumulation of its cyclic dimer, cinnabarinic acid (CA). The initial rate of formation of the phenoxazinone CA accounted for approximately 75% of the initial rate of oxidation of 3HAA, taking into account that 2 mol of 3HAA are required to form 1 mol of CA. Consumption of 3HAA under anaerobic conditions (where alkyl radicals are produced from AAPH) was considerably slower and did not result in detectable formation of CA. Addition of superoxide dismutase enhanced autoxidation of 3HAA as well as the initial rates of peroxyl radical-induced oxidation of 3HAA and formation of CA by approximately 40-50%, whereas inclusion of xanthine/xanthine oxidase decreased the rate of oxidation of 3HAA by approximately 50% and inhibited formation of CA almost completely, suggesting that superoxide anion radical (O2.-) was formed and reacted with reaction intermediate(s) to curtail formation of CA. Formation of CA was also observed when 3HAA was added to performed compound I of horseradish peroxidase (HRPO) or catalytic amounts of either HRPO, myeloperoxidase, or bovine liver catalase together with glucose/glucose oxidase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enzymic conversion of 3-hydroxanthranilic acid into cinnabarinic acid by the nuclear fraction of rat liver

1. An enzyme solely localized in the nuclear fraction of rat liver was found to convert 3-hydroxyanthranilic acid into a red product that was isolated and crystallized from the reaction mixture. The product was identified as cinnabarinic acid (2-amino-3-oxo-3H-phenoxazine-1,9-dicarboxylic acid) by comparing its properties with synthetic cinnabarinic acid. 2. The enzyme had optimum pH at 7·2. Heavy-metal ions like Ag⁺, Hg²⁺, MoO₄²⁻, Fe²⁺ and Cu²⁺ were inhibitory; Mn²⁺ activated the reaction to a considerable extent. 3. The reaction was inhibited by mercaptoethanol, GSH and cysteine, and activated by p-hydroxymercuribenzoate and sodium arsenite, which may suggest the involvement of disulphide groups in the reaction.     

Simultaneous measurement of tryptophan and related compounds by liquid chromatography/electrospray ionization tandem mass spectrometry

We have expanded a liquid chromatographic-tandem mass spectrometric method that measures 3-hydroxykynurenine and 3-hydroxyanthranilic acid in addition to tryptophan and kynurenine both intra- and extracellularly. After reversed phase HPLC separation, the compounds were detected in the MS positive multiple reaction monitoring mode. We found a good linear response for each tryptophan metabolite. The lower limit of quantification for each compound ranged from 0.01 to 0.1 microM. The extraction efficiencies from spiked cell samples and culture medium ranged between 83 and 111% and the overall coefficient of variation of analyses was less than 7%. Using our method, we found tryptophan metabolites in the cells and the culture medium of LN229 human glioma cells were stimulated by interferon-gamma, a known inducer of indoleamine 2,3-dioxygenase. The intracellular concentrations of kynurenine, 3-hydroxykynurenine and 3-hydroxyanthranilic acid were higher than those in the medium. This is the first report of a method for the simultaneous determination of tryptophan and its metabolic products both intra- and extracellularly.     

4-Methyl-3-hydroxyanthranilic acid activating enzyme from actinomycin-producing Streptomyces chrysomallus

A 4-methyl-3-hydroxyanthranilic acid (4-MHA) activating enzyme was purified 24-fold from a crude protein extract of Streptomyces chrysomallus . The enzyme catalyzes both 4-MHA-dependent ATP/PPi exchange and the formation of the corresponding adenylate. No AMP was formed during the reaction, indicating that no covalent binding of 4-MHA takes place. Besides 4-MHA, the enzyme also catalyzes the formation of adenylates from 3-hydroxyanthranilic acid (3-HA), anthranilic acid (AA), benzoic acid (BA), 3-hydroxybenzoic acid (3-HB), 4-methyl-3-hydroxybenzoic acid (4-MHB), 4-methyl-3-methoxybenzoic acid (4- MMB ), and 4-aminobenzoic acid (4-AB). No such adenylates were formed from 2-aminophenol (2-AP), 2-hydroxybenzoic acid (2-HB), 3-hydroxykynurenine (3-HK), and tryptophan (Trp). 3-HA, 4-MHB, and 4-AB were among the structural analogues of 4-MHA that were the most effective for adenylate synthesis. In the case of 3-HA, considerable AMP release was observed, most probably due to nonenzymatic hydrolysis of the corresponding adenylate. A molecular weight between 53 000 and 57 000 was estimated. The specific activity of the enzyme was correlated with the titer of antibiotic in the cultures, and feeding experiments with whole mycelium of S. chrysomallus showed that 4-MHB was a strong inhibitor of actinomycin synthesis in vivo. The data strongly suggest that the enzyme is involved in the biosynthesis of actinomycin.     

Cloning of human 3-hydroxyanthranilic acid dioxygenase in Escherichia coli: characterisation of the purified enzyme and its in vitro inhibition by Zn2+

3-hydroxyanthranilic acid oxygenase (3-HAO) catalyses the conversion of 3-hydroxyanthranilic acid to quinolinic acid. Because of the involvement of quinolinic acid in the initiation of neurodegenerative phenomena, we have cloned human 3-HAO in Escherichia coli, overexpressed and purified it with the aim of studying its enzymatic activity and for future structural studies. The recombinant human protein, obtained in E. coli, retains its enzymatic activity which can occur only in the presence of Fe(II); several other metals have been tested but in no case the formation of the product has been observed. On the contrary, two of the ions tested inhibit the catalytic reaction and one of them, Zn2+, could be of physiological relevance. A circular dichroism analysis has also been performed, showing that the secondary structure is mainly of the beta type, with a minority of alpha.     

Effects of the 3-hydroxyanthranilic acid analogue NCR-631 on anoxia-, IL-1β- and LPS-induced hippocampal pyramidal cell lossin vitro

Summary The kynurenine pathway intermediate 3-hydroxyanthranilic acid (3-HANA) is converted by 3-HANA 3,4-dioxygenase (3-HAO) to the putative neuropathogen quinolinic acid (QUIN). In the present study, the neuroprotective effects of the 3-HANA analogue and 3-HAO inhibitor NCR-631 was investigated using organotypic cultures of rat hippocampus. An anoxic lesion was induced by exposing the cultures to 100% N₂ for 150 min, resulting in a pronounced loss of pyramidal neurons, as identified using NMDA-R1 receptor subunit immunohistochemistry. NCR-631 provided a concentration-dependent protective effect against the anoxia. NCR-631 was also found to counteract the loss of pyramidal neurons in two models of neuroinflammatory-related damage; incubation with either LPS (10 ng/ml) or IL-1 (10 IU/ml). The findings suggest that NCR-631 has neuroprotective properties and that it may be a useful tool to study the role of kynurenines in neurodegeneration.Abbreviations EAA excitatory amino acid - 3-HANA 3-hydroxyanthranilic acid - 3-HAO 3-hydroxyanthranilic acid 3,4-dioxygenase - IL-1 interleukin-1 - KYNA kynurenic acid - LPS lipopolysaccaride - NCR-631 4,6-dibromo-3hydroxyanthranilic acid - NMDA N-methyl-d-aspartate - QUIN quinolinic acid     

Increased levels of serum lipofuscin derived from 3-hydroxyanthranilic acid in patients with chronic renal failure

Normal Caucasian male sera incubated with 3-hydroxyanthranilic acid to generate soluble lipofuscin were studied together with unincubated serum samples from uremic Caucasian males, using the methods of Schwertner & Hawthorne in order to identify a fluorescent substance found by them to increase in uremic sera. Ethanol extracts of uremic sera, of normal sera containing this soluble lipofuscin and of same normal serum blanks were prepared. Reversed-phase thin-layer chromatograms of the extracts developed with methanol-water (40:60, v./v.), displayed one significant spot per sample, with RF values of 0.89 +/- 0.02. The spots showed blue fluorescence in 366 nm ultraviolet light. Aqueous solutions of the spots from uremic sera and from 3-hydroxyanthranilic acid-incubated normal sera produced closely similar fluorescence excitation shoulders and maxima at approximately 321 nm and emission maxima at 402 +/- 3 nm with significantly higher intensities than the normal. Thin-layer chromatograms of the ethanol extracts were also prepared on silica gel G developed with ethanol. The uremic, the 3-hydroxyanthranilic acid-incubated normal sera and the normal blank sera showed identical patterns in 366 nm light. The findings demonstrate that serum lipofuscin derived from 3-hydroxyanthranilic acid either in vivo or in vitro yields the fluorescent substance or component separated by ethanol extraction and reversed-phase thin-layer chromatography and that this serum lipofuscin present at low concentration in normal sera increases in uremic sera.