Hepatobiliary circulation and dominant urinary excretion of homogentisic acid in a mouse model of alkaptonuria.

Altered activity of specific enzymes in phenylalanine-tyrosine (phe-tyr) metabolism results in incomplete breakdown of various metabolite substrates in this pathway. Increased biofluid concentration and tissue accumulation of the phe-tyr pathway metabolite homogentisic acid (HGA) is central to pathophysiology in the inherited disorder alkaptonuria (AKU). Accumulation of metabolites upstream of HGA, including tyrosine, occurs in patients on nitisinone, a licenced drug for AKU and hereditary tyrosinaemia type 1, which inhibits the enzyme responsible for HGA production. The aim of this study was to investigate the phe-tyr metabolite content of key biofluids and tissues in AKU mice on and off nitisinone to gain new insights into the biodistribution of metabolites in these altered metabolic states. The data show for the first time that HGA is present in bile in AKU (mean [±SD] = 1003[±410] µmol/L; nitisinone-treated AKU mean [±SD] = 45[±23] µmol/L). Biliary tyrosine, 3(4-hydroxyphenyl)pyruvic acid (HPPA) and 3(4-hydroxyphenyl)lactic acid (HPLA) are also increased on nitisinone. Urine was confirmed as the dominant elimination route of HGA in untreated AKU, but with indication of biliary excretion. These data provide new insights into pathways of phe-tyr metabolite biodistribution and metabolism, showing for the first time that hepatobiliary excretion contributes to the total pool of metabolites in this pathway. Our data suggest that biliary elimination of organic acids and other metabolites may play an underappreciated role in disorders of metabolism. We propose that our finding of approximately 3.8 times greater urinary HGA excretion in AKU mice compared with patients is one reason for the lack of extensive tissue ochronosis in the AKU mouse model.

Ochronosis in a murine model of alkaptonuria is synonymous to that in the human condition.

OBJECTIVE: Alkaptonuria (AKU) is a rare genetic disease which results in severe early onset osteoarthropathy. It has recently been shown that the subchondral interface is of key significance in disease pathogenesis. Human surgical tissues are often beyond this initial stage and there is no published murine model of pathogenesis, to study the natural history of the disease. The murine genotype exists but it has been reported not to demonstrate ochronotic osteoarthropathy consistent with the human disease. Recent anecdotal evidence of macroscopic renal ochronosis in a mouse model of tyrosinaemia led us to perform histological analysis of tissues of these mice that are known to be affected in human AKU. DESIGN: The homogentisate 1,2-dioxygenase Hgd(+/)(-)Fah(-)(/)(-) mouse can model either hereditary tyrosinaemia type I (HT1) or AKU depending on selection conditions. Mice having undergone Hgd reversion were sacrificed at various time points, and their tissues taken for histological analysis. Sections were stained with haematoxylin eosin (H&E) and Schmorl's reagent. RESULTS: Early time point observations at 8 months showed no sign of macroscopic ochronosis of tissues. Macroscopic examination at 13 months revealed ochronosis of the kidneys. Microscopic analysis of the kidneys revealed large pigmented nodules displaying distinct ochre colouration. Close microscopic examination of the distal femur and proximal fibula at the subchondral junctions revealed the presence of numerous pigmented chondrocytes. CONCLUSIONS: Here we present the first data showing ochronosis of tissues in a murine model of AKU. These preliminary histological observations provide a stimulus for further studies into the natural history of the disease to provide a greater understanding of this class of arthropathy.

The role of calcified cartilage and subchondral bone in the initiation and progression of ochronotic arthropathy in alkaptonuria.

OBJECTIVE: Alkaptonuria is a genetic disorder of tyrosine metabolism, resulting in elevated circulating concentrations of homogentisic acid. Homogentisic acid is deposited as a polymer, termed ochronotic pigment, in collagenous tissues, especially cartilages of weight-bearing joints, leading to a severe osteoarthropathy. We undertook this study to investigate the initiation and progression of ochronosis from the earliest detection of pigment through complete joint failure. METHODS: Nine joint samples with varying severities of ochronosis were obtained from alkaptonuria patients undergoing surgery and compared to joint samples obtained from osteoarthritis (OA) patients. Samples were analyzed by light and fluorescence microscopy, 3-dimensional scanning electron microscopy (SEM), and the quantitative backscattered electron mode of SEM. Cartilage samples were mechanically tested by compression to determine Young's modulus of pigmented, nonpigmented, and OA cartilage samples. RESULTS: In alkaptonuria samples with the least advanced ochronosis, pigment was observed intracellularly and in the territorial matrix of individual chondrocytes at the boundary of the subchondral bone and calcified cartilage. In more advanced ochronosis, pigmentation was widespread throughout the hyaline cartilage in either granular composition or as blanket pigmentation in which there is complete and homogenous pigmentation of cartilage matrix. Once hyaline cartilage was extensively pigmented, there was aggressive osteoclastic resorption of the subchondral plate. Pigmented cartilage became impacted on less highly mineralized trabeculae and embedded in the marrow space. Pigmented cartilage samples were much stiffer than nonpigmented or OA cartilage as revealed by a significant difference in Young's modulus. CONCLUSION: Using alkaptonuria cartilage specimens with a wide spectrum of pigmentation, we have characterized the progression of ochronosis. Intact cartilage appears to be resistant to pigmentation but becomes susceptible following focal changes in calcified cartilage. Ochronosis spreads throughout the cartilage, altering the mechanical properties. In advanced ochronosis, there is aggressive resorption of the underlying calcified cartilage leading to an extraordinary phenotype in which there is complete loss of the subchondral plate. These findings should contribute to better understanding of cartilage-subchondral interactions in arthropathies.

PPAR agonists modulate human osteoclast formation and activity in vitro.

Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear steroid hormone superfamily and exist in three isoforms: PPARalpha, beta and gamma, each with specific functions. In this study, we have investigated the expression of PPARs by human osteoclast precursors and osteoclasts generated in vitro. In addition, the effects of fibrates and isoform-specific PPAR agonists on osteoclast formation and resorption in vitro were determined. Human peripheral blood mononuclear cells (PBMCs) were stimulated with human recombinant RANKL and M-CSF to generate osteoclasts. RNA was extracted at days 0, 7, 14 and 21 and RT-PCR for all three PPAR isoforms demonstrated their expression throughout this culture period. To determine the effect on osteoclast formation, PPAR agonists (10(-8) M to 10(-5) M) were added from the beginning of the culture until day 14 and the number of multinucleated osteoclasts counted. The effect of PPAR agonists on osteoclast function was similarly determined by treating mature, multinucleated osteoclasts cultured on dentine wafers with PPAR agonists (10(-8) M to 10(-5) M) for 7 days and quantifying resorption. Bezafibrate and fenofibrate, which non-discriminately activate all PPAR isoforms, significantly inhibited the formation of multinucleated osteoclasts from PBMC in vitro. Bezafibrate treatment of mature osteoclast resulted in 50% inhibition (at 10(-8) M and 10(-7) M) of resorption, yet fenofibrate had no significant effect. Activation of individual PPARs with isoform-specific agonist (GW9578, L165041 and ciglitizone which preferentially activate PPARalpha, beta and gamma respectively) resulted in significant dose-dependent inhibition of multinucleated osteoclast formation. Divergent effects on osteoclast resorption were observed; GW9578 had no significant effect on resorption, whereas ciglitizone and L165041 dose-dependently inhibited and stimulated resorption, respectively. These data show for the first time expression of all three PPAR isoforms throughout the development and maturation period of osteoclasts generated from human PBMCs. In addition, we demonstrate that isoform-specific PPAR agonists have strong effects on multinucleation and highly variable effects on bone resorption. In conclusion, this study highlights the potential of PPARs as therapeutic targets in diseases with accelerated osteoclast formation and resorption.

Calculi and intracellular ochronosis in the submandibular tissues from a patient with alkaptonuria.

Alkaptonuria (AKU) is a rare autosomal recessive condition caused by deficiency of the enzyme homogentisate 1,2 dioxygenase, resulting in widespread deposition of oxidised homogentisic acid (HGA) polymer, primarily in joint tissues but also in other connective tissues. Macroscopic pigmentation of connective tissues in AKU is well documented and is the end point of a process that is not understood. Deposition in less common regions may provide clues to the pigment formation process. This is the first report of detection of ochronotic pigment in acinar cells and lumina in the submandibular gland of a patient with AKU. Deposition was noted in the apical region of the cells. A lobar duct presented a large calculus with unusual deposits possibly associated with calcium salts. This report highlights the effect that local and intracellular factors may have on converting HGA into polymeric derivatives in the absence of an extracellular matrix.