Nils Janzen

Rapid steroid hormone quantification for congenital adrenal hyperplasia (CAH) in dried blood spots using UPLC liquid chromatography-tandem mass spectrometry.

Newborn screening for congenital adrenal hyperplasia (CAH) is usually done by quantifying 17α-hydroxyprogesterone using immunoassay. However, this test produces high rates of false positive results caused by cross reacting steroids. Therefore we have developed a selective and specific method with a short run time (1.25 min) for quantification of 17α-hydroxyprogesterone, 21-deoxycortisol, 11-deoxycortisol, 11-deoxycorticosterone and cortisol from dried blood spots. The extraction procedure is very simple and steroid separation is ensured on a BEH C18 column and an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Analysis was done in positive ionization mode (ESI+) and recorded in multiple reaction monitoring mode (MRM). The method gave linear results for all steroids over a range of 5-200 (cortisol: 12.5-500)nmol/L with coefficients of regression >0.992. Absolute recovery was >64.1%. Across the analytical range the inter-assay coefficient of variation (CV) was <3%. Newborn blood samples of patients with confirmed 21-CAH and 11-CAH could clearly be distinguished from samples of unaffected newborns falsely positive on immunoassay. The method is not influenced by cross reactions as found on immunoassay. Analysis of dried blood spots shows that this method is sensitive and fast enough to allow rapid analysis and can therefore improve the newborn screening program.

Preeclampsia and HELLP Syndrome: Impaired Mitochondrial Function in Umbilical Endothelial Cells

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.

Rapid quantification of conjugated and unconjugated bile acids and C27 precursors in dried blood spots and small volumes of serum

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 ursodeoxycholic 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.

Monitoring tyrosinaemia type I: Blood spot test for nitisinone (NTBC)

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 3.2mm 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.

EDTA in Dried Blood Spots Leads to False Results in Neonatal Endocrinologic Screening

BACKGROUND Blood samples for neonatal screening for inborn errors of metabolism are collected and shipped on standardized filter paper cards. Occasionally these samples are contaminated with EDTA, which is often used for anticoagulation. EDTA may interfere with newborn screening tests based on lanthanide fluorescence and thus lead to false-negative or false-positive results. METHODS We used tandem mass spectrometry (MS/MS) to detect EDTA in dried blood spots by use of an extra experiment that was integrated into the standard MS/MS neonatal screening and did not require an additional sample spot, nor extra time or work. We analyzed the influence of different blood sampling procedures on lanthanide fluorescence tests for thyroid-stimulating hormone (TSH) and 17-hydroxyprogesterone (17-OHP). RESULTS EDTA was increased in 138 of 190 000 newborn screening samples, 27 of which caused false- positive results in the immunoassay for 17-OHP. No false-negative TSH results were found. False-positive results in the 17-OHP test occurred when EDTA concentrations were >2.0 g/L; the TSH test, however, produced false negatives only when EDTA concentrations were >3.0 g/L. Using EDTA-containing devices the procedure of blood collection significantly influenced the concentration of the anticoagulant. CONCLUSION Addition of EDTA quantification into standard MS/MS tests is a simple and useful method to avoid false-positive or false-negative neonatal screening results in lanthanide fluorescence-based tests.

Rapid diagnosis of hypoglycin A intoxication in atypical myopathy of horses.

Hypoglycin A (2-amino-3-(2-methylidenecyclopropyl)propanoic acid) is the plant toxin shown to cause atypical myopathy in horses. It is converted in vivo to methylenecyclopropyl acetic acid, which is transformed to a coenzyme A ester that subsequently blocks beta oxidation of fatty acids. Methylenecyclopropyl acetic acid is also conjugated with carnitine and glycine. Acute atypical myopathy may be diagnosed by quantifying the conjugates of methylenecyclopropyl acetic acid plus a selection of acyl conjugates in urine and serum. We describe a new mass spectrometric method for sample volumes of <0.5 mL. Samples were extracted with methanol containing 5 different internal standards. Extracts were analyzed by ultra–high-performance liquid chromatography–tandem mass spectrometry focusing on 11 metabolites. The total preparation time for a series of 20 samples was 100 min. Instrument run time was 14 min per sample. For the quantification of carnitine and glycine conjugates of methylenecyclopropyl acetic acid in urine, the coefficients of variation for intraday quantification were 2.9% and 3.0%, respectively. The respective values for interday were 9.3% and 8.0%. Methylenecyclopropyl acetyl carnitine was detected as high as 1.18 µmol/L in serum (median: 0.46 µmol/L) and 1.98 mmol/mol creatinine in urine (median: 0.79 mmol/mol creatinine) of diseased horses, while the glycine derivative accumulated up to 1.97 mmol/mol creatinine in urine but was undetectable in most serum samples. In serum samples from horses with atypical myopathy, the intraday coefficients of variation for C4–C8 carnitines and glycines were ≤4.5%. Measured concentrations exceeded those in healthy horses by ~10 to 1,400 times.

Cyclosporine A: impact on mitochondrial function in endothelial cells

Illsinger S, Janzen N, Lücke T, Bednarczyk J, Schmidt K‐H, Hoy L, Sander J, Das AM. Cyclosporine A: impact on mitochondrial function in endothelial cells.
Clin Transplant 2011: 25: 584–593.

Quantification of Methylenecyclopropyl Compounds and Acyl Conjugates by UPLC-MS/MS in the Study of the Biochemical Effects of the Ingestion of Canned Ackee (Blighia sapida) and Lychee (Litchi chinensis)

Consumption of ackee (Blighia sapida) and lychee (Litchi chinensis) fruit has led to severe poisoning. Considering their expanded agricultural production, toxicological evaluation has become important. Therefore, the biochemical effects of eating 1 g/kg canned ackee, containing 99.2 μmol/kg hypoglycin A, and 5 g/kg canned lychee, containing 1.3 μmol/kg hypoglycin A, were quantified in a self-experiment. Using ultra-high-performance liquid chromatography/mass spectrometry, hypoglycin A, methylenecyclopropylacetyl-glycine, and methylenecyclopropylformyl-glycine, as well as the respective carnitine conjugates, were found in urine after ingesting ackee. Hypoglycin A and its glycine derivative were also present in urine after eating lychee. Excretion of physiological acyl conjugates was significantly increased in the ackee experiment. Ingestion of ackee led to up to 15.1 nmol/L methylenecyclopropylacetyl-glycine and traces of methylenecyclopropylformyl-carnitine in the serum. These compounds were not found in the serum after eating lychee. Hypoglycin A accumulated in the serum in both experiments.

Tandemmassenspektrometrie : Beitrag zum Neugeborenenscreening auf angeborene Störungen des Stoffwechsels

ZusammenfassungHintergrund. Die Tandemmassenspektrometrie wurde als neue Analysetechnik für die Früherkennung angeborener Stoffwechselstörungen im Neugeborenenscreening erprobt. Ergebnisse. Die Untersuchung von 128.225 Blutproben von Neugeborenen deckte 11-mal eine Phenylketonurie (PKU) und 3-mal eine Hyperphenylalaninämie sowie 4 Fälle von Citrullinämie und 1 Tyrosinämie auf. Eine Fehlfunktion der Dehydrogenase der mittellangkettigen Fettsäuren (MCAD-Defekt) erwies sich mit 17 Fällen als häufig. Bei 2 Kindern wurde eine Abbaustörung der langkettigen hydroxylierten Fettsäuren (LCHAD-Defekt) gefunden, bei 1 weiteren ein Defekt des trifunktionalen Proteins. Unter den Organazidämien zeigten der 3-Methylcrotonyl-CoA-Karboxylasemangel und die Isovalerianazidämie mit je 4 nachgewiesenen Fällen eine unerwartet hohe Inzidenz. Die Quote der notwendigen Zweitanforderungen von Blut zur Kontrolle betrug 0,38%. Sie war damit niedriger als die bisher für das PKU-Screening allein angegebene. Schlussfolgerung. Die Tandemmassenspektrometrie kann nach diesen Erfahrungen den präventiven Nutzen des Neugeborenenscreenings weiter erhöhen.SummaryBackground. As part of our neonatal screening program we applied tandem mass spectrometry on 128.225 dried blood samples for detection of inborn errors of metabolism. Results. Eleven patients with phenylketonuria and three patients with hyperphenylalaninemia were detected. Tyrosinemia and citrullinemia were found in one and four newborns, respectively. Analysis of free carnitine and acylcarnitines resulted in the diagnosis of medium chain acyl-CoA dehydrogenase (MCAD) deficiency in 17 newborns. Other detected defects of fatty acid oxidation were long-chain-3-hydroxyacyl-CoA dehydrogenase (LCHAD) deficiency (two cases) and the defect of the trifunctional protein (one case). Each, isovaleric acidemia and 3-methylcrotonyl-CoA carboxylase deficiency were found in four individuals, which may indicate a considerably higher incidence of these diseases than previously reported. The recall rate for all parameters measured by tandem mass spectrometry was 0.38%. This was significantly lower than the recall rate of 0.5% for PKU screening alone prior to the introduction of the new technique. Conclusion. We expect tandem mass spectrometry to be used in many neonatal screening laboratories in the near future.

Quantification of hypoglycin A as butyl ester

L-α-amino-methylenecyclopropyl propionic acid (Hypoglycin A, HGA) has been found to be the toxic compound in fruits of the Sapindaceae family causing acute intoxication when ingested as food or feed. Clinical symptoms are consistent with acquired multiple acyl-CoA dehydrogenase deficiency (MADD). Ultra performance liquid chromatography-tandem mass spectrometry was used to measure HGA after butylation. Sample volumes were 10μL for serum and 20μL for urine. Internal standard for HGA was d3-leucine, samples were plotted on a 7-point linear calibration curve. Coefficients of variation were <15% at 0.01μmol HGA/L and ≤4.1% at 10μmol/L. R(2) values for linearity were ≥0.995. In order to quantify non-metabolized HGA together with some of its metabolites plus a spectrum of acyl glycines and acyl carnitines typical for acquired MADD in one single analysis HGA measurement was integrated into a method which we previously developed for metabolites of HGA and acyl conjugates. The new method is suitable for biochemical diagnosis of Ackee fruit poisoning or atypical myopathy in horses and for forensic purposes in cases of suspected HGA poisoning.