Urinary phenylacetylglutamine as dosing biomarker for patients with urea cycle disorders.

UNLABELLED: We have analyzed pharmacokinetic data for glycerol phenylbutyrate (also GT4P or HPN-100) and sodium phenylbutyrate with respect to possible dosing biomarkers in patients with urea cycle disorders (UCD). STUDY DESIGN: These analyses are based on over 3000 urine and plasma data points from 54 adult and 11 pediatric UCD patients (ages 6-17) who participated in three clinical studies comparing ammonia control and pharmacokinetics during steady state treatment with glycerol phenylbutyrate or sodium phenylbutyrate. All patients received phenylbutyric acid equivalent doses of glycerol phenylbutyrate or sodium phenylbutyrate in a cross over fashion and underwent 24-hour blood samples and urine sampling for phenylbutyric acid, phenylacetic acid and phenylacetylglutamine. RESULTS: Patients received phenylbutyric acid equivalent doses of glycerol phenylbutyrate ranging from 1.5 to 31.8 g/day and of sodium phenylbutyrate ranging from 1.3 to 31.7 g/day. Plasma metabolite levels varied widely, with average fluctuation indices ranging from 1979% to 5690% for phenylbutyric acid, 843% to 3931% for phenylacetic acid, and 881% to 1434% for phenylacetylglutamine. Mean percent recovery of phenylbutyric acid as urinary phenylacetylglutamine was 66.4 and 69.0 for pediatric patients and 68.7 and 71.4 for adult patients on glycerol phenylbutyrate and sodium phenylbutyrate, respectively. The correlation with dose was strongest for urinary phenylacetylglutamine excretion, either as morning spot urine (r = 0.730, p < 0.001) or as total 24-hour excretion (r = 0.791 p<0.001), followed by plasma phenylacetylglutamine AUC(24-hour), plasma phenylacetic acid AUC(24-hour) and phenylbutyric acid AUC(24-hour). Plasma phenylacetic acid levels in adult and pediatric patients did not show a consistent relationship with either urinary phenylacetylglutamine or ammonia control. CONCLUSION: The findings are collectively consistent with substantial yet variable pre-systemic (1st pass) conversion of phenylbutyric acid to phenylacetic acid and/or phenylacetylglutamine. The variability of blood metabolite levels during the day, their weaker correlation with dose, the need for multiple blood samples to capture trough and peak, and the inconsistency between phenylacetic acid and urinary phenylacetylglutamine as a marker of waste nitrogen scavenging limit the utility of plasma levels for therapeutic monitoring. By contrast, 24-hour urinary phenylacetylglutamine and morning spot urine phenylacetylglutamine correlate strongly with dose and appear to be clinically useful non-invasive biomarkers for compliance and therapeutic monitoring.

Newborn screening compared to clinical identification of biochemical genetic disorders.

A group of 28 patients with inherited metabolic disease (homocystinuria galactosaemia, maple syrup urine disease and biotinidase deficiency) diagnosed by screening were compared with a group of 17 similar patients identified clinically. The rate of hospitalization was similar for the two groups. The patients diagnosed clinically showed a higher incidence of mental retardation and their parents experienced greater stress and found greater difficulty in meeting their child's needs.

Tyrosine supplementation in phenylketonuria: diurnal blood tyrosine levels and presumptive brain influx of tyrosine and other large neutral amino acids.

UNLABELLED: Tyrosine supplementation has not consistently been found to improve neuropsychologic function in phenylketonuria (PKU), possibly because of failure to achieve adequate levels of tyrosine in the brain. OBJECTIVES: To evaluate blood levels achieved after tyrosine supplementation in treated PKU and calculate brain influxes of tyrosine and other large neutral amino acids before and with tyrosine supplementation. STUDY DESIGN: Ten subjects with PKU receiving a phenylalanine-restricted diet were studied over 48 hours; each received tyrosine supplementation (300 mg/kg) on day 2. Plasma phenylalanine and tyrosine were measured every 2 hours, and all free amino acids were measured every 6 hours. Brain influxes of tyrosine and other large neutral amino acids were calculated. RESULTS: Plasma tyrosine levels were low normal at baseline. With supplementation there was a substantial but unsustained rise in plasma tyrosine. Calculated brain influx of tyrosine was 27% +/- 19% of normal before supplementation, increasing to 90% +/- 58% of normal with supplementation. Nevertheless, calculated influx remained less than 70% of normal at 50% of the time points. The calculated brain influxes of all other large neutral amino acids except tryptophan were 20% to 40% of normal before and with tyrosine supplementation. CONCLUSIONS: Tyrosine supplementation in the diet for PKU produces marked but nonsustained increases in plasma tyrosine levels, with calculated brain influx that often remains suboptimal. This could explain the lack of consistent neuropsychologic benefit with tyrosine supplementation.

Urinary lactate excretion to monitor the efficacy of treatment of type I glycogen storage disease.

The purpose of this study was to investigate the usefulness of urinary lactate measurements to assess the adequacy of dietary treatment in patients with type I glycogen storage disease (GSD-I). We determined the correlation of urine and blood lactate concentrations in 21 GSD-I patients during 24-h admissions to the General Clinical Research Center (GCRC) during which hourly blood samples and aliquots of every void were obtained. In all but 1 patient, we found a good correlation between blood lactate concentrations and urinary lactate excretion. One patient did not excrete lactate in significant amounts despite elevated blood lactate concentrations. In 17 patients, the highest blood lactate concentrations occurred during the night. Markedly elevated nighttime average blood lactate concentrations above 3.5 mmol/l resulted in a urinary lactate concentration above the normal limit of 0.067 mmol/mmol creatinine in the first morning urine specimen. Mildly elevated nighttime blood lactate concentrations (between 2.2 and 3.5 mmol/l) led to urinary lactate concentrations that were either normal or moderately elevated. All patients with normal blood lactate concentrations during the night also had normal first morning urinary lactate concentrations. The degree of urinary lactate excretion in relation to blood lactate concentrations varied by individual. Urinary filter paper specimens, collected at home during the night and in the morning and mailed to the laboratory, were used to monitor the dietary compliance of 5 GSD-I patients at home over a period of 6 to 9 weeks prior to their GCRC admissions. These data suggested variable degrees of dietary control. In conclusion, the urinary lactate concentration is a useful parameter to monitor therapy of GSD-I patients at home. To be interpretable, the baseline urinary lactate concentration in relation to the blood lactate concentration has to be determined.

Quality of life assessment in adults with type 1 Gaucher disease.

The effect of enzyme replacement therapy on health-related quality of life in 25 adults with type 1 Gaucher disease was investigated over a 2-year period. Quality of life was assessed using the SF-36 Health Survey (SF-36). Psychological functioning was assessed using the Symptom Checklist--90R. The results indicated significant improvement in 7 of 8 SF scale scores beginning at 18 months of therapy (P < 0.05 to 0.001). The SF scale showing improvement first was Vitality (energy level and fatigue) at 6 months of therapy (P < 0.01). The SF-36 scales showing the largest improvements were Role-Physical and Social Functioning (P < 0.001). Compared to the general US adult population, the study population's health profile was significantly lower prior to starting therapy but by 24 months of therapy there were no differences between the two. No differences were found in psychological functioning compared to a US adult normative group at the start of therapy. However, within the study population there was significant improvement in mood and global functioning and fewer psychological symptoms reported at 24 months of therapy. The findings indicate that enzyme replacement therapy for type 1 Gaucher disease has a positive impact on health-related quality of life from the patient's perspective.

A GC/MS/MS screening method for multiple organic acidemias from urine specimens.

A gas chromatography tandem mass spectrometry method using an ion trap GC/MS system was developed to quickly screen urine samples for 14 organic acids associated with multiple organic acidemias. The following organic acids are used as diagnostic markers: methylmalonic acid, glutaric acid, 2-ketoisocaproic acid, succinylacetone, 3-methylcrotonylglycine, tiglylglycine, isovalerylglycine, fumaric acid, butyrylglycine, propionylglycine, hexanoylglycine, adipic acid, suberic acid, and sebacic acid. 2-ketocaproic acid is used as an internal standard. The samples are prepared using a solid-phase extraction and converted to trimethylsilyl derivatives. The extraction efficiency for the 14 compounds is between 57 and 106%. A derivatized standard mixture of the 14 markers is run prior to the patient samples to determine the accurate absolute and relative retention times. The samples are then injected and the product ion spectra monitored. For data analysis, one characteristic product ion plot is extracted for each of the 14 marker compounds, and the presence of a peak with the expected retention time is determined. The areas of the product ion peaks are compared with the reference range determined from 30 normal controls. Ten samples of patients with known organic acidemias were measured. For all patients, diagnostic peaks at the expected retention times of at least five times the upper limit of the reference range were detected. The method, with its relatively fast sample preparation, short 10.0 min run time and simple data analysis, is suitable for use as a quick metabolic screen of very sick patients in whom there is concern regarding the possibility of a treatable inborn error.

Quantification of glutaric acid by isotope dilution mass spectrometry for patients with glutaric acidemia type I: selected ion monitoring vs. selected ion storage.

An isotope dilution mass spectrometric assay for the quantification of glutaric acid in urine and serum samples was developed. The performance of a quadrupole mass filter (QMF) gas chromatography-mass spectrometry (GC-MS) instrument, operated in the selected ion monitoring mode, and a quadrupole ion trap (QIT) GC/MS instrument, operated in the selected ion storage mode, was compared. Both instruments gave linear standard curves with glutaric acid concentrations between 0.19 and 3.8 microM. The average coefficients of correlation were 0.9998 and 0.9993 for the QMF and the QIT system, respectively. There was good agreement between the glutaric acid concentrations measured with the two instruments. The run-to-run precision was between 1.2 and 3.7% and between 6.2 and 8.6%, the average recovery of glutaric acid in urine and serum samples was 96 and 103% with the QMF and QIT instrument, respectively. We conclude that although the QMF has a slightly better performance, both instruments can be used to reliably measure glutaric acid concentrations from urine and serum patient samples.

Protein status of infants with phenylketonuria undergoing nutrition management.

OBJECTIVES: The objectives of this study were to determine if Phenex-1, amino-acid modified medical food with iron maintained normal indices of protein status in infants with phenylketonuria (PKU) and to investigate factors that influence plasma amino acid concentrations. METHODS: A study was conducted for six months in 35 infants with classical PKU diagnosed in the neonatal period. Diet diaries and plasma amino acid concentrations were obtained monthly. Blood for analysis of plasma albumin, blood urea nitrogen (BUN), retinol binding protein (RBP) and transthyretin was obtained at one, three and six months of study. RESULTS: Mean (+/-SEM) total daily intake of medical food and nutrients was 79+/-4 g; 17.3+/-0.6 g protein, 660+/-18 kcal, 255+/-10 mg phenylalanine (Phe), and 1423+/-56 mg tyrosine (Tyr). Mean concentrations of plasma amino acids, except cystine (during entire study), glycine (first month) and Phe were in the normal range. Mean concentrations of plasma Phe were in the treatment range (120 to 360 micromol/L). Plasma concentrations of arginine, methionine, Phe, tryptophan, Tyr, and valine were positively correlated with intakes at various months of study. Concentrations of aspartic and glutamic acids, Phe, and Tyr were positively correlated and 17 amino acids were negatively correlated with the interval between feeding and blood draw. At six months of study, concentration of plasma albumin was 4.1+/-0.1 g/dL, RBP was 3.74+/-0.2 mg/dL, transthyretin was 17.9+/-0.9 mg/dL, and urea nitrogen was 11.9+/-0.5 mg/dL. CONCLUSION: During study, all mean plasma indices of protein status were in normal reference ranges. Phenex-1 supports normal mean plasma amino acid, albumin, RBP, transthyretin, and BUN concentrations when fed in adequate amounts.

Reversal of severe hypertrophic cardiomyopathy and excellent neuropsychologic outcome in very-long-chain acyl-coenzyme A dehydrogenase deficiency.

Very-long-chain acyl-coenzyme A dehydrogenase (VLCAD) deficiency is a disorder of fatty acid beta oxidation that reportedly has high rates of morbidity and mortality. We describe the outcome of a 5-year-old girl with VLCAD deficiency who was first seen at 5 months of age with severe hypertrophic cardiomyopathy, hepatomegaly, encephalopathy, and hypotonia. Biochemical studies indicated VLCAD deficiency caused by a stable yet inactive enzyme. Molecular genetic analysis of her VLCAD gene revealed a T1372C (F458L) missense mutation and a 1668 ACAG 1669 splice site mutation. After initial treatment with intravenous glucose and carnitine, the patient has thrived on a low-fat diet supplemented with medium-chain triglyceride oil and carnitine and avoidance of fasting. Her ventricular hypertrophy resolved significantly over 1 year, and cognitively, she is in the superior range for age. Clinical recognition of VLCAD deficiency is important because it is one of the few directly treatable causes of cardiomyopathy in children.

Infantile phosphofructokinase deficiency with arthrogryposis: clinical benefit of a ketogenic diet.

We report a 2-year-old boy with phosphofructokinase deficiency presenting in the newborn period with congenital arthrogryposis and severe myopathy, who has had significant improvement on a ketogenic diet since its institution at 4 months of age. We provide a rationale for use of this treatment and hypothesize it may be beneficial in other patients with phosphofructokinase deficiency and progressive muscular involvement. Confirmation awaits further clinical trials in carefully selected patients.

Cardiac mitochondrial dysfunction in Leigh syndrome.

An infant with Leigh syndrome and associated cardiomyopathy is described. Abnormal activities of mitochondrial respiratory complexes III and V and a change in mtDNA at nt 8993 were detected in heart and skeletal muscle but not in liver.

Clinical approach to genetic cardiomyopathy in children.

BACKGROUND: Cardiomyopathy (CM) remains one of the leading cardiac causes of death in children, although in the majority of cases, the cause is unknown. To have an impact on morbidity and mortality, attention must shift to etiology-specific treatments. The diagnostic evaluation of children with CM of genetic origin is complicated by the large number of rare genetic causes, the broad range of clinical presentations, and the array of specialized diagnostic tests and biochemical assays. METHODS AND RESULTS: We present a multidisciplinary diagnostic approach to pediatric CM of genetic etiology. We specify criteria for abnormal left ventricular systolic performance and structure that suggest CM based on established normal echocardiographic measurements and list other indications to consider an evaluation for CM. We provide a differential diagnosis of genetic conditions associated with CM, classified as inborn errors of metabolism, malformation syndromes, neuromuscular diseases, and familial isolated CM disorders. A diagnostic strategy is offered that is based on the clinical presentation: biochemical abnormalities, encephalopathy, dysmorphic features or multiple malformations, neuromuscular disease, apparently isolated CM, and pathological specimen findings. Adjunctive treatment measures are recommended for severely ill patients in whom a metabolic cause of CM is suspected. A protocol is provided for the evaluation of moribund patients. CONCLUSIONS: In summary, we hope to assist pediatric cardiologists and other subspecialists in the evaluation of children with CM for a possible genetic cause using a presentation-based approach. This should increase the percentage of children with CM for whom a diagnosis can be established, with important implications for treatment, prognosis, and genetic counseling.

Oxidative phosphorylation defect associated with primary adrenal insufficiency.

An 18-month-old girl with an oxidative phosphorylation defect had neonatal onset of chronic lactic acidosis, lipid storage myopathy, bilateral cataracts, and primary adrenal insufficiency. Chronic lactic acidosis responded to treatment with dichloroacetate. Sequential muscle biopsies demonstrated resolution of the lipid storage myopathy associated with the return to normal muscle free carnitine levels. This case demonstrates a new clinical phenotype associated with a defect in oxidative phosphorylation and the need to consider mitochondrial disorders in the differential diagnosis of primary adrenal insufficiency in childhood.

Infantile onset of hereditary ascending spastic paralysis with bulbar involvement.

Primary lateral sclerosis and hereditary spastic paraparesis are both rare neurodegenerative disorders characterized by progressive weakness and spasticity of the lower limbs, with involvement of the corticospinal tracts and sparing of anterior horn cells. We describe a consanguineous family in which three sons developed progressive paralysis of the lower extremities in infancy with subsequent involvement of the upper extremities and bulbar muscles but cognitive sparing. This family presents the nosologic difficulty of distinguishing between hereditary spastic paraparesis and primary lateral sclerosis. We suggest that the diagnosis in this family is hereditary primary lateral sclerosis. This is the first instance of familial occurrence of primary lateral sclerosis.

Bilateral infantile cataractogenesis in a patient with deficiency of complex I, a mitochondrial electron transport chain enzyme.

Progressive bilateral cataracts developed in infancy in a 5-month-old girl with deficiency of complex I, a mitochondrial electron transport chain enzyme. In the newborn period, she had severe lactic acidosis and the diagnosis of complex I deficiency was confirmed by mitochondrial respiratory chain assay on muscle biopsy. By 5 months, she had completely opaque nuclear sclerotic cataracts, with loss of fixation and the red reflex. She underwent bilateral, sequential cataract extraction. The lens aspirate was submitted for cytologic analysis and electron microscopy, which revealed increased intracellular glycogen and swollen mitochondria. To our knowledge the association of complex I deficiency with cataracts in infancy has not been reported previously. The diagnosis of a respiratory chain enzyme defect in infancy is an indication for early ophthalmic evaluation to identify cataracts that may result in visual loss. Conversely, the recognition of cataracts in infants with unexplained neurologic disease or metabolic acidosis may necessitate further evaluation for metabolic etiologies, including mitochondrial disorders.