Homogentisic acid

Homogentisic Acid-Based Whole-Cell Biosensor for Detection of Alkaptonuria Disease

Clinicians require simple quantitative tools for the detection of homogentisic acid in alkaptonuria patients, a rare inherited disorder of amino acid metabolism. In this study, we report a whole-cell biosensor for homogentisic acid to detect alkaptonuria disease through the expression of green fluorescence protein. The assay system utilizes a promoter sequence (hmgA) isolated from the Pseudomonas aeruginosa genome. To increase the sensitivity, the sensor module harboring phmgA::GFP was further transformed into various transposon mutants debilitated in steps involved in the metabolism of phenylalanine and tyrosine via homogentisic acid as a central intermediate. The proposed biosensor was further checked for analytical features such as sensitivity, selectivity, linearity, and precision for the quantification of homogentisic acid in spiked urine samples. The limit of detection for the developed biosensor was calculated to be 3.9 μM, which is comparable to that of the various analytical techniques currently in use. The sensor construct showed no interference from all of the amino acids and its homolog molecules. The accuracy and precision of the proposed biosensor were validated using high-performance liquid chromatography (HPLC) with satisfactory results.

Homogentisic Acid and Gentisic Acid Biosynthesized Pyomelanin Mimics: Structural Characterization and Antioxidant Activity

Pyomelanin mimics from homogentisic acid (HGA) and gentisic acid (GA) were biosynthesized by the oxidative enzyme T. versicolor laccase at physiological pH to obtain water soluble melanins. The pigments show brown-black color, broad band visible light absorption, a persistent paramagnetism and high antioxidant activity. The EPR approach shows that at least two different radical species are present in both cases, contributing to the paramagnetism of the samples. This achievement can also shed light on the composition of the ochronotic pigment in the Alkaptonuria disease. On the other hand, these soluble pyomelanin mimics, sharing physico-chemical properties with eumelanin, can represent a suitable alternative to replace the insoluble melanin pigment in biotechnological applications.

Alkaptonuria

Alkaptonuria is a hereditary disease resulted from accumulation of homogentisic acid within the body due to deficiency of homogentisic acid oxidase. The main clinical feature is dark brown color of urine caused by high urinary output of homogentisic acid. There are no other symptoms or signs of the disease until the fourth decade of life when ochronosis is developed. Life-long accumulation of abnormal metabolites becomes overt in form of severe spondylosis, peripheral arthropathy, tendon rupture, bone osteoporosis as well as aortic valve stenosis and skin pigmentation. The features of the disease are associated with affinity of homogentisic acid to the connective tissue and its effect on collagen structure. Only symptomatic treatment is applied in case of alkaptonuria and ochronosis.

Homogentisic acid induces autophagy alterations leading to chondroptosis in human chondrocytes: Implications in Alkaptonuria

Alkaptonuria (AKU) is an ultra-rare genetic disease caused by a deficient activity of the enzyme homogentisate 1,2-dioxygenase (HGD) leading to the accumulation of homogentisic acid (HGA) on connective tissues. Even though AKU is a multi-systemic disease, osteoarticular cartilage is the most affected system and the most damaged tissue by the disease. In chondrocytes, HGA causes oxidative stress dysfunctions, which induce a series of not fully characterized cellular responses. In this study, we used a human chondrocytic cell line as an AKU model to evaluate, for the first time, the effect of HGA on autophagy, the main homeostasis system in articular cartilage. Cells responded timely to HGA treatment with an increase in autophagy as a mechanism of protection. In a chronic state, HGA-induced oxidative stress decreased autophagy, and chondrocytes, unable to restore balance, activated the chondroptosis pathway. This decrease in autophagy also correlated with the accumulation of ochronotic pigment, a hallmark of AKU. Our data suggest new perspectives for understanding AKU and a mechanistic model that rationalizes the damaging role of HGA.

Homogentisic acid induces cytoskeleton and extracellular matrix alteration in alkaptonuric cartilage

Alkaptonuria (AKU) is an ultra-rare disease caused by the deficient activity of homogentisate 1,2-dioxygenase enzyme, leading the accumulation of homogentisic acid (HGA) in connective tissues implicating the formation of a black pigmentation called "ochronosis." Although AKU is a multisystemic disease, the most affected tissue is the articular cartilage, which during the pathology appears to be highly damaged. In this study, a model of alkaptonuric chondrocytes and cartilage was realized to investigate the role of HGA in the alteration of the extracellular matrix (ECM). The AKU tissues lost its architecture composed of collagen, proteoglycans, and all the proteins that characterize the ECM. The cause of this alteration in AKU cartilage is attributed to a degeneration of the cytoskeletal network in chondrocytes caused by the accumulation of HGA. The three cytoskeletal proteins, actin, vimentin, and tubulin, were analyzed and a modification in their amount and disposition in AKU chondrocytes model was identified. Cytoskeleton is involved in many fundamental cellular processes; therefore, the aberration in this complex network is involved in the manifestation of AKU disease.