Hannover, Germany
Hannover, Germany

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Janzen N.,Hannover Medical School | Janzen N.,Screening Laboratory Hanover | Sander S.,Screening Laboratory Hanover | Terhardt M.,Screening Laboratory Hanover | And 6 more authors.
Journal of Lipid Research | Year: 2010

The aim of the study was to develop a method for fast and reliable diagnosis of peroxisomal diseases and to facilitate differential diagnosis of cholestatic hepatopathy. For the quantification of bile acids and their conjugates as well as C27 precursors di-and trihydroxycholestanoic acid (DHCA, THCA), in small pediatric blood samples we combined HPLC separation on a reverse-phase C18 column with ESI-MS/MS analysis in the negative ion mode. Analysis was done with good precision (CV 3,7%-11.1%) and sufficient sensitivity (LOQ: 11-91 nmol/L) without derivatization. Complete analysis of 17 free and conjugated bile acids from dried blood spots and 10 μL serum samples, respectively, was performed within 12 min. Measurement of conjugated primary bile acids plus DHCA and THCA as well as ursode-oxycholic acid was done in 4.5 min. In blood spots of healthy newborns, conjugated primary bile acids were found in the range of 0.01 to 2.01 μmol/L. Concentrations of C27 precursors were below the detection limit in normal controls. DHCA and THCA were specifically elevated in cases of peroxysomal defects and one Zellweger patient. Copyright © 2010 by the American Society for Biochemistry and Molecular Biology, Inc.


Illsinger S.,Hannover Medical School | Goken C.,Hannover Medical School | Brockmann M.,Hannover Medical School | Thiemann I.,Hannover Medical School | And 8 more authors.
Annals of Transplantation | Year: 2011

Background: Tacrolimus has a wide spectrum of adverse effects, including neurotoxic and vascula events. Vascular dysfunction due to interference of tacrolimus with mitochondrial function in endothelial cells may contribute to these adverse reactions. Material/Methods: We evaluated the impact of clinically relevant tacrolimus concentrations after 48 hours on energy metabolism in cultured human umbilical vein endothelial cells (HUVEC): Global fatty acid oxidation (FAO), activities of respiratory chain complexes I-V (RC), citratesynthase (CS), glycolytic enzymes and energy rich phosphates were measured. Results: RC-complexes II+III were significantly compromised at 100 nmol/L and CS at 10, 25 and 50 nmol/L, while global FAO was not significantly impaired. Cellular lactate-dehydrogenase (LDH)-, hexokinase- and phosphofructokinase-activities were not altered; AMP levels increased after 48 hours at 200 nmol/L while energy charges remained stable. No cellular toxicity, assessed by light microscopy and LDH leakage was observed even at highest tacrolimus concentrations. Conclusions: Tacrolimus partially impaired mitochondrial function in HUVEC at the level of RC-complexes II+III and CS. Part of tacrolimus toxicity and vascular dysfunction may arise from these metabolic alterations. To some extent, energy balance could be maintained by FAO and cytosolic energy production; energy consumption might be economized. Although only demonstrated for endothelial cells, it is conceivable that such effects will alter energy metabolism in different tissues with high oxidative demands.


Janzen N.,Ruhr University Bochum | Janzen N.,Screening Laboratory Hanover | Riepe F.G.,University of Kiel | Peter M.,Screening Laboratory Hanover | And 8 more authors.
Hormone Research in Paediatrics | Year: 2012

Background: 21-Hydroxylase deficiency (21-OHD) is the target disease of newborn screening for congenital adrenal hyperplasia (CAH). We describe the additional detection of patients suffering from 11β-hydroxylase deficiency (11-OHD) by second-tier testing. Method: Over a period of 5 years, screening for CAH was done in a total of 986,098 newborns by time-resolved immunoassay (DELFIA®) for 17α-hydroxyprogesterone (17-OHP). Positive samples were subsequently analyzed in an LC-MS/MS second-tier test including 17-OHP, cortisol, 11-deoxycortisol, 4-androstenedione and 21-deoxycortisol. Results: In addition to 78 cases of 21-OHD, 5 patients with 11-OHD were identified. Diagnostic parameters were a markedly elevated concentration of 11-deoxycortisol in the presence of a low level of cortisol. Androstenedione was also increased. In contrast to 21-OHD, concentrations of 21-deoxycortisol were normal. Conclusion: Steroid profiling in newborn blood samples showing positive results in immunoassays for 17-OHP allows for differentiating 21-OHD from 11-OHD. This procedure may not detect all cases of 11-OHD in the newborn population because there may be samples of affected newborns with negative results for 17-OHP in the immunoassay. Copyright © 2012 S. Karger AG, Basel.


Janzen N.,Hannover Medical School | Janzen N.,Screening Laboratory Hanover | Illsinger S.,Hannover Medical School | Meyer U.,Hannover Medical School | And 5 more authors.
Archives of Medical Research | Year: 2011

Background and Aims: Galactokinase (GALK) deficiency is an autosomal recessive disorder causing cataract formation that can be prevented or mitigated by early diagnosis and galactose-restricted diet. The aim of this retrospective study was to explore whether GALK-deficiency meets the criteria for neonatal mass screening programs. Methods: From 2000 until 2010, the Screening Laboratory Hannover performed newbornscreening in 1,950,927 infants from Germany for galactosemia by measuring galactose-1-phosphate-uridyl-transferase and total galactose concentration (free galactose plus galactose-1-phosphate), including automatic screening for GALK deficiency. Results: Eleven cases were found with elevated galactose levels accompanied by normal transferase activity. Nine of 11 cases were informative; the diagnosis was established by demonstrating deficient activity of the GALK enzyme in erythrocytes. To our knowledge, screening did not produce any false negative results. All patients were treated with a galactose-restricted diet from the neonatal period or infancy. Three of nine patients suffered from congenital cataracts or eventual development of cataracts, despite normal galactose concentrations in blood. Conclusions: Newborn screening for GALK deficiency prevents or at least mitigates cataract formation. As screening for GALK deficiency is technically simple, it seems to be reasonable to include this disorder in routine screening programs by simultaneous determination of transferase activity and quantification of galactose plus galactose-1-phosphate in dried blood spots. © 2011 IMSS.


Illsinger S.,Hannover Medical School | Janzen N.,Hannover Medical School | Janzen N.,Screening Laboratory Hanover | Sander S.,Screening Laboratory Hanover | And 10 more authors.
Journal of Perinatal Medicine | Year: 2011

Aim: The aim of this study was to investigate the impact of gestational age on energy metabolism in human umbilical vein endothelial cells (HUVECs) of preterm and term neonates. Methods: Activities of respiratory chain (RC) complexes I-V, citrate synthase (CS), overall mitochondrial fatty acid oxidation (FAO), carnitine palmitoyltransferase 2 (CPT2), glycolytic enzymes as well as energy-rich phosphates in HUVECs from uncomplicated term and preterm pregnancies were measured. Neonatal acylcarnitine profiles were analyzed postpartum. Results: Activities of RC complexes II+III, IV, V, and CS were higher in HUVECs from immature pregnancies. Overall FAO did not change, whereas CPT2 activity was higher in term neonates. RC complexes II-V and CS correlated inversely to gestational age, as well as CPT2 activity within the term cohort. Phosphofructokinase activity increased with maturation; lactate dehydrogenase and hexokinase as well as energy-rich phosphates remained constant. In blood, long-chain acylcarnitines were higher in term neonates. Conclusions: Gestational age-dependent differences of energy-providing pathways in HUVECs were shown. Alterations of RC complexes with gestational age may be an adaptive process to cope with metabolic stress during birth; reduced oxidative phosphorylation and high glycolytic activity make HUVECs less susceptible to peripartum hypoxic damage. We hypothesize that HUVECs of premature neonates are metabolically maladapted to birth, which may be responsible for perinatal complications. © 2011 by Walter de Gruyter Berlin Boston.


Wieacker I.,Otto Von Guericke University of Magdeburg | Peter M.,Screening Laboratory Hanover | Borucki K.,Otto Von Guericke University of Magdeburg | Empting S.,Otto Von Guericke University of Magdeburg | And 2 more authors.
Journal of Pediatric Endocrinology and Metabolism | Year: 2015

Careful monitoring of the therapy is crucial for patients with congenital adrenal hyperplasia (CAH) in order to prevent the effects of increased androgen production as well as life-threatening salt-wasting crisis. The key metabolite, 17α-hydroxyprogesterone (17-OHP) can be detected in serum, saliva or dried blood. In clinical practice there are challenges due to discomfort of venous blood sampling and complicated retrieval of saliva during infancy. Furthermore, the immunoassay method is limited in its specificity due to cross-reactions. In this observational study we prospectively examined over a period of 5 years, 20 patients with CAH due to 21-hydroxylase deficiency using standard immunoassays for serum samples (radioimmunoassay and enzyme immunoassay) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) in dried blood spots. Bland-Altman plots show goodness of agreement between both the methods for the desirable therapeutic concentration range of 17-OHP. LC-MS/MS is characterized by a high accuracy in the therapeutic concentration range of 17-OHP <100 nmol/L (r=0.91). Dried blood samples are convenient and reliable specimen for 17-OHP measured by LC-MS/MS. This method could be used for home monitoring of hydrocortisone replacement therapy both in salt-waster and simple virilizer CAH. © 2015 by De Gruyter.


Sander J.,Screening Laboratory Hanover | Janzen N.,Screening Laboratory Hanover | Janzen N.,Hannover Medical School | Terhardt M.,Screening Laboratory Hanover | And 6 more authors.
Clinica Chimica Acta | Year: 2011

Background: Quantification of nitisinone, 2-(nitro-4-trifluoromethylbenzoyl)1,3-cyclohexanedione (NTBC) has been repeatedly described. Nevertheless monitoring of NTBC has not yet become part of routine therapy surveillance in tyrosinaemia type I (OMIM 276700).We developed a blood spot test to facilitate collection and transport of samples. Furthermore, the test material can be used for determination of other parameters like tyrosine and succinylacetone. Method: For quantification of NTBC in blood spots filter paper discs of 3.2. mm diameter were extracted with 150. μL methanol containing mesotrione as internal standard (IS). Analysis was done by UPLC-MS/MS on a Xevo mass spectrometer (ESI+), (MRM). Parent ions were 330.05 for NTBC and 340.05 for IS, daughter ions were m/. z 217.95 and m/. z 125.95 for NTBC, and m/. z 227.95 and m/. z 103.95 for IS. Results: The calibration curve for NTBC in blood spots was linear from 0.1. μmol/L to 100. μmol/L. Recovery exceeded 73.1%, CV intraday and interday were below 9.6%. Instrumental run time was 2.5. min. Sensitivity of the method was 0.1. μmol/L. NTBC concentrations in plasma were higher than in blood spots by a factor of 1.56. ±. 0.13. Conclusion: As demonstrated in patients with tyrosinaemia type I quantification of NTBC by UPLC-MS/MS in blood spots is feasible and gives valuable information for monitoring NTBC treatment. © 2010 Elsevier B.V.


Das A.M.,Hannover Medical School | Steuerwald U.,Screening Laboratory Hanover | Illsinger S.,Hannover Medical School
Journal of Biomedicine and Biotechnology | Year: 2010

Inherited neuromuscular disorders affect approximately one in 3,500 children. Structural muscular defects are most common; however functional impairment of skeletal and cardiac muscle in both children and adults may be caused by inborn errors of energy metabolism as well. Patients suffering from metabolic myopathies due to compromised energy metabolism may present with exercise intolerance, muscle pain, reversible or progressive muscle weakness, and myoglobinuria. In this review, the physiology of energy metabolism in muscle is described, followed by the presentation of distinct disorders affecting skeletal and cardiac muscle: glycogen storage diseases types III, V, VII, fatty acid oxidation defects, and respiratory chain defects (i.e., mitochondriopathies). The diagnostic work-up and therapeutic options in these disorders are discussed. Copyright © 2010 Anibh M. Das et al.


Illsinger S.,Hannover Medical School | Janzen N.,Hannover Medical School | Janzen N.,Screening Laboratory Hanover | Sander S.,Screening Laboratory Hanover | And 9 more authors.
Reproductive Sciences | Year: 2010

Preeclampsia (PE) and hemolysis, elevated liver enzymes, and low platelet count (HELLP) syndrome have been linked to congenital fetal disorders of mitochondrial fatty acid oxidation (FAO). Different incidences may argue for the association of noncongenital alterations of mitochondrial energy metabolism with PE/HELLP syndrome. We studied human umbilical vein endothelial cells [HUVEC] as selected part of the feto-placental unit from uncomplicated (n = 46) and diseased (n = 27; 17 PE and 10 HELLP) pregnancies by measuring the overall FAO, carnitine palmitoyltransferase 2 (CPT2), respiratory chain (RC) complexes I-V, citratesynthase (CS), lactatedehydrogenase (LDH), hexokinase (HK), phosphofructokinase (PFK), and energy rich phosphates. Maternal and infantile acylcarnitines in blood were investigated post partum. Overall FAO, RC complexes II-V, and CS were significantly compromised in HUVEC from complicated pregnancies; impairment of complexes I + III was not significant. CPT2 and energy charges were unaffected. Lactatedehydrogenase and PFK from complicated pregnancies were upregulated, and HK remained constant. In blood, carnitine was elevated in diseased women and their children, acylcarnitines were higher in affected infants. Impaired mitochondrial function in HUVEC is associated with PE/HELLP syndrome and may be involved in the pathophysiology of these diseases.


PubMed | Screening Laboratory Hanover
Type: Journal Article | Journal: Clinica chimica acta; international journal of clinical chemistry | Year: 2010

Quantification of nitisinone, 2-(nitro-4-trifluoromethylbenzoyl)1,3-cyclohexanedione (NTBC) has been repeatedly described. Nevertheless monitoring of NTBC has not yet become part of routine therapy surveillance in tyrosinaemia type I (OMIM 276700). We developed a blood spot test to facilitate collection and transport of samples. Furthermore, the test material can be used for determination of other parameters like tyrosine and succinylacetone.For quantification of NTBC in blood spots filter paper discs of 3.2mm diameter were extracted with 150 L methanol containing mesotrione as internal standard (IS). Analysis was done by UPLC-MS/MS on a Xevo mass spectrometer (ESI+), (MRM). Parent ions were 330.05 for NTBC and 340.05 for IS, daughter ions were m/z 217.95 and m/z 125.95 for NTBC, and m/z 227.95 and m/z 103.95 for IS.The calibration curve for NTBC in blood spots was linear from 0.1 mol/L to 100 mol/L. Recovery exceeded 73.1%, CV intraday and interday were below 9.6%. Instrumental run time was 2.5 min. Sensitivity of the method was 0.1 mol/L. NTBC concentrations in plasma were higher than in blood spots by a factor of 1.56 0.13.As demonstrated in patients with tyrosinaemia type I quantification of NTBC by UPLC-MS/MS in blood spots is feasible and gives valuable information for monitoring NTBC treatment.

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