LC-MS/MS-based quantification of cholesterol and related metabolites in dried blood for the screening of inborn errors of sterol metabolism.

Smith-Lemli-Opitz syndrome (SLOS) is an inherited metabolic disease in the cholesterol biosynthesis pathway which is characterised by accumulation of 7- and 8-dehydrocholesterol and by reduced cholesterol concentrations in all tissues and body fluids. With this study, we developed a new, rapid, robust and high-throughput tandem mass spectrometric method as routine application for the selective SLOS screening and therapy monitoring in serum and dried blood. After protein precipitation of 10 µL serum or 4.7 mm dried blood spot, the sum of 7- and 8-dehydrocholesterol (DHC) was analysed by rapid chromatography combined with tandem mass spectrometry. Method comparison with GC-MS was performed for 46 serum samples. A comparison between serum and corresponding dried blood spots for DHC and cholesterol was performed with 40 samples from SLOS patients. Concentrations of DHC and cholesterol were analysed in 2 dried blood samples from newborns with SLOS and 100 unaffected newborns. Intra- and inter-assay variabilities ranged between 3.7 and 17.7% for serum and dried blood spots. Significant correlations between the new LC-MS/MS method and GC-MS were determined for DHC (r = 0.937, p < 0.001) and for cholesterol (r = 0.946, p < 0.001). Significant coefficients of correlation between serum and dried blood spot samples above 0.8 were calculated for both analytes. A cut-off value of 5.95 for the ratio of DHC/cholesterol (multiplied by 1000) was found to distinguish newborns diagnosed with SLOS from normal newborns in a retrospective analysis after 5 years. The developed method enables a rapid quantification of the sum parameter 7- and 8-DHC in newborns and SLOS patients under therapy in serum as well as dried blood spot samples.

Unrestricted fruits and vegetables in the PKU diet: a 1-year follow-up.

Phenylketonuria (PKU) therapy demands phenylalanine (Phe) calculation. In most countries, almost all food is taken into account, even fruits and vegetables. We investigated whether unrestricted consumption of fruits and vegetables negatively influences metabolic control. Nineteen PKU children (2-10 years) started with 2 weeks of free or restricted fruit and vegetable intake. After 2 weeks, the regime changed from free to restricted or restricted to free (cross-over design). Over the first 4 weeks, dried blood Phe concentration was measured, fruit and vegetable consumption recorded and nutrient intake calculated from diet records. Thereafter the diet was changed to free use of fruits and vegetables for all patients. Six and 12 months later, diet and Phe concentrations were monitored. Median Phe intake increased significantly by 65 mg/day (week 4, P<0.001), 68 mg/day (month 6, P<0.001) and 70 mg/day (month 12, P<0.001). Dried blood Phe concentrations remained stable (P=0.894), as did the frequency of Phe concentrations above the recommended range (P=0.592). In conclusion, PKU diet liberalization for fruits and vegetables seems unproblematic.

PKU patients on a relaxed diet may be at risk for micronutrient deficiencies.

OBJECTIVE: To investigate micronutrient supply in phenylketonuria (PKU) patients on a relaxed diet. SUBJECTS/METHODS: Sixty-seven patients (6-45 years) with a phenylalanine tolerance ≥ 600 mg/day were included in the study. From a 3-day diet record, protein supply as well as consumption of essential amino acids and several micronutrients were assessed and compared with the current recommendations and data for the healthy population. RESULTS: Protein supply and consumption of all essential amino acids were sufficient in all patients. Supply of micronutrients depended on dietary regime. Patients with a total protein supply of 120% or more of the recommended amount and at least 0.5 g protein per kg body weight from amino-acid mixture (AAM) were sufficiently supplied with all investigated micronutrients. All patients without AAM supplement showed severe micronutrient deficiencies in their diet records. CONCLUSION: PKU patients under a relaxed diet are at risk of an insufficient nutrient supply, if they have first no substitution with AAM, second a protein supply less than 0.5 g per kg body weight from AAM or third a total protein supply less than 120% of the recommendations. Therefore, close monitoring, specific dietary counseling and potential supplementation is mandatory to prevent micronutrient deficiencies in PKU patients.

[How safe are dimenhydrinate suppositories?]. / Wie sicher sind Dimenhydrinat-Suppositorien?

HISTORY: A 13-month-old girl suffered from 3 generalized tonic-clonic seizures for several minutes within a total period of 9 hours. History revealed that the child received a total of 5 dimenhydrinate containing suppositories à 40 mg during the previous 2 days (i. e. 23 mg dimenhydrinate per kg body weight) due to enteritis with vomiting. The first seizure occurred 10 hours after the last administration. INVESTIGATIONS: The plasma level of diphenhydramin was 230 µg/l approximately one hour after the first seizure. Electroencephalography showed no pathological signs, an MRI scan of the brain was normal except of several small gliotic spots and body temperature was regularly. TREATMENT AND COURSE: Two stationary occurring seizures were stopped with 5 mg diazepam rectally. Continued surveillance and an EEG two days later showed age-appropriate normal findings. There were no further seizures in the next 4 years. CONCLUSION: Infants have the risk to develop dimenhydrinate intoxication, especially in cases where suppositories were given repeatedly because of intermittent defecation.

Nutritional Changes and Micronutrient Supply in Patients with Phenylketonuria Under Therapy with Tetrahydrobiopterin (BH(4)).

BACKGROUND: Since 2008 patients with BH(4)-sensitive phenylketonuria can be treated with sapropterin dihydrochloride (Kuvan®) in addition to the classic phenylalanine (Phe) restricted diet. The aim of this study was to evaluate the nutritional changes and micronutrient supply in patients with phenylketonuria (PKU) under therapy with tetrahydrobiopterin (BH(4)). SUBJECTS AND METHODS: 19 children with PKU (4-18 years) and potential BH(4)-sensitivity were included, 14 completed the study protocol. Dried blood Phe concentrations as well as detailed dietary records were obtained throughout the study at preassigned study days. RESULTS: Eight patients could increase their Phe tolerance from 629 ± 476 mg to 2131 ± 1084 mg (P = 0.006) under BH(4) while maintaining good metabolic control (Phe concentration in dried blood 283 ± 145 µM vs. 304 ± 136 µM, P = 1.0), therefore proving to be BH(4)-sensitive. They decreased their consumption of special low protein products and fruit while increasing their consumption of high protein foods such as processed meat, milk and dairy products. Intake of vitamin D (P = 0.016), iron (P = 0.002), calcium (P = 0.017), iodine (P = 0.005) and zinc (P = 0.046) significantly declined during BH(4) treatment while no differences in energy and macronutrient supply occurred. CONCLUSION: BH(4)-sensitive patients showed good metabolic control under markedly increased Phe consumption. However, the insufficient supply of some micronutrients needs consideration. Long-term multicenter settings with higher sample sizes are necessary to investigate the changes of nutrient intake under BH(4) therapy to further evaluate potential risks of malnutrition. Supplementation may become necessary.

Tetrahydrobiopterin (BH4) in PKU: effect on dietary treatment, metabolic control, and quality of life.

BACKGROUND: Tetrahydrobiopterin (BH(4))-sensitive phenylketonuria (PKU) can be treated with sapropterin dihydrochloride. We studied metabolic control and health-related quality of life (HRQoL) in PKU patients treated with BH(4). SUBJECTS AND METHODS: Based on the review of neonatal BH(4) test results and mutation analysis in 41 PKU patients, 19 were identified as potentially BH(4)-sensitive (9 females, 10 males, age 4-18 years). We analyzed phenylalanine (phe) concentrations in dried blood samples, nutrition protocols, and HRQoL questionnaires (KINDL(®)) beginning from 1 year before, during the first 42 days, and after 3 months of BH(4) therapy. RESULTS: Eight BH(4)-sensitive patients increased their phe tolerance (629 ± 476 vs. 2131 ± 1084 mg, p = 0.006) while maintaining good metabolic control (phe concentration in dried blood 283 ± 145 vs. 304 ± 136 µM, p = 1.0). Six of them were able to stop dietary protein restriction entirely. BH(4)-sensitive patients had average HRQoL scores that were comparable to age-matched healthy children. There was no improvement in HRQoL scores after replacing classic dietary treatment with BH(4) supply, although personal reports given by the patients and their parents suggest that available questionnaires are inappropriate to detect aspects relevant to inborn metabolic disorders. DISCUSSION: BH(4) can allow PKU patients to increase their phe consumption significantly or even stop dietary protein restrictions. Unexpectedly, this does not improve HRQoL as assessed with KINDL(®), partly due to high scores even before BH(4) therapy. Specific questionnaires should be developed for inborn metabolic disorders.

Unrestricted consumption of fruits and vegetables in phenylketonuria: no major impact on metabolic control.

BACKGROUND/OBJECTIVES: The treatment of phenylketonuria (PKU) requires consistent restriction of protein intake from natural sources. Therefore, protein from all foods has to be accounted for, even the small amounts in fruits and vegetables. We studied whether free consumption of fruits and vegetables containing less than 75 mg phenylalanine (phe) per 100 g affects metabolic control in children with PKU. SUBJECTS/METHODS: Fourteen children (2-10 years) were included in a cross-over study, with a two-week period of conventional treatment (accounting for protein from fruits and vegetables) and a two-week period with free fruit and vegetable consumption. The instruction to follow liberal fruit and vegetable consumption in the first or second study period was randomized. Detailed daily dietary records were obtained throughout the study. Phe and nutrient content was calculated. Dried-blood phe concentration was monitored daily. RESULTS: Although total phe intake increased by an average of 58 mg per day (P=0.037) during the 2 weeks of free fruit and vegetable consumption, dried-blood phe concentrations were unchanged. Total intake of fruits and vegetables did not increase, but patients instead used the higher phe tolerance to consume more of other foods, which were calculated and accounted for. CONCLUSION: Free consumption of fruits and vegetables does not impair metabolic control in PKU patients over a 2-week period.

Hallermann-Streiff Syndrome: No Evidence for a Link to Laminopathies.

Hallermann-Streiff syndrome (HSS) is a rare inherited disorder characterized by malformations of the cranium and facial bones, congenital cataracts, microphthalmia, skin atrophy, hypotrichosis, proportionate short stature, teeth abnormalities, and a typical facial appearance with prominent forehead, small pointed nose, and micrognathia. The genetic cause of this developmental disorder is presently unknown. Here we describe 8 new patients with a phenotype of HSS. Individuals with HSS present with clinical features overlapping with some progeroid syndromes that belong to the laminopathies, such as Hutchinson-Gilford progeria syndrome (HGPS) and mandibuloacral dysplasia (MAD). HGPS is caused by de novo point mutations in the LMNA gene, coding for the nuclear lamina proteins lamin A and C. MAD with type A and B lipodystrophy are recessive disorders resulting from mutations in LMNA and ZMPSTE24, respectively. ZMPSTE24 in addition to ICMT encode proteins involved in posttranslational processing of lamin A. We hypothesized that HSS is an allelic disorder to HGPS and MAD. As the nuclear shape is often irregular in patients with LMNA mutations, we first analyzed the nuclear morphology in skin fibroblasts of patients with HSS, but could not identify any abnormality. Sequencing of the genes LMNA, ZMPSTE24 and ICMT in the 8 patients with HSS revealed the heterozygous missense mutation c.1930C>T (p.R644C) in LMNA in 1 female. Extreme phenotypic diversity and low penetrance have been associated with the p.R644C mutation. In ZMPSTE24 and ICMT, no pathogenic sequence change was detected in patients with HSS. Together, we found no evidence that HSS is another laminopathy.

Transient hyperammonemia due to L-asparaginase therapy in children with acute lymphoblastic leukemia or non-Hodgkin lymphoma.

The standard treatment protocol for acute lymphoblastic leukemia (ALL) and non-Hodgkin lymphoma (NHL) in childhood includes intravenous therapy with asparaginase (Asp), which may cause hyperammonemia. In this study, all patients receiving asparaginase therapy at the Hospital for Children and Adolescents of the University of Leipzig between January 2002 and December 2007 were reviewed for the occurrence of hyperammonemia. Fifty-four patients were identified (22 girls, 32 boys; mean age 5.8 years). Blood ammonia concentrations were determined in 4 patients due to suspicious clinical signs. All showed hyperammonemia with NH(3) concentrations between 260 and 700 µmol/L. They received specific acute detoxification therapy consisting in protein restriction, administration of benzoic acid, glucose/insulin. All 4 recovered completely. All patients receiving therapeutic regimes that include asparaginase (Asp) should be monitored for the development of transient hyperammonemia.