Activity Of Homogentisic Acid Oxidase Research Articles

Amyloidosis in alkaptonuria.

Alkaptonuria (AKU) is an ultra-rare inborn error of metabolism developed from the lack of homogentisic acid oxidase activity, causing homogentisic acid (HGA) accumulation that produces an HGA-melanin ochronotic pigment, of hitherto unknown composition. Besides the accumulation of HGA, the potential role and presence of unidentified proteins has been hypothesized as additional causal factors involved in ochronotic pigment deposition. Evidence has been provided on the presence of serum amyloid A (SAA) in several AKU tissues, which allowed classifying AKU as a novel secondary amyloidosis. In this paper, we will briefly review all direct and indirect lines of evidence related to the presence of amyloidosis in AKU. We also report the first data on abnormal SAA serum levels in a cohort of AKU patients.

Alkaptonuria is a novel human secondary amyloidogenic disease

Alkaptonuria (AKU) is an ultra-rare disease developed from the lack of homogentisic acid oxidase activity, causing homogentisic acid (HGA) accumulation that produces a HGA-melanin ochronotic pigment, of unknown composition. There is no therapy for AKU. Our aim was to verify if AKU implied a secondary amyloidosis. Congo Red, Thioflavin-T staining and TEM were performed to assess amyloid presence in AKU specimens (cartilage, synovia, periumbelical fat, salivary gland) and in HGA-treated human chondrocytes and cartilage. SAA and SAP deposition was examined using immunofluorescence and their levels were evaluated in the patients' plasma by ELISA. 2D electrophoresis was undertaken in AKU cells to evaluate the levels of proteins involved in amyloidogenesis. AKU osteoarticular tissues contained SAA-amyloid in 7/7 patients. Ochronotic pigment and amyloid co-localized in AKU osteoarticular tissues. SAA and SAP composition of the deposits assessed secondary type of amyloidosis. High levels of SAA and SAP were found in AKU patients' plasma. Systemic amyloidosis was assessed by Congo Red staining of patients' abdominal fat and salivary gland. AKU is the second pathology after Parkinson's disease where amyloid is associated with a form of melanin. Aberrant expression of proteins involved in amyloidogenesis has been found in AKU cells. Our findings on alkaptonuria as a novel type II AA amyloidosis open new important perspectives for its therapy, since methotrexate treatment proved to significantly reduce in vitro HGA-induced A-amyloid aggregates.

Ochronosis and lumbar disc herniation

Alkaptonuria is a rare, autosomal recessive metabolic disorder in which the homogentisic acid oxidase activity is absent. Its incidence is as low as 0.001%. Ochronosis is the pigmentation of connective tissues and this pigmentation leads to degenerative changes in alkaptonuric patients. Alkaptonuria most prominently involves the lumbar region, but lumbar disc herniation as the presenting feature of alkaptonuria is not common. Only a few patients required surgical intervention. Herewith we report an alkaptonuric patient, who was operated on for lumbar disc herniation. His discectomy material was black and the metabolic disorder was diagnosed retrospectively. This metabolic disease is often recognized on physical re-examination after the black disc material was seen during the operation. Therefore urinalysis for homogentisic acid should be performed in all patients with degenerative changes of the vertebral column. The results of disc surgery in this patient group is successful.

Tissue Distribution of 2-(2-Nitro-4-trifluoromethylbenzoyl)cyclohexane-1,3-dione (NTBC): Effect on Enzymes Involved in Tyrosine Catabolism and Relevance to Ocular Toxicity in the Rat

Administration of a single oral dose of 2-(2-nitro-4-trifluoromethylbenzoyl)cyclohexane-1,3-dione (NTBC) to rats produced a marked tyrosinemia in the plasma and aqueous humor. The tyrosinemia was both time- and dose-dependent with the duration being more marked at the higher doses. The dose-response curve was very steep with a single dose of 1.5 μmol NTBC/kg (0.5 mg/kg) and above producing maximal concentrations of tyrosine in plasma of about 2500 nmol/ml and in aqueous humor of about 3500–4000 nmol/ml at 24 hr after dosing. Analysis of the key hepatic enzymes involved in tyrosine catabolism showed that 4-hydroxyphenylpyruvate dioxygenase (HPPD) was markedly inhibited soon after dosing at either 0.3 or 30 μmol/kg (0.1 or 10 mg/kg) NTBC and that the activity recovered very slowly. In response to the tyrosinemia, the activity of tyrosine aminotransferase (TAT) in the liver was induced about twofold, while the activity of homogentisic acid oxidase (HGO) was not affected. Daily oral administration of NTBC for 6 weeks induced lesions to the cornea of the eye, with a dose of 0.3 μmol/kg/day producing about a 38% incidence and a higher dose of 30 μmol/kg/day a 75% incidence. Administration of a single oral dose of [14C]NTBC at either 0.3 or 30 μmol/kg led to selective retention of radiolabel in the liver and to a lesser extent in the kidneys and the Harderian gland. Concentrations of radioactivity in the liver and kidneys remained constant over 4 days and after the lower NTBC dose were about 2 nmol/g wet wt and 0.9 nmol/g wet wt, respectively. Subcellular fractionation of the liver showed that the majority of the radiolabel, >90%, was associated reversibly with the cytosol fraction. No retention of radiolabel was detected in the cornea, the site of toxicity. Our studies indicate that NTBC binds to protein in rat liver cytosol, inhibits the hepatic cytosolic enzyme HPPD, and causes a marked and sustained tyrosinemia. We suggest that this marked and sustained ocular tyrosinemia produced by NTBC in the rat is responsible for the corneal lesions since similar corneal lesions are produced by feeding rats a high tyrosine diet.

Purification of the homogentisic acid oxidase from mammalian liver

Alkaptonuria is a rare human autosomal recessive disease caused by the lack of homogentisic acid oxidase (HTO) activity. This enzyme, produced mainly in liver, has been purified and characterized from bovine liver. The aim of this work was to attempt a new purification approach for this enzyme from rabbit liver and to compare its characteristics with the enzyme isolated from human embryonal liver and livers of mice suffering with alkaptonuria. The crude enzyme extracts from livers of rabbit, human embryo and alkaptonuric mice were purified on Sephadex G-100, DEAE-cellulose and sucrose gradients. The pH and temperature optima were determined, as was electrophoretic mobility on SDS-P. We show that HTO activity was completely absent in the liver of the alkaptonuric mouse. In contrast, we purified HTO from rabbit liver 54-fold and determined some basic biochemical characteristics of this enzyme: the pH optimum was approximately 5 and the temperature optimum was 30°C. Although the molecular mass of the enzyme was estimated to be 450–480 kDa, it could be degraded to two proteins with Mw 230 and 200 kDa. The K M of rabbit HTO was estimated to be 1 × 10 −5 M and the activity of HTO was decreased by the salt. Similar biochemical features were obtained with HTO obtained from the human embryo and normal mouse liver. In this study, we biochemically characterized HTO from rabbit liver. We show the inhibitory effect of salt on HTO activity. While this protein could be found in normal murine liver and human embryonal liver, the liver of alkaptonuric mice completely lacked HTO activity.

The Human Gene for Alkaptonuria (AKU) Maps to Chromosome 3q

Alkaptonuria (AKU; McKusick no. 203500) is a rare autosomal recessive disorder caused by the lack of homogentisic acid oxidase activity. Patients excrete large amounts of homogentisic acid in their urine and a black ochronotic pigment is deposited in their cartilage and collagenous tissues. Ochronosis is the predominant clinical complication of the disease leading to ochronotic arthropathy, dark urine, pigment changes of the skin, and other clinical features. A mutation causing alkaptonuria in the mouse has mapped to chromosome 16. Considering conserved synteny, we were able to map the human gene to chromosome 3q in six alkaptonuria pedigrees of Slovak origin.

Ochronosislike Pigmentation From Hydroquinone Bleaching Creams

To the Editor.— I was interested to read the article by Hoshaw et al 1 in the January 1985Archivesdescribing ochronosislike pigmentation from hydroquinone bleaching creams in American blacks. We have recently had the opportunity to evaluate a similar case. In reviewing the literature concerning the biochemical pathway involved in phenylalanine and tyrosine catabolism, I found the article written by Shepartz 2 in 1953. Shepartz has shown that hydroquinone inhibits the activity of homogentisic acid oxidase in vitro. This same enzyme has markedly decreased activity in alkaptonuria (Figure). I would like to propose the following hypothesis: Topically applied hydroquinone inhibits the activity of homogentisic acid oxidase in the skin, resulting in the local accumulation of homogentisic acid, which polymerizes to form ochronotic pigment. In effect, this process would mimic the cutaneous manifestation of endogenous ochronosis. This explanation is supported by the observation that hydroquinone-induced exogenous ochronosis is clinically and

Electron Microscopic and Isozyme Study of a Case of Ochronosis

Hereditary ochronosis in very few cases has been examined electron microscopically or histochemically. In this disease homogentisic acid, a normal intermediary of tyrosine metabolism, forms in excessive amounts. This is believed to be due to absence or defective activity of homogentisic acid oxidase, an enzyme system necessary to break the benzene ring and to further break it down to fumaric and acetoacetic acids. Ochronotic pigment, a polymerized form of homogentisic acid, deposits mainly in mesenchymal tissues. There has been a question whether the pigment originates from the collagenous tissues, or deposits passively, where in contrast to melanin it induces degenerative changes.

Alkaptonuria and Gilbert's syndrome: Report of two affected siblings and hepatic ultrastructure in one sibling

Siblings affected with two hereditable liver enzyme deficiency disorders, alkaptonuria and idiopathic unconjugated hyperbilirubinemia (Gilbert's syndrome), are described. A biopsy specimen of liver from one sibling showed no enzyme activity of homogentisic acid oxidase or bilirubin glucuronyl transferase. Electron micrographs of the liver demonstrated a marked increase in the profiles of the smooth endoplasmic reticulum, frequent dilation of the Golgi apparatus and a peculiar formation of collagen bundles within the space of Disse. The previously reported disruption of the hepatocytic microvilli in Gilbert's syndrome was not found in this patient. Study of the pedigree suggested autosomal dominant inheritance of the Gilbert's syndrome in these siblings. Siblings affected with two hereditable liver enzyme deficiency disorders, alkaptonuria and idiopathic unconjugated hyperbilirubinemia (Gilbert's syndrome), are described. A biopsy specimen of liver from one sibling showed no enzyme activity of homogentisic acid oxidase or bilirubin glucuronyl transferase. Electron micrographs of the liver demonstrated a marked increase in the profiles of the smooth endoplasmic reticulum, frequent dilation of the Golgi apparatus and a peculiar formation of collagen bundles within the space of Disse. The previously reported disruption of the hepatocytic microvilli in Gilbert's syndrome was not found in this patient. Study of the pedigree suggested autosomal dominant inheritance of the Gilbert's syndrome in these siblings.