Alkaptonuria and Ochronosis – Experience from Slovakia Alkaptonúria a Ochronóza – Skúsenosti Zo Slovenska

Acta Fac. Pharm. Univ. Comen. LXI, 2014 (1), p. 29–35 ISSN 1338-6786 (online) and ISSN 0301-2298 (print version) DOI 10.2478/afpuc-2014-0001

ACTA FACULTATIS PHARMACEUTICAE UNIVERSITATIS COMENIANAE

ALKAPTONURIA AND OCHRONOSIS – EXPERIENCE FROM SLOVAKIA ALKAPTONÚRIA A OCHRONÓZA – SKÚSENOSTI ZO SLOVENSKA

Original research article Rovenský, J.1, Urbánek, T.1, Imrich, R.2

1National Institute of Rheumatic Diseases, Piešťany, Slovak Republic 1Národný ústav reumatických chorôb, Piešťany, Slovenská republika 2Center for Molecular Medicine, Institute of Experimental Endocrinology, / 2Centrum molekulárnej medicíny, Ústav experimentálnej endokrino- Slovak Academy of Sciences, Bratislava, Slovak Republic lógie, Slovenská akadémia vied, Bratislava, Slovenská republika

Received February 14, 2014, accepted April 2, 2014

Abstract Alkaptonuria is a rare inherited genetic disorder of phenylalanine and tyrosine metabolism. This is an autosomal recessive con- dition that is caused due to a defect in the enzyme homogentisate 1,2-dioxygenase, which participates in the degradation of tyrosine. As a result, homogentisic acid and its oxide accumulate in the blood and are excreted in urine in large amounts. The polymer of homogentisic acid called alkapton impregnates bradotrophic tissues.

Slovak Alakptonúria je zriedkavé hereditárne ochorenie, ktoré je spôsobené poruchou metabolizmu fenylalanínu a tyrozínu. Je to auto- abstract zomálne recesívne ochorenie spôsobené defektom enzýmu 1,2-dioxygenázy, ktorá sa podieľa na degradácii tyrozínu. Výsledkom je, že kyselina homogentisová a jej oxid sa akumulujú v krvi a sú vylučované močom. Polymér kyseliny homogentisovej, ktorý sa nazýva alkaptón, impregnuje bradytropné tkanivá.

Keywords alkaptonuria; ochronosis; homogentisic acid in urine Kľúčové slová: alkaptonúria; ochronóza; kyselina homogentisová v moči

DEFINITION Alkaptonuria is an inherited disorder of metabolism of aro- alkaptonuria is an inherited metabolic disease and that defi- matic amino acids phenylalanine and tyrosine that is caused ciency of the enzyme that metabolises homogentisic acid is a due to the lack of activity of the enzyme homogentisate result of a defective gene. This concept was later proved cor- 1,2-dioxygenase (HGD). The homogentisic acid is not me- rect. His article about alkaptonuria was published in Lancet in tabolised – it accumulates in the body and is excreted into 1902 (Garrod, 1902). At that time it was a bold statement when urine. The polymer – the ochronotic pigment – impregnates one considers how little was known about enzymes, human bradytrophic tissues. genetics and intermediary metabolism. His knowledge was later summarised in his book Inborn Errors of Metabolism (Gar- CLINICAL SIGNS rod, 1909). In 1958, the study by La Du et al. provided a biochemical evidence of the defect in alkaptonuria (La Du et al., Alkaptonuria is characterised by: 1958). They demonstrated the absence of homogentisic acid • homogentisic acid accumulation in the body metabolising enzyme activity in liver homogenates from a • presence of homogentisic acid in urine patient with alkaptonuria. He found that the defect is related • visible, functionally benign symptoms of eyes and ears to one enzyme – 1,2-dioxygenase of the homogentisic acid. • debilitating changes in the locomotor system. He suggested that the people affected do not synthesise the enzyme. The gene responsible for alkaptonuria was identified HISTORY in 1993 by Pollak et al. and it was localised to chromosome 3q2 (Pollak et al., 1993). In alkaptonuria, the homogentisic Scientists found alkaptonuria in the Egyptian mummy acid is not broken down due to the lack of enzyme activity Harwa dated to around 1500 BC. The name alkaptonuria that metabolises the homogentisic acid; it accumulates in was first used in 1859 in a patient whose urine contained the body and is excreted in the urine. The homogentisic acid a reducing compound (alkapton) later identified as the ho- builds an oxidised polymer in the body, which is stored in the mogentisic acid. In 1902, Garrod postulated a hypothesis that form of blue-black deposits in tissues (ochronosis).

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AETIOLOGY AND PATHOGENESIS as the countries where the highest incidence of alkaptonuria (1 in 19 000 inhabitants) was found. The world prevalence is Phenylalanine and tyrosine are the simplest aromatic amino one case in 250 000–1 000 000 inhabitants. Increased preva- acids derived from alanine. Phenylalanine is an essential ami- lence in Slovakia is explained by the fact that patients come no acid that cannot be synthesised by our body. This is not from mountainous areas where the population evolved as true for tyrosine. The body can synthesise tyrosine, but only genetic isolates with frequent marriages of parents related to if there is sufficient amount of phenylalanine – a precursor of each other. However, it should be noted that the active way of tyrosine and the enzyme involved in the conversion of phe- looking for patients, which has been a tradition in Slovakia for nylalanine to tyrosine. Phenylalanine and tyrosine are closely more than 50 years and dates back to the first case described related; phenylalanine is converted to tyrosine in the liver and by Siťaj, also contributed to the sample size. to phenylpyruvate in the kidneys. Aromatic amino acids have Siťaj, Červeňanský and Urbánek described ochronosis, its a common intermediary metabolism. The conversion of phe- development in patients with alkaptonuria and the de- nylalanine to tyrosine and further metabolism are controlled tailed clinical picture as well as manifestations in the joints by a complex enzymatic system. The failure to convert phe- (Siťaj, 1947; Siťaj and Lagier, 1973; Urbánek and Siťaj, 1955; nylalanine to tyrosine is known as phenylketonuria – one of Červeňanský et al., 1959). They published the results in a the most common recessive inborn diseases (approximately monograph titled Alkaptonuria and ochronosis (1956), which 1 affected in 10 000 newborns). Phenylketonuria is caused was the first in the world literature (Siťaj et al., 1956). Their by the lack of the enzyme phenylalanine hydroxylase, which pioneering work is still being cited. Sršeň et al. from the re- converts phenylalanine to tyrosine or its cofactor tetra search laboratory of clinical genetics in Martin followed up hydrobiopterrine. Alkaptonuria arises as a result of the lack of on their epidemiological studies and continued in the ge- another enzyme – 1,2-dioxygenase of the homogentisic acid netic analysis of patients with alkaptonuria in Slovakia (Sršeň (homogentisate 1,2-dioxygenase; HGD). This enzyme is a part and Neuwirth, 1974; Sršen, 1984; Sršeň et al., 1996; Sršeň et al., of the degradation pathway of aromatic amino acids pheny- 2002). Institutions participating in the molecular characterisa- lalanine and tyrosine. Homogentisate excreted in the urine tion of mutations in the Slovak population include: Institute is then oxidised by oxygen in the air to brownish-black pig- of Molecular Physiology and Genetics SAS, Bratislava and ment – alkapton. Other symptoms later in life include ochro- Faculty of Natural Sciences of Comenius University in Brati- nosis – pigmentation of the connective tissue (cartilage). The slava (Zaťková et al., 2000a; Zaťková et al., 2000b; Zaťková et mechanism of ochronosis is the oxidation of homogentisate al., 2000c). Most of the observed families came from sites in polyphenoloxidase resulting in the production of benzochi- Slovakia studied by Siťaj et al. (Siťaj, 1947; Siťaj et al., 1956; non acetate, which polymerises and binds to macromolecules Siťaj and Lagier, 1973). Later work was continued by Rovenský of the connective tissue. Exogenous ochronosis results from and coworkers (Rovenský and Urbánek, 2000; Rovenský and prolonged treatment of ulcers with carbol and some other Urbánek, 2003) and the genetic aspects by Bošák in coopera- chemicals. Endogenous ochronosis is due to alkaptonuria. tion with laboratories in Bratislava (Bošák, 2010; Zaťková et al., 2000a; Zaťková et al., 2000b). EPIDEMIOLOGY AND GENETIC ASPECTS In 1958, La Du with co-workers found that the biochemical essence of alkaptonuria is the lack of biological activity of the The observation of alkaptonuria inheritance described by enzyme HGD in the liver, thus confirming the original assump- Garrod was studied closely by Hogben et al. (1932) who con- tion of Garrod that it is a metabolic disorder (La Du, 1958). firmed that alkaptonuria is inherited in an autosomal reces- HGD is an enzyme that is involved in the catabolism of phe- sive manner. They found that about half of the affected indi- nylalanine and tyrosine. It has a molecular weight of 50 kDa viduals came from kin marriages. and consists of 445 amino acids. It is specific for homogentisic The list of scientists who significantly contributed to the acid and does not oxidase related substances such as gen- knowledge of the clinical manifestations of alkaptonuria and tisic acid and phenylacetic acid. The gene that encodes the ochronosis includes Siťaj, Červeňanský, Urbánek, Hüttl and HGD enzyme was cloned in 1996, thereby opening the era others (Siťaj, 1947; Siťaj et al., 1956; Siťaj and Lagier, 1973; of molecular genetics of alkaptonuria (Granadino et al., 1997). Urbánek and Siťaj, 1955; Červeňanský et al., 1959; Hüttl et al., We now know that the HGD gene consists of 14 exons (cod- 1966). In 1947, Siťaj diagnosed and described the first case of ing parts of the gene) and 13 introns (non-coding part of the alkaptonuria (Siťaj, 1947). Till 1953, Siťaj and his team collect- gene). HGD gene has a tissue-specific expression, particularly ed a sample of 102 patients – members of 15 families – while in the liver, kidneys, small and large intestine, prostate and at that time approximately 100 cases were described in the the brain (Fernandez-Canón, 1996). Increased activity in the whole world, mostly isolated case reports. Between 1956 and liver and kidneys has been attributed to metabolic activity of 1960, they registered other affected families increasing the to- these organs. In the brain, HGD probably participates in the tal number to 28 and the number of patients with alkaptonu- degradation of amino acids derived from neurotransmitters, ria increased to 182, of whom 43 had ochronosis. Slovakia which often contain aromatic amino acids. The HGD gene and the Dominican Republic became known in the literature was localised on the long arm of chromosome number 3 in

30 Rovenský, J., Urbánek, T., Imrich, R. humans (3q) (Pollak et al., 1993; Janocha, et al., 1994). In the blue sclerae in osteopsatyrosis should be excluded. The diag- following years, its location on the 3rd chromosome has been nosis of alkaptonuria is based on the characteristic findings in further clarified to the locus 3q21-23 using the technique of urine. Alkaptonuria patients do not seek medical help due to polymerase chain reaction and fluorescent in situ hybridisa- difficulties with viewing these spots – they are without sub- tion (Bošák, 2010). jective complications. Molecular genetic studies of alkaptonuria yielded a surprising In parallel with the ocular manifestations, ochronotic chang- finding that the defect of the HGD enzyme is not caused by a es can be found in the hearing organ. Colour changes of the single mutation, as originally expected, but by several muta- auricle are visible in the 10th to 15th year of life. Detailed his- tions in coding and non-coding sequences of the HGD gene. tological examination of the temporal bone performed by By 2000, more than 40 different mutations that cause alkap- Brunner revealed accumulation of the ochronotic pigment tonuria were identified in more than 100 patients from sev- in the bone and its membranous parts (Brunner, 1929). The eral countries around the world (Porfirio et al., 2000; Zaťková changes taking place in the ears are slow and patients are et al., 2000). Currently, there are at least 67 known mutations. alerted to the blue-grey colour of the ear by their relatives. Most mutations change the sense of the genetic information On the cartilage, painless, hard, rough lumps can be seen (missense), which involves changing one nucleotide in the firmly connected with the basis and shining through the DNA molecule. The most common mutation in Europe, which delicate skin as dark-blue-violet colour. The first rough ridges represents about 20% of all mutations, is Met368Val (sub- appear on the lower arm of the anthelix, and later through- stitution of methionine instead of valine at position 368); in out the anthelix, in the fossa triangularis, cavum conchae, the Dominican Republic, it is Cys120Trp; and in Slovakia, it is cymbal and the tragus. In advanced cases, sometimes auri- IVS5 +1G→A (Bošák, 2010). cle deformation can be found. The external auditory canal is without changes, earwax is dark brown, drum is dark, dull, THE CLINICAL PICTURE often inverted, with an atypical reflex, with bluish tint, and in most cases calcium incrustations are present. Patients may The first signs of alkaptonuria can even be seen in the new- also suffer from hearing loss type hypoacusis mixta with a born. Their urine darkens when exposed to the air and leaves stronger involvement of the perceptive apparatus. Symp- brownish black spots on their diapers. The spots are high- toms of alkaptonuria hearing organ are specific and often lighted and cannot be washed away when alkaline soap is lead to the diagnosis of this disease. used. The dark earwax is also a characteristic that can be seen Also, changes in the skin typical of alkaptonuric ochronosis after birth. Dark urine and ear wax remained the only clinical include mainly brownish or bluish pigmentation of the skin symptoms of alkaptonuria for many years. In the meantime, under the arm, in the face, neck and hands, and rarely on the a much more serious process takes place in the organism af- nails. Given their visibility, they may be relevant for the early fected by this metabolic disorder. The ochronotic pigment is diagnosis of alkaptonuric ochronosis. produced by oxidative polymerisation of the homogentisic Ochronotic pigment is deposited also in the internal organs. acid; it accumulates in bradytrophic tissues and stains them In the field of cardiovascular organs, it is the myocardium and dark brown. In principle, it is a benign process, which goes on blood vessels. Statistically significant myocardial disorders unnoticed for a long time. were not found, but earlier atherosclerotic changes in the The first signs of deposition of the ochronotic pigment can be aorta were observed. Urolithiasis was found in more than half detected accidentally during professional examination of the of the patients and rare cases of nephropathy were seen. anterior segment of the eye. The ochronotic pigmentation of From a clinical point of view, the most serious process takes the ocular structures is present in approximately 70% of pa- place in the joints and is called ochronotic arthropathy. Basi- tients. In addition to the sclera, lumps of the ochronotic pig- cally, it is a degenerative process with a known genesis and ment can be found in the conjunctiva and cornea. Since simi- with an increased risk of disability. The basic clinical mani- lar pigmentation of the cornea is not present in other medical festation of ochronotic arthropathy from the beginning is conditions, this finding is regarded as pathognomonic for related to the spine. The first subjective difficulties appear at alkaptonuric ochronosis. Skinsnes described a patient who the end of the third decade of life. Gender is significant with underwent enucleation of his only eye (the other one was lost a predominance of men relative to women 2:1. Objective due to injury) due to a pigment stain in the sclera deemed as findings include flattening of thoracic kyphosis and lumbar melanosarcoma (Skinsnes, 1948). After an unrelated death of lordosis, mild rigidity with a tendency to deterioration. Later, the patient, however, the autopsy revealed that this was an in an advanced stage, the contours of the spine worsen with alkaptonuric ochronosis with ocular pigmentation. Pigment irregular spinous processes and complete ankylosis of the en- spots on the sclera are mostly visible. They appear usually in tire lumbar and thoracic spine. The spine is rigid, irregular and the third decade of life in two-thirds of patients with alkap- the contours do not change when bending forward. Cervical tonuric ochronosis. In the advanced stage, they can be seen spine maintains its mobility for a relatively long time despite with the naked eye. When found, chronic poisoning with significant sciagraphic changes. In an advanced stage, dorsal phenolic substances, arsenic and lead, Addison’s disease, and flexing and rotational movements become limited, while the

31 Acta Fac. Pharm. Univ. Comen. LXI, 2014 (1), p. 29–35 ALKAPTONURIA AND OCHRONOSIS – EXPERIENCE FROM SLOVAKIA

head moves forward. As a result of degenerative changes to X-ray image of the shoulder joints shows even at an early the plates in the narrowing intervertebral space, body height stage signs of ossificating enthesopathy. Siťaj proves with an decreases up to 8 cm in 20 years. analysis of 42 patients with ochronosis that the calcareous Using X-ray of the spine, characteristic calcification of in- deposits in the shoulder rotators are present in more than tervertebral discs can be diagnosed. Osteolytic and hyper- 25% of patients (Siťaj et al., 1956). In the next stage, around plastic changes and secondary reactive bone formation can the 50th year of life, patients develop degenerative changes be found on the vertebral bodies. Osteophytes are created, with exostosis on the bottom of the joint fossa, and later with sometimes even massive bone bridges of the type of ankylos- cystoid translucency, usures and destruction on the humerus ing hyperostosis. Calcification of some peripheral bundles of head. This finding is completely different from genuine osteo- the connective rings may be similar to pseudosyndesmotic arthrosis and pathognomonic for ochronotic arthropathy of bridges. Even in the early stages, hollow formations in the the shoulder. plates are formed; this is called the vacuum phenomenon. The hip joints are affected only in later stages of ochronosis In the intervertebral joints, the gap is narrowed and reactive and approximately in a third of patients. The course is faster subchondral sclerosis is present. than in coxarthrosis and results in an almost complete restric- Sometimes, calcification is found in the ligaments between tion of the mobility. the spinous processes. Occasionally, osteoporotic vertebral The X-ray examination shows a severe, in some patients, fractures are found. On the other hand, thickening of bone destructive coxarthrosis. Červeňanský et al. describe ochro- structure is not unusual. This resembles Paget osteitis. notic enthesopathy in the hip area and highlight the selec- While the spine is affected in all patients with ochronotic tive deposition of the ochronotic pigment in the tendons arthropathy, peripheral joints are often, but not always, af- (Červeňanský et al., 1959). fected. Based on the analysis of 26 patients with ochronotic Similarity of alkaptonuric ochronosis with other diseases. The arthropathy, it may be noted that small joints are spared and metabolic disorders in ochronotic arthropathy of the spine large joints are affected in the following order: knee (64%), and large joints of the limbs include osteoporosis. It is assumed shoulders (42.3%) and hips (34.6%). that this is a secondary form of osteoporosis due to immobili- The finding in the knee is basically of arthritic nature. It dif- sation of severely affected individuals. Barel et al. describe an fers from genuine osteoarthrosis by an earlier start (average affected family with alkaptonuria, phenylketonuria and con- of 39 years), faster progress and larger deformations. Hydrops genital cataract (Barel et al., 1960). Occasionally, alkaptonuria occurred in 30.4% of our patients. Based on a series of investi- occurs concurrently with psoriasis. In 1955, Urbánek and Siťaj gations, Hüttl et al. have found that the synovial effusion is of described a unique coincidence of alkaptonuric ochronosis a non-inflammatory, irritating and degenerative nature. The and Bechterev’s disease in a 51-year-old man (Urbánek and effusion has a yellowish colour that remains unchanged even Siťaj, 1955). The patient came from a family in which four of after prolonged standing in air, suggesting a low concentra- five siblings had ochronotic arthropathy. Based on an analysis tion of homogentisic acid (Hüttl et al., 1966). From the noso- of clinical and X-ray findings in the spine, it could be assumed graphic point of view, it is important to find histiocytes with that ochronotic arthropathy and Bechterev’s disease interact. brown-violet and blue-black cytoplasmic inclusions, which In our patient, typical ochronotic changes were present, es- can be assumed to be the phagocytosed ochronotic pigment. pecially calcifications of the intervertebral discs, less marked The finding of histiocytes with pigment inclusions in the cy- in comparison with other healthy patients with ochronosis in toplasm was described by Hüttl et al. for the first time in the advanced disease stages. It may be that the premature rigid- world literature (Hüttl et al., 1966). The X-ray image in ochro- ity of the spine due to the Bechterev’s disease prevented age- notic arthropathy shows similar changes as in osteoarthrosis, related development of ochronotic changes. On the other and this is often asymmetric. The characteristic sign is the hand, despite standard Bechterev’s disease symptoms (af- formation of free calcified and ossificated pea sized or even fected sacroiliac joints, paraspinal ligament ossification and larger bodies of a diverse shape. These are signs of ochronotic obliteration of intervertebral joints), the patient had dispro- chondromatosis. Occasionally, narrow strips of calcified soft portionately low pain throughout the course of the disease. tissue parts of the extremities are detected that resemble The long-term observation of a large number of patients with Thieberg–Weissenbach syndrome. The main difference be- ochronosis revealed that the relatively subtle pain is charac- tween genuine arthrosis and ochronosis of the knee is in a teristic for ochronotic arthropathy (Siťaj et al., 1956). more rapid progression and in advanced findings in relation Japanese authors Kihara et al. described the coexistence of to the age of the patient with ochronosis. ochronosis and rheumatoid arthritis in a 64-year-old woman In the shoulder joints in the early stages of ochronosis, there (Kihara et al., 1994). Magnetic resonance imaging of interver- are painful episodes of the type of humeroscapular periar- tebral discs found the typical changes suggestive for ochro- thropathy that are probably related to the deposition of pig- notic arthropathy. At the same time, symptoms of rheuma- ment and lime deposits tendons in the rotator. Gradually, the toid wrist arthritis were identified with positive rheumatoid mobility gets limited due to the retraction of the joint capsule, factor and nodules, which histologically were compatible destruction of the cartilage and the adjacent bone structures. with the diagnosis of RA. The authors state at the end that the

32 Rovenský, J., Urbánek, T., Imrich, R. pre-existing ochronotic arthropathy could have masked the birth, has a very typical red colour. Similarly, inhaematuria or manifestation of RA and made it quite difficult to diagnose. haemoglobinuria urine has a pinkish red colour. The analysis The conclusion of these two case reports is that the process of the urinary sediment is of decisive importance for the di- of ochronosis slows down the development of inflammatory agnosis. In bilirubinuria, the urine has a reddish-brown colour symptoms of Bechterev’s disease and rheumatoid arthritis (like black beer) and in melanuria a dark brown colour. Urine and makes it more benign. is always coloured when it is fresh, with the exception of alkaptonuria, when the urine darkens in contact with the air after DIAGNOSIS hours or immediately after the addition of alkali. Ochronosis may be endogenous (on the basis of alkaptonuria) Diagnosis of alkaptonuria is based on the proof of homogen- and exogenous, caused by the contact with certain chemicals. tisic acid in urine. It does not occur in a healthy person. Labo- Several authors have described yellowish pigmentation of the ratory evidence of homogentisic acid is based on its reducing skin and cartilage caused by carbolic acid (phenol), which was properties. In practise, the test using the Fehling’s solution, previously used to treat leg ulcers and other cases. This exog- which is used in diabetes, proved valuable. While diabetic enous “karbolochronosis” was not associated with spondylosis urine with Fehling’s solution brings brick-red clot, alkaptonu- and arthropathy. More recently, exogenous ochronosis in the ric urine changes the colour after the addition of the Fehling’s skin and sclera associated with myxedema caused by long- solution to grey-black. term use of resorcinol was described. Anderson described For screening, alkalinisation of the urine with 10% NaOH can the pigmentation of the eye conjunctiva and cornea due to be used. After instillation of NaOH to the tube with the urine, a working in the production of hydroquinone (Anderson, 1947). dark ring is created and then the entire sample of urine darkens. The sclera was dark and spots on the cornea caused blindness. Earlier quantitative methods for determining homogentisic Sugar and Waddell found a pigmentation of the sclera and acid in the urine used the ability of the acid to reduce silver, cartilage similar to ochronosis after long-term use of atebrin phosphomolybdic acid or iodine. The drawback of these (Sugar and Waddell, 1946). Skinsnes describes a case of incor- methods was the fact that they may determine also other re- rect melanosarcoma diagnosis in a patient with ochronosis ducing compounds present in the urine. This bias appeared that led to a tragic outcome – enucleation of a single eye of in lower concentration of homogentisic acid in the urine of the patient (see above) (Skinsnes, 1948). patients with alkaptonuria. Certain marked improvement Differentiating ochronotic changes in the spine from other brought the extraction of homogentisic acid into ether and spondylopathies is crucial to find calcified intervertebral discs, subsequent iodometric determination. In 1961, Seegmiller which are pathognomonic for alkaptonuric ochronosis. Spon- et al. developed a spectrophotometric enzymatic determi- dylopathies in chondrocalcinosis could cause certain prob- nation of homogentisic acid in plasma and urine using the lems in differential diagnosis, but this disease is characterised purified homogentisic acid oxygenase, which enabled the by painful process with episodes of attacks of inflammatory authors to specifically determine 1 µg of the acid (Seegmiller nature, by calcifications of small joints, especially at the wrists, et al., 1961). The oxidatively produced maleylacetic acid is de- and the spondylopathy has an easier course with no ten- termined spectrophotometrically at 330 nm. Electrophoretic dency to ankylosis. In rare cases, a distinction from calcified method of determining homogentisic acid was established discs in hemochromatosis can be considered. When in doubt, by Trnavská (1962). At present, the quantitative determina- analysis of the urine for the presence of homogentisic acid is tion of the homogentisic acid in urine is based on liquid chro- decisive, as well as a comprehensive view of the patient and matography and capillary electrophoresis. evaluation of eye, ear and skin. Alkaptonuric ochronosis has The diagnosis of ochronosis is based on finding pigment a characteristic polytope symptomatology that usually does spots on ocular structures, on the blue-grey discolouration of not cause greater differential difficulties. Rather, it is neces- the auricles and skin in the armpit and on the X-ray findings sary to exclude other diseases that may require more urgent in the calcified intervertebral discs. In the advanced stage of intervention before the full clinical picture develops. the disease, irregularly protruding spinous processes of the thoracic and lumbar spine are typical of ochronotic arthropa- THERAPY thy and the finding of pigmented inclusions in the cells of the synovial effusion is specific. As yet, no causal treatment is available for alkaptonuria; ther- Differential diagnosis. The fresh urine of a patient with alkap- apeutic interventions essentially are in three directions: tonuria has a normal pale yellow colour and after prolonged -- reduction of the excretion of homogentisic acid into the standing in air or in contact with alkali media (soaps, etc.) urine, darkens to dark grey to black. This sign of alkaptonuria is spe- -- restriction of the onset of ochronosis, cific and it will almost always distinguish it from some other -- therapeutic and preventive interventions to influence diseases associated with changes of urine colour. For exam- ochronotic arthropathy. ple, the urine in the hereditary disease congenital erythropoi- In recent decades, various dietary interventions have been tried etic porphyriai (m. Günther), which also begins shortly after to reduce the formation of homogentisic acid and its excretion

33 Acta Fac. Pharm. Univ. Comen. LXI, 2014 (1), p. 29–35 ALKAPTONURIA AND OCHRONOSIS – EXPERIENCE FROM SLOVAKIA

into urine including vitamins, hormones and other substances requires more careful studies of the distribution of isomerase with uncertain and in some cases only a transient effect. and other enzymes following the metabolic pathway. When it comes to limiting the production of the ochronotic Therapeutic and preventive interventions aimed at influenc- pigment and reducing the risk of ochronosis with its disas- ing ochronotic arthropathy are essentially identical to those trous consequences especially on the musculoskeletal system, used in the treatment of the degenerative disease of the spine a positive effect is attributed to vitamin C (Morava et al., 2003; and limb joints. These include non-steroidal anti-rheumatic Turgay et al., 2009). Some studies, however, did not confirm drugs, physical therapy, rehabilitation with balneotherapy, the positive effect of vitamin C and there is a lack of long-term prevention of the creation of deformities and rheumosurgi- controlled clinical studies targeting this problem (Phornphut- cal interventions if needed. The follow-up of all newborn chil- kul et al., 2002). Progress in therapy leads to a direct pharma- dren diagnosed with alkaptonuria, their constant monitoring, cological reduction of the homogentisic acid. Theoretically, it diet and life style guidance, proper selection of sports and would be possible to use nitisinone, a triketone herbicide that especially a suitable profession are very important. quickly and reversibly binds 4-hydroxyphenylpyruvate dioxy- In this regard, the research and practise of the workgroup genase, which catalyses the formation of homogentisic acid of Prof. Sršeň at the Medical faculty of the Comenius Univer- from 4-hydroxyphenylpyruvate. This treatment has, however, sity in Martin are of particular importance. In children with important adverse effects such as increased plasma concen- alkaptonuria, mild restriction of daily intake of proteins rich trations of tyrosine and subsequent irritation of the cornea in phenylalanine and tyrosine, as well as the application of (Suwannarat et al., 2005). Application of nitisinone in alkap- ascorbic acid supplemented with vitamins E, A and selenium tonuria has so far been experimental. is recommended. In addition, the recommendations include Alkaptonuria as a congenital metabolic disease is now well a daily regimen saving the sites predilected to be affected – defined. Gene therapy has moved from the phase of model large joints and the spine with corresponding selection and experiments to the phase of clinical application in humans implementation of a profession. and one can hope that prospectively it will be introduced in Somatic gene therapy is progressing from the phase of exper- the therapy of alkaptonuria. The solution would be the re- imental models to the stage of clinical application in humans placement of the missing homogentisate dioxygenase, but and one can only hope that treatment will be introduced for the application of recombinant homogentisate dioxygenase alkaptonuria in the near future.

References

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