Energy metabolism in umbilical endothelial cells from preterm and term neonates.

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.

A new method for quantifying causative and diagnostic markers of methylenecyclopropylglycine poisoning.

BACKGROUND: Up to now quantification of hypoglycin A in serum and urine in the range of nmols to µmols per liter plus the measurement of accumulated acyl conjugates have been used for the diagnosis of poisoning by fruits or seeds ofSapindaceae in humans and animals. A second poison, methylenecyclopropylglycine, however, is known to occur in this material. The objective of our study was to develop and evaluate a method for the quantification of this compound suitable for test materials obtained from animals and man. METHOD: Methylenecyclopropylglycine was extracted from serum and urine of a volunteer by a methanolic solution containing labeled methylenecyclopropylglycine as internal standard. UPLC-MS/MS analysis was performed after butylation. RESULTS: Lower limits of detection and quantification were found at 0.5 and 2.5 nmol/L respectively in both urine and serum for each of two isomers, linearity of results (r2 > 0.998) was demonstrated for the range of 0.5-500 nmol/L in urine and serum.The method was applied to urine and serum of horses poisoned by Acer seeds, methylenecyclopropylglycine was found in addition to hypoglycin A. Methylenecyclopropylformyl glycine, a metabolite of methylenecyclopropylglycine, however, was present in much higher concentrations than methylenecyclopropylglycine in all but one samples. CONCLUSIONS: Quantification of methylenecyclopropylglycine can be successfully integrated into our established analytical procedure used for clinical diagnosis of Sapindaceae poisoning. The extended method will improve disease evaluation in humans and animals.

Detection of MCPG metabolites in horses with atypical myopathy.

Atypical myopathy (AM) in horses is caused by ingestion of seeds of the Acer species (Sapindaceae family). Methylenecyclopropylacetyl-CoA (MCPA-CoA), derived from hypoglycin A (HGA), is currently the only active toxin in Acer pseudoplatanus or Acer negundo seeds related to AM outbreaks. However, seeds or arils of various Sapindaceae (e.g., ackee, lychee, mamoncillo, longan fruit) also contain methylenecyclopropylglycine (MCPG), which is a structural analogue of HGA that can cause hypoglycaemic encephalopathy in humans. The active poison formed from MCPG is methylenecyclopropylformyl-CoA (MCPF-CoA). MCPF-CoA and MCPA-CoA strongly inhibit enzymes that participate in ß-oxidation and energy production from fat. The aim of our study was to investigate if MCPG is involved in Acer seed poisoning in horses. MCPG, as well as glycine and carnitine conjugates (MCPF-glycine, MCPF-carnitine), were quantified using high-performance liquid chromatography-tandem mass spectrometry of serum and urine from horses that had ingested Acer pseudoplatanus seeds and developed typical AM symptoms. The results were compared to those of healthy control horses. For comparison, HGA and its glycine and carnitine derivatives were also measured. Additionally, to assess the degree of enzyme inhibition of ß-oxidation, several acyl glycines and acyl carnitines were included in the analysis. In addition to HGA and the specific toxic metabolites (MCPA-carnitine and MCPA-glycine), MCPG, MCPF-glycine and MCPF-carnitine were detected in the serum and urine of affected horses. Strong inhibition of ß-oxidation was demonstrated by elevated concentrations of all acyl glycines and carnitines, but the highest correlations were observed between MCPF-carnitine and isobutyryl-carnitine (r = 0.93) as well as between MCPA- (and MCPF-) glycine and valeryl-glycine with r = 0.96 (and r = 0.87). As shown here, for biochemical analysis of atypical myopathy of horses, it is necessary to take MCPG and the corresponding metabolites into consideration.

Fast and direct quantification of adrenal steroids by tandem mass spectrometry in serum and dried blood spots.

We present a fast and reproducible method for steroid analysis (corticosterone, deoxycorticosterone, progesterone, 17alpha-hydroxyprogesterone, 11-deoxycortisol, 21-deoxycortisol, androstenedione, testosterone, dihydrotestosterone and cortisol) in small volumes of serum and in dried blood spot samples by LC-MS/MS. No derivatisation was needed. LC separation was achieved by using an Atlantis C18 column and water-methanol-formic acid gradient as a mobile phase and a flow rate of 250 microL/min over a run time of 6 min. Steroids were measured in MRM mode with electrospray interface (positive ion mode). Validation showed excellent precision, sensitivity, recovery and linearity with coefficients of determination r2>0.992.

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.

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.

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.

Propionic acidemia: unusual course with late onset and fatal outcome.

A 4 1/2-year-old girl with a so far unremarkable medical history became comatose during a simple infection. She showed severe metabolic acidosis without elevation of lactate. In blood the branched-chain amino acids were increased. In urine ketone-bodies, increased 3-OH-isovaleric and 3-OH propionic acid excretion were detected, while methylmalonate was not found. The profile of acylcarnitines revealed increased propionylcarnitine. Despite restriction of protein supply, high-caloric nutrition, correction of acidosis, and supplementation of biotin and carnitine, the girl died 2 days after admission due to arrhythmia of the heart. In skin fibroblasts the activity of propionyl-coenzyme A carboxylase (PCC) was markedly decreased. Mutation analysis confirmed the diagnosis of propionic acidemia (PA) with compound heterozygosity for 2 new missense mutations L417W/Q293E in the PCCA gene, with the mother carrying the Q293E and the father the L417W mutation. Late-onset PA should be included in the differential diagnosis of unclear coma. Determination of the acylcarnitines using tandem mass spectrometry as well as organic acids in urine is recommended.

Towards newborn screening for ornithine transcarbamylase deficiency: fast non-chromatographic orotic acid quantification from dried blood spots by tandem mass spectrometry.

BACKGROUND: Orotic acid (OA) is the key parameter in the detection of ornithine transcarbamylase deficiency (OTC-D). Inclusion of OA into newborn screening compatibility with existing analytical procedures is necessary. METHODS: OA was eluted from dried blood spots with methanol containing deuterated [1,3-(15)N2] OA as internal standard. Quantification by tandem mass spectrometry was accomplished without chromatographic separation. Samples were measured in MRM mode for the masses m/z 155.1 → 111 for OA and 157.1 → 113 for d2 OA. RESULTS: OA was determined in a wide range of concentrations with high precision, LOD and LOQ being 0.21 and 0.65 µmol/L, respectively. Values correlated well with those obtained after chromatography. Pretreatment of samples with HCl-butanol regularly used for acylcarnitine measurement did not significantly affect quantitative results. Inclusion of the new method into the standard newborn screening procedure did not alter the results for acylcarnitines or amino acids; the total time per analysis, however, was increased from 1.15 to 1.85 min. OA levels of 707 unaffected newborns ranged from 0.28 to 3.73 µmol/L. Five newborns with OTC-D showed concentrations of 89.7-211.1 µmol/L. In newborns with severe citrullinaemia we found values in the range of 4.99-127.7 µmol/L. CONCLUSIONS: This new method can be used as a standalone measurement of OA but it can also easily be implemented into standard newborn screening techniques as a useful supplement. In this case the method allows detection of newborns with OTC deficiency without an extra analytical run.

UPLC-MS/MS analysis of C5-acylcarnitines in dried blood spots.

BACKGROUND: Metabolic screening including newborn screening requires further differentiation of C5-acylcarnitines in order to separate different metabolic disorders and to detect interferents like pivalic acid originating from antibiotics. METHODS: For individual quantification of C5-acylcarnitine isoforms in dried blood spots we combined UPLC using a C18 column and gradient elution with tandem mass spectrometry in ESI+mode. RESULTS: Results were linear, coefficients of determination (R(2))>0.9977, intra- and inter-assay coefficients of variations <5.2%, recovery 96.8-105.2%, limits of detection and quantitation <0.2 µmol/L. Out of 29.309 blood samples of the isolated population of the Faroe Islands 56 exceeded the cut-off of 0.5 µmol/L for C5-acylcarnitine; 45 of which could be retested using the method described. Pivaloylcarnitine was identified in 43 samples, isovalerylcarnitine was found in two samples. CONCLUSIONS: The method was developed to allow direct re-analysis of samples showing elevated concentrations of C5-acylcarnitines in a metabolic screening program based on quantification of acylcarnitines after butylation. The technique should be especially useful in newborn screening for exclusion of false positives and for differentiation between isovaleric acidemia and 2-methylbutyryl-CoA dehydrogenase deficiency.

Neonatal screening: identification of children with 11ß-hydroxylase deficiency by second-tier testing.

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 11ß-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.

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.

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.

Newborn screening for hepatorenal tyrosinemia: Tandem mass spectrometric quantification of succinylacetone.

BACKGROUND: False-positive and false-negative results occur in current newborn-screening programs for hepatorenal tyrosinemia, which measure tyrosine concentrations in blood spots, sometimes in combination with other metabolites, including succinylacetone. We present our experience with a newly described method for succinylacetone quantification in routine newborn screening. METHODS: Succinylacetone was extracted from blood spots that had already been extracted with absolute methanol for acylcarnitine and amino acid analysis. The solvent was acetonitrile-water (80:20 by volume) containing formic acid, hydrazine hydrate, and 100 nmol/L 5,7-dioxooctanoic acid as internal standard. Analysis was performed by tandem mass spectrometry in a separate run. RESULTS: Of 61,344 samples, 99.6% had succinylacetone concentrations < or =5 micromol/L. With a cutoff of 10 micromol/L, no false-positive results were obtained. In 2 patients, the succinylacetone concentrations in the dried blood spots from the 36th and 56th hours of life were 152 and 271 micromol/L, respectively, and the tyrosine concentrations were 54 and 129 micromol/L. Hepatorenal tyrosinemia was subsequently confirmed in both patients. Retrospective analysis of the neonatal screening samples of 2 additional known patients revealed increased succinylacetone concentrations of 46 and 169 micromol/L, respectively. CONCLUSIONS: Tandem mass spectrometric quantification directly from residual blood spots is a useful method for the early detection of hepatorenal tyrosinemia in newborn-screening programs.

Neonatal screening for defects of the mitochondrial trifunctional protein.

Long-chain l-3-hydroxyacyl-CoA dehydrogenase (LCHAD) deficiency has been included in the routine neonatal screening program by the German screening commission. As tandem mass spectrometry (TMS) does not discriminate between the different defects of the mitochondrial trifunctional protein (MTP) screening for isolated LCHAD deficiency includes the detection of long-chain 3-ketoacyl-CoA thiolase and complete MTP deficiencies as well. We identified 11 patients with abnormalities of the MTP out of 1.2 million newborns screened in our laboratory during the last 6 years. Treatment was started on the day the screening result was obtained (day 3 to day 9 of life). Seven of these newborns developed satisfactorily during an observation period of up to 64 months. They had isolated LCHAD deficiency, four of them caused by the typical mutation (1528 G>C), three others had no molecular genetic analysis done or were shown to have previously unknown mutations. Four children did not survive, two of them showing complete deficiency of MTP and two showing deficiency of long-chain 3-ketoacyl-CoA thiolase. We conclude that, despite the rarity of the disease, screening for MTP deficiencies is justified based on the following criteria: improved quality of life for patients with isolated LCHAD deficiency, absence of stigmatisation of babies showing mild variants without necessity of treatment, no significant increase of the total number of false positive screening results, no false negative results to our knowledge. Finally, extension of analysis to MTP deficiencies is achieved without additional costs for screening laboratories already using TMS.

Neonatal screening for citrullinaemia.

UNLABELLED: In a period of 40 months (1st March 1999 to 30th June 2002) 610,000 blood samples were analysed in one screening centre for citrulline as a pilot study for neonatal screening using tandem mass spectrometry. Persistent hypercitrullinaemia (Cit >1.5 mg/dl or 85.5 micro mol/l, not corrected for recovery) was identified in 15 newborns. Four children were diagnosed with classical neonatal onset citrullinaemia and eight with persisting asymptomatic hypercitrullinaemia. In two asymptomatic newborns and in one symptomatic preterm patient, argininosuccinate lyase deficiency was identified as the cause of moderately elevated levels of citrulline (cases not described in this paper). Citrulline concentrations were only temporarily mildly elevated in two newborns and in these the results of the original neonatal screening were therefore regarded as false-positive; we did not find any other false-positives. The screening result allowed the introduction of immediate specific treatment in two cases of citrullinaemia and may have prevented metabolic decompensation in those with presumed mild citrullinaemia. In one child who developed severe hyperammonaemia on the 2nd day of life, sequelae could not be avoided. CONCLUSION: neonatal screening for citrullinaemia is more complex than expected and, with the actual logistics, results may be obtained too late in severe forms.