Ochronotic arthropathy in the context of spondyloarthritis differential diagnosis: a case-based review.

Alkaptonuria is a disease often forgotten because of its rarity. Its pathogenic mechanism is the deficiency of one of the enzymes of the tyrosine degradation pathway-homogentisate-1, 2-dioxygenase, which sequelae is accumulation and deposition of its metabolite homogentisic acid in connective tissues and urine. Alkaptonuria presents as a clinical triad-darkening urine upon prolonged exposure to air, pigmentation of connective tissues and debilitating arthropathy. We present a case report of a 67-year old patient with alkaptonuria who presented with the clinical triad, but was mistakenly diagnosed as having ankylosing spondylitis in the past. Currently there is no treatment for the disease hence the management strategy was focused on symptoms control with analgesics, physical therapy, dietary modification, vitamin C supplementation, and joint arthroplasty. Alkaptonuria's clinical features are extensively described in the literature and despite the fact that it is a rare disease, due to the similar radiographic changes with spondyloarthropathies, it should be included in the differential diagnosis in young patients presenting with severe joint involvement. Early recognition of the disease is necessary since its natural evolution is joint destruction leading to significant reduction in the quality of life. Alkaptonuria's articular features in the spine and peripheral tissues are well described using the classical imaging techniques. Musculoskeletal ultrasonography shows a characteristic set of findings in the soft tissues, including synovium, cartilage, tendons and entheses.

Efficacy of Phenylalanine- and Tyrosine-Restricted Diet in Alkaptonuria Patients on Nitisinone Treatment: Case Series and Review of Literature.

INTRODUCTION: Nitisinone used in alkaptonuria (AKU) can result in keratopathy due to strongly increased tyrosine levels. METHODS: This study aimed to investigate nutritional status and changes in plasma tyrosine and phenylalanine and urinary homogentisic acid (u-HGA) levels in 8 adult AKU patients (mean age, 56.3 ± 4.7 years) who were on tyrosine/phenylalanine-restricted diet together with 2 mg/day nitisinone. RESULTS: The treatment period was 23.4 ± 6.9 months. Daily dietary protein intake was restricted to 0.8-1.0 g/kg/day. Daily tyrosine intake was restricted to 260-450 mg/day for females and 330-550 mg/day for males. Tyrosine/phenylalanine-free amino acid supplements accounted for an average of 56.1% of daily protein intake. The following assessments were performed: anthropometric and plasma tyrosine level measurements every 2 months; ophthalmological examination every 6 months, and nutritional laboratory analyses and measurements of plasma amino acids and u-HGA once in a year. It was targeted to keep the plasma tyrosine level <500 µmol/L. The plasma tyrosine level was <100 µmol/L before the treatment in all patients and around a mean of 582.5 ± 194.8 µmol/L during the treatment. The diet was rearranged if a plasma tyrosine level of >700 µmol/L was detected. The u-HGA level before and after the 1st year of treatment was 1,429.3 ± 1,073.4 mmol/mol creatinine and 33.6 ± 9.5 mmol/mol creatinine, respectively. None of the patients developed keratopathy or experienced weight loss and protein or micronutrient deficiency. CONCLUSION: AKU patients should receive tyrosine/phenylalanine-restricted diet for reducing plasma tyrosine level to the safe range. Tyrosine/phenylalanine-free amino acid supplements can be safely used to enhance dietary compliance. Keratopathy and nutrient deficiency should be frequently monitored.

HPLC with electrochemical detection for determining homogentisic acid and its application to urine from rats fed tyrosine-enriched food.

A highly sensitive method for determining urine homogentisic acid (HGA) is required to provide adequate diagnosis and therapy for alkaptonuria in early stages. In this study, we developed a highly sensitive high-performance liquid chromatography with electrochemical detection (HPLC-ECD) for determining HGA in urine. In order to obtain a chromatogram of HGA by HPLC-ECD, an oxidation current was monitored at +0.5 V vs. Ag/AgCl. The peak heights of HGA showed linearity (r = 0.999) ranging from 4.2 ng/mL to 168 ng/mL, and the detection limit was 1.2 ng/mL (signal-to-noise ratio, S/N = 3). In recovery tests using human control urine spiked with an HGA standard, the recoveries of HGA were more than 93.2 %, and the relative standard deviations (n = 6) were less than 1.9 %. As an in vivo application using male Wistar rats, the level of urine HGA, which was metabolized from tyrosine in tyrosine-enriched food, was determined by this HPLC-ECD method. The determination of HGA in urine by this HPLC-ECD method requires only 0.1 mL of a rat urine specimen and simple sample preparation consisting of dilution and filtration.

Dietary restriction of tyrosine and phenylalanine lowers tyrosinemia associated with nitisinone therapy of alkaptonuria.

Alkaptonuria (AKU) is caused by homogentisate 1,2-dioxygenase deficiency that leads to homogentisic acid (HGA) accumulation, ochronosis and severe osteoarthropathy. Recently, nitisinone treatment, which blocks HGA formation, has been effective in AKU patients. However, a consequence of nitisinone is elevated tyrosine that can cause keratopathy. The effect of tyrosine and phenylalanine dietary restriction was investigated in nitisinone-treated AKU mice, and in an observational study of dietary intervention in AKU patients. Nitisinone-treated AKU mice were fed tyrosine/phenylalanine-free and phenylalanine-free diets with phenylalanine supplementation in drinking water. Tyrosine metabolites were measured pre-nitisinone, post-nitisinone, and after dietary restriction. Subsequently an observational study was undertaken in 10 patients attending the National Alkaptonuria Centre (NAC), with tyrosine >700 µmol/L who had been advised to restrict dietary protein intake and where necessary, to use tyrosine/phenylalanine-free amino acid supplements. Elevated tyrosine (813 µmol/L) was significantly reduced in nitisinone-treated AKU mice fed a tyrosine/phenylalanine-free diet in a dose responsive manner. At 3 days of restriction, tyrosine was 389.3, 274.8, and 144.3 µmol/L with decreasing phenylalanine doses. In contrast, tyrosine was not effectively reduced in mice by a phenylalanine-free diet; at 3 days tyrosine was 757.3, 530.2, and 656.2 µmol/L, with no dose response to phenylalanine supplementation. In NAC patients, tyrosine was significantly reduced (P = .002) when restricting dietary protein alone, and when combined with tyrosine/phenylalanine-free amino acid supplementation; 4 out of 10 patients achieved tyrosine <700 µmol/L. Tyrosine/phenylalanine dietary restriction significantly reduced nitisinone-induced tyrosinemia in mice, with phenylalanine restriction alone proving ineffective. Similarly, protein restriction significantly reduced circulating tyrosine in AKU patients.

Alkaptonuria: A Case Report With Diagnostic Challenge.

Alkaptonuria is a rare autosomal recessive metabolic disorder caused by deficiency of homogentisic acid (HGA) oxidase, the only enzyme capable of catabolizing HGA. Deficiency of this enzyme leads to excess HGA which deposits in the connective tissue. We present a case of a 64-year-old woman who was referred to the dermatology clinic for a full body mole check and skin cancer screening. Clinically she had blue/gray pigmentation of the external ear and sclera. Also she had a domed papule on the left cheek with punctate gray pigmentation which was biopsied. Histopathological examination showed a benign dermal nevus and nonpolarizable, yellow-brown, irregular shaped fibers. Subsequent organic acid screen showed markedly elevated urinary HGA, diagnostic of alkaptonuria. On specific inquiry, the patient revealed she had a history of bilateral Achilles tendon rupture, black urine, arthritis, and external ear discoloration for many years. The pigmented material was then considered to be HGA deposition within the dermal collagen fibers. However, without the appropriate clinical data and confirmatory lab findings, the pigmented fragments on skin biopsy represent a diagnostic challenge. Measures like low protein diet and ascorbic acid supplementation will slow down the disease progression and potential complications later in life; however, there is no definitive treatment for the disease. We emphasize the prompt recognition of the clinical signs and symptoms as well as the importance of the microscopic findings.

Osteoarticular cells tolerate short-term exposure to nitisinone-implications in alkaptonuria.

Alkaptonuria (AKU) is a rare genetic disease resulting in severe, rapidly progressing, early onset multi-joint osteoarthropathy. A potential therapy, nitisinone, is being trialled that reduces the causative agent; homogentisic acid (HGA) and in a murine model has shown to prevent ochronosis. Little is currently known about the effect nitisinone has on osteoarticular cells; these cells suffer most from the presence of HGA and its polymeric derivatives. This led us to investigate nitisinone's effect on chondrocytes and osteoblast-like cells in an in vitro model. Human C20/A4 immortalized chondrocytes, and osteosarcoma cells MG63 cultured in DMEM, as previously described. Confluent cells were then plated into 24-well plates at 4 × 10(4) cells per well in varying concentrations of nitisinone. Cells were cultured for 7 days with medium changes every third day. Trypan blue assay was used to determine viability and the effect of nitisinone concentration on cells. Statistical analysis was performed using analysis of variance, and differences between groups were determined by Newman-Keuls post-test. Analysis of C20/A4 chondrocyte and MG63 osteoblast-like cell viability when cultured in different concentrations of nitisinone demonstrates that there is no statistically significant difference in cell viability compared to control cultures. There is currently no literature surrounding the use of nitisinone in human in vitro models, or its effect on chondrocytes or osteoblast like cells. Our results show that nitisinone does not appear detrimental to cell viability of chondrocytes or osteoblast-like cells, which adds to the evidence that this therapy could be useful in treating AKU.

Ochronotic arthropathy as a paradigm of metabolically induced degenerative joint disease. A case-based review.

Alkaptonuria is a rare, hereditary metabolic disorder in which a deficiency in the homogentisate 1,2-dioxygenase enzyme results in an accumulation of homogentisic acid. Deposition of excess homogentisic acid in different intra- and extra-articular structures with high content of connective tissue causes brownish-black pigmentation and weakening, ultimately resulting in tissue degeneration and finally osteoarthritis. Ochronotic arthropathy is considered a rapidly progressive, disabling condition in which weight-bearing joints and the thoracolumbar spine are predominantly affected. Patients often require multiple joint replacements, such as in the case of the patient presented here. At present, there is no definitive cure for ochronosis, and management is predominantly symptomatic.

Effect of combination of 1064 nm Q-switched Nd:YAG and fractional carbon dioxide lasers for treating exogenous ochronosis.

We describe three cases of exogenous ochronosis of the malar areas due to long-term application of skin-lightening agents for melasma, effectively treated by combination of Q-switched Nd:YAG and the fractional carbon dioxide lasers. None of these lasers has been reported to be used to effectively treat ochronosis before. The Q-switched Nd:YAG laser is capable of disintegrating dermal ochronotic fibers, thereby facilitating their phagocytosis and elimination via lymphatics. The fractional carbon dioxide laser is believed to assist transepidermal elimination of the onchronotic material. We believe successful treatment of ochronosis is possible when both mechanisms are applied.

Alkaptonuria.

A 69-year-old woman presented with a 30-year history of lower back and large joint pain of the hips and shoulders. On examination blue-grey, pigmented macules were present over the cartilaginous portions of the ears and on the sclera. Past medical history included aortic stenosis. Urine homogentisic acid level was elevated, which is diagnostic for alkaptonuria. Alkaptonuria is an autosomal recessive disorder that results in deficiency of homogentisic acid oxidase and in the accumulation of homogentisic acid in connective tissue. Disease can result in blue-grey pigmentation of the cartilage, sclerae, face, and hands as well as severe arthropathy and cardiac valve disease. Treatment is limited at this time. Promising early reports of the use of nitisinone have prompted ongoing trials of this therapeutic agent.

Hemolysis in a patient with alkaptonuria and chronic kidney failure.

In alkaptonuria, the absence of homogentisic acid oxidase results in the accumulation of homogentisic acid (HGA) in the body. Fatal disease cases are infrequent, and death often results from kidney or cardiac complications. We report a 24-year-old alkaptonuric man with severe decreased kidney function who developed fatal metabolic acidosis and intravascular hemolysis. Hemolysis may have been caused by rapid and extensive accumulation of HGA and subsequent accumulation of plasma soluble melanins. Toxic effects of plasma soluble melanins, their intermediates, and reactive oxygen side products are increased when antioxidant mechanisms are overwhelmed. A decrease in serum antioxidative activity has been reported in patients with chronic decreased kidney function. However, despite administration of large doses of an antioxidant agent and ascorbic acid and intensive kidney support, hemolysis and acidosis could not be brought under control and hemolysis led to the death of the patient.

The probable involvement of soluble and deposited melanins, their intermediates and the reactive oxygen side-products in human diseases and aging.

The plasma soluble melanins (PSM) form spontaneously in vitro and in vivo and their formation involves oxidative polymerization and copolymerization of dopa, catecholamines, homogentisic acid, 3-hydroxyanthranilic acid, p-aminophenol, p-phenylenediamine, and other end(ex)ogenous ortho and para polyhydroxy-, (poly)hydroxy(poly)amino- and polyamino-phenyl compounds. The build up of PSM is visible within 2-3 h after the start of incubation at 37 degrees C with 1 mg/ml of plasma. PSM also form similarly in blood and these processes cause hemolysis. The mean quantity of PSM in normal human plasma is 1.61+/-0.1 (S.D.) mg/ml (n = 20) and in normal human urine is 1.1+/-1.2 g/24 h collection (n = 8). They contribute to the yellow color of plasma and urine. Antioxidants delay the formation of PSM. The deposited melanins also form from these precursors. Reactive oxygen side products (ROSP) are generated during and after melanogenesis. Melanins in vivo are generally associated with proteins or with proteins and lipids. The PSM-protein-lipid complexes are called plasma soluble lipofuscins (PSL), because they have histochemical and fluorescence properties similar to those of solid lipofuscins. The soluble and deposited melanins (SDM) and their intermediates have similar toxic chemical reactivities. The oxidizing quinoid (they can produce partially and completely substituted conjugates) and the semiquinoid free radical intermediates are also moieties in most human melanin structures. Soluble melanins formed from dopa, or dopamine, or norepinephrine in weak alkaline solution have been shown to be toxic to human CD4+ lymphoblastic cells (MT-2) at higher than 10 microg/ml concentrations. Alkaptonuria with high levels of homogentisic acid in the plasma is a potentially fatal disease, exhibiting the toxic effects of the homogentisic acid melanin (soluble and deposited), its intermediates and the ROSP. Patients with alkaptonuria develop arthritis and often suffer from other diseases too, including cardiovascular disease (frequent cause of death) and kidney disease. Pheochromocytoma, with high levels of catecholamines in the plasma is another potentially fatal disease. The catecholamine PSM of pheochromocytoma have very light yellow or practically no colors, due to the concentrations and chemical structures. Pheochromocytomas can cause hypertension, cardiovascular disease (frequent cause of death), kidney disease, stroke, cancer, amyloid formation and can mimic many other diseases, including acute pancreatitis, carcinoid, neuroblastoma, psychiatric illness, hypercalcemia, retinal vascular lesions, and diabetes mellitus. Pheochromocytoma is potentially fatal even in patients without hypertension. Following trauma and surgery, heavily pigmented eyes are apt to experience greater inflammation than lightly pigmented eyes. In Parkinson's disease those neurons are lost first in the substantia nigra and locus ceruleus which contain the greatest amounts of neuromelanins. The antihypertensive alphamethyldopa causes Parkinson's syndrome. It forms PSM in a short time in vitro. The side effects of L-dopa (immobility episodes alternate with normal or involuntary movements; psychotic abnormalities) suggest that the SDM, their intermediates and the ROSP present naturally in vivo are involved in the cause of Parkinson's disease and Alzheimer's disease. There is a large overlap between these two diseases. (ABSTRACT TRUNCATED)

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.