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Alkaptonuria (AKU) is a rare genetic disorder where oxidised homogentisic acid accumulates in connective tissues, leading to multisystem disease. The clinical evaluation Alkaptonuria Severity Score Index (cAKUSSI) is a composite score that assesses the extent of AKU disease. However, some components assess similar disease features, are difficult to measure reliably or cannot be measured in resource-limited environments. cAKUSSI data from the 4-year SONIA 2 randomised controlled trial, which investigated nitisinone treatment in adults with AKU, were analysed (

In an open‐label, controlled study of nitisinone in alkaptonuria (SONIA 2), patients were advised to lower dietary protein intake to reduce serum tyrosine (s‐Tyr) levels and the risk of keratopathy. A body weight increase was observed in the nitisinone‐treated patients but not in the control group. To investigate the effectiveness and consequence of protein restriction in patients with alkaptonuria, a post‐hoc analysis of SONIA 2 was performed. One hundred and thirty‐eight patients were randomised (nitisinone: n = 69, controls: n = 69). Comparison of baseline and Month 12 data on 24‐h urinary excretion of HGA (u‐HGA24) and urea (u‐urea24, used as an approximate protein intake measure), tyrosine and body weight were performed using paired t tests. Comparisons of data between groups were made using 2‐sample t tests. We found that u‐urea24 decreased more in nitisinone‐treated than controls. The study centre with lowest average s‐Tyr and u‐urea24 (nitisinone arm) at Month 12 also had lowest keratopathy incidence (3.1%), while the centre with highest values showed the highest (14.6%). S‐Tyr was generally high in those with keratopathy, but those without keratopathy had similar elevated values. A similar pattern across centres was seen for body weight changes, with a statistically significant weight increase in nitisinone‐treated patients at centres with lower u‐urea24 values. Therefore, in nitisinone‐treated patients, protein restriction led to increased body weight but may also have lowered the risk of developing keratopathies. If introduced, a protein‐restricted diet should be supervised by a dietician and, when appropriate, include amino acid supplements deficient in tyrosine and phenylalanine, to avoid malnutrition and undesired weight increase.

Background Outcomes from studies employing nitisinone 10 mg and 2 mg in alkaptonuria were compared. Patients and methods Sixty‐nine patients in each of the nitisinone (10 mg daily) and controls of suitability of nitisinone in alkaptonuria 2 (SONIA 2), as well as 37 and 23 in nitisinone (2 mg daily) and control cohorts at the National Alkaptonuria Centre (NAC), respectively, were followed up for 4 years. Severity of alkaptonuria (AKU) was assessed by the AKU Severity Score Index (AKUSSI). 24‐h urine homogentisic acid (uHGA24), serum HGA (sHGA), serum tyrosine (sTYR) and serum nitisinone (sNIT) were also analysed at each time point. Dietetic support was used in the NAC, but not in SONIA 2. Safety outcomes were also compared. All statistical analyses were post hoc. Results The slope of the AKUSSI was 0.55, 0.19, 0.30, and 0.06 per month in the control NAC, nitisinone NAC, control SONIA 2, and nitisinone SONIA 2 cohorts, respectively. The intersection of the slopes on the x‐axis was −132, −411, −295, and − 1460 months, respectively. The control and nitisinone slope comparisons were statistically significant both in the NAC (p

Four patients, from three families, with alkaptonuria receiving 4‐hydroxyphenylpyruvate dioxygenase‐inhibiting nitisinone therapy, which lowers homogentisic acid and increases tyrosine, developed vitiligo. Three of the four patients were receiving nitisinone 2 mg daily, while the fourth was on 10 mg daily. All four patients were either receiving or had received transiently proton‐pump inhibitors as therapy for dyspepsia. The ages of the patients were 35, 42, 40, and 67 years, respectively. Three patients were men and one was a woman. All four patients were either taking a proton‐pump inhibitor or had been taking one at some point. Three of the four were of South Asian and one of Caucasian background. The three patients with South Asian background also had either a personal or family history of autoimmune disease. Distressing vitiligo, initially in an acrofacial distribution, developed unexpectedly in these four patients, before then progressing to involve other parts of the body. Potential factors in the appearance of vitiligo in this setting, including nitisinone and other drug therapy, are explored and responses to the appearance of vitiligo are discussed.

Mutations in SQSTM1 are strongly associated with Paget's disease of bone (PDB), but little is known about the clinical characteristics of those with early disease. Radionuclide bone scans, biochemical markers of bone turnover, and clinical characteristics were analyzed in SQSTM1 mutation carriers who took part in the Zoledronic acid in the Prevention of Paget's disease (ZiPP) study. We studied 222 individuals, of whom 54.9% were female, with mean ± SE age of 50.1 ± 0.6 years. Twelve SQSTM1 mutations were observed, including p.Pro392Leu, which was present in 141 of 222 (63.5%) subjects. Bone scan examination revealed evidence of PDB in 20 subjects (9.0%), ten of whom (50%) had a single affected site. Participants with lesions were older than those without lesions but the difference was not significant (53.6 ± 9.1 versus 49.8 ± 8.9; p = .07). The mean age of participants with lesions was not significantly different from the age at which their parents were diagnosed with PDB (55 years versus 59 years, p = .17). All individuals with lesions were asymptomatic. Serum concentrations of total alkaline phosphatase (ALP) normalized to the upper limit of normal in each center were higher in those with lesions (0.75 ± 0.69 versus 0.42 ± 0.29 arbitary units; p

BackgroundAlkaptonuria (AKU) is a disorder of tyrosine/protein metabolism leading to accumulation of homogentisic acid. Clinical management historically recommended reducing dietary protein intake, especially in childhood, which has since been discredited in the literature. For the first time, analysis of baseline cross-sectional nutritional surveillance data from a large cohort of AKU patients is presented, which has clinical implications in all aspects of treatment planning.MethodSeventy-four patients (mean 55 years) admitted to the National Alkaptonuria Centre (NAC), underwent a global nutritional assessment, which included objective anthropometry, bioimpedance measures, habitual nutritional intake using a 7-day food diary and key nutritional biomarkers, including 24 hours urinary nitrogen, serum albumin, total protein and total 25-hydroxy vitamin D. All data was compared with cohort norms or recommended nutrient intakes for health (RNI). The potential beneficial impact of protein and anti-inflammatory nutrients such as vitamin C, selenium, and zinc were statistically interrogated against the AKU severity score index (AKUSSI)-a validated measure of disease progression stratified by age.ResultsFifty percent of AKU patients reported some level of protein restriction at some point in their lives. In comparison with national data sets, AKU patients present with significantly lower than predicted mid-upper arm circumference, grip strength, BMI, total energy and protein intake, and higher than predicted percentage body fat. They therefore meet the ESPEN criteria as "clinically undernourished." Severity fluctuates over the life course. No statistical association is identified between protein intake, expressed as %RNI or g/kg, or anti-inflammatory nutrients, including vitamin C as a high dose supplement on the severity of the disease, when correlated against the validated AKUSSI score.ConclusionAKU patients are at risk of protein depletion associated with a "perfect storm" of risk factors: historical, poorly evidenced recommendations to reduce total protein intake; limited mobility as the condition progresses, compromising muscle integrity; frequent hospital admissions for major surgery associated with multiple joint replacements, creating pinch points of high metabolic demand and the potential impact of the disease itself. As this is the first time this risk has been identified, the authors consider the dietetic implications of nitisinone treatment, which requires dietary protein control to manage the acquired tyrosinaemia. The lack of statistically significant evidence to support dietary manipulation of any kind to impede disease progression in AKU is demonstrated.

For over two decades, nitisinone (NTBC) has been successfully used to manipulate the tyrosine degradation pathway and save the lives of many children with hereditary tyrosinaemia type 1. More recently, NTBC has been used to halt homogentisic acid accumulation in alkaptonuria (AKU) with evidence suggesting its efficacy as a disease modifying agent. NTBC-induced hypertyrosinaemia has been associated with cognitive impairment and potentially sight-threatening keratopathy. In the context of a non-lethal condition (ie, AKU), these serious risks call for an evaluation of the wider impact of NTBC on the tyrosine pathway. We hypothesised that NTBC increases the tyrosine pool size and concentrations in tissues. In AKU mice tyrosine concentrations of tissue homogenates were measured before and after treatment with NTBC. In humans, pulse injection with l-[13 C9 ]tyrosine and l-[d8 ]phenylalanine was used along with compartmental modelling to estimate the size of tyrosine pools before and after treatment with NTBC. We found that NTBC increased tyrosine concentrations in murine tissues by five to nine folds. It also significantly increased the tyrosine pool size in humans (P

BACKGROUND: 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. METHODS: 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 >700μmol/L who had been advised to restrict dietary protein intake and where necessary, to use tyrosine/phenylalanine-free amino acid supplements. RESULTS: Elevated tyrosine (813μmol/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 389.3μmol/L, 274.8μmol/L and 144.3μmol/L with decreasing phenylalanine doses. In contrast, tyrosine was not effectively reduced in mice by a phenylalanine-free diet; at 3 days tyrosine was 757.3μmol/L, 530.2μmol/L and 656.2μmol/L, with no dose response to phenylalanine supplementation. In NAC patients, tyrosine was significantly reduced (p=0.002) when restricting dietary protein alone, and when combined with tyrosine/phenylalanine-free amino acid supplementation; 4 out of 10 patients achieved tyrosine

BackgroundThe homogentisic acid-lowering therapy nitisinone is being evaluated for the treatment of alkaptonuria (AKU) at the National Centre for AKU. Beyond hypertyrosinemia, the wider metabolic consequences of its use are largely unknown. The aim of this work was to evaluate the impact of nitisinone on the serum metabolome of patients with AKU after 12 and 24 months of treatment.MethodsDeproteinized serum from 25 patients with AKU (mean age[±SD] 51.1 ± 14.9 years, 12 male) was analyzed using the 1290 Infinity II liquid chromatography system coupled to a 6550 quadrupole time-of-flight mass spectrometry (Agilent, UK). Raw data were processed using a batch targeted feature extraction algorithm and an accurate mass retention time database containing 469 intermediary metabolites (MW 72-785). Matched entities (±10 ppm theoretical accurate mass and ±0.3 minutes retention time window) were filtered based on their frequency and variability (ResultsEight metabolites increased in abundance (log2 fold change [FC] 2.1-15.2, P 2 FC 1.5-15.5, P ConclusionsEvaluation of the serum metabolome of patients with AKU showed a significant difference in the abundance of several metabolites following treatment with nitisinone, including a number that have not been previously reported; several of these were not related to the tyrosine metabolic pathway.SynopsisNitisinone therapy has a significant impact on several metabolites beyond the tyrosine metabolic pathway, several of which appear to be related to the redox state of the cell.

Damage mechanisms in unidirectional (UD) and bi-directional (BD) woven carbon fiber reinforced polymer (CFRP) laminates subjected to four point flexure, both in static and fatigue loadings, were studied. The damage progression in composites was monitored by observing the slopes of the load vs. deflection data that represent the stiffness of the given specimen geometry over a number of cycles. It was observed that the unidirectional composites exhibit gradual loss in stiffness whereas the bidirectional woven composites show a relatively quicker loss during stage II of fatigue damage progression. Both, the static and the fatigue failures in unidirectional carbon fiber reinforced polymer composites originates due to generation of cracks on compression face while in bidirectional woven composites the damage ensues from both the compression and the tensile faces. These observations are supported by a detailed fractographic analysis.

High density mineralised protrusions (HDMP) from the tidemark mineralising front into hyaline articular cartilage (HAC) were first described in Thoroughbred racehorse fetlock joints and later in Icelandic horse hock joints. We now report them in human material. Whole femoral heads removed at operation for joint replacement or from dissection room cadavers were imaged using magnetic resonance imaging (MRI) dual echo steady state at 0.23 mm resolution, then 26-μm resolution high contrast X-ray microtomography, sectioned and embedded in polymethylmethacrylate, blocks cut and polished and re-imaged with 6-μm resolution X-ray microtomography. Tissue mineralisation density was imaged using backscattered electron SEM (BSE SEM) at 20 kV with uncoated samples. HAC histology was studied by BSE SEM after staining block faces with ammonium triiodide solution. HDMP arise via the extrusion of an unknown mineralisable matrix into clefts in HAC, a process of acellular dystrophic calcification. Their formation may be an extension of a crack self-healing mechanism found in bone and articular calcified cartilage. Mineral concentration exceeds that of articular calcified cartilage and is not uniform. It is probable that they have not been reported previously because they are removed by decalcification with standard protocols. Mineral phase morphology frequently shows the agglomeration of many fine particles into larger concretions. HDMP are surrounded by HAC, are brittle, and show fault lines within them. Dense fragments found within damaged HAC could make a significant contribution to joint destruction. At least larger HDMP can be detected with the best MRI imaging ex vivo.

Evidence shows that raised cobalt (Co), chromium (Cr), and nickel (Ni) whole blood concentrations correlate with poor device outcome in patients following metal-on-metal (MoM) hip arthroplasty. To understand the local and systemic pathological effects of these raised metal concentrations it is important to define their distribution between whole blood, plasma, and urine. The metals were measured by Inductively Coupled Plasma Mass Spectrometry (ICPMS). Two hundred and five plasma, 199 whole blood, and 24 sets of urine samples were analyzed from 202 patients with Co–Cr alloy MoM hip prostheses implanted between 8 months to 12 years (mean 6.0 years) prior to analysis. Plasma Co (median 39.1 nmol/L) showed significantly positive 1:1 correlation with whole blood Co (median 45.9 nmol/L; R2 = 0.98, p

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.

Alkaptonuria (AKU) is due to excessive homogentisic acid accumulation in body fluids due to lack of enzyme homogentisate dioxygenase leading in turn to varied clinical manifestations mainly by a process of conversion of HGA to a polymeric melanin-like pigment known as ochronosis. A potential treatment, a drug called nitisinone, to decrease formation of HGA is available. However, successful demonstration of its efficacy in modifying the natural history of AKU requires an effective quantitative assessment tool. We have described two potential tools that could be used to quantitate disease burden in AKU. One tool describes scoring the clinical features that includes clinical assessments, investigations and questionnaires in 15 patients with AKU. The second tool describes a scoring system that only includes items obtained from questionnaires used in 44 people with AKU. Statistical analyses were carried out on the two patient datasets to assess the AKU tools; these included the calculation of Chronbach's alpha, multidimensional scaling and simple linear regression analysis. The conclusion was that there was good evidence that the tools could be adopted as AKU assessment tools, but perhaps with further refinement before being used in the practical setting of a clinical trial.

The cardiovascular manifestations of alkaptonuria relate to deposition of ochronotic pigment within heart valves, endocardium, aortic intima and coronary arteries. We assessed 16 individuals with alkaptonuria for cardiovascular disease, including full electrocardiographic and echocardiographic assessment. The self reported prevalence of valvular heart disease and coronary artery disease was low. There was a significant burden of previously undiagnosed aortic valve disease, reaching a prevalence of over 40% by the fifth decade of life. The aortic valve disease was found to increase in both prevalence and severity with advancing age. In contrast to previous reports, we did not find a significant burden of mitral valve disease or coronary artery disease. These findings are important for the clinical follow-up of patients with alkaptonuria and suggest a role for echocardiographic surveillance of patients above 40 years old.

Progress in research into rare diseases is challenging. This paper discusses strategies to identify individuals with the rare genetic disease alkaptonuria (AKU) within the general population. Strategies used included a questionnaire survey of general practitioners, a dedicated website and patient network contact, targeted family screening and medical conference targeting. Primary care physicians of the UK were targeted by a postal survey that involved mailing 11,151 UK GPs; the response rate was 18.2%. We have identified 75 patients in the UK with AKU by the following means: postal survey (23), targeted family screening (11), patient networks and the website (41). Targeting medical conferences (AKU, rare diseases, rheumatology, clinical biochemistry, orthopaedics, general practitioners) did not lead to new identification in the UK but helped identify overseas cases. We are now aware of 626 patients worldwide including newly identified non-UK people with AKU in the following areas: Slovakia (208), the rest of Europe (including Turkey) (79), North America (including USA and Canada) (110), and the rest of the world (154). A mechanism for identifying individuals with AKU in the general population-not just in the UK but worldwide-has been established. Knowledge of patients with AKU, both in the UK and outside, is often confined to establishing their location in a particular GP practice or association with a particular medical professional. Mere identification, however, does not always lead to full engagement for epidemiological research purposes or targeting treatment since further barriers exist.

A comparative evaluation of fatigue and fracture behaviour of commercially produced cast and forged rail wheels has been made using specimens extracted from various locations of the wheel quadrant. A systematic investigation in the web and rim regions of the wheel quadrant with various notch orientations showed that the forged material exhibited a better intrinsic resistance to fatigue crack growth than the cast material. Since linear elastic fracture mechanics (LEFM) based fracture toughness could not be validated for both the cast and forged wheel material, elastic plastic fracture mechanics (EPFM) based characteristic fracture toughness was used. Results showed that fracture resistance of the forged material is superior to that of the cast material. Cast wheel specimens exhibited unstable crack extension in comparison to substantial stable tearing in forged specimens. Microstructural and fractographic analyses showed that the cast wheel material contained large amounts of inclusions. The poor fracture resistance of cast wheel material is therefore attributed to the inferior material quality.

Alkaptonuria (AKU), the first human disorder recognised to conform to Mendelian inheritance, is accompanied by early onset, rapidly progressing osteoarthropathy. Novel anatomical and microanatomica...

In this work, an attempt is made to model the ductile fracture behaviour of two Cu-strengthened high strength low alloy (HSLA) steels through the understanding of their deformation behaviour. The variations in deformation behaviour are imparted by prior deformation of steels to various predetermined strains. The variations in parameters such as yield strength and true uniform elongation with prior deformation is studied and was found to be analogous to that of initiation fracture toughness determined by independent method. A unique method is used to measure the crack tip deformation characterized by stretch zone depth that also depicted a similar trend. Fracture toughness values derived from the stretch zone depth measurements were found to vary in the same fashion as the experimental values. A semiempirical relationship for obtaining ductile fracture toughness from basic deformation parameters is derived and model is demonstrated to estimate initiation ductile fracture toughness accurately.

Alkaptonuria is a rare, inherited defect of homogentisic acid 1,2-dioxygenase that leads to the widespread deposition of polymeric homogentisic acid, and clinical symptoms from degeneration of joints and the aortic valve. Pathological descriptions are few and mainly those of late-stage changes related to joint or valve failure. In this review, the macroscopic and histopathological changes in the tissues in alkaptonuria are illustrated by the detailed autopsy study of a 74-year-old female who died from disseminated ovarian carcinoma. The pathology is discussed in the context of the literature and in relation to potential pathogenic mechanisms of tissue damage. This review highlights the heterogeneity of some of the manifestations. In symptomatic patients, degenerative changes in synovial and intervertebral joints are usually well advanced, while early changes include diffuse cartilage pigmentation and chondrocyte necrosis. The initial stage of pigment deposition in the cardiovascular system may be influenced by intravascular pressure and flow disturbances, whereas more intense pigmentation affects fibrolipid components of atheromatous plaques. Pigmentation of the aortic and mitral valve cusps and valve rings is a result of intracellular and extracellular pigment deposition and is associated with calcification and clinically significant aortic stenosis.

A comparison is made of two methods by which one may derive molecular weight distribution and degree of long-chain branching using only the measured solution viscosity of a branched polyethylene whole polymer and its GPC trace. These are (a) Drott and Mendelson method and (b) Ram and Miltz procedure. In each case, the purpose of the method is to devise a means by which one may establish a relationship between solution viscosity and molecular weight for use in conjunction with the GPC universal calibration relationship of Benoit et al. The effectiveness of these theoretical approaches is evaluated by comparison with the true D and degree of long-chain branching data obtained using our complete iterative analysis method. Such a detailed comparison using low, moderate, and highly branched resins leads to a conclusion that both the techniques provide very good MWD and branching data and, further, that they may be considered interchangeable for most resins. For highly branched resins, the Ram and Miltz method, which is slightly more sensitive to the presence of a high degree of long-chain branching, is preferred. In practice, the Drott and Mendelson method has the advantage of using less computer time and providing a direct measure of degree of long-chain branching, and thus is likely to be used most frequently.

Melt How data has been determined for a series of fractionated and whole low density polyethylenes which has been characterized in terms of their molecular weights and degree of long-chain branching, (LCB). The resulting data indicate that low LCB influences melt flow both through a reduction in molecular size and an increased level of intermolecular interaction. Die swell measurements on whole polymers indicate an increase in melt elasticity with increase in degree of LCB for samples of similar melt flow (MI). Comparison of GPC data with observed die swell characteristics indicates that die swell is a molecular size dependent property and independent of intermolecular entanglement effects, suggesting that the measurement of elastic properties of LDPE melts will provide a means of determining relative degrees of LCB for commercial resins.

An accurate GPC calibration is essential if computer techniques are to be utilized in obtaining the molecular weight distribution and degree of long-chain branching from an intrinsic viscosity and GPC trace of a polymer. The use of the National Bureau of Standards Linear Polyethylene Standard Reference Material, SRM 1475, to calibrate GPC is described. Employing this calibration, the Mark–Houwink relationship for linear polyethylene in 1,2,4-trichlorobenzene was established utilizing narrow molecular weight fractions derived through fractionation of SRM 1475 and other polymers. This Mark–Houwink equation was subsequently employed for the evaluation of high molecular weight fractions which were then used to extend the GPC calibration to the high molecular weight region not covered by SRM 1475. An iterative technique was used to obtain coincidence of the measured intrinsic viscosity and the viscosity calculated from the GPC data. The accuracy of the GPC calibration was demonstrated by obtaining coincidence of the measured and calculated viscosity of high and low molecular weight polymers of both narrow and broad polydispersity.