Glutamine and hyperammonemic crises in patients with urea cycle disorders.

UNLABELLED: Blood ammonia and glutamine levels are used as biomarkers of control in patients with urea cycle disorders (UCDs). This study was undertaken to evaluate glutamine variability and utility as a predictor of hyperammonemic crises (HACs) in UCD patients. METHODS: The relationships between glutamine and ammonia levels and the incidence and timing of HACs were evaluated in over 100 adult and pediatric UCD patients who participated in clinical trials of glycerol phenylbutyrate. RESULTS: The median (range) intra-subject 24-hour coefficient of variation for glutamine was 15% (8-29%) as compared with 56% (28%-154%) for ammonia, and the correlation coefficient between glutamine and concurrent ammonia levels varied from 0.17 to 0.29. Patients with baseline (fasting) glutamine values >900 µmol/L had higher baseline ammonia levels (mean [SD]: 39.6 [26.2]µmol/L) than patients with baseline glutamine ≤ 900 µmol/L (26.6 [18.0]µmol/L). Glutamine values >900 µmol/L during the study were associated with an approximately 2-fold higher HAC risk (odds ratio [OR]=1.98; p=0.173). However, glutamine lost predictive significance (OR=1.47; p=0.439) when concomitant ammonia was taken into account, whereas the predictive value of baseline ammonia ≥ 1.0 upper limit of normal (ULN) was highly statistically significant (OR=4.96; p=0.013). There was no significant effect of glutamine >900 µmol/L on time to first HAC crisis (hazard ratio [HR]=1.14; p=0.813), but there was a significant effect of baseline ammonia ≥ 1.0 ULN (HR=4.62; p=0.0011). CONCLUSIONS: The findings in this UCD population suggest that glutamine is a weaker predictor of HACs than ammonia and that the utility of the predictive value of glutamine will need to take into account concurrent ammonia levels.

Validation of the finding of hypertrophy of the clava in infantile neuroaxonal dystrophy/PLA2G6 by biometric analysis.

INTRODUCTION: Infantile neuroaxonal dystrophy (INAD), an autosomal recessive neurodegenerative disorder due to PLA2G6 mutation, is classified both as a PLA2G6-associated neurodegeneration (PLAN) disorder and as one of the neurodegeneration with brain iron accumulation (NBIA) disorders. Age of onset and clinical presentation in INAD is variable. Typically described imaging features of cerebellar atrophy, cerebellar cortex bright FLAIR signal, and globus pallidus iron deposition are variable or late findings. We characterize clinical and neuroimaging phenotypes in nine children with confirmed PLA2G6 mutations and show a useful imaging feature, clava hypertrophy, which may aid in earlier identification of patients. Measurements of the clava confirm actual enlargement, rather than apparent enlargement due to volume loss of the other brain stem structures. METHODS: A retrospective clinical and MRI review was performed. Brain stem measurements were performed and compared with age-matched controls. RESULTS: We identified nine patients, all with novel PLA2G6 gene mutations. MRI, available in eight, showed clava hypertrophy, regardless of age or the absence of other more typically described neuroimaging findings. Brain autopsy in our cohort confirmed prominent spheroid bodies in the clava nuclei. CONCLUSION: Clava hypertrophy is an important early imaging feature which may aid in indentification of children who would benefit from specific testing for PLA2G6 mutations.

A randomized, placebo-controlled, double-blind study of sapropterin to treat ADHD symptoms and executive function impairment in children and adults with sapropterin-responsive phenylketonuria.

Symptoms of attention deficit-hyperactivity disorder (ADHD), particularly inattention, and impairments in executive functioning have been reported in early and continuously treated children, adolescents, and adults with phenylketonuria (PKU). In addition, higher blood phenylalanine (Phe) levels have been correlated with the presence of ADHD symptoms and executive functioning impairment. The placebo-controlled PKU ASCEND study evaluated the effects of sapropterin therapy on PKU-associated symptoms of ADHD and executive and global functioning in individuals who had a therapeutic blood Phe response to sapropterin therapy. The presence of ADHD inattentive symptoms and executive functioning deficits was confirmed in this large cohort of 206 children and adults with PKU, of whom 118 responded to sapropterin therapy. In the 38 individuals with sapropterin-responsive PKU and ADHD symptoms at baseline, sapropterin therapy resulted in a significant improvement in ADHD inattentive symptoms in the first 4 weeks of treatment, and improvements were maintained throughout the 26 weeks of treatment. Sapropterin was well-tolerated with a favorable safety profile. The improvements in ADHD inattentive symptoms and aspects of executive functioning in response to sapropterin therapy noted in a large cohort of individuals with PKU indicate that these symptoms are potentially reversible when blood Phe levels are reduced.

Elevated phenylacetic acid levels do not correlate with adverse events in patients with urea cycle disorders or hepatic encephalopathy and can be predicted based on the plasma PAA to PAGN ratio.

BACKGROUND: Phenylacetic acid (PAA) is the active moiety in sodium phenylbutyrate (NaPBA) and glycerol phenylbutyrate (GPB, HPN-100). Both are approved for treatment of urea cycle disorders (UCDs) - rare genetic disorders characterized by hyperammonemia. PAA is conjugated with glutamine in the liver to form phenylacetyleglutamine (PAGN), which is excreted in urine. PAA plasma levels ≥ 500 µg/dL have been reported to be associated with reversible neurological adverse events (AEs) in cancer patients receiving PAA intravenously. Therefore, we have investigated the relationship between PAA levels and neurological AEs in patients treated with these PAA pro-drugs as well as approaches to identifying patients most likely to experience high PAA levels. METHODS: The relationship between nervous system AEs, PAA levels and the ratio of plasma PAA to PAGN were examined in 4683 blood samples taken serially from: [1] healthy adults [2], UCD patients of ≥ 2 months of age, and [3] patients with cirrhosis and hepatic encephalopathy (HE). The plasma ratio of PAA to PAGN was analyzed with respect to its utility in identifying patients at risk of high PAA values. RESULTS: Only 0.2% (11) of 4683 samples exceeded 500 µg/ml. There was no relationship between neurological AEs and PAA levels in UCD or HE patients, but transient AEs including headache and nausea that correlated with PAA levels were observed in healthy adults. Irrespective of population, a curvilinear relationship was observed between PAA levels and the plasma PAA:PAGN ratio, and a ratio>2.5 (both in µg/mL) in a random blood draw identified patients at risk for PAA levels>500 µg/ml. CONCLUSIONS: The presence of a relationship between PAA levels and reversible AEs in healthy adults but not in UCD or HE patients may reflect intrinsic differences among the populations and/or metabolic adaptation with continued dosing. The plasma PAA:PAGN ratio is a functional measure of the rate of PAA metabolism and represents a useful dosing biomarker.

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.

Early-onset LBSL: how severe does it get?

AIM: Leukoencephalopathy with brainstem and spinal cord involvement and lactate elevation (LBSL) is known as a relatively mild leukoencephalopathy. We investigated the occurrence of severe variants of LBSL with extensive brain magnetic resonance imaging (MRI) abnormalities. METHOD: MRIs of approximately 3,000 patients with an unknown leukoencephalopathy were retrospectively reviewed for extensive signal abnormalities of the cerebral and cerebellar white matter, posterior limb of the internal capsule, cerebellar peduncles, pyramids, and medial lemniscus. Clinical data were retrospectively collected. RESULTS: Eleven patients fulfilled the MRI criteria (six males); six had DARS2 mutations. Clinical and laboratory findings did not distinguish between patients with and without DARS2 mutations, but MRI did. Patients with DARS2 mutations more often had involvement of structures typically affected in LBSL, including decussatio of the medial lemniscus, anterior spinocerebellar tracts, and superior and inferior cerebellar peduncles. Also, involvement of the globus pallidus was associated with DARS2 mutations. Earliest disease onset was neonatal; earliest death at 20 months. INTERPRETATION: This study confirms the occurrence of early infantile, severe LBSL, extending the known phenotypic range of LBSL. Abnormality of specific brainstem tracts and cerebellar peduncles are MRI findings that point to the correct diagnosis.

Ammonia control in children with urea cycle disorders (UCDs); phase 2 comparison of sodium phenylbutyrate and glycerol phenylbutyrate.

UNLABELLED: Twenty four hour ammonia profiles and correlates of drug effect were examined in a phase 2 comparison of sodium phenylbutyrate (NaPBA) and glycerol phenylbutyrate (GPB or HPN-100), an investigational drug being developed for urea cycle disorders (UCDs). STUDY DESIGN: Protocol HPN-100-005 involved open label fixed-sequence switch-over from the prescribed NaPBA dose to a PBA-equimolar GPB dose with controlled diet. After 7 days on NaPBA or GPB, subjects underwent 24-hour blood sampling for ammonia and drug metabolite levels as well as measurement of 24-hour urinary phenyacetylglutamine (PAGN). Adverse events (AEs), safety labs and triplicate ECGs were monitored. RESULTS: Eleven subjects (9 OTC, 1 ASS, 1 ASL) enrolled and completed the switch-over from NaPBA (mean dose=12.4 g/d or 322 mg/kg/d; range=198-476 mg/kg/d) to GPB (mean dose=10.8 mL or 0.284 mL/kg/d or 313 mg/kg/d; range=192-449 mg/kg/d). Possibly-related AEs were reported in 2 subjects on NaPBA and 4 subjects on GPB. All were mild, except for one moderate AE of vomiting on GPB related to an intercurrent illness. No clinically significant laboratory or ECG changes were observed. Ammonia was lowest after overnight fast, peaked postprandially in the afternoon to early evening and varied widely over 24h with occasional values >100 µmol/L without symptoms. Ammonia values were ~25% lower on GPB vs. NaPBA (p≥0.1 for ITT and p<0.05 for per protocol population). The upper 95% confidence interval for the difference between ammonia on GPB vs. NaPBA in the ITT population (95% CI 0.575, 1.061; p=0.102) was less than the predefined non-inferiority margin of 1.25 and less than 1.0 in the pre-defined per-protocol population (95% CI 0.516, 0.958; p<0.05). No statistically significant differences were observed in plasma phenylacetic acid and PAGN exposure during dosing with GPB vs. NaPBA, and the percentage of orally administered PBA excreted as PAGN (66% for GPB vs. 69% for NaPBA) was very similar. GPB and NaPBA dose correlated best with urinary-PAGN. CONCLUSIONS: These findings suggest that GPB is at least equivalent to NaPBA in terms of ammonia control, has potential utility in pediatric UCD patients and that U-PAGN is a clinically useful biomarker for dose selection and monitoring.

Pyruvate dehydrogenase phosphatase 1 (PDP1) null mutation produces a lethal infantile phenotype.

Pyruvate dehydrogenase phosphatase deficiency has previously only been confirmed at the molecular level in two brothers and two breeds of dog with exercise intolerance. A female patient, who died at 6 months, presented with lactic acidemia in the neonatal period with serum lactate levels ranging from 2.5 to 17 mM. Failure of dichloroacetate to activate the PDH complex in skin fibroblasts was evident, but not in early passages. A homozygous c.277G > T (p.E93X) nonsense mutation in the PDP1 gene was identified in genomic DNA and immunoblotting showed a complete absence of PDP1 protein in mitochondria. Native PDHC activity could be restored by the addition of either recombinant PDP1 or PDP2. This highlights the role of PDP2, the second phosphatase isoform, in PDP1-deficient patients for the first time. We conclude that the severity of the clinical course associated with PDP1 deficiency can be quite variable depending on the exact nature of the molecular defect.

Diurnal rhythm: effects on hepatic regeneration and hepatic regenerative stimulator substance.

The effect of a controlled lighting schedule on the activity of a weanling rat liver extract that stimulates DNA synthesis in regenerating adult rat liver, and on the response of the test animals to the extract, has been investigated. Both activity of the extract and endogenous DNA synthesis in the weanling animals follow the same distinct diurnal rhythm. Reversal of the lighting schedule reverses the rhythm of endogenous DNA synthesis but activity of the extract no longer correlates with the peak of DNA synthesis. Diurnal rhythm also has a striking effect on DNA synthesis in the regenerating test animal, but the extract increases DNA synthesis to the same relative degree, regardless of the time of day the hepatectomy is performed.

A novel mitochondrial DNA mutation in COX1 leads to strokes, seizures, and lactic acidosis.

Cytochrome c oxidase (COX) is the terminal enzyme of the respiratory chain, with subunits originating both from the mitochondrial and nuclear genome. An eleven-year-old female presented initially with a seizure followed two months later with tonic-clonic seizures, weakness and aphasia. MRI of the cerebral hemispheres showed multiple infarcts. Previous history suggested gross and fine motor control deficits with learning difficulties. A muscle biopsy showed a specific decrease of COX staining in all fibres and pleomorphic mitochondria. Respiratory chain studies confirmed an isolated complex IV defect in muscle, whilst fibroblasts showed an initial COX activity below normal which rapidly came up to the normal range on culture. Sequencing of mtDNA revealed an heteroplasmic m.7023G>A mutation in the COX1 gene, with levels of 96% in muscle, 70% in blood and 50% in the initial skin fibroblast culture dropping to 10% in later passages. The mutation was present in a critical region of the COX1 gene, the V374M change being close to the two histidine residues His376 and His378 co-ordinating with the heme a and a (3), and His367 which co-ordinates a magnesium ion. This case highlights that a MELAS-like syndrome can occur with isolated COX deficiency.

Craniosynostosis associated with partial duplication of 15q and deletion of 2q.

We report on an infant with multiple congenital anomalies including complex craniosynostosis associated with an unbalanced karyotype, 46,XY,-2,+der(2),t(2;15)(q37;q26)pat. The previous report of a child with cloverleaf skull and partial duplication of 15q25----qter and the Man-on-Mouse Homology map suggests that a critical segment for synostosis of sutures may be in this region.

DHCR7 genotypes of cousins with Smith-Lemli-Opitz syndrome.

Smith-Lemli-Opitz syndrome (SLOS) is an autosomal recessive disorder of cholesterol biosynthesis caused by mutations of the 7-dehydrocholesterol reductase gene (DHCR7). We report on three cousins with SLOS, all of whom were found to be compound heterozygotes for the common splice site mutation IVS8-1G-->C and the missense mutation T289I. DNA analysis of one set of parents demonstrated that the father carried the missense mutation and the mother carried the IVS8-1G-->C mutation. By extension, the two unrelated mothers were both heterozygous for IVS8-1G-->C. This finding supports the notion of a high carrier frequency of the IVS8-1G-->C null mutation in Northern European Caucasians.

Premature atherosclerosis with photomyoclonic epilepsy, deafness, diabetes mellitus, nephropathy, and neurodegenerative disorder in two brothers: a new syndrome?

We describe two brothers with sensorineural deafness, diabetes mellitus, progressive neurological deterioration with photomyoclonic epilepsy, and progressive deterioration in renal function, resulting in death in the third decade of life. Autopsy showed diffuse atherosclerosis and arteriolosclerosis of the systemic vasculature. There was no evidence of these abnormalities in the patients' 2 sisters or either parent. Mitochondrial enzyme analysis documented partial deficiencies of Complex III and IV of the respiratory chain. This deficiency was expressed in skin fibroblasts, kidney and liver but not in muscle. This suggests that the disease-causing mutation is either in the mitochondrial or nuclear DNA. Various modes of inheritance are considered, including maternal, autosomal recessive, or X-linked recessive. We suggest this is a new genetic syndrome characterized by an underlying metabolic disease and premature atherosclerosis, possibly of mitochondrial origin.

The expanding clinical phenotype of the tRNA(Leu(UUR)) A-->G mutation at np 3243 of mitochondrial DNA: diabetic embryopathy associated with mitochondrial cytopathy.

We describe a family which demonstrates and expands the extreme clinical variability now known to be associated with the A-->G transition at nucleotide position 3243 of the mitochondrial DNA. The propositus presented at birth with clinical manifestations consistent with diabetic embryopathy including anal atresia, caudal dysgenesis, and multicystic dysplastic kidneys. His co-twin was normal at birth, but at 3 months of life, presented with intractable seizures later associated with developmental delay. The twins' mother developed diabetes mellitus type I at the age of 20 years and gastrointestinal problems at 22 years. Since age 19 years, the maternal aunt has had recurrent strokes, seizures, mental deterioration and deafness, later diagnosed as MELAS syndrome due to the tRNA(Leu(UUR)) A-->G mutation. A maternal uncle had diabetes mellitus type I, deafness, and normal intellect, and died at 35 years after recurrent strokes. This pedigree expands the known clinical phenotype associated with tRNA(Leu(UUR)) A-->G mutation and raises the possibility that, in some cases, diabetic embryopathy may be due to a mitochondrial cytopathy that affects both the mother's pancreas (and results in diabetes mellitus and the metabolic dysfunction associated with it) and the embryonic/fetal and placental tissues which make the embryo more vulnerable to this insult.

Molecular basis of lipoamide dehydrogenase deficiency in Ashkenazi Jews.

We studied 13 patients with lipoamide dehydrogenase (LAD) deficiency, originating from seven Ashkenazi Jewish families. Their disease was characterized by recurrent attacks of vomiting, abdominal pain, and encephalopathy accompanied by elevated liver transaminases, prolonged prothrombin time, and occasionally associated with lactic and ketoacidemia or with myoglobinuria. Two patients who presented neonatally suffered from residual neurological damage with attention deficit hyperactive disorder, mild ataxia, motor incoordination, muscle hypotonia, and weakness. Nine patients who presented in early childhood or later suffered from exertional fatigue between decompensation episodes but were otherwise asymptomatic. Two patients died because of intractable metabolic acidosis and multi-organ failure. In all patients LAD activity was reduced to 8 to 21% of the control in muscle or lymphocytes. In four patients LAD protein in muscle was reduced to 20 to 60% of the control. Direct sequencing of the cDNA of the LAD gene showed that 12 of the 14 mutated alleles carried the G229C mutation and two carried an insertion mutation 105insA (Y35X). The patients who presented neonatally and had more severe sequelae were compound heterozygotes for the two mutations; patients who presented in early childhood or later were homozygous for the G229C mutation. Using an allele-specific oligonucleotide hybridization technique, nine heterozygotes for the G229C mutation were identified among 845 anonymous individuals of Ashkenazi Jewish origin disclosing a carrier rate of 1:94. Because of the significant morbidity associated with the disease, screening for the G229C mutation among Ashkenazi Jewish couples should be considered.

Early-infantile galactosialidosis: prenatal presentation and postnatal follow-up.

Galactosialidosis (GS) is an autosomal recessive condition caused by combined deficiency of the lysosomal enzymes beta-galactosidase and alpha-neuraminidase. The combined deficiency has been found to result from a defect in protective protein/cathepsin A (PPCA), an intralysosomal protein which protects these enzymes from premature proteolytic processing. The most severe form of GS, the early-infantile form, results in early onset of edema, ascites, visceromegaly, and skeletal dysplasia. We report a case of early-infantile GS in a male infant who presented with nonimmune fetal hydrops (NIH), "coarse" facial appearance, massive fluid-filled inguinal hernias, multiple telangiectasia, and diffuse hypopigmentation; he subsequently developed visceromegaly. The diagnosis of GS was confirmed biochemically and the defect in PPCA characterized at the protein level. Examination of fetal peripheral blood smears sampled at 30 weeks gestation demonstrated vacuolation of lymphocytes, suggesting blood film examination may be a useful screening tool for cases of NIH where a metabolic disorder is suspected. Skeletal radiography at birth demonstrated punctate epiphyses of the femora, calcanei, and sacrum. We present a discussion of and differential diagnosis for this radiographic finding. To the best of our knowledge, this is the first case of early-infantile GS presenting with stippled epiphyses.

Evaluation and counseling of teratogenic risk: the motherisk approach.

The authors present a new approach for evaluation and counseling of teratogenic risk, The Motherisk program in Toronto presently deals with 60 inquiries per day from the public and health professionals. Most calls are answered through the telephone, and 16-20 women per week are scheduled for our clinic following exposure to known or suspected teratogens, to new drugs with sparse information, to chronic drug therapy, or drugs of abuse. Also seen in clinic are women who want to be counseled or whose physicians want them to meet us due to high levels of anxiety. Since its inception in 1985 the Program has been very effective in preventing unnecessary terminations of otherwise wanted pregnancies. In addition to the clinical service we followup the outcome of these prospectively collected exposures, thus creating a large data base necessary to verify safety/risk of various agents. During the last 2 years Motherisk has completed singly, or in collaboration, large prospective studies on gestational exposure to lithium, antiepileptics, cocaine, and fluexitime. To study the reproductive risks of cocaine we compared first-trimester exposure to cocaine with two control groups. Infants exposed to cocaine did not have higher rates of any adverse perinatal outcome. Their cognitive function at 18 months of age was identical to the control infants. Meta analysis of all studies assessing gestational risks of cocaine reveals that the results of these studies were dependent on the type of comparison conducted. When babies exposed to cocaine were compared with middle class nonusers, the cocaine groups seemed to be more often different from their controls.(ABSTRACT TRUNCATED AT 250 WORDS)

The molecular basis of Tay-Sachs disease: mutation identification and diagnosis.

Tay-Sachs disease is the prototype of lysosomal storage disease. While it was first described over a century ago, the defective enzyme was not identified until 1969, making possible the development of enzyme-based diagnostic and carrier screening techniques. This led to the establishment of the successful international Tay-Sachs screening program, primarily for the high risk Ashkenazi Jewish population. In the past five years the development of recombinant DNA technology has allowed researchers to characterize 95-99% of the mutations causing Tay-Sachs disease in this high risk ethnic group. Knowledge of the exact mutations responsible for the disease coupled with the powerful polymerase chain reaction technique has now made DNA-based screening and diagnosis possible. While the enzyme-based test has proven to be reliable and economical, it cannot differentiate variant phenotypes and requires the presence of specialized testing centers. Although the DNA-based test is presently less economical, it can provide carrier couples with their exact genotype and thus, predict the general phenotype of an unborn child. Furthermore, as the catalogue of mutations leading to human disease increases, more economical DNA methodologies will be developed. In the future it would be expected that a laboratory using a single DNA-based technology could diagnose and screen for a myriad of human diseases including Tay-Sachs disease.

Newborn phenylketonuria (PKU) Guthrie (BIA) screening and early hospital discharge.

Recent policies of early discharge of postpartum mothers and their infants has raised concerns of possible decreased sensitivity in Guthrie bacterial inhibition assay (BIA) phenylketonuria (PKU) screening resulting in missed cases. In order to assess the potential impact of early discharge from hospital on neonatal screening for PKU and its variants, we performed 18 standard BIA screening tests on 11 newborn infants with the disease. Blood spot samples were collected from 1 to 24 h after birth and were analyzed at the Ontario Ministry of Health newborn screening laboratory according to the routine screening protocol. Except for one 4-hour postnatal sample from an infant with 'non-PKU mild hyperphenylalaninemia' (MHP) all blood samples showed phenylalanine levels > or = 240 mumol/l, irrespective of the age of the baby. During our 29 year experience with neonatal PKU screening (3.9 million infants tested), employing a cutoff blood phenylalanine of 240 mumol/l in blood spots obtained at > or = 24 h of age, only two biological false negative (one confirmed) tests were discovered in infants subsequently shown to have classical PKU: another three false negative tests were discovered in sibs of infants with MHP. The sensitivity of the screening test was 99.2% for infants with classical and mild PKU. Ascertainment of patients with MHP is unknown and is very likely incomplete. Over a 3-year period (1992-4) the specificity of the test was 99.9% for those screened after 24 h. The positive predictive value was 12.8%. Although early discharge may have an impact on other screened diseases, we conclude, from our studies, that early discharge may not affect the detection of infants with classical and mild (atypical) PKU, but would probably increase the number of infants with MHP missed using the BIA and a cutoff level of 240 mumol/l. Because of our experience and that of others, we recommend that neonates be at least 12 h of age before initial BIA PKU screening be carried out. To confirm this recommendation further prospective studies should be initiated.

Influence of packaging on the aroma stability of strawberry syrup during shelf life.

Different types of packaging (glass bottle, PVC, and PET) were compared for the preservation of aroma quality of a strawberry syrup during shelf life. Esters, alcohols, and aldehydes were analyzed by solid-phase micro-extraction (SPME) and solvent extraction. During storage, hydrolysis of esters in acids and alcohols led to a modification of the aroma profile which can be explained by the replacement of "fruity" and "fresh" notes by "dairy note" in the syrup. Aroma compounds that are responsible for fruity notes, such as methyl cinnamate, methyl anthranilate, and methyl dihydrojasmonate, were strongly reduced after 90 days. This could be explained by a selective interaction of these compounds with the polymer matrix (PET or PVC). After 330 days, a later and important decrease of the "fruity notes" occurred in both PETs; so PVC2 and the glass bottle were found to be able to maintain a balanced aroma for long-term storage.