Characterization of the pigment from homogentisic acid and urine and tissue from an alkaptonuria patient.

When urine samples from alkaptonuria patients are allowed to stand, they turn black, presumably owing to the oxidation of homogentisic acid to a melanin-like substance. We report the characterization of the pigments formed by polymerization of (a) the components in the urine from a patient with alkaptonuria and (b) homogentisic acid. The absorption spectra and electron spin resonance signals of these pigments are similar to those of eumelanins. Irradiation of the pigments with nitroblue tetrazolium caused reduction of the tetrazolium; this was partially inhibited by superoxide dismutase. Irradiation of Ehrlich ascites carcinoma cells with the pigments from homogentisic acid or urine caused cell lysis. Since this lysis was inhibited by catalase, we have concluded that it was mediated by H2O2. A similar pigment was also extracted from the tissue from an alkaptonuria patient. It is suggested that the degeneration of tissue in vivo may be due to the deposition of melanin-like pigments in the tissues, probably in combination with metal ions.     

Alkaptonuria in an orangutan (Pongo pygmaeus)

Alkaptonuria has been diagnosed in a laboratory born, five year old, female orangutan. In man, this relatively rare amino acid metabolic disorder is characterized by arthritis, pigmentation of cartilage, and darkening of the urine upon standing. The color change is due to oxidation of homogentisic acid not normally found in the urine. The condition in man is a simple Mendelian recessive trait characterized by the absence of homogentisic acid oxidase activity. At two years of age, the affected orangutan was noted to void urine of normal color, which upon standing turned a very dark maroon color. Clinically the animal appeared normal; and the results of a urinalysis, hemogram, and survey radiographs confirmed this evaluation. Nonspecific laboratory determinations used to diagnose alkaptonuria in man were positive. Homogentisic acid in the urine was confirmed by paper chromatography, and a quantitative evaluation was made by a commercial laboratory. The orangutan has shown no clinical change or evidence of ochronosis during the past three years, and quarterly survey radiographs show no evidence of osteoarthropathy. The affected orangutan has half brothers and sisters in the colony but no full sibling. Both parents are young, however, and are being paired in an attempt to produce siblings. It is hoped that additional animals with this metabolic defect will become available for use as potential models for studying this inborn error of metabolism.     

Detection of Novel Visible-Light Region Absorbance Peaks in the Urine after Alkalization in Patients with Alkaptonuria

Background

Alkaptonuria, caused by a deficiency of homogentisate 1,2-dioxygenase, results in the accumulation of homogentisic acid (2,5-dihydroxyphenylacetic acid, HGA) in the urine. Alkaptonuria is suspected when the urine changes color after it is left to stand at room temperature for several hours to days; oxidation of homogentisic acid to benzoquinone acetic acid underlies this color change, which is accelerated by the addition of alkali. In an attempt to develop a facile screening test for alkaptonuria, we added alkali to urine samples obtained from patients with alkaptonuria and measured the absorbance spectra in the visible light region.

Methods

We evaluated the characteristics of the absorption spectra of urine samples obtained from patients with alkaptonuria (n = 2) and compared them with those of urine specimens obtained from healthy volunteers (n = 5) and patients with phenylketonuria (n = 3), and also of synthetic homogentisic acid solution after alkalization. Alkalization of the urine samples and HGA solution was carried out by the addition of NaOH, KOH or NH4OH. The sample solutions were incubated at room temperature for 1 min, followed by measurement of the absorption spectra.

Results

Addition of alkali to alkaptonuric urine yielded characteristic absorption peaks at 406 nm and 430 nm; an identical result was obtained from HGA solution after alkalization. The absorbance values at both 406 nm and 430 nm increased in a time-dependent manner. In addition, the absorbance values at these peaks were greater in strongly alkaline samples (NaOH- KOH-added) as compared with those in weakly alkaline samples (NH4OH-added). In addition, the peaks disappeared following the addition of ascorbic acid to the samples.

Conclusions

We found two characteristic peaks at 406 nm and 430 nm in both alkaptonuric urine and HGA solution after alkalization. This new quick and easy method may pave the way for the development of an easy method for the diagnosis of alkaptonuria.     

Homogentisic acid autoxidation and oxygen radical generation: implications for the etiology of alkaptonuric arthritis

The metabolic disorder, alkaptonuria, is distinguished by elevated serum levels of 2,5-dihydroxyphenylacetic acid (homogentisic acid), pigmentation of cartilage and connective tissue and, ultimately, the development of inflammatory arthritis. Oxygen radical generation during homogentisic acid autoxidation was characterized in vitro to assess the likelihood that oxygen radicals act as molecular agents of alkaptonuric arthritis in vivo. For homogentisic acid autoxidized at physiological pH and above, yielding superoxide (O2-)2 and hydrogen peroxide (H2O2), the homogentisic acid autoxidation rate was oxygen dependent, proportional to homogentisic acid concentration, temperature dependent and pH dependent. Formation of the oxidized product, benzoquinoneacetic acid was inhibited by the reducing agents, NADH, reduced glutathione, and ascorbic acid and accelerated by SOD and manganese-pyrophosphate. Manganese stimulated autoxidation was suppressed by diethylenetriaminepentaacetic acid (DTPA). Homogentisic acid autoxidation stimulated a rapid cooxidation of ascorbic acid at pH 7.45. Hydrogen peroxide was among the products of cooxidation. The combination of homogentisic acid and Fe3+-EDTA stimulated hydroxyl radical (OH.) formation estimated by salicylate hydroxylation. Ferric iron was required for the reaction and Fe3+-EDTA was a better catalyst than either free Fe3+ or Fe3+-DTPA. SOD accelerated OH. production by homogentisic acid as did H2O2, and catalase reversed much of the stimulation by SOD. Catalase alone, and the hydroxyl radical scavengers, thiourea and sodium formate, suppressed salicylate hydroxylation. Homogentisic acid and Fe3+-EDTA also stimulated the degradation of hyaluronic acid, the chief viscous element of synovial fluid. Hyaluronic acid depolymerization was time dependent and proportional to the homogentisic acid concentration up to 100 microM. The level of degradation observed was comparable to that obtained with ascorbic acid at equivalent concentrations. The hydroxyl radical was an active intermediate in depolymerization. Thus, catalase and the hydroxyl radical scavengers, thiourea and dimethyl sulfoxide, almost completely suppressed the depolymerization reaction. The ability of homogentisic acid to generate O2-, H2O2 and OH. through autoxidation and the degradation of hyaluronic acid by homogentisic acid-mediated by OH. production suggests that oxygen radicals play a significant role in the etiology of alkaptonuric arthritis.     

Experimental studies on the pathogenesis of ochronotic arthropathy. The effects of homogentisic acid on adult and fetal articular chondrocyte morphology, proliferative capacity and synthesis of proteoglycans in vitro

Lapine adult and fetal articular chondrocytes in monolayer culture were employed as an experimental model for studying the effects of homogentisic acid on chondrocyte morphology, proliferation and synthesis of proteoglycans. Concentrations of homogentisic acid in the range from 0.1 to 100 micrograms/ml were investigated and a cytotoxic effect was detected with concentrations of 5 micrograms/ml and above. Thus, with adult articular chondrocytes this effect was seen between 36 and 48 h of subculture with 5 micrograms/ml or more of homogentisic acid present from the beginning of subculture. In fetal articular chondrocyte cultures a concentration of 1 microgram/ml elicited a statistically significant reduction (13%) in cell proliferation without altering sulphated macromolecular synthesis. Experiments with 3H-thymidine autoradiography to measure the pulse labeling index (LI) in fetal chondrocytes in vitro showed that the 5 micrograms/ml concentration of homogentisic acid present for 21 h from the beginning of subculture gave a LI of 2%, compared with 25% for controls. Thus, homogentisic acid can reduce chondrocyte proliferative capacity before morphological alterations can be detected by light microscopy. No significant alterations in the synthesis of proteoglycans were detected at concentrations below the cytotoxic level. The homogentisic acid-induced cytotoxicity and reduction of proliferation in chondrocytes represents a possible pathway by which cartilage is damaged in ochronotic arthropathy.     

Brown pigments produced by Yarrowia lipolytica result from extracellular accumulation of homogentisic acid

Yarrowia lipolytica produces brown extracellular pigments that correlate with tyrosine catabolism. During tyrosine depletion, the yeast accumulated homogentisic acid, p-hydroxyphenylethanol, and p-hydroxyphenylacetic acid in the medium. Homogentisic acid accumulated under all aeration conditions tested, but its concentration decreased as aeration decreased. With moderate aeration, equimolar concentrations of alcohol and p-hydroxyphenylacetic acid (1:1) were detected, but with lower aeration the alcohol concentration was twice that of the acid (2:1). p-Hydroxyphenylethanol and p-hydroxyphenylacetic acid may result from the spontaneous disproportionation of the corresponding aldehyde, p-hydroxyphenylacetaldehyde. The catabolic pathway of tyrosine in Y. lipolytica involves the formation of p-hydroxyphenylacetaldehyde, which is oxidized to p-hydroxyphenylacetic acid and then further oxidized to homogentisic acid. Brown pigments are produced when homogentisic acid accumulates in the medium. This acid can spontaneously oxidize and polymerize, leading to the formation of pyomelanins. Mn(2+) accelerated and intensified the oxidative polymerization of homogentisic acid, and lactic acid enhanced the stimulating role of Mn(2+). Alkaline conditions also accelerated pigment formation. The proposed tyrosine catabolism pathway appears to be unique for yeast, and this is the first report of a yeast producing pigments involving homogentisic acid.     

Endogenous mutagens derived from amino acids

L-Cysteine, glutathione and the therapeutically used L-cysteine precursor, N-acetyl-L-cysteine, induced strong mutagenic effects in Salmonella typhimurium (reversion of the his- strains TA97, TA92 and TA104), when tested in the presence of subcellular kidney preparations. The tyrosine metabolites, levodopa (an ortho-hydroquinone) and homogentisic acid (a para-hydroquinone) reverted various his- strains as well. This mutagenicity did not require the presence of mammalian enzymes, and was relatively weak. The induction of gene mutations was also studied in mammalian cells (V79 Chinese hamster cells), using acquisition of resistance toward 6-thioguanine as the marker. L-Cysteine and N-acetyl-L-cysteine were found to be inactive, levodopa was weakly mutagenic, and homogentisic acid was strongly mutagenic (enhancing the mutation frequency 135-fold above background at an exposure concentration of 50 microM). This finding is striking as the urinary concentration of homogentisic acid is about 1000 times higher in patients with a genetic defect in homogentisic acid 1,2-dioxygenase (alkaptonuria). Genotoxic and carcinogenic effects of other amino acids and metabolites, reported in the literature, are discussed as well.     

Biosynthesis of phytoquinones. Homogentisic acid: a precursor of plastoquinones, tocopherols and α-tocopherolquinone in higher plants, green algae and blue–green algae

1. By means of (14)C tracer experiments and isotope competition experiments the roles of d-tyrosine, p-hydroxyphenylpyruvic acid, p-hydroxyphenylacetic acid, phenylacetic acid, homogentisic acid and homoarbutin (2-methylquinol 4-beta-d-glucoside) in the biosynthesis of plastoquinones, tocopherols and alpha-tocopherolquinone by maize shoots was investigated. It was established that d-tyrosine, p-hydroxyphenylpyruvic acid and homogentisic acid can all be utilized for this purpose, whereas p-hydroxyphenylacetic acid, phenylacetic acid and homoarbutin cannot. Studies on the mode of incorporation of d-tyrosine, p-hydroxyphenylpyruvic acid and homogentisic acid showed that their nuclear carbon atoms and the side-chain carbon atom adjacent to the nucleus give rise (as a C(6)-C(1) unit) to the p-benzoquinone rings and nuclear methyl groups (one in each case) of plastoquinone-9 and alpha-tocopherolquinone and the aromatic nuclei and nuclear methyl groups (one in each case) of gamma-tocopherol and alpha-tocopherol. 2. By using [(14)C]-homogentisic acid it has been shown that homogentisic acid is also a precursor of plastoquinone, tocopherols and alpha-tocopherolquinone in the higher plants Lactuca sativa and Rumex sanguineus, the green algae Chlorella pyrenoidosa and Euglena gracilis and the blue-green alga Anacystis nidulans.     

The production of homogentisic acid out of phenylacetic acid byAspergillus niger

Summary With the aid of the replacement culture technique it was found that a strain ofAspergillus niger was able to produce homogentisic acid out of phenylacetic acid with a yield upto 15.7 % of the theoretical amount.Evidence is in favour of the view that homogentisic acid is a normal intermediate in the oxidation of the said substrate.     

Brown Pigments Produced by Yarrowia lipolytica Result from Extracellular Accumulation of Homogentisic Acid

Yarrowia lipolytica produces brown extracellular pigments that correlate with tyrosine catabolism. During tyrosine depletion, the yeast accumulated homogentisic acid, p-hydroxyphenylethanol, and p-hydroxyphenylacetic acid in the medium. Homogentisic acid accumulated under all aeration conditions tested, but its concentration decreased as aeration decreased. With moderate aeration, equimolar concentrations of alcohol and p-hydroxyphenylacetic acid (1:1) were detected, but with lower aeration the alcohol concentration was twice that of the acid (2:1). p-Hydroxyphenylethanol and p-hydroxyphenylacetic acid may result from the spontaneous disproportionation of the corresponding aldehyde, p-hydroxyphenylacetaldehyde. The catabolic pathway of tyrosine in Y. lipolytica involves the formation of p-hydroxyphenylacetaldehyde, which is oxidized to p-hydroxyphenylacetic acid and then further oxidized to homogentisic acid. Brown pigments are produced when homogentisic acid accumulates in the medium. This acid can spontaneously oxidize and polymerize, leading to the formation of pyomelanins. Mn²⁺ accelerated and intensified the oxidative polymerization of homogentisic acid, and lactic acid enhanced the stimulating role of Mn²⁺. Alkaline conditions also accelerated pigment formation. The proposed tyrosine catabolism pathway appears to be unique for yeast, and this is the first report of a yeast producing pigments involving homogentisic acid.     

Pyomelanin is produced by Shewanella algae BrY and affected by exogenous iron

Melanin production by Shewanella algae BrY occurred during late- and (or) post-exponential growth in lactate basal salts liquid medium supplemented with tyrosine or phenylalanine. The antioxidant ascorbate inhibited melanin production but not production of the melanin precursor homogentisic acid. In the absence of ascorbate, melanin production was inhibited by the 4-hydroxyphenylpyruvate dioxygenase inhibitor sulcotrione and by concentrations of Fe >or= 0.38 mmol L(-1). These data support the hypothesis that pigment production by S. algae BrY was a result of the conversion of tyrosine or phenylalanine to homogentisic acid, which was excreted, auto-oxidized, and self-polymerized to form pyomelanin. Pyomelanin production by S. algae BrY may play an important role in the biogeochemical cycling of Fe in the environment.     

Homogentisic acid is the product of MelA, which mediates melanogenesis in the marine bacterium Shewanella colwelliana D.

Shewanella colwelliana D is a marine procaryote which produces a diffusible brown pigment that correlates with melA gene expression. Previously, melA had been cloned, sequenced, and expressed in Escherichia coli; however, the reaction product of MelA had not been identified. This report identifies that product as homogentisic acid, provides evidence that the pigment is homogentisic acid-melanin (pyomelanin), and suggests that MelA is p-hydroxyphenylpyruvate hydroxylase. This is the first report of pyomelanin in an obligate marine bacterium.     

Evaluation of filter paper collection of urine samples for detection and measurement of organic acidurias by capillary electrophoresis

There is little doubt that mental retardation has been prevented in most babies diagnosed by newborn screening programs for inborn errors and the cost-benefit ratios of these programs have been reported as highly positive. In a previous work we optimised a CE method for quick profiling of organic acidurias, which characterize a large number of inborn errors, so that it permits the separation, detection and even identification in less than 15 min of 22 organic acids in urine samples related to a wide range of metabolic disorders. In the present work we have studied the adequacy of filter paper collection of urine samples to simplify this step, always difficult in babies, when it is not performed by training personnel. The studied parameters were: media and conditions for re-extraction to give the best sensitivity and a more simple procedure when the samples are measured by CE, interferences coming from the diaper, recoveries obtained, possible correction of recoveries with creatinine and stability of the compounds. The whole method we report has the advantages of easy sample collection, easy shipping or delivery, and rapid analysis. Moreover, this method of collection and analysis allows the identification and quantitation of fumaric, methylmalonic, N-acetylaspartic, pyroglutamic and homogentisic acids, as well as glutaric acid for which screening is considered especially advisable.     

Catabolic ring-cleavage of tyrosine in plant cell cultures

Summary Ten species of plants from 8 families, grown as sterile cell cultures, were examined for their ability to degrade the aromatic ring of l-tyrosine and two of its metabolites, homogentisic acid and 3,4-dihydroxyphenylalanine (DOPA). All cultures showed low levels of tyrosine degradation (0.3–2.6% in 24 h) and high levels of homogentisic acid degradation (9.3–31.0% in 24 h). Cultures of Amaranthus caudatus L. resembled the other nine species in possessing a moderate capacity for DOPA degradation (0.3–11.1% in 24 h).     

A two-component hydroxylase involved in the assimilation of 3-hydroxyphenyl acetate in Pseudomonas putida

The complete catabolic pathway involved in the assimilation of 3-hydroxyphenylacetic acid (3-OH-PhAc) in Pseudomonas putida U has been established. This pathway is integrated by the following: (i) a specific route (upper pathway), which catalyzes the conversion of 3-OH-PhAc into 2,5-dihydroxyphenylacetic acid (2,5-diOH-PhAc) (homogentisic acid, Hmg), and (ii) a central route (convergent route), which catalyzes the transformation of the Hmg generated from 3-OH-PhAc, l-Phe, and l-Tyr into fumarate and acetoacetate (HmgABC). Thus, in a first step the degradation of 3-OH-PhAc requires the uptake of 3-OH-PhAc by means of an active transport system that involves the participation of a permease (MhaC) together with phosphoenolpyruvate as the energy source. Once incorporated, 3-OH-PhAc is hydroxylated to 2,5-diOH-PhAc through an enzymatic reaction catalyzed by a novel two-component flavoprotein aromatic hydroxylase (MhaAB). The large component (MhaA, 62,719 Da) is a flavoprotein, and the small component (MhaB, 6,348 Da) is a coupling protein that is essential for the hydroxylation of 3-OH-PhAc to 2,5-diOH-PhAc. Sequence analyses and molecular biology studies revealed that homogentisic acid synthase (MhaAB) is different from the aromatic hydroxylases reported to date, accounting for its specific involvement in the catabolism of 3-OH-PhAc. Additionally, an ABC transport system (HmgDEFGHI) involved in the uptake of homogentisic acid and two regulatory elements (mhaSR and hmgR) have been identified. Furthermore, the cloning and the expression of some of the catabolic genes in different microbes presented them with the ability to synthesize Hmg (mhaAB) or allowed them to grow in chemically defined media containing 3-OH-PhAc as the sole carbon source (mhaAB and hmgABC).     

Catabolism of homophthalic acid by a soil bacterium

A microorganism capable of degrading homophthalic acid as a sole source of carbon was isolated from soil. The strain was tentatively identified as Pseudomonas sp. Oxygen uptake studies were carried out with possible intermediates. Assays for several different enzymes were performed. Homophthalic acid may be metabolized by this bacterium via p-hydroxyphenyl acetic acid and homogentisic acid intermediates.     

Homogentisic acid affects human osteoblastic functionality by oxidative stress and alteration of the Wnt/β-catenin signaling pathway

Alkaptonuria (AKU) is a rare disease correlated with deficiency of the enzyme homogentisate 1,2 dioxygenase, which causes homogentisic acid (HGA) accumulation. HGA is subjected to oxidation/polymerization reactions, leading to the production of a peculiar melanin-like pigmentation (ochronosis) after chronic inflammation, which is considered as a triggering event for the generation of oxidative stress. Clinical manifestations of AKU are urine darkening, sclera pigmentation, early severe osteoarthropathy, and cardiovascular and renal complication. Despite major clinical manifestations of AKU being observed in the bones and skeleton, the molecular and functional parameters are so far unknown in AKU. In the present study, we used human osteoblasts supplemented with HGA as a AKU cellular model. We observed marked oxidative stress, and for the first time, we were able to correlate HGA deposition with an impairment in the Wnt/β-catenin signaling pathway, opening a range of possible therapeutic strategies for a disease still lacking a known cure.     

Metabolism of carnitine in phenylacetic acid-treated rats and in patients with phenylketonuria

The effect of metabolites accumulating in phenylketonuria (PKU) was investigated on carnitine metabolism in rats and in patients with PKU. Of phenylacetic acid (PEAA), phenylpyruvic acid and homogentisic acid the PEAA was found to be the most effective in inhibiting carnitine biosynthesis in rats. Following 60 min, a single intraperitoneal dose of PEAA the relative conversion rate, i. e. the hydroxylation, of tracer [Me-(3)H]butyrobetaine to [Me-(3)H]carnitine decreased from 62.2+/-6.00% to 39.4+/-5.11% (means+/-S.E.M., P<0.01) in the liver, in the only organ doing this conversion in rats. The conversion of loading amount of unlabeled butyrobetaine to carnitine was also markedly reduced. The impaired hydroxylation of butyrobetaine was reflected by a reduced free and total carnitine levels in the liver and a reduced total carnitine concentration in the plasma. PEAA decreased the hepatic level of glutamic acid and alpha-ketoglutaric acid (alpha-KG), suggesting a mechanism for the reduced flux through the butyrobetaine hydroxylase enzyme, because alpha-KG is an obligatory co-enzyme. In the plasma and urine of PKU patients on unrestricted diet, markedly decreased total carnitine levels were detected. In the liver of PEAA-treated rats and urine of PKU patients, a novel carnitine derivative, phenacetyl-carnitine was verified by HPLC and gas chromatography-mass spectrometry.     

Novel vitamin E forms in leaves of Kalanchoe daigremontiana and Phaseolus coccineus

In the present study, we isolated novel tocochromanols from green leaves of Kalanchoe daigremontiana and primary leaves of etiolated seedlings of Phaseolus coccineus that were identified as β-, γ-, and δ-tocomonoenols with unsaturation at the terminal isoprene unit of the side chain. The content of γ-tocomonoenol in leaves of etiolated bean increased gradually with the age of seedlings, reaching 50% of the γ-tocopherol level in 40-day-old plants. The content of this compound in leaves was increased by short illumination of etiolated plants and by addition of homogentisic acid, a biosynthetic precursor of tocopherols. These data indicated that γ-tocomonoenol is synthesized de novo from homogentisic acid and tetrahydro-geranylgeraniol diphosphate, a phytol precursor. Based on these results, a biosynthetic pathway of tocomonoenols is proposed.