Stability of 5-aminolevulinic acid on dried urine filter paper for a diagnostic marker of tyrosinemia type I

The chemical diagnosis of tyrosinemia type I generally involves the detection of succinylacetone (SA) in patient urine. However, 5-aminolevulinate (5ALA), which accumulates due to succinylacetone's inhibition of porphyrin synthesis, can also be used as diagnostic metabolites. Here we examined the stabilities of these markers on dried urine filter paper. After two weeks at room temperature, the succinylacetone was 10% of its original level, but over 80% of 5-aminolevulinate remained. Thus, although insufficient succinylacetone was recovered from dried urine filter paper to diagnose tyrosinemia type I, 5-aminolevulinate was readily detected, permitting the diagnosis.     

Gas chromatographic–mass spectrometric screening for organic acidemias using dried urine filter paper: determination of α-ketoacids

There are several organic acid disorders that require information on α-ketoacids, such as maple syrup urine disease or α-ketoadipic acidemia. The recovery, stability and diagnostic availability of α-ketoacids in dried urine filter paper analyzed by GC–MS with oxime-trimethylsilyl derivatization was studied for organic acidemia screening. The recovery of all nine types of α-ketoacids tested, but for phenylpyruvate, 2-ketoadipate, and p-OH-phenylpyruvate, from filter paper samples was acceptable. The stability of pyruvate, branched-chain α-ketoacids, α-ketoadipate and α-ketoglutarate was stable for at least 28 days, although some α-ketoacids such as succinylacetone were unstable. It indicated it was difficult to diagnose only tyrosinemia type 1 among nine specimens from organic acidemia patients tested. The method could be applied to global organic acidemia screening.     

A GC/MS validated method for the nanomolar range determination of succinylacetone in amniotic fluid and plasma: an analytical tool for tyrosinemia type I

A sensitive and accurate stable isotope dilution GC/MS assay was developed and validated for the quantification of succinylacetone (SA) in plasma and amniotic fluid (AF). SA is pathognonomic for tyrosinemia type I, a genetic disorder caused by a reduced activity of fumarylacetoacetate hydrolase (FAH). In untreated patients, SA can easily be measured in plasma and urine because the expected concentrations are in the micromol/L range. Due to a founder effect, the province of Quebec has an unusually high prevalence of tyrosinemia type I, hence, the quantification of SA in AF or plasma of treated patients in the nmol/L range becomes very useful. The method utilizes 13C5-SA as an internal standard and a three-step sample treatment consisting of oximation, solvent extraction and TMCS derivatization. The assay was validated by recording the ion intensities of m/z 620 for SA and m/z 625 for ISTD in order to demonstrate the precision of measurements, the linearity of the method, limit of quantification and detection (LOQ and LOD), specificity, accuracy, as well as metabolite stability. Values for the intra-day assays ranged from 0.2 to 3.2% while values for the inter-day assays ranged from 1.9 to 5.6% confirming that the method has good precision. A calibration plot using SA detected by GC/MS gave excellent linearity with a correlation coefficient of 0.999 over the injected concentration range of 5-2000 nmol/L. LOQ and LOD were 3 and 1 nmol/L, respectively. The usefulness of this method was demonstrated by SA quantification in an AF sample of an affected fetus and in plasma of patients treated with NTBC. The results demonstrate that this novel GC/MS method may be a valuable tool for metabolic evaluation and clinical use.     

Fumarylacetoacetase measurement as a mass-screening procedure for hereditary tyrosinemia type I.

Fluorometric quantitative tyrosine determination on dried-blood spots is the primary neonatal screening test used for tyrosinemia type I (HT) in the province of Quebec. Succinylacetone determination on these same spots is used as the complementary test when the tyrosine level is higher than a given threshold. This procedure has proved to be less discriminant over the past few years because of changes in newborn feeding and because of early discharge of newborns from the nursery. We have developed an enzyme-linked immunosorbent assay (ELISA) to measure the deficient enzyme in HT in dried-blood spots. Fumarylacetoacetase (FAH) (E.C.3.7.1.2) was measured retrospectively by an ELISA on 25 dried-blood samples from proven patients with HT and prospectively in 72,000 specimens received in the neonatal screening program. In this pilot project, FAH was measured first, and, if necessary, succinylacetone was determined as the complementary test. All 25 samples from proven patients and specimens from four other patients detected in the pilot study have shown almost complete absence of FAH in dried-blood samples. At a cutoff level of 12.5% of normal adult blood spotted on the same type of paper, only 30 other cases disclosed FAH levels low enough to warrant succinylacetone measurement but had no detectable succinylacetone. The false-positive rate is thus 1:2,400 with this primary ELISA. However, blood transfusion in newborns prior to blood collection on filter paper may yield false-negative tests, since FAH is present in erythrocytes of normal donors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tandem mass spectrometric assay of succinylacetone in urine for the diagnosis of hepatorenal tyrosinemia

We describe an isotope dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of succinylacetone (SA) in urine for the diagnosis of hepatorenal tyrosinemia (HT1). The method used 15N-labeled 5(3)-methyl-3(5)-isoxazole propionic acid as internal standard. Urine samples were oximated with hydroxylamine hydrochloride at 80 degrees C, extracted by solvent-solvent extraction, and followed by derivatization of the butyl ester. The butylated isoxazole derivatives of SA and its internal standard were detected and quantified using positive ion electrospray LC-MS/MS with selected reaction monitoring. The turnaround time between injections was 10 min. Calibration curves were linear over the range of 0.0633-63.3 micromol/L. The intra- and interday assay variations were less than 7%. Mean recoveries of SA at three different concentrations ranged from 96 to 109%. During the course of this study, we identified 12 new patients with HT1 and applied this method to follow up the treatment of 4 of these patients as well as previously diagnosed HT1 patients.     

Metabolic studies in a mouse model of hepatorenal tyrosinemia: absence of perinatal abnormalities.

Radiation induced chromosomal deletions at the albino locus in the mouse, lethal when homozygous, cause abnormalities of expression of several unlinked liver specific genes. Recently, the gene encoding FAH was shown to be included in the deletions. Since in humans FAH mutations cause tyrosinemia type I, deletion homozygous mice were suspected of having tyrosinemia. Studies of plasma amino acids did not confirm this suspicion. Also, succinylacetone levels were normal in fetal and newborn livers of deletion homozygotes. The present evidence, therefore, does not support the assumption that the earlier described ultrastructural and enzyme abnormalities in deletion homozygotes are secondary effects of tyrosinemia caused by the deletion of FAH.     

Assessing the DNA methylation status of single cells with the comet assay

The comet assay (single cell gel electrophoresis) is a cost-effective, sensitive, and simple technique that is traditionally used for analyzing and quantifying DNA damage in individual cells. The aim of this study was to determine whether the comet assay could be modified to detect changes in the levels of DNA methylation in single cells. We used the difference in methylation sensitivity of the isoschizomeric restriction endonucleases HpaII and MspI to demonstrate the feasibility of the comet assay to measure the global DNA methylation level of individual cells. The results were verified with the well-established cytosine extension assay. We were able to show variations in DNA methylation after treatment of cultured cells with 5-azacytidine and succinylacetone, an accumulating metabolite in human tyrosinemia type I.     

Determination of succinylacetone in dried blood spots and liquid urine as a dansylhydrazone by liquid chromatography tandem mass spectrometry

Succinylacetone (SA) is a specific marker for the inherited metabolic disease, hepatorenal tyrosinemia. We developed a stable-isotope dilution liquid chromatography tandem mass spectrometry for the determination of SA in dried blood spots (DBS) and liquid urine using a (13)C(4)-SA as internal standard. SA was extracted, converted to the butyl ester and derivatized with dansylhydrazine (Dns-H). Calibration curves in DBS and urine calibrators were linear up to 100 and 30 microM, respectively. At a signal-to-noise ratio of 3, the limits of detection in DBS and urine were 0.2 and 0.005 microM, respectively. Total run time was 5 min. Intra- and inter-assay precision expressed as coefficient of variation were better than 9.1% with more than 96% recovery. The method was applied retrospectively and prospectively for the diagnosis of hepatorenal tyrosinemia and for follow-up of patients under treatment.     

The human fumarylacetoacetase gene: characterisation of restriction fragment length polymorphisms and identification of haplotypes in tyrosinemia type 1 and pseudodeficiency

Summary Deficiency of human fumarylacetoacetase (FAH) activity results in hereditary tyrosinemia type I. Using the restriction enzymes BglII, KpnI and StuI and a 1.3-kb cDNA probe for the FAH gene, we have found 6 restriction fragment length polymorphisms (RFLPs). These RFLPs were utilised in 3 tyrosinemia families in which one or both parents are carriers of both a tyrosinemia and a pseudodeficiency gene for FAH. Full information was obtained in two of these families. The polymorphisms identified 6 haplotypes. The haplotype distribution was significantly different in 32 unrelated tyrosinemia patients compared with a reference population of 100 individuals. The combined polymorphism information content was 0.77.     

Analysis of nitrate and nitrite in water and urine by capillary zone electrophoresis

A capillary zone electrophoresis method for the separation and analysis of nitrate and nitrite in water and urine was developed. No interference in the electropherogram from other anions is observed by using a polyacrylamide-coated column with a modified phosphate buffer at pH 3 for the separation, and UV absorption at 214 nm for the detection. The method does not require sample pretreatment or the use of organic solvents. The limit of detection for each analyte (S/N = 3), using a 75 μm I.D. capillary, is 0.5 μg/ml. Urine samples require 40-fold dilution in order to maintain migration time reproducibility to within 1% relative standard deviation.     

On rat renal aminolevulinate transport and metabolism in experimental Fanconi syndrome

Hereditary tyrosinemia, an autosomal recessive disease of human infants, is characterized by severe liver disease, a renal Fanconi syndrome, and urinary excretion of large quantities of both aminolevulinate (ALA) and succinylacetone (SA). The latter is a metabolic end-product of tyrosine catabolism in affected individuals, produced by both liver and kidney, and is a potent inhibitor of aminolevulinate dehydratase (ALAD) in liver. This inhibition has been assumed to result in release of large amounts of aminolevulinate from liver into the circulation, with subsequent urinary excretion. In the present report we examine the effects of succinylacetone on rat renal cortical tubular handling of ALA and the relationship to tubular heme content, demonstrating a marked impairment of each. In contrast, maleic acid was found to have no effect on either renal ALAD or heme content. Thus, we conclude that renal handling of ALA in SA-treated rat renal cortex may indicate a contribution by the kidney to the increased net ALA excretion observed in hereditary tyrosinemia.     

Oral loading of homogentisic acid in controls and in obligate heterozygotes for hereditary tyrosinemia type I.

Homogentisic acid (HGA) (50 mg/kg) was given orally to 22 obligate heterozygotes for hereditary tyrosinemia type 1 (HT) and to 11 controls. After 1 h the mean +/- standard error (SE) plasma level of HGA was 30.42 +/- 1.41 micrograms/ml in carriers and 19.29 +/- 1.62 in controls. Mean +/- SE fasting delta-amino-levulinate dehydratase (delta-ALD) was 40.05 +/- 1.79 m microM/min/g Hb in carriers, much lower than the 60.81 +/- 5.11 found in controls. After 3 h this difference in levels of delta-ALD remained, with mean +/- SE values of 25.70 +/- 2.89 m microM/min/g Hb in carriers, compared with 48.83 +/- 5.37 in controls. Three-hour mean +/- SE excretion of fumarylacetone "equivalent" [FAc] in urine in carriers, 51.597 +/- 5.580 micrograms/mg/creatinine, was significantly higher than the 27.941 +/- 5.916 in controls. Three-hour excretion of succinylacetone "equivalent" [SAc] was also significantly higher in the urine of carriers. FAc in 3-h urine was identified by thin-layer chromatography and confirmed by gas chromatography/mass spectrometry. Multivariate stepwise discriminant analysis showed that the inclusion order of significant variables was as follows: HGA levels at 1 hr, fasting level of delta-ALD, residual level of HGA at 3 h, and 3-h excretion of [FAc]. Non-significant variables were HGA tolerance, levels of delta-ALD at 3 h, sex, and 3-h excretion of [SAc].(ABSTRACT TRUNCATED AT 250 WORDS)     

Hereditary tyrosinemia type 1 metabolites impair DNA excision repair pathways

Hereditary tyrosinemia type 1 is an autosomal recessive metabolic disorder, which is caused by a defective fumarylacetoacetate hydrolase enzyme, and consequently metabolites such as succinylacetone and p-hydroxyphenylpyruvate accumulate. We used a modified comet assay to determine the effect of these metabolites on base- and nucleotide excision repair pathways. Our results indicate that the metabolites affected the repair mechanisms differently, since the metabolites had a bigger detrimental effect on BER than on NER.     

Capillary electrophoresis with contactless conductivity detection for the determination of carnitine and acylcarnitines in clinical samples

A capillary electrophoresis method with contactless conductivity detection was developed for the quantification of carnitine and six acylcarnitines in plasma and urine samples. The running buffer employed consisted of 500 mmol/L acetic acid, 1.0 mmol/L hydroxypropyl-β-cyclodextrin and 0.05% Tween at a pH of 2.6. Under these conditions, the isomeric valproyl- and octanoyl-carnitines could be distinguished. The linearity was in the range from 5.0 to 200.0 μmol/L with correlation coefficients between 0.9992 and 0.9997. The limits of detection were between 1.0 and 3.2 μmol/L. Intra- and inter-day precisions as %RSD were better than 10%. The method allows for direct determination without derivatisation or extraction processes. The method was applied for the quantification of carnitine and acetylcarnitine in plasma pre- and post-exercise, and to measure valproylcarnitine in plasma and urine of patients undergoing valproate therapy.     

Direct and fast capillary zone electrophoretic method for the determination of Gleevec and its main metabolite in human urine

A capillary zone electrophoretic (CZE) method was investigated for the determination of Gleevec and its main metabolite (N-demethylated piperazine derivative) in human urine using a fused-silica capillary (75 microm I.D.x60 cm total length, 10 cm effective length). The separation was performed with an hydrodynamic injection time of 10 s (0.5 p.s.i.) a voltage of -25 kV, a capillary temperature of 25 degrees C and a 100 mM phosphoric acid adjusted to pH 2 with the addition of triethanolamine. Under these conditions, the analysis takes about 5 min. A linear response over the 0.4-30.0 mg l(-1) concentration range was investigated for two compounds. A dilution of the sample was the only step necessary before the electrophoresis analysis. Detection limits of 0.1 mg l(-1) for Gleevec and its metabolite (S/N=3) were obtained. The developed method is easy, rapid and sensitive and has been applied to determine Gleevec and its main metabolite in clinical urine samples.     

A murine model for type III tyrosinemia: lack of immunologically detectable 4-hydroxyphenylpyruvic acid dioxygenase enzyme protein in a novel mouse strain with hypertyrosinemia.

We have characterized a new mutant strain of mouse that has hypertyrosinemia. The blood tyrosine level was persistently high, and increased amounts of 4-hydroxyphenylpyruvic acid and its derivatives were excreted into the urine. Succinylacetone was not detected in urine samples from these mice. All the animals were apparently healthy, and there was no evidence of hepatorenal dysfunction. The hypertyrosinemia was transmitted through an autosomal recessive inheritance. Analyses of hepatic enzymes related to tyrosine metabolism revealed that 4-hydroxyphenylpyruvic acid dioxygenase activity was virtually absent, while fumarylacetoacetase and tyrosine aminotransferases (cytosolic and mitochondrial forms) were normal in these mutant mice. Immunoblot analysis of 4-hydroxyphenylpyruvic acid dioxygenase protein in the liver indicated that the subunit protein of the enzyme was absent. It would appear that hypertyrosinemia in this mutant strain was caused by a genetic defect in 4-hydroxyphenylpyruvic acid dioxygenase. These features are similar to type III tyrosinemia in humans. Analysis of this mutant strain of mouse is expected to provide valuable information on the pathogenesis of human type III tyrosinemia and can also serve as a useful system for studies on tyrosine metabolism.     

Quantification of urinary 5-aminolevulinic acid by gas chromatography-mass spectrometry

In this report, we describe a method for the specific quantification of urinary 5-aminolevulinic acid. It is based on gas chromatographic mass spectrometric measurement of the trimethylsilyl ester of the ethylacetoacetate pyrrole derivative of 5-aminolevulinic acid. Selected ion monitoring (SIM) was used for quantification using 3-hydroxymyristic acid as an internal standard. The detection limit of this method was 0.1 nmol/ml. This method was applied to the determination of urinary 5-aminolevulinic acid from a tyrosinemia type I patient and normal subjects, and 21.4 mmol/mol creatinine and 0.54+/- 0.49 mmol/mol creatinine (n = 7), respectively, were detected. Less than 0.2 ml urine was sufficient for the determination of 5-aminolevulinic acid in healthy subjects.     

Iron mobilization by succinylacetone methyl ester in rats. A model study for hereditary tyrosinemia and porphyrias characterized by 5-Aminolevulinic acid overload

Accumulation of 5-aminolevulinic acid (ALA) is an event characteristic of porphyrias that may contribute to their pathological manifestations. To investigate effects of ALA independent of porphyrin accumulation we treated rats with the methyl ester of succinylacetone, an inhibitor of 5-aminolevulinic acid dehydratase that accumulates in the porphyric-like syndrome hereditary tyrosinemia. Acute 2-day treatment of fasted rats with succinylacetone methyl ester (SAME) promoted a 27% increase in plasma ALA. This increase in plasma ALA was accompanied by augmentation of the level of total nonheme iron in liver (37%) and brain (20%). Mobilization of iron was also indicated by 49% increase in plasma iron and a 77% increase in plasma transferrin saturation. Liver responded with a mild (12%) increase in ferritin. Under these acute conditions, some indications of oxidative stress were evident: a 15% increase in liver reactive protein carbonyls, and a 42% increase in brain subcellular membrane TBARS. Brain also showed a 44% increase in CuZnSOD activity, consistent with observations in treatment with ALA. Overall, the data indicate that SAME promotes ALA-driven changes in iron metabolism that could lead to increased production of free radicals. The findings support other evidence that accumulation of ALA in porphyrias and hereditary tyrosinemia may induce iron-dependent biological damage that contributes to neuropathy and hepatoma.     

Determination of catecholamines and metanephrines in urine by capillary electrophoresis-electrospray ionization-time-of-flight mass spectrometry

A method successfully coupling capillary electrophoretic separation to time-of-flight mass spectrometric (TOFMS) detection for the simultaneous analysis of catecholamines (dopamine, norepinephrine, and epinephrine) and their O-methoxylated metabolites (3-methoxytyramine, normetanephrine, and metanephrine) is described. The inner capillary wall was coated with polyvinyl alcohol in order to obtain baseline resolution of catecholamines and metanephrines and to ensure reproducibility without extensive restorative washing of the capillary. Using electrokinetic injection, detection limits of 0.3 microM for dopamine and norepinephrine, 0.2 microM for 3-methoxytyramine and normetanephrine, and 0.1 microM for epinephrine and metanephrine were achieved with standard solutions. The usefulness of this approach was demonstrated by applying the developed method to the analysis of a spot collection of human urine from a healthy volunteer. The catecholamines and metanephrines were removed from the urine samples and preconcentrated by simultaneous SPE on cation-exchange sorbents. The recoveries of all analytes, with the exception of epinephrine (75%), were over 80%. Catecholamines and metanephrines in the urine samples were quantitated using 3,4-dihydroxybenzylamine as an internal standard. Submicromolar concentrations, consistent with the catecholamine and metanephrine levels reported for normal human urine, were detected.