Laboratory monitoring of patients with hereditary tyrosinemia type I.

BACKGROUND: The prognosis of patients with Hereditary Tyrosinemia Type 1 (HT-1) has greatly improved with early detection through newborn screening and the introduction of nitisinone (NTBC) therapy. A recent guideline calls for periodic monitoring of biochemical markers and NTBC levels to tailor treatment; however, this is currently only achieved through a combination of clinical laboratory tests. We developed a multiplexed assay measuring relevant amino acids, succinylacetone (SUAC), and NTBC in dried blood spots (DBS) to facilitate treatment monitoring. METHODS: Tyrosine, phenylalanine, methionine, NTBC and SUAC were eluted from DBS with methanol containing internal standards for each analyte and analyzed by liquid chromatography tandem mass spectrometry over 6.5 min in the multiple reaction monitoring positive mode. RESULTS: Pre-analytical and analytical factors were studied and demonstrated a reliable assay. Chromatography resolved an unknown substance that falsely elevates SUAC concentrations and was present in all samples. To establish control and disease ranges, the method was applied to DBS collected from controls (n = 284) and affected patients before (n = 2) and after initiation of treatment (n = 29). In the treated patients SUAC concentrations were within the normal range over a wide range of NTBC levels. CONCLUSIONS: This assay enables combined, accurate measurement of revelevant metabolites and NTBC in order to simplify treatment monitoring of patients with HT-1. In addition, the use of DBS allows for specimen collection at home to facilitate more standardization in relation to drug and dietary treatment.

Hypersuccinylacetonaemia and normal liver function in maleylacetoacetate isomerase deficiency.

BACKGROUND: A high level of succinylacetone (SA) in blood is a sensitive, specific newborn screening marker for hepatorenal tyrosinemia type 1 (HT1, MIM 276700) caused by deficiency of fumarylacetoacetate hydrolase (FAH). Newborns with HT1 are usually clinically asymptomatic but show liver dysfunction with coagulation abnormalities (prolonged prothrombin time and/or high international normalised ratio). Early treatment with nitisinone (NTBC) plus dietary restriction of tyrosine and phenylalanine prevents the complications of severe liver disease and neurological crises. METHODS AND RESULTS: Six newborns referred for hypersuccinylacetonaemia but who had normal coagulation testing on initial evaluation had sequence variants in the GSTZ1 gene, encoding maleylacetoacetate isomerase (MAAI), the enzyme preceding FAH in tyrosine degradation. Initial plasma SA levels ranged from 233 to 1282 nmol/L, greater than normal (<24 nmol/L) but less than the initial values of patients with HT1 (16 944-74 377 nmol/L, n=15). Four individuals were homozygous for c.449C>T (p.Ala150Val). One was compound heterozygous for c.259C>T (p.Arg87Ter) and an intronic sequence variant. In one, a single heterozygous GSTZ1 sequence variant was identified, c.295G>A (p.Val99Met). Bacterial expression of p.Ala150Val and p.Val99Met revealed low MAAI activity. The six individuals with mild hypersuccinylacetonaemia (MHSA) were not treated with diet or nitisinone. Their clinical course has been normal for up to 13 years. CONCLUSIONS: MHSA can be caused by sequence variants in GSTZ1. Such individuals have thus far remained asymptomatic despite receiving no specific treatment.

Succinylacetone as primary marker to detect tyrosinemia type I in newborns and its measurement by newborn screening programs.

Tyrosinemia type I (TYR I) is caused by autosomal recessive fumarylacetoacetate hydrolase deficiency and is characterized by development of severe liver disease in infancy and neurologic crises. If left untreated, most patients die of liver failure in the first years of life. Intervention with medication is effective when initiated during the first month of life. This improvement in the treatment of TYR I patients influenced the decision to include TYR I in the US Secretary of the Department of Health and Human Services' (HHS) Recommended Uniform Screening Panel. However, while tyrosine is routinely measured in newborn screening (NBS) by tandem mass spectrometry (MS/MS), elevated tyrosine levels are not specific to TYR I. To improve the specificity of NBS for TYR I, several assays were developed to measure succinylacetone (SUAC) in dried blood spots (DBS). SUAC is a pathognomonic marker of TYR I, and its detection by NBS MS/MS is possible. This review of the current status of NBS for TYR I in the US is the result of discussions at the HHS Secretary's (Discretionary) Advisory Committee on Heritable Disorders in Newborns and Children about the inconsistent implementation of effective NBS for TYR I in the US. We sought to understand the different TYR I screening practices in US NBS programs. Results indicate that 50 out of 51 NBS programs in the US screen for TYR I, and a successful SUAC performance evaluation scheme is available from the Centers for Disease Control and Prevention. Programmatic and methodological barriers were identified that prevent widespread adoption of SUAC measurements in NBS laboratories. However, since SUAC detection is currently the best approach to NBS for TYR I, a further delay of the addition of SUAC measurement into NBS procedures is discouraged. SUAC measurement should improve both the false positive and false negative rate in NBS for TYR I thereby yielding the desired benefits for affected patients at no expense to the overall population served.

Tyrosinemia type 1: an overview of nursing care.

Tyrosinemia type 1 (TT1) is an inherited metabolic disease that can be fatal when not detected early by newborn screening. In the past, children with TT1 had a poor prognosis due to organ failure and neurologic crisis during infancy. Recent improvements in newborn screening have changed the prognosis of affected children. Measurement of succinylacetone by tandem mass spectrometry provides early identification and the opportunity to manage TT1 as a chronic disease. Treatment includes genetic counseling, dietary management, pharmacotherapy, metabolic crisis prevention, and whole organ transplant. Nursing care is critical to successful management when it is based on a clear understanding of the pathophysiology. This overview of nursing care will provide specific recommendations to reduce complications and enhance the quality of life for children with TT1.

Plasma succinylacetone is persistently raised after liver transplantation in tyrosinaemia type 1.

BACKGROUND: Tyrosinaemia type 1 (HT1) is a rare disorder leading to accumulation of toxic metabolites such as succinylacetone (SA) and a high risk of hepatocellular carcinoma. Children with HT1 traditionally required liver transplantation (OLT) and while the need for this has been reduced by the introduction of nitisinone some still require OLT. SA inhibits the enzyme porphobilinogen (PBG) synthase and its activity can be used as a marker of active SA. Elevated urinary SA post OLT has been reported previously. This study describes a novel finding of elevated plasma SA following OLT for HT1. METHODS: A retrospective analysis was performed of patients treated for HT1 at our institution from 1989-2010. RESULTS: Thirteen patients had an OLT for HT1. In patients who received nitisinone prior to OLT, mean urinary and plasma SA were elevated prior to treatment but normalised by the time of OLT (p ≤ 0.01). Mean PBG synthase activity increased from 0.032 to 0.99 nkat/gHb (ref range 0.58-1.25) at the time of OLT (p < 0.01). Mean urinary SA in patients not treated with nitisinone was also elevated prior to OLT; plasma levels and PBG synthase activity were not available prior to OLT for this group. Following OLT, mean urinary and plasma SA were elevated in all for the duration of follow-up and associated with low-normal PBG synthase activity. CONCLUSION: Urinary and plasma SA levels are elevated following OLT for HT1. Low-normal PBG synthase activity suggests the plasma SA may be active. The clinical significance of this is unclear.

Dried blood spot analysis by digital microfluidics coupled to nanoelectrospray ionization mass spectrometry.

Dried blood spot (DBS) samples on filter paper are surging in popularity as a sampling and storage vehicle for a wide range of clinical and pharmaceutical applications. For example, a DBS sample is collected from every baby born in the province of Ontario, Canada, for quantification of approximately one hundred analytes that are used to screen for 28 conditions, including succinylacetone (SA), a marker for hepatorenal tyrosinemia. Unfortunately, the conventional methods used to evaluate DBS samples for newborn screening and other applications are tedious and slow, with limited options for automated analysis. In response to this challenge, we have developed a method to couple digital microfluidics (DMF) to nanoelectrospray ionization mass spectrometry (nESI-MS) for SA quantification in DBS samples. The new system is formed by sandwiching a pulled glass capillary emitter between the two DMF substrates such that the capillary emitter is immobilized without external seals or gaskets. Moreover, we introduce a new feedback control system that enables high-fidelity droplet manipulation across DBS samples without manual intervention. The system was validated by application to on-chip extraction, derivatization, and analysis of SA and other analytes from DBS samples, with comparable performance to gold-standard methods. We propose that the new methods described here can potentially contribute to a new generation of analytical techniques for quantifying analytes in DBS samples for a wide range of applications.

LC-MS/MS method for simultaneous determination on a dried blood spot of multiple analytes relevant for treatment monitoring in patients with tyrosinemia type I.

Tyrosinemia type 1 is caused by deficiency of fumarylacetoacetate hydrolase. The enzymatic defect impairs the conversion of fumarylacetoacetate to fumarate, causing accumulation of succinylacetone which induces severe liver and kidney dysfunction along with mutagenic changes and hepatocellular carcinoma. Treatment is based on nitisinone (NTBC), an enzymatic inhibitor which suppresses succinylacetone production. NTBC, which has dramatically changed the disease course improving liver and kidney functions and reducing risk of liver cancer, causes a side effect of the increase of tyrosine levels. Treatment is therefore based on the combination of NTBC with a protein-restricted diet to prevent the potential toxicity of excessive tyrosine accumulation. Long-term therapy requires a careful monitoring in blood of NTBC levels along with other disease biomarkers, which include succinylacetone, and a selected panel of circulating aminoacids. We have developed a straightforward and fast MS/MS method for the simultaneous determination of NTBC, succinylacetone, tyrosine, phenylalanine, and methionine on a dried blood spot requiring a 2 min run. A single assay suitable for quantitative evaluation of all biochemical markers is of great advance over conventional methods, especially in pediatric patients, since it reduces laboratory costs and blood sampling, is less invasive and particularly suitable for pediatric patients, and allows easier storage and shipping.

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.

Newborn screening for Tyr-I: two years' experience of the New York State program.

In 2 years, the New York newborn screening program has analyzed approximately 500,000 samples for succinylacetone (SUAC), the biomarker for Tyrosinemia, type I. There have been five screen-positive results. Two of these results were considered borderline, and a repeat specimen was requested. In three cases, an immediate referral was made to a specialty care center. Two of those three cases were confirmed for Tyr-I.

Maternal and fetal tyrosinemia type I.

A 22 year-old woman with tyrosinemia type I (HT1) married her first cousin who is heterozygous for the same FAH mutation for which the patient is homozygous. During her pregnancy she was treated with diet (prescribed tyrosine intake 300 mg/day), and nitisinone (60 mg/day). Median plasma tyrosine levels were 560 µmol/L (range: 375-838, n = 21) and nitisinone 51 µmol/L (range: 41-57, n = 3) during pregnancy. She gave birth to a clinically healthy girl affected with tyrosinemia type 1. Birth was normal (birth weight 2615 g) and the baby had normal liver function, normal plasma alpha-fetoprotein concentrations, low urinary excretion of phenolic acids and no detectable succinylacetone. At birth, the baby had hypertyrosinemia (860 µmol/L in blood cord) and nitisinone levels of 14 µmol/L. Following molecular confirmation of the diagnosis of HT1 specific treatment began on day 15 by which time she had detectable urinary succinylacetone.

Significant increase of succinylacetone within the first 12 h of life in hereditary tyrosinemia type 1.

INTRODUCTION: In most countries, hereditary tyrosinemia type 1 is not included in routine newborn screening. DISCUSSION: We present the case of a female newborn with prenatal diagnosis of hereditary tyrosinemia type 1 and clear identification of this disorder by succinylacetone measurement in cord blood and peripheral blood immediately after birth. Succinylacetone was 44 micromol/L (norm <5 micromol/L) and increased within 12 h to 87.5 micromol/L. CONCLUSION: With the high toxic potential of downstream metabolites, these data clearly point out the necessity of early nitisinone treatment to prevent symptomatic disease.

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.

Clinical utilization of dried blood spot nitisinone (NTBC) and succinylacetone (SA) concentrations in hereditary tyrosinaemia type 1 - A UK centre experience.

BACKGROUND: Dried blood spot monitoring of nitisinone and succinylacetone in hereditary tyrosinaemia type 1 patients is not widely available in the United Kingdom. Currently, biochemical monitoring utilizes urinary succinylacetone, blood spot tyrosine and phenylalanine monitoring, which can lack in convenience and accuracy, respectively. METHODS: We report the development of a dried blood spot assay for nitisinone and succinylacetone and analysed retrospective clinical and biochemical data for hereditary tyrosinaemia type 1 patients from a single UK centre. RESULTS: A total of 13 hereditary tyrosinaemia type 1 patients were evaluated. Eleven presented with liver dysfunction (two with associated renal tubulopathy) and two were detected by early sibling screening. All patients (age 0.03-22 months) were commenced on a tyrosine-/phenylalanine-restricted diet and nitisinone at diagnosis. Ten patients were on twice daily dosing and three were on single daily dosing at the start of monitoring. One patient from each dosing group swapped between dosing regimens at 20 years of age and 8 months of age, respectively. A total of 684 dried blood spot samples were analysed; 80% of nitisinone concentrations were between 9.2 and 27 µmol/L when succinylacetone was <0.3 µmol/L. Patients on twice daily dosing regimens had significantly higher nitisinone concentration compared with those on once daily dosing (P < 0.0001). The median dose required in the twice daily doing group was significantly lower when compared with once daily dosing. CONCLUSIONS: Dried blood spot monitoring for nitisinone and succinylacetone concentrations in hereditary tyrosinaemia type 1 patients is a rapid and convenient method which allows physicians to individualize treatment plans and observe adherence to treatment.

Preliminary proficiency testing results for succinylacetone in dried blood spots for newborn screening for tyrosinemia type I.

BACKGROUND: Succinylacetone (SUAC) is the primary metabolite accumulated in tyrosinemia type I--an inborn error of metabolism that, if untreated, can cause death from liver failure during the first months of life. Newborn screening laboratories measure SUAC in dried blood spot (DBS) samples to detect asymptomatic tyrosinemia type I. We used panels of SUAC-enriched DBSs to compare and evaluate the performance of these screening tests. METHODS: We prepared sets of DBS materials enriched with predetermined SUAC concentrations and distributed samples of these materials, along with a screening practices questionnaire, to laboratories that perform SUAC tests. We compared their reported SUAC concentrations and questionnaire responses to identify screening practices that affect SUAC test outcomes. RESULTS: Data from 2 pilot surveys showed large differences among laboratories in SUAC recoveries, reproducible within-laboratory recoveries, and stable performance of the DBS materials. Results from 257 proficiency test analyses contained a total of 6 false-negative misclassifications. Reported recoveries of added SUAC ranged from 0 to >200%. Low-biased SUAC recoveries were associated with 1 method used by 5 laboratories. All laboratories that reported SUAC recoveries > or =100% used DBS matrix calibrators. CONCLUSIONS: The wide ranges of SUAC concentrations reported for pilot and proficiency testing specimens demonstrate a need to harmonize quantitative results among laboratories. Although DBS matrix calibrators are important for optimizing SUAC recoveries, the preparation of these calibrators is not standardized among laboratories. Certified DBS-based SUAC calibrators are needed for accuracy and harmonization.

The Effect of Various Doses of Phenylalanine Supplementation on Blood Phenylalanine and Tyrosine Concentrations in Tyrosinemia Type 1 Patients.

Tyrosinemia type 1 (TT1) treatment with 2-(2-nitro-4-trifluormethyl-benzyl)-1,3-cyclohexanedione (NTBC) and a phenylalanine-tyrosine restricted diet is associated with low phenylalanine concentrations. Phenylalanine supplementation is prescribed without comprehensive consideration about its effect on metabolic control. We investigated the effect of phenylalanine supplementation on bloodspot phenylalanine, tyrosine, NTBC and succinylacetone. Eleven TT1 patients received 0, 20 and 40 mg/kg/day phenylalanine supplementation with the phenylalanine-tyrosine free L-amino acid supplements. Bloodspots were collected before breakfast, midday and evening meal. Differences between study periods, sample times and days within a study period were studied using (generalized) linear mixed model analyses. Twenty and 40 mg/kg/day phenylalanine supplementation prevented daytime phenylalanine decreases (p = 0.05) and most low phenylalanine concentrations, while tyrosine concentrations increased (p < 0.001). Furthermore, NTBC and succinylacetone concentrations did not differ between study periods. To conclude, 20 mg/kg/day phenylalanine supplementation can prevent most low phenylalanine concentrations without increasing tyrosine to concentrations above the target range or influencing NTBC and succinylacetone concentrations, while 40 mg/kg/day increased tyrosine concentrations to values above the targeted range. Additionally, this study showed that the effect of phenylalanine supplementation, and a possible phenylalanine deficiency, should be assessed using pre-midday meal blood samples that could be combined with an overnight fasted sample when in doubt.

Improved method to determine succinylacetone in dried blood spots for diagnosis of tyrosinemia type 1 using UPLC-MS/MS.

We describe an improved diagnostic method for tyrosinemia type 1 based on quantifying succinylacetone in dried blood spots by ultra-performance liquid chromatography tandem mass spectrometry. Succinylacetone extracted from a single 3/16 inch disk of specimen collection paper containing a dried blood spot was derivatized with dansylhydrazine, separated on an Acquity UPLC BEH C(18) column (2.1 x 50 mm, 1.7 microm) and detected by electrospray ionization tandem mass spectrometry. Succinylacetone derivative eluted at 0.6 min with a complete run time of 1 min. Using a 13C4 labeled succinylacetone as an internal standard, the calibration plot was linear up to 100 micromol/L with a detection limit (S/N = 3) of 0.2 micromol/L. Intra-day (n = 13) and inter-day (n = 10) variations were better than 10%. The cutoff level of succinylacetone in dried blood spots from healthy infants obtained by the current method was 0.63 micromol/L (n = 151). In dried blood spots from patients with established tyrosinemia type 1 (n = 11), concentration of succinylacetone was 6.4-30.8 micromol/L.

Simultaneous quantification of succinylacetone and nitisinone for therapeutic drug monitoring in the treatment of Tyrosinemia type 1.

We present a straightforward and robust method for simultaneous quantification of succinylacetone and nitisinone in plasma using LC-ESI-MS/MS. The method has been developed for routine therapeutic drug monitoring in hepatorenal tyrosinemia type 1 (HT1) patients undergoing nitisinone treatment. Previous methods are based on separate analyses of succinylacetone and nitisinone, often using the potentially harmful compound hydrazine for derivatization of the former. In the present procedure, succinylacetone is derivatized in a single-step using butanolic HCl. Analyte extraction and sample clean-up is carried out by simple protein precipitation. The linear range for both analytes is 0.1 up to 125µM, covering the vast majority of encountered levels in real-life samples. The sensitivity and limit of quantification allows measurement of succinylacetone in the therapeutical range for HT1 patients. Stability studies show that succinylacetone is highly sensitive to storage conditions, whereas nitisinone shows little to no degradation. Correct sample handling is therefore important for reliable results when monitoring succinylacetone concentrations.

Tandem mass spectrometric determination of succinylacetone in dried blood spots enables presymptomatic detection in a case of hepatorenal tyrosinaemia.

Tyrosinaemia type I, or fumarylacetoacetase deficiency, causes hepatorenal damage by accumulation of fumarylacetoacetate. Patients are generally in good condition at birth, but are at risk of developing serious metabolic crises with liver failure and hepatic coma. An early start of treatment with NTBC and a tyrosine-balanced diet can prevent harm to the patients. The application of tandem mass spectrometry to newborn screening allows for easy determination of tyrosine to detect the presence of hypertyrosinaemia in the neonate, but most patients with tyrosinaemia type I do not present with high tyrosine levels at the time of newborn screening. We report on a 7-week-old girl presenting with acute hepatopathy and severe coagulopathy due to tyrosinaemia type I. The metabolic screening, which was performed by tandem mass spectrometry at the age of 48 h, had revealed normal values for tyrosine and methionine that were well within ranges observed in the general population and equally normal ratios of methionine/tyrosine and tyrosine/serine. In this patient even lowering the cut-off levels for tyrosine and methionine would not have provided better sensitivity. Residual blood spots from the newborn screening filter paper were retrospectively analysed using a specific mass-spectrometric method for the detection of succinylacetone and revealed a 5-fold elevated succinylacetone concentration. This indicates that identification of all newborns with hepatorenal tyrosinaemia is only possible by determination of succinylacetone as part of the newborn screening process.

Newborn screening for Tyrosinemia type 1 using succinylacetone - a systematic review of test accuracy.

BACKGROUND: Tyrosinemia type 1 is an autosomal recessive disorder of amino acid metabolism. Without treatment, death in childhood is common. Treatment with nitisinone and dietary restrictions are associated with improved outcomes; some studies suggest better outcomes when treatment begins at an asymptomatic stage. Newborn screening allows for earlier identification, but there is uncertainty regarding the test accuracy of the current method: succinylacetone measurement in dried blood spots using tandem mass spectrometry. METHODS: We conducted a systematic review of literature published up to January 2016. Two reviewers independently assessed titles, abstracts, full texts, and conducted quality appraisals. A single reviewer extracted data, which was checked by a second reviewer. RESULTS: Ten studies provided test accuracy data: five studies reporting screening experiences and five case-control studies. Sensitivity (29 cases in total) and specificity (34,403 controls in total) were 100% in the case-control studies, but could not be calculated in the studies reporting screening experiences due to a lack of follow-up of screen-negative babies. Positive predictive values in the screening experience studies ranged from 66.7% (2 true positive cases, 1 false positive case from ~500,000 people screened) to 100% (8 true positive cases from 856,671 people screened); negative predictive values could not be calculated. Positive and negative predictive values cannot be calculated from case-control studies. CONCLUSIONS: Screening for Tyrosinemia type 1 using tandem mass spectrometry measurement of succinylacetone from dried blood spots appears to be promising. Confirmation of test accuracy data should be obtained from studies that include a two-year follow-up of individuals who screen negative.