Characterizing the alkaptonuria joint and spine phenotype and assessing the effect of homogentisic acid lowering therapy in a large cohort of 87 patients.

A large alkaptonuria (AKU) cohort was studied to better characterize the poorly understood spondyloarthropathy of rare disease AKU. Eighty-seven patients attended the National Alkaptonuria Centre (NAC) between 2007 and 2020. Seven only attended once. Fifty-seven attended more than once and received nitisinone 2 mg daily. Twenty-three attended at least twice without receiving nitisinone. Assessments included questionnaire analysis, 18F Positron emission tomography computerised tomography (PETCT), as well as photographs of ochronotic pigment in eyes and ears at baseline when 2 mg nitisinone was commenced and yearly thereafter. Blood and urine samples were collected for chemical measurement. The prevalence of ochronosis, as well as pain, PETCT and combined pain and PETCT scores, was greatly increased at 90.5%, 85.7%, 100%, and 100%, respectively. Joint pain scores were greatest in proximal joints in upper and lower limbs. PETCT joint scores were higher in proximal joints in upper limb but higher in distal joints in the lower limb. Spine pain scores were highest in lumbar, followed by cervical, thoracic, and cervical regions at 77.4%, 59.5%, 46.4%, and 25%, respectively. PETCT spine scores were highest in thoracic followed by lumbar, cervical, and sacroiliac regions at 74.4%, 70.7%, 64.6%, and 47.8% respectively; ochronosis associated closely with spondyloarthropathy scores (R = .65; P < .0001). Nitisinone reversed ochronosis significantly, with a similar pattern of decreased joint and spine disease. Spondyloarthropathy is a highly prevalent feature in this NAC cohort. Ochronosis appears to be associated with spondyloarthropathy. Nitisinone decreases ochronosis and had a similar nonsignificant effect pattern on spondyloarthropathy.

Characterising the arthroplasty in spondyloarthropathy in a large cohort of eighty-seven patients with alkaptonuria.

Arthroplasty in the spondyloarthropathy (SPOND) of alkaptonuria (AKU) in incompletely characterised. The aim was to improve the understanding of arthroplasty in AKU through a study of patients attending the National Alkaptonuria Centre (NAC). Eighty-seven patients attended the NAC between 2007 and 2020. Seven only attended once. Fifty-seven attended more than once and received nitisinone 2 mg daily. Twenty-three attended at least twice without receiving nitisinone. Assessments including questionnaire analysis eliciting details of arthroplasty and other surgical treatments for SPOND, 18 FPETCT and CT densitometry at the neck of hip and lumbar spine, as well as photographs of the eyes and ears were acquired from patients attending the National Alkaptonuria Centre (NAC) at baseline when 2 mg nitisinone was commenced, and yearly thereafter. Photographs were scored to derive ochronosis scores. Blood and urine samples were collected for chemical analyses. The prevalence of arthroplasty was 36.8%, similar in males and females, occurring especially in the knees, hips and shoulders. Multiple arthroplasties were found in 29 patients (33.3%) in this cohort. Incident arthroplasty was 6.5% in the nitisinone group and 7.1% in the no-nitisinone group. Incident arthroplasty was 11.3% in the group with baseline arthroplasty and 3.51% in the group without. A strong association of arthroplasty with SPOND (R = 0.5; P << .0001) and ochronosis (R = 0.54; P < .0001) was seen. Nitisinone had no significant effect on incident arthroplasty. Arthroplasty due to ochronosis and SPOND is common in AKU. Nitisinone decreased ochronosis but had no effect on arthroplasty in this cohort.

Efficacy of low dose nitisinone in the management of alkaptonuria.

AIM: To study the efficacy of low dosage of nitisinone in alkaptonuria. BACKGROUND: Alkaptonuria (AKU) is a rare genetic disease which induces deposition of homogentisic acid (HGA) in connective inducing premature arthritis, lithiasis, cardiac valve disease, fractures, muscle and tendon ruptures and osteopenia. Recent studies showed that nitisinone decreases HGA and is a beneficial therapy in AKU. This treatment induces an increase in tyrosine levels which can induces adverse effects as keratopathy. METHODS: We described the evolution HGA excretion and tyrosine evolution in 3 AKU patients treated by very low dosage of nitisinone with regards to their daily protein intakes. We also described the first pregnancy in an AKU patient treated by nitisinone. RESULTS: We found mild clinical signs of alkaptonuria on vertebra MRI in two young adults and homogentisate deposition in teeth of a 5 years old girl. Very low dose of nitisinone (10% of present recommended dose: 0.2 mg/day) allowed to decrease homogentisic acid by >90% without increasing tyrosine levels above 500 µmol/ in these three patients. INTERPRETATIONS: The analysis of the follow-up data shows that, in our three patients, a low-dosage of nitisinone is sufficient to decrease urinary HGA without increasing plasma tyrosine levels above the threshold of 500 µmol/L.

Assessing the effect of nitisinone induced hypertyrosinaemia on monoamine neurotransmitters in brain tissue from a murine model of alkaptonuria using mass spectrometry imaging.

OBJECTIVE: Nitisinone induced hypertyrosinaemia is a concern in patients with Alkaptonuria (AKU). It has been suggested that this may alter neurotransmitter metabolism, specifically dopamine and serotonin. Herein mass spectrometry imaging (MSI) is used for the direct measurement of 2,4-diphenyl-pyranylium tetrafluoroborate (DPP-TFB) derivatives of monoamine neurotransmitters in brain tissue from a murine model of AKU following treatment with nitisinone. METHODS: Metabolite changes were assessed using MSI on DPP-TFB derivatised fresh frozen tissue sections directing analysis towards primary amine neurotransmitters. Matched tail bleed plasma samples were analysed using LC-MS/MS. Eighteen BALB/c mice were included in this study: HGD-/- (n = 6, treated with nitisinone-4 mg/L, in drinking water); HGD-/- (n = 6, no treatment) and HGD+/- (n = 6, no treatment). RESULTS: Ion intensity and distribution of DPP-TFB derivatives in brain tissue for dopamine, 3-methoxytyramine, noradrenaline, tryptophan, serotonin, and glutamate were not significantly different following treatment with nitisinone in HGD -/- mice, and no significant differences were observed between HGD-/- and HGD+/- mice that received no treatment. Tyrosine (10-fold in both comparisons, p = 0.003; [BALB/c HGD-/- (n = 6) and BALB/c HGD+/- (n = 6) (no treatment) vs. BALB/c HGD-/- (n = 6, treated)] and tyramine (25-fold, p = 0.02; 32-fold, p = 0.02) increased significantly following treatment with nitisinone. Plasma tyrosine and homogentisic acid increased (ninefold, p = < 0.0001) and decreased (ninefold, p = 0.004), respectively in HGD-/- mice treated with nitisinone. CONCLUSIONS: Monoamine neurotransmitters in brain tissue from a murine model of AKU did not change following treatment with nitisinone. These findings have significant implications for patients with AKU as they suggest monoamine neurotransmitters are not altered following treatment with nitisinone.

Nitisinone arrests ochronosis and decreases rate of progression of Alkaptonuria: Evaluation of the effect of nitisinone in the United Kingdom National Alkaptonuria Centre.

QUESTION: Does Nitisinone prevent the clinical progression of the Alkaptonuria? FINDINGS: In this observational study on 39 patients, 2 mg of daily nitisinone inhibited ochronosis and significantly slowed the progression of AKU over a three-year period. MEANING: Nitisinone is a beneficial therapy in Alkaptonuria. BACKGROUND: Nitisinone decreases homogentisic acid (HGA), but has not been shown to modify progression of Alkaptonuria (AKU). METHODS: Thirty-nine AKU patients attended the National AKU Centre (NAC) in Liverpool for assessments and treatment. Nitisinone was commenced at V1 or baseline. Thirty nine, 34 and 22 AKU patients completed 1, 2 and 3 years of monitoring respectively (V2, V3 and V4) in the VAR group. Seventeen patients also attended a pre-baseline visit (V0) in the VAR group. Within the 39 patients, a subgroup of the same ten patients attended V0, V1, V2, V3 and V4 visits constituting the SAME Group. Severity of AKU was assessed by calculation of the AKU Severity Score Index (AKUSSI) allowing comparison between the pre-nitisinone and the nitisinone treatment phases. RESULTS: The ALL (sum of clinical, joint and spine AKUSSI features) AKUSSI rate of change of scores/patient/month, in the SAME group, was significantly lower at two (0.32 ±â€¯0.19) and three (0.15 ±â€¯0.13) years post-nitisinone when compared to pre-nitisinone (0.65 ±â€¯0.15) (p < .01 for both comparisons). Similarly, the ALL AKUSSI rate of change of scores/patient/month, in the VAR group, was significantly lower at one (0.16 ±â€¯0.08) and three (0.19 ±â€¯0.06) years post-nitisinone when compared to pre-nitisinone (0.59 ±â€¯0.13) (p < .01 for both comparisons). Combined ear and ocular ochronosis rate of change of scores/patient/month was significantly lower at one, two and three year's post-nitisinone in both VAR and SAME groups compared with pre-nitisinone (p < .05). CONCLUSION: This is the first indication that a 2 mg dose of nitisinone slows down the clinical progression of AKU. Combined ocular and ear ochronosis progression was arrested by nitisinone.

Clinical and biochemical assessment of depressive symptoms in patients with Alkaptonuria before and after two years of treatment with nitisinone.

OBJECTIVE: Concerns exist over hypertyrosinaemia that is observed following treatment with nitisinone. It has been suggested that tyrosine may compete with tryptophan for uptake into the central nervous system, and or inhibit tryptophan hydroxylase activity reducing serotonin production. At the National Alkaptonuria (AKU) Centre nitisinone is being used off-licence to treat AKU, and there is uncertainty over whether hypertyrosinaemia may alter mood. Herein results from clinical and biochemical assessments of depression in patients with AKU before and after treatment with nitisinone are presented. PATIENTS AND METHODS: 63 patients were included pre-nitisinone treatment, of these 39 and 32 patients were followed up 12 and 24 months after treatment. All patients had Becks Depression Inventory-II (BDI-II) assessments (scores can range from 0 to 63, the higher the score the more severe the category of depression), and where possible urinary monoamine neurotransmitter metabolites and serum aromatic amino acids were measured as biochemical markers of depression. RESULTS: Mean (±standard deviation) BDI-II scores pre-nitisinone, and after 12 and 24 months were 10.1(9.6); 9.8(10.0) and 10.5(9.9) (p ≥ 0.05, all visits). Paired scores (n = 32), showed a significant increase at 24 months compared to baseline 10.5(9.9) vs. 8.6 (7.8) (p = 0.03). Serum tyrosine increased at least 6-fold following nitisinone (p ≤ 0.0001, all visits), and urinary 3-methoxytyramine (3-MT) increased at 12 and 24 months (p ≤ 0.0001), and 5-hydroxyindole acetic acid (5-HIAA) decreased at 12 months (p = 0.03). CONCLUSIONS: BDI-II scores were significantly higher following 24 months of nitisinone therapy in patients that were followed up, however the majority of these patients remained in the minimal category of depression. Serum tyrosine and urinary 3-MT increased significantly following treatment with nitisinone. In contrast urinary 5-HIAA did not decrease consistently over the same period studied. Together these findings suggest nitisinone does not cause depression despite some observed effects on monoamine neurotransmitter metabolism.

Daily variation of NTBC and its relation to succinylacetone in tyrosinemia type 1 patients comparing a single dose to two doses a day.

INTRODUCTION: In hereditary tyrosinemia type 1 (HT1) patients, the dose of NTBC that leads to the absence of toxic metabolites such as succinylacetone (SA) is still unknown. Therefore, the aims of this study were to investigate the variation and concentrations of 2-(2-nitro-4-trifluormethyl-benzyl)-1,3-cyclohexanedione (NTBC) during the day in relation to the detection of SA, while comparing different dosing regimens. METHODS: All patients were treated with NTBC (mean 1.08 ± 0.34 mg/kg/day) and a low phenylalanine-tyrosine diet. Thirteen patients received a single dose of NTBC and five patients twice daily. Home bloodspots were collected four times daily for three consecutive days measuring NTBC and SA concentrations. Statistical analyses were performed by using mixed model analyses and generalized linear mixed model analyses to study variation and differences in NTBC concentrations and the correlation with SA, respectively. RESULTS: NTBC concentrations varied significantly during the day especially if NTBC was taken at breakfast only (p = 0.026), although no significant difference in NTBC concentrations between different dosing regimens could be found (p = 0.289). Momentary NTBC concentrations were negatively correlated with SA (p < 0.001). Quantitatively detectable SA was only found in subjects with once daily administration of NTBC and associated with momentary NTBC concentrations <44.3 µmol/l. DISCUSSION: NTBC could be less stable than previously considered, thus dosing NTBC once daily and lower concentrations may be less adequate. Further research including more data is necessary to establish the optimal dosing of NTBC.

Suitability Of Nitisinone In Alkaptonuria 1 (SONIA 1): an international, multicentre, randomised, open-label, no-treatment controlled, parallel-group, dose-response study to investigate the effect of once daily nitisinone on 24-h urinary homogentisic acid excretion in patients with alkaptonuria after 4 weeks of treatment.

BACKGROUND: Alkaptonuria (AKU) is a serious genetic disease characterised by premature spondyloarthropathy. Homogentisate-lowering therapy is being investigated for AKU. Nitisinone decreases homogentisic acid (HGA) in AKU but the dose-response relationship has not been previously studied. METHODS: Suitability Of Nitisinone In Alkaptonuria 1 (SONIA 1) was an international, multicentre, randomised, open-label, no-treatment controlled, parallel-group, dose-response study. The primary objective was to investigate the effect of different doses of nitisinone once daily on 24-h urinary HGA excretion (u-HGA24) in patients with AKU after 4 weeks of treatment. Forty patients were randomised into five groups of eight patients each, with groups receiving no treatment or 1 mg, 2 mg, 4 mg and 8 mg of nitisinone. FINDINGS: A clear dose-response relationship was observed between nitisinone and the urinary excretion of HGA. At 4 weeks, the adjusted geometric mean u-HGA24 was 31.53 mmol, 3.26 mmol, 1.44 mmol, 0.57 mmol and 0.15 mmol for the no treatment or 1 mg, 2 mg, 4 mg and 8 mg doses, respectively. For the most efficacious dose, 8 mg daily, this corresponds to a mean reduction of u-HGA24 of 98.8% compared with baseline. An increase in tyrosine levels was seen at all doses but the dose-response relationship was less clear than the effect on HGA. Despite tyrosinaemia, there were no safety concerns and no serious adverse events were reported over the 4 weeks of nitisinone therapy. CONCLUSIONS: In this study in patients with AKU, nitisinone therapy decreased urinary HGA excretion to low levels in a dose-dependent manner and was well tolerated within the studied dose range. TRIAL REGISTRATION NUMBER: EudraCT number: 2012-005340-24. Registered at ClinicalTrials.gov: NCTO1828463.

Tyrosinemia type 1 and irreversible neurologic crisis after one month discontinuation of nitisone.

Tyrosinemia type I is an autosomal recessively inherited metabolic disease of tyrosine metabolism due to the deficiency of fumarylacetoacetate hydrolase. Clinical manifestations include hepatic failure, cirrhosis, hepatocellular carcinoma, renal fanconi syndrome, and neurologic crisis. With the introduction of 2-(2-nitro-4-trifluoro-methylbenzyol)-1,3 cyclohexanedione treatment the prognosis improved with reduced rate of complications. "Neurologic crisis" of tyrosinemia type I is a rare complication seen after discontinuation of treatment characterized with anorexia, vomiting, and hyponatremia in the initial phase continuing with paresthesia and paralysis of the extremities and the diaphragm. Here, we report a tyrosinemia type I patient who admitted to the hospital with nonspecific symptoms such as vomiting, anorexia, weakness, and restlessness only after one month discontinuation of nitisone and diagnosed as neurological crisis.

Single dose NTBC-treatment of hereditary tyrosinemia type I.

NTBC (2-(2-nitro-4-trifluoromethylbenzoyl)-1,3cyclohexanedione) is the mainstay of treatment in tyrosinemia type 1 (HT 1). The current recommendation is to divide the total daily dose of NTBC into two doses. We monitored the plasma NTBC concentrations in a series of seven patients who were changed from multiple divided doses to a single daily dose of NTBC. Two additional patients were started on a single daily dose of NTBC after the diagnosis of HT 1 was established. In three patients, NTBC kinetics were performed over 6 and 24 hours, respectively. The use of multiple divided doses or a single daily dose did not significantly affect plasma NTBC concentrations or the mean daily dose needed to attain therapeutic plasma NTBC concentrations. Moreover, kinetic studies demonstrated that plasma NTBC concentrations were completely stable over a period of 24 hours with a single dose regimen, as expected given the known NTBC plasma half life of 54 hours. Although these preliminary results need to be confirmed in more patients, our findings show that administration of NTBC in a single daily dose may be as effective as a multiple-dose regimen in reaching therapeutic plasma NTBC concentrations and suppressing succinylacetone formation in patients with HT 1. In fact, single dose treatment may increase patients' compliance with the drug treatment and improve metabolic control.

Casein Glycomacropeptide: An Alternative Protein Substitute in Tyrosinemia Type I.

Tyrosinemia type I (HTI) is treated with nitisinone, a tyrosine (Tyr) and phenylalanine (Phe)-restricted diet, and supplemented with a Tyr/Phe-free protein substitute (PS). Casein glycomacropeptide (CGMP), a bioactive peptide, is an alternative protein source to traditional amino acids (L-AA). CGMP contains residual Tyr and Phe and requires supplementation with tryptophan, histidine, methionine, leucine, cysteine and arginine. AIMS: a 2-part study assessed: (1) the tolerance and acceptability of a low Tyr/Phe CGMP-based PS over 28 days, and (2) its long-term impact on metabolic control and growth over 12 months. METHODS: 11 children with HTI were recruited and given a low Tyr/Phe CGMP to supply all or part of their PS intake. At enrolment, weeks 1 and 4, caregivers completed a questionnaire on gastrointestinal symptoms, acceptability and ease of PS use. In study part 1, blood Tyr and Phe were assessed weekly; in part 2, weekly to fortnightly. In parts 1 and 2, weight and height were assessed at the study start and end. RESULTS: Nine of eleven children (82%), median age 15 years (range 8.6-17.7), took low Tyr/Phe CGMP PS over 28 days; it was continued for 12 months in n = 5 children. It was well accepted by 67% (n = 6/9), tolerated by 100% (n = 9/9) and improved gastrointestinal symptoms in 2 children. The median daily dose of protein equivalent from protein substitute was 60 g/day (range 45-60 g) with a median of 20 g/day (range 15 to 30 g) from natural protein. In part 2 (n = 5), a trend for improved blood Tyr was observed: 12 months pre-study, median Tyr was 490 µmol/L (range 200-600) and Phe 50 µmol/L (range 30-100); in the 12 months taking low Tyr/Phe CGMP PS, median Tyr was 430 µmol/L (range 270-940) and Phe 40 µmol/L (range 20-70). Normal height, weight and BMI z scores were maintained over 12 months. CONCLUSIONS: In HTI children, CGMP was well tolerated, with no deterioration in metabolic control or growth when studied over 12 months. The efficacy of CGMP in HTI needs further investigation to evaluate the longer-term impact on blood Phe concentrations and its potential influence on gut microflora.

Clinical practice. NTBC therapy for tyrosinemia type 1: how much is enough?

UNLABELLED: Four patients with tyrosinemia type 1 (ages 6-32 months) were treated with 2-(2-nitro-4-trifluoro-methylbenzoyl)-1,3-cyclohexandion (NTBC) at Cairo University Children's Hospital, Egypt and followed up for 12-27 months. The recommended average dose of NTBC is 1 mg/kg/day. They were started on the following doses: 0.8, 0.58, 0.5, and 0.625 mg/kg/day, respectively. Two months after start of therapy, succinylacetone was undetectable in patients 1, 2, and 4, while in case 3, it was 5.4 microM. Her NTBC dose was increased from 0.5 to 0.65 mg/kg/day, and succinylacetone was undetectable 1 month later. They were kept on NTBC doses ranging from 0.55 to 0.65 mg/kg/day. These doses allowed catch up growth, normalization of synthetic liver functions, steep drop in serum alpha fetoprotein, reduction in phosphate loss in urine, normalization of serum calcium, phosphate, and alkaline phosphatase, and healing of active rickets. Succinylacetone was undetectable in urine on these doses. IN CONCLUSION: Doses of NTBC, lower than recommended, may be helpful in treatment of tyrosinemia, on condition that succinylacetone production is suppressed, and AFP is maintained normal or showing a progressive decrease. This cost-effective dose may allow treatment of affected children from economically underprivileged countries, but longer follow up periods are needed.

Efficacy and safety of once-daily nitisinone for patients with alkaptonuria (SONIA 2): an international, multicentre, open-label, randomised controlled trial.

BACKGROUND: Alkaptonuria is a rare, genetic, multisystem disease characterised by the accumulation of homogentisic acid (HGA). No HGA-lowering therapy has been approved to date. The aim of SONIA 2 was to investigate the efficacy and safety of once-daily nitisinone for reducing HGA excretion in patients with alkaptonuria and to evaluate whether nitisinone has a clinical benefit. METHODS: SONIA 2 was a 4-year, open-label, evaluator-blind, randomised, no treatment controlled, parallel-group study done at three sites in the UK, France, and Slovakia. Patients aged 25 years or older with confirmed alkaptonuria and any clinical disease manifestations were randomly assigned (1:1) to receive either oral nitisinone 10 mg daily or no treatment. Patients could not be masked to treatment due to colour changes in the urine, but the study was evaluator-blinded as far as possible. The primary endpoint was daily urinary HGA excretion (u-HGA24) after 12 months. Clinical evaluation Alkaptonuria Severity Score Index (cAKUSSI) score was assessed at 12, 24, 36, and 48 months. Efficacy variables were analysed in all randomly assigned patients with a valid u-HGA24 measurement at baseline. Safety variables were analysed in all randomly assigned patients. The study was registered at ClinicalTrials.gov (NCT01916382). FINDINGS: Between May 7, 2014, and Feb 16, 2015, 139 patients were screened, of whom 138 were included in the study, with 69 patients randomly assigned to each group. 55 patients in the nitisinone group and 53 in the control group completed the study. u-HGA24 at 12 months was significantly decreased by 99·7% in the nitisinone group compared with the control group (adjusted geometric mean ratio of nitisinone/control 0·003 [95% CI 0·003 to 0·004], p<0·0001). At 48 months, the increase in cAKUSSI score from baseline was significantly lower in the nitisinone group compared with the control group (adjusted mean difference -8·6 points [-16·0 to -1·2], p=0·023). 400 adverse events occurred in 59 (86%) patients in the nitisinone group and 284 events occurred in 57 (83%) patients in the control group. No treatment-related deaths occurred. INTERPRETATION: Nitisinone 10 mg daily was well tolerated and effective in reducing urinary excretion of HGA. Nitisinone decreased ochronosis and improved clinical signs, indicating a slower disease progression. FUNDING: European Commission Seventh Framework Programme.

Natural Protein Tolerance and Metabolic Control in Patients with Hereditary Tyrosinaemia Type 1.

In a longitudinal retrospective study, we aimed to assess natural protein (NP) tolerance and metabolic control in a cohort of 20 Hereditary Tyrosinaemia type I (HTI) patients. Their median age was 12 years ([3.2-17.7 years], n = 11 female, n = 8 Caucasian, n = 8 Asian origin, n = 2 Arabic and n = 2 Indian). All were on nitisinone (NTBC) with a median dose of 0.7 g/kg/day (range 0.4-1.5 g/kg/day) and were prescribed a tyrosine (Tyr)/phenylalanine (Phe)-restricted diet supplemented with Tyr/Phe-free L-amino acids. Data were collected on clinical signs at presentation, medical history, annual dietary prescriptions, and blood Phe and Tyr levels from diagnosis until transition to the adult service (aged 16-18 years) or liver transplantation (if it preceded transition). The median age of diagnosis was 2 months (range: 0 to 24 months), with n = 1 diagnosed by newborn screening, n = 3 following phenylketonuria (PKU) screening and n = 7 by sibling screening. Five patients were transplanted (median age 6.3 years), and one died due to liver cancer. The median follow-up was 10 years (3-16 years), and daily prescribed NP intake increased from a median of 5 to 24 g/day. Lifetime median blood Tyr (370 µmol/L, range 280-420 µmol/L) and Phe (50 µmol/L, 45-70 µmol/L) were maintained within the target recommended ranges. This cohort of HTI patients were able to increase the daily NP intake with age while maintaining good metabolic control. Extra NP may improve lifelong adherence to the diet.

Hepatorenal Tyrosinaemia: Impact of a Simplified Diet on Metabolic Control and Clinical Outcome.

Background: Tyrosinaemia type 1 is a rare inherited metabolic disease caused by an enzyme defect in the tyrosine degradation pathway. It is treated using nitisinone and a low-protein diet. In a workshop in 2013, a group of nutritional specialists from Germany, Switzerland and Austria agreed to advocate a simplified low-protein diet and to allow more natural protein intake in patients with tyrosinaemia type 1. This retrospective study evaluates the recommendations made at different treatment centers and their impact on clinical symptoms and metabolic control. Methods: For this multicenter study, questionnaires were sent to nine participating treatment centers to collect data on the general therapeutic approach and data of 47 individual patients treated by those centers. Results: Dietary simplification allocating food to 3 categories led to increased tyrosine and phenylalanine blood concentrations without weighing food. Phenylalanine levels were significantly higher in comparison to a strict dietary regimen whereas tyrosine levels in plasma did not change. Non-inferiority was shown for the simplification and liberalization of the diet. Compliance with dietary recommendations was higher using the simplified diet in comparison to the stricter approach. Age correlates negatively with compliance. Conclusions: Simplification of the diet with increased natural protein intake based on three categories of food may be implemented in the diet of patients with tyrosinaemia type 1 without significantly altering metabolic control. Patient compliance is strongly influencing tyrosine blood concentrations. A subsequent prospective study with a larger sample size is necessary to get a better insight into the effect of dietary recommendations on metabolic control.

Oxidative Stress, Glutathione Metabolism, and Liver Regeneration Pathways Are Activated in Hereditary Tyrosinemia Type 1 Mice upon Short-Term Nitisinone Discontinuation.

Hereditary tyrosinemia type 1 (HT1) is an inherited condition in which the body is unable to break down the amino acid tyrosine due to mutations in the fumarylacetoacetate hydrolase (FAH) gene, coding for the final enzyme of the tyrosine degradation pathway. As a consequence, HT1 patients accumulate toxic tyrosine derivatives causing severe liver damage. Since its introduction, the drug nitisinone (NTBC) has offered a life-saving treatment that inhibits the upstream enzyme 4-hydroxyphenylpyruvate dioxygenase (HPD), thereby preventing production of downstream toxic metabolites. However, HT1 patients under NTBC therapy remain unable to degrade tyrosine. To control the disease and side-effects of the drug, HT1 patients need to take NTBC as an adjunct to a lifelong tyrosine and phenylalanine restricted diet. As a consequence of this strict therapeutic regime, drug compliance issues can arise with significant influence on patient health. In this study, we investigated the molecular impact of short-term NTBC therapy discontinuation on liver tissue of Fah-deficient mice. We found that after seven days of NTBC withdrawal, molecular pathways related to oxidative stress, glutathione metabolism, and liver regeneration were mostly affected. More specifically, NRF2-mediated oxidative stress response and several toxicological gene classes related to reactive oxygen species metabolism were significantly modulated. We observed that the expression of several key glutathione metabolism related genes including Slc7a11 and Ggt1 was highly increased after short-term NTBC therapy deprivation. This stress response was associated with the transcriptional activation of several markers of liver progenitor cells including Atf3, Cyr61, Ddr1, Epcam, Elovl7, and Glis3, indicating a concreted activation of liver regeneration early after NTBC withdrawal.

Activation of nuclear factor E2-related factor 2 in hereditary tyrosinemia type 1 and its role in survival and tumor development.

In tyrosinemia type 1 (HT1), accumulation of toxic metabolites results in oxidative stress and DNA damage, leading to a high incidence of hepatocellular carcinomas. Nuclear factor erythroid-2 related factor 2 (Nrf2) is a key transcription factor important for cellular protection against oxidative stress and chemical induced liver damage. To specifically address the role of Nrf2 in HT1, fumarylacetoacetate hydrolase (Fah)/Nrf2(-/-) mice were generated. In acute HT1, loss of Nrf2 elicited a strong inflammatory response and dramatically increased the mortality of mice. Following low grade injury, Fah/Nrf2(-/-) mice develop a more severe hepatitis and liver fibrosis. The glutathione and cellular detoxification system was significantly impaired in Fah/Nrf2(-/-) mice, resulting in increased oxidative stress and DNA damage. Consequently, tumor development was significantly accelerated by loss of Nrf2. Potent pharmacological inducers of Nrf2 such as the triterpenoid analogs 1[2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole have been developed as cancer chemoprevention agents. Pretreatment with 1[2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole dramatically protected Fah(-/-) mice against fumarylacetoacetate (Faa)-induced toxicity. Our data establish a central role for Nrf2 in the protection against Faa-induced liver injury; the Nrf2 regulated cellular defense not only prevents acute Faa-induced liver failure but also delays hepatocarcinogenesis in HT1.

Unusual first presentation of a metabolic disorder.

An 8-month-old boy presented to hospital with a fever, irritability and 'back arching'. On examination, he demonstrated profound opisthotonic posturing and had tonsillitis. He had a full septic screen and was treated with broad spectrum antibiotics. Blood tests showed a transaminitis, raised alpha fetoprotein and deranged clotting. The clotting abnormalities and raised alpha fetoprotein persisted post discharge and an abdominal ultrasound showed steatosis, splenomegaly and bilateral increased renal cortical reflectivity. A full metabolic screen revealed type 1 tyrosinaemia. The opisthotonic posturing, a major part of this child's presentation, has not been reported as a presenting feature of tyrosinaemia. It was part of a 'neurological crisis' caused by tyrosinaemia and exacerbated by the intercurrent infection. These are known to occur in tyrosinaemia but not commonly as the first presentation. This represents an unusual presentation of a metabolic condition which, without intervention, can lead to severe hepatic, renal and neurodevelopmental complications.

Severe neurological crisis in a patient with hereditary tyrosinaemia type I after interruption of NTBC treatment.

Neurological crises do not occur in patients with tyrosinaemia type I treated with NTBC. We report an 8 month-old boy with severe neurological crisis after interruption of NTBC treatment including progressive ascending polyneuropathy and diaphragmatic paralysis, arterial hypertension, respiratory distress requiring mechanical ventilation who later also developed impaired liver function and tubulopathy. After re-introduction of NTBC the patient slowly regained normal neurological functions and recovered completely.

5-Nitro-2-(3-phenylpropylamino)benzoic acid is a GPR35 agonist.

GPR35 is a Gi/o- and G16-coupled receptor abundantly expressed in gastrointestinal tissues and immune cells. Kynurenic acid (a tryptophan metabolite and ionotropic glutamate receptor antagonist) and zaprinast (a phosphodiesterase inhibitor) are GPR35 agonists. Here, we show that the chloride channel blocker 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) is also a GPR35 agonist. NPPB activates the GPR35-Gi/o and GPR35-G16 pathways in human embryonic kidney 293 (HEK293) cells and induces intracellular calcium mobilization in a concentration-dependent manner in HEK293 cells coexpressing human, rat or mouse GPR35 and the chimeric G protein G(qi5). These results suggest a novel pharmacological activity of NPPB and will provide useful information to search for more potent and selective GPR35 agonists.