Free carnitine concentrations and biochemical parameters in medium-chain acyl-CoA dehydrogenase deficiency: Genotype-phenotype correlation.

Biallelic variants in the ACADM gene cause medium-chain acyl-CoA dehydrogenase deficiency (MCADD). This study reports on differences in the occurrence of secondary free carnitine (C0) deficiency and different biochemical phenotypes related to genotype and age in 109 MCADD patients followed-up at a single tertiary care center during 22 years. C0 deficiency occurred earlier and more frequently in c.985A>G homozygotes (genotype A) compared to c.985A>G compound heterozygotes (genotype B) and individuals carrying variants other than c.985A>G and c.199C>T (genotype D) (median age 4.2 vs. 6.6 years; p < 0.001). No patient carrying c.199C>T (genotype C) developed C0 deficiency. A daily dosage of 20-40 mg/kg carnitine was sufficient to maintain normal C0 concentrations. Compared to genotype A as reference group, octanoylcarnitine (C8) was significantly lower in genotypes B and C, whereas C0 was significantly higher by 8.28 µmol/L in genotype C (p < 0.05). In conclusion, C0 deficiency is mainly found in patients with pathogenic genotypes associated with high concentrations of presumably toxic acylcarnitines, while individuals carrying the variant c.199C>T are spared and show consistently mild biochemical phenotypes into adulthood. Low-dose carnitine supplementation maintains normal C0 concentrations. However, future studies need to evaluate clinical benefits on acute and chronic manifestations of MCADD.

Diagnostic Challenges Using a 2-Tier Strategy for Methylmalonic Acidurias: Data from 1.2 Million Dried Blood Spots.

BACKGROUND: The detection of methylmalonic acid (MMA) by second-tier analysis has been shown to reduce the number of false positives in newborn screening (NBS) for genetically determined methylmalonic acidurias (MMAuria). In addition to genetic conditions, MMA is an indicator of vitamin B12 status, thus applicable to detect maternal vitamin B12 deficiency in the newborns screened. METHODS: Biochemical and clinical follow-up data of a 7.5-year pilot study with 1.2 million newborns screened were reviewed. RESULTS: Among 1,195,850 NBS samples, 3,595 (0.3%) fulfilled criteria for second-tier analysis of MMA. In 37 (0.003%; 1/32,000) samples, elevated concentrations of MMA were detected, resulting in diagnostic workup at a metabolic center in 21 newborns. In 6 infants (1/199,000), genetic conditions were established, 1 infant with cobalamin C deficiency (CblC) showed only a moderate elevation of MMA. The remaining 15 newborns (1/79,000) displayed significantly lower concentrations of MMA and were evaluated for maternal vitamin B12 deficiency. In 9 mothers, vitamin B12 deficiency was verified, and 6 showed no indication for vitamin B12 deficiency. Treatment with vitamin B12 normalized biochemical parameters in all 15 infants. CONCLUSIONS: Applying a 2-tier strategy measuring MMA in NBS identified genetic conditions of MMAuria. It was possible to separate severe, early-onset phenotypes from maternal vitamin B12 deficiency. However, the detection of CblC deficiency with mildly elevated MMA interferes with impaired vitamin B12 status of unknown relevance and thus burdens possibly healthy newborns. Regarding maternal vitamin B12 deficiency, testing and supplementing mothers-to-be is preferable. This might decrease straining follow-up of newborns and improve quality and overall perception of NBS.

Novel pharmacological chaperones that correct phenylketonuria in mice.

Phenylketonuria (PKU) is caused by inherited phenylalanine-hydroxylase (PAH) deficiency and, in many genotypes, it is associated with protein misfolding. The natural cofactor of PAH, tetrahydrobiopterin (BH(4)), can act as a pharmacological chaperone (PC) that rescues enzyme function. However, BH(4) shows limited efficacy in some PKU genotypes and its chemical synthesis is very costly. Taking an integrated drug discovery approach which has not been applied to this target before, we identified alternative PCs for the treatment of PKU. Shape-focused virtual screening of the National Cancer Institute's chemical library identified 84 candidate molecules with potential to bind to the active site of PAH. An in vitro evaluation of these yielded six compounds that restored the enzymatic activity of the unstable PAHV106A variant and increased its stability in cell-based assays against proteolytic degradation. During a 3-day treatment study, two compounds (benzylhydantoin and 6-amino-5-(benzylamino)-uracil) substantially improved the in vivo Phe oxidation and blood Phe concentrations of PKU mice (Pah(enu1)). Notably, benzylhydantoin was twice as effective as tetrahydrobiopterin. In conclusion, we identified two PCs with high in vivo efficacy that may be further developed into a more effective drug treatment of PKU.

Stability of acylcarnitines and free carnitine in dried blood samples: implications for retrospective diagnosis of inborn errors of metabolism and neonatal screening for carnitine transporter deficiency.

OBJECTIVE: Electrospray ionization-tandem mass spectrometry (ESI-MS/MS) is increasingly used in newborn screening programs. Acylcarnitine profiles from dried blood spots (DBS) are used to detect fatty acid oxidation disorders, carnitine cycle disorders, and organic acidurias. Stored dried blood is also a valuable source for postmortem investigations to unravel the cause of unexplained death in early childhood. However, diagnostic uncertainties arising from the unknown stability of acylcarnitines and free carnitine during prolonged storage have not yet been studied in a systematic manner. METHODS: Whole blood spiked with acylcarnitines was stored either at -18 degrees C or at room temperature up to 1000 days. At regular time intervals 3.2 mm spots of these samples were extracted with 150 microL of methanol. Free carnitine and acylcarnitines were converted to their corresponding butyl esters and analyzed by ESI-MS/MS. RESULTS: At -18 degrees C acylcarnitines are stable for at least 330 days. If stored for prolonged periods at room temperature (>14 days), acylcarnitines are hydrolyzed to free carnitine and the corresponding fatty acids. The velocity of decay is logarithmic and depends on the chain length of the acylcarnitines. Short-chain acylcarnitines hydrolyze quicker than long-chain acylcarnitines. CONCLUSION: The data indicate that stored filter cards should only be used for retrospective quantitation of acylcarnitines if appropriate correction for sample decay during storage is applied. Free carnitine increases upon storage but can reliably be quantitated under standardized derivatization conditions. Furthermore, carnitine transporter (OCTN2) deficiency can reliably be diagnosed by examining acylcarnitine profiles, which can supplement free carnitine levels as a discriminatory marker.

Age-related reference values for serum selenium concentrations in infants and children.

BACKGROUND: Children are at particular risk for selenium deficiency, which has potentially serious medical implications. Reliable age-specific reference values for serum selenium concentrations in children are sparse, but are essential for the identification of selenium deficiency and decisions regarding selenium supplementation. METHODS: Using electrothermal atomic absorption spectrometry, we analyzed serum selenium concentrations from 1010 apparently healthy children (age range, 1 day to 18 years) and from 60 patients on a protein-restricted diet because of inborn errors of metabolism. Reference intervals were defined according to recommended guidelines. RESULTS: Medians for serum selenium concentrations showed a statistically significant age dependency: a decrease from the age <1 month (0.64 micromol/L) to 4 months (0.44 micromol/L); an increase to 0.62 micromol/L in the 4-12 months age group; constant values in children between 1 and 5 years of age (0.90 micromol/L); and an additional slight increase to reach a plateau between 5 and 18 years (0.99 micromol/L). Of 43 children older than 1 year and on a protein-restricted diet, 87% showed serum selenium concentrations below the 2.5 percentile. CONCLUSIONS: Because of nutritional changes, serum selenium concentrations are significantly higher in older children than in infants under 1 year of age. The application of age-adjusted reference values may provide more specific criteria for selenium supplementation. Long-term protein restriction in children is reflected by a failure to achieve higher serum selenium concentrations with increasing age.