The Neonatal QRS Complex and Its Association with Left Ventricular Mass.

To evaluate QRS complex features during the first month of life and the association with echocardiographic measurements of left ventricular mass in neonates. Prospective cohort study of neonates with electrocardiography (ECG) and echocardiography performed during the first month of life. Left ventricular mass index (LVMI) was determined by echocardiography and the correlation with electrocardiographic markers of LVMI outliers (≥ 98th percentile) were analyzed. We included 17,450 neonates (52% boys; median age at examination 11 days) and found an increase in median QRS duration and LVMI during the first month of life (54 vs. 56 ms and 24.7 vs. 28.6 g/m2 at days 0-4 and 25-30, respectively; both p < 0.001). All investigated ECG features (QRS duration, QRS area in V1/V6, maximum amplitudes of S-V1/R-V6, and the Sokolow-Lyon voltage product) showed no to low correlation with LVMI, resulting in low sensitivities (0-9.0%), but high specificities (97.2-98.1%), and area under the curve values close to the identity line (0.49-0.61) for identifying LVMI outliers. Adjustment of outlier definition for LVMI and threshold for QRS features had no significant effect on sensitivity. We present reference values for QRS complex features and their association with LVMI in neonates from a large, unselected, population-based cohort. The QRS complex gradually evolved during the first month of life but had a low correlation with LVMI. Our results indicate a poor diagnostic value of using ECG features to identify LVMI outliers in neonates.Trial Registry Copenhagen Baby Heart, NCT02753348, https://clinicaltri-als.gov/ct2/show/NCT02753348?cond=Copenhagen+Baby+Heart&draw=2&rank=1 , deidentified individual participant data will not be made available.

The Wolff-Parkinson-White pattern in neonates: results from a large population-based cohort study.

AIMS: Wolff-Parkinson-White (WPW) syndrome is a conduction disorder characterized by an accessory electrical pathway between the atria and ventricles, which may predispose to supraventricular tachycardia (SVT) and sudden cardiac death. It can be seen as an isolated finding or associated with structural heart disease. Our aims were to determine the prevalence of a WPW pattern in a large and unselected cohort of neonates and to describe the electro- and echocardiographic characteristics as well as the natural history during early childhood. METHODS AND RESULTS: Electrocardiograms and echocardiograms of neonates (aged 0-30 days) from a large, prospective, population-based cohort study were included. Neonates with a WPW pattern were identified and matched 1:4 to controls. Localization of the accessory pathway was assessed by different algorithms. Among 17 489 neonates, we identified 17 (76% boys) with a WPW pattern consistent with a prevalence of 0.1%. One neonate had moderate mitral regurgitation while other echocardiographic parameters were similar between cases and controls (all P > 0.05). The accessory pathways were primarily predicted to be left-sided. At follow-up (available in 14/17 children; mean age 3.2 years) the pre-excitation pattern persisted in only four of the children and none of the children had experienced any episodes of SVT. CONCLUSION: The prevalence of a WPW pattern in our cohort of unselected neonates was 0.1%. The WPW pattern was more frequent in boys and generally not associated with structural heart disease, and the accessory pathways were primarily left-sided. At follow-up, the WPW pattern had disappeared in most of the children suggesting either an intermittent nature or that normalization occurs. CLINICAL TRIAL REGISTRATION: Copenhagen Baby Heart, NCT02753348.

The Impact of Maternal Age on the Neonatal Electrocardiogram.

BACKGROUND: Previous studies have suggested an increased prevalence of congenital heart disease among children born to women aged ≥35 years. In recent decades, the mother's age at childbirth has increased dramatically in industrialized countries. It has not been investigated if increasing maternal age affects the neonatal cardiac electrical system. METHODS: The Copenhagen Baby Heart Study is a prospective general population study that performed cardiac evaluation in newborns. Electrocardiograms were analyzed with a computerized algorithm. RESULTS: We included 16,518 newborns with normal echocardiograms (median age 11 days; range 0-30 days; 52% boys). Median maternal age at delivery was 31 years; 790 newborns were born to mothers aged between 16 and 24 years, 11,403 between 25 and 34 years, 4,279 between 35 and 44 years, and 46 newborns had mothers aged between 45 and 54 years. The QRS axis and maximum R-wave amplitude in V1 (R-V1) differed across the four maternal age groups (both p < 0.01), with absolute differences of 3.5% (114 vs. 110°) and 12% (1,152 vs. 1,015 µV), respectively, between newborns with the youngest and oldest mothers. Associations between maternal age and the QRS axis and R-V1 remained significant after multifactorial adjustment. Heart rate, PR interval, QRS duration, uncorrected QT interval, QTcBazett, and maximum amplitudes of S-V1, R-V6, and S-V6 were not associated with maternal age (all p > 0.05). CONCLUSION: We observed a significant association between maternal age and the neonatal QRS axis and R-V1. However, the absolute differences were relatively small and maternal age is unlikely to have a clinically significant effect on the neonatal cardiac electrical system.

Gestational Age and Neonatal Electrocardiograms.

OBJECTIVES: Interpretation of the neonatal electrocardiogram (ECG) is challenging due to the profound changes of the cardiovascular system in this period. We aimed to investigate the impact of gestational age (GA) on the neonatal ECG and create GA-specific reference values. METHODS: The Copenhagen Baby Heart Study is a prospective general population study that offered cardiac evaluation of neonates. ECGs and echocardiograms were obtained and systematically analyzed. GA, weight, height, and other baseline variables were registered. RESULTS: We included 16 462 neonates (52% boys) with normal echocardiograms. The median postnatal age was 11 days (range 0 to 30), and the median GA was 281 days (range 238 to 301). Analyzing the ECG parameters as a function of GA, we found an effect of GA on almost all investigated ECG parameters. The largest percentual effect of GA was on heart rate (HR; 147 vs 139 beats per minute), the QRS axis (103° vs 116°), and maximum R-wave amplitude in V1 (R-V1; 0.97 vs 1.19 mV) for GA ≤35 vs ≥42 weeks, respectively. Boys had longer PR and QRS intervals and a more right-shifted QRS axis within multiple GA intervals (all P < .01). The effect of GA generally persisted after multifactorial adjustment. CONCLUSIONS: GA was associated with significant differences in multiple neonatal ECG parameters. The association generally persisted after multifactorial adjustment, indicating a direct effect of GA on the developing neonatal cardiac conduction system. For HR, the QRS axis, and R-V1, the use of GA-specific reference values may optimize clinical handling of neonates.

[Waiting Times For Admission Into A Psychotherapeutic Inpatient Treatment - Results of A Prospective Study In South-Württemberg]. / Wartezeiten auf die Aufnahme in eine Psychosomatische Klinik ­ Ergebnisse einer prospektiven Studie in Südwürttemberg.

INTRODUCTION: Waiting times for the admission into a so called psychosomatic hospital in Germany prevent the necessary immediate treatment. They lead to further incapacity for work and chronic manifestation of the disease. It is reported that most psychosomatic hospitals have waiting times, but there are no studies on data on that. Therefore, it was the aim of this study to access prospectively in a defined region, how long it takes for the patients to get an outpatient preliminary talk and thereafter, how long they have to wait for their admission. METHODS: 7 hospitals out of the region of South-Württemberg took part on this study, 2 of them had bigger day hospitals. Data were assessed prospectively in 2015 over 9 months, in total 916 admissions were assessed. RESULTS: The waiting time until a preliminary talk, in which the indication for inpatient treatment was secured, was in the mean 25 days (SD=31). The waiting time after this talk until admission was 56 days (SD=47). Patients who waited for a day treatment had to wait even longer. An urgency remark, given by the therapist of the preliminary talk, as well as a private illness insurance led to shorter waiting times. The diagnosis had no influence on the waiting time. CONCLUSIONS: The waiting times are substantial and imply a burden for the patient and also for the health care system. It is recommended to assess and publish these waiting times on a regularly basis. Politics, but also the actors in the health care system should discuss if and how this deficit can be changed.

Neuroprotective effects of bilobalide are accompanied by a reduction of ischemia-induced glutamate release in vivo.

Neuroprotective properties of bilobalide, a specific constituent of Ginkgo extracts, were tested in a mouse model of stroke. After 24h of middle cerebral artery occlusion (MCAO), bilobalide reduced infarct areas in the core region (striatum) by 40-50% when given at 10mg/kg 1h prior to MCAO. Neuroprotection was also observed at lower doses, or when the drug was given 1h past stroke induction. Sensorimotor function in mice was improved by bilobalide as shown by corner and chimney tests. When brain metabolism in situ was monitored by microdialysis, MCAO caused a rapid disappearance of extracellular glucose in the striatum which returned to baseline levels after reperfusion. Extracellular levels of glutamate were increased by more than ten-fold in striatal tissue, and by four- to fivefold in hippocampal tissue (penumbra). Bilobalide did not affect glucose levels but strongly attenuated glutamate release in both core and penumbra regions. Bilobalide was equally active when given locally via the microdialysis probe and also reduced ischemia-induced glutamate release in vitro in brain slices. We conclude that bilobalide is a strong neuroprotectant in vivo at doses that can be used therapeutically in humans. The mechanism of action evidently involves reduction of glutamate release, thereby reducing excitotoxicity.

Neurotransmitters and energy metabolites in amyloid-bearing APP(SWE)xPSEN1dE9 Mouse Brain.

Alzheimer's disease is characterized by amyloid peptide formation and deposition, neurofibrillary tangles, synaptic loss and central cholinergic dysfunction, dysfunction of energy metabolism, and dementia; however, the interactions between these hallmarks remain poorly defined. We studied a well characterized mouse model of amyloid deposition, the doubly transgenic APP(SWE)xPSEN1dE9 mouse. At 10 to 14 months of age, these mice had high levels of amyloid peptides (6.6 microg/g wet weight) and widespread amyloid plaques. Extracellular levels of acetylcholine (ACh) were determined by microdialysis in the hippocampus and were comparable with nontransgenic mice from the same colony. In the open field, both mouse strains responded with a 3-fold increase of hippocampal ACh release. Exploratory behavior of the transgenic mice appeared normal. Infusion of scopolamine evoked 5- to 6-fold increases of ACh levels in both mouse strains. High-affinity choline uptake and cholinesterase activities were identical in both mouse lines. Extracellular levels of glucose and glycerol were similar in control and transgenic mice, whereas lactate levels were slightly (p = 0.06) and glutamate levels significantly (p = 0.02) lower in transgenic mice. Exploration caused increases of glucose and lactate, whereas infusion of scopolamine (1 microM) increased glucose but not lactate. Glutamate levels were increased by scopolamine, whereas glycerol remained constant under all the conditions. We conclude that amyloid peptide production and plaque deposition causes minor changes in cholinergic function and energy metabolites in transgenic mice in vivo. Amyloid peptide formation and/or deposition may not be sufficient for long-term cholinergic or metabolic dysfunction.

Metabolic and transmitter changes in core and penumbra after middle cerebral artery occlusion in mice.

Middle cerebral artery occlusion (MCAO) is a popular model in experimental stroke research and causes prominent ischemic damage in the forebrain. To characterize metabolic changes induced by MCAO, we have induced permanent MCAO in mice that were implanted with a microdialysis probe in either striatum or hippocampus. Immediately after the onset of ischemia, glucose levels dropped to <10% of basal values in the striatum while they dropped to 50%, and recovered thereafter, in hippocampus. Extracellular levels of glutamate rose 80-fold in the striatum but only 10-fold, and in a transient fashion, in hippocampus. In striatum, release of acetylcholine briefly increased, then dropped to very low values. Both glycerol and choline levels increased strongly during ischemia in the striatum reflecting membrane breakdown. In hippocampus, glycerol increased transiently while the increase of choline levels was moderate. Taken together, these observations delineate metabolic changes in ischemic mouse brain with the striatum representing the core area of ischemia. In comparison, the dorsal hippocampus was identified as a brain area suitable for monitoring metabolic responses in the penumbra region.

Effects of rivastigmine and donepezil on brain acetylcholine levels in acetylcholinesterase-deficient mice.

PURPOSE: Alzheimer s disease is characterized by a dysfunction of central cholinergic systems and is treated by inhibitors of acetylcholinesterase (AChE). This study tests the effect of two AChE inhibitors in therapeutic use, rivastigmine and donepezil, in mice that are devoid of AChE (AChE-/- mice). Rivastigmine is an inhibitor of both AChE and butyrylcholinesterase (BChE) whereas donepezil is a selective inhibitor of AChE. METHODS: We have used in vivo microdialysis to investigate the effects of the two drugs on the extracellular concentration of acetylcholine (ACh) in the hippocampus of AChE-/- mice. RESULTS: Extracellular ACh levels in the hippocampus were 30-fold elevated in AChE-/- mice compared to wild-type (AChE+/+) animals. Infusion of rivastigmine (1 and 10 microM) caused a further doubling of ACh levels in AChE-/- mice within 90-120 min. In contrast, infusion of donepezil (1 microM) did not affect hippocampal ACh levels in AChE-/- mice although it increased ACh levels more than twofold in wild-type mice. CONCLUSIONS: In the absence of AChE, rivastigmine enhances the levels of extracellular ACh by inhibiting BChE. This finding may be of therapeutic relevance because BChE activity is preserved, but AChE activity is strongly decreased, in late-stage Alzheimer s disease.

Role of glycine receptors and glycine release for the neuroprotective activity of bilobalide.

Bilobalide, a constituent of Ginkgo biloba, has neuroprotective properties. Its mechanism of action is unknown but it was recently found to interact with neuronal transmission mediated by glutamate, gamma-aminobutyric acid (GABA) and glycine. The goal of this study was to test the interaction of bilobalide with glycine in assays of neuroprotection. In rat hippocampal slices exposed to N-methyl-D-aspartate (NMDA), release of choline indicates breakdown of membrane phospholipids. NMDA-induced choline release was almost completely blocked in the presence of bilobalide (10 microM). Glycine (10-100 microM) antagonized the inhibitory action of bilobalide in this assay. In a second assay of excitotoxicity, we measured tissue water content as an indicator of cytotoxic edema formation in hippocampal slices which were exposed to NMDA. In this assay, edema formation was suppressed by bilobalide but bilobalide's action was attenuated in the presence of glycine and of D-serine (100 microM each). To investigate bilobalide's interaction with glycine receptors directly, we determined 36chloride flux in rat cortico-hippocampal synaptoneurosomes. Glycine (100 microM) was inactive in this assay indicating an absence of functional glycine-A receptors in this preparation. [3H]Glycine was used to assess binding at the glycine binding site of the NMDA receptor but bilobalide was found to be inactive in this assay. Finally, [3H]glycine release was monitored in hippocampal slices exposed to oxygen-glucose deprivation. In this model, glycine release was induced by ischemia, an effect that was strongly reduced by bilobalide. We conclude that bilobalide does not interact with glycine receptors in neurochemical assays but it significantly reduces the release of glycine under ischemic conditions. This effect likely contributes to bilobalide's neuroprotective effects in assays of excitotoxicity and ischemia.

Choline availability and acetylcholine synthesis in the hippocampus of acetylcholinesterase-deficient mice.

Mice deficient for acetylcholinesterase (AChE) have strongly increased extracellular levels of acetylcholine (ACh) in the dorsal hippocampus [Hartmann, J., Kiewert, C., Duysen, E.G., Lockridge, O., Greig, N.H., Klein, J., 2007. Excessive hippocampal acetylcholine levels in acetylcholinesterase-deficient mice are moderated by butyrylcholinesterase activity. J. Neurochem. 100, 1421-1429]. Using microdialysis, we found that increased ACh levels are accompanied by decreased levels of extracellular choline which were 1.60 microM in AChE-deficient mice and 4.36 microM in wild-type mice. Addition of choline (10 microM) to the perfusion fluid, while ineffective in wild-type animals, more than doubled extracellular ACh levels in AChE-deficient mice. High-affinity choline uptake (HACU), as measured ex vivo in corticohippocampal synaptosomes, was more than doubled in AChE-deficient mice. Inhibition of HACU by hemicholinium-3 (HC-3) in vivo reduced extracellular levels of ACh by 60% in wild-type mice but by more than 90% in AChE-deficient mice. Decreased ACh levels caused by infusion of HC-3 or tetrodotoxin (TTX) were accompanied by increased levels of free choline. Infusion of scopolamine (1 microM) caused a fivefold increase of ACh levels in wild-type animals but only a 50% increase in AChE-deficient mice. In conclusion, absence of AChE causes dynamic changes in the ratio of choline to ACh. High levels of extracellular ACh are accompanied by reduced levels of extracellular choline, and ACh release becomes strongly dependent on choline availability. Similar changes may take place in patients chronically exposed to AChE inhibitors.

Excessive hippocampal acetylcholine levels in acetylcholinesterase-deficient mice are moderated by butyrylcholinesterase activity.

Central cholinergic systems are involved in a plethora of brain functions and are severely and selectively damaged in neurodegenerative diseases such as Alzheimer's disease and dementia with Lewy bodies. Cholinergic dysfunction is treated with inhibitors of acetylcholinesterase (AChE) while the role of butyrylcholinesterase (BChE) for brain cholinergic function is unclear. We have used in vivo microdialysis to investigate the regulation of hippocampal acetylcholine (ACh) levels in mice that are devoid of AChE (AChE-/- mice). Extracellular ACh levels in the hippocampus were 60-fold elevated in AChE-/- mice compared with wild-type (AChE+/+) animals. In AChE-/- mice, calcium-free conditions reduced hippocampal ACh levels by 50%, and infusion of tetrodotoxin by more than 90%, indicating continuous ACh release. Infusion of a selective AChE inhibitor (BW284c51) caused a dose-dependent, up to 16-fold increase of extracellular ACh levels in AChE+/+ mice but did not change ACh levels in AChE-/- mice. In contrast, infusion of a selective inhibitor of BChE (bambuterol) caused up to fivefold elevation of ACh levels in AChE-/- mice, but was without effect in AChE+/+ animals. These results were corroborated with two other specific inhibitors of AChE and BChE, tolserine and bis-norcymserine, respectively. We conclude that lack of AChE causes dramatically increased levels of extracellular ACh in the brain. Importantly, in the absence of AChE, the levels of extracellular ACh in the brain are controlled by the activity of BChE. These results point to a potential usefulness of BChE inhibitors in the treatment of central cholinergic dysfunction in which brain AChE activity is typically reduced.

Role of GABAergic antagonism in the neuroprotective effects of bilobalide.

Bilobalide, a constituent of Ginkgo biloba, has neuroprotective properties. Its mechanism of action is unknown but it was recently found to block GABA(A) receptors. The goal of this study was to test the potential role of a GABAergic mechanism for the neuroprotective activity of bilobalide. In rat hippocampal slices exposed to NMDA, release of choline indicates breakdown of membrane phospholipids. NMDA-induced choline release was almost completely blocked in the presence of bilobalide (10 microM) and under low-chloride conditions. Bicuculline (100 microM), a competitive antagonist at GABA(A) receptors, reduced NMDA-induced choline release to a small extent (-23%). GABA (100 microM) partially antagonized the inhibitory action of bilobalide. Exposure of hippocampal slices to NMDA also caused edema formation as measured by increases of tissue water content. NMDA-induced edema formation was suppressed by bilobalide and by low-chloride conditions. Bicuculline exerted partial protection (by 30%) while GABA reduced bilobalide's effect by about one third. To investigate bilobalide's interaction with GABA(A) receptors directly, we measured binding of [(35)S]-TBPS to rat cortical membranes. TBPS binding was competitively inhibited by bilobalide in the low micromolar range (IC(50)=3.7 microM). As a functional test, we determined (36)chloride flux in rat corticohippocampal synaptoneurosomes. GABA (100 microM) significantly increased (36)chloride flux (+65%), and this increase was blocked by bilobalide, but with low potency (IC(50): 39 microM). We conclude that, while antagonism of GABA(A) receptors may contribute to bilobalide's neuroprotective effects, additional mechanisms must be postulated to fully explain bilobalide's actions.

NGP1-01 is a brain-permeable dual blocker of neuronal voltage- and ligand-operated calcium channels.

Calcium overload of neurons leads to cell death and is a key feature in neurodegenerative diseases. The polycyclic amine NGP1-01 blocks L-type voltage operated calcium channels in cardiomyocytes. Here, we tested whether NGP1-01 blocks neuronal calcium channels. NGP1-01 (1 microM) inhibited depolarization-induced calcium influx by 78% in cortical neurons preloaded with fura-2 AM, with a potency similar to nimodipine. NGP1-01 (1 microM) also inhibited N-methyl-D: -aspartate (NMDA)-induced (1 mM) calcium influx by 52%, only slightly less potent than memantine. Using in vivo-microdialysis, we monitored choline release during NMDA infusion as a measure of excitotoxic membrane breakdown. Intraperitoneal injection of NGP1-01 (40 mg/kg) reduced NMDA-induced membrane breakdown by 31% (P < 0.01) while memantine (10 mg/kg) reduced choline release by 40%. Our results demonstrate that NGP1-01 simultaneously blocks both major neuronal calcium channels and is sufficiently brain-permeable. We conclude that NGP1-01 is a promising lead structure for a new class of dual-mechanism neuroprotective agents.

Glycerophosphocholine is elevated in cerebrospinal fluid of Alzheimer patients.

Experimental and clinical studies give evidence for breakdown of membrane phospholipids during neurodegeneration. In the present study, we measured the levels of glycerophosphocholine (GPCh), phosphocholine (PCh), and choline, that is, water-soluble metabolites of phosphatidylcholine (PtdCho), in human cerebrospinal fluid (CSF). Among 30 cognitively normal patients the average CSF levels of GPCh, phosphocholine and choline were 3.64, 1.28, and 1.93 microM, respectively; metabolite levels did not change with increasing age. When compared with age-matched controls, patients with Alzheimer's disease had elevated levels of all choline metabolites: GPCh was significantly increased by 76% (P<0.01), phosphocholine by 52% (P<0.05), and free choline (Ch) by 39%. Six patients with vascular dementia had lower choline and elevated phosphocholine levels, when compared to controls, but normal levels of GPCh. These data demonstrate that Alzheimer's disease is accompanied by an increased PtdCho hydrolysis in the brain. PtdCho breakdown seems to be mediated by phospholipase A2 and leads to significantly elevated levels of GPCh in CSF.

Stimulation of hippocampal acetylcholine release by hyperforin, a constituent of St. John's Wort.

Extracts of the medicinal plant St. John's Wort (Hypericum perforatum) are widely used in the therapy of affective disorders and have been reported to exert antidepressant, anxiolytic, and cognitive effects in experimental and clinical studies. We here report that hyperforin, the major active constituent of the extract, increases the release of acetylcholine from rat hippocampus in vivo as determined by microdialysis. Hippocampal acetylcholine levels were increased by 50-100% following the systemic administration of pure hyperforin at doses of 1 and 10 mg/kg. The effect was almost completely suppressed by local perfusion with calcium-free buffer or with tetrodotoxin (1 microM). We conclude that hyperforin releases hippocampal acetylcholine by an indirect mechanism of action which is calcium-dependent and requires intact neuronal communication and cell firing. Our findings suggest therapeutic efficacy of St. John's Wort extracts in central cholinergic dysfunction.

Effects of septal grafts on acetylcholine release from rat hippocampus after 192 IgG-saporin lesion.

The cholinergic inputs to the rat hippocampus were lesioned by intraseptal injections of 192 IgG-saporin. After 15 days, fetal septal cells were grafted into the hippocampus. Thirteen months later, hippocampal acetylcholine (ACh) release was studied by microdialysis. Lesioning reduced basal ACh release (100%) to 20% of normal, which was compensated for by the graft (71%). Infusion of the serotonin uptake inhibitor citalopram (100 microM) enhanced ACh release to the same extent (% of basal release) in all rat groups. Systemic injection of 8-OH-DPAT (0.5 mg/kg, SC), an agonist of 5-HT1A receptors, caused a smaller ACh release than citalopram. Acetylcholinesterase (AChE) staining and densitometric quantification revealed that the lesion-induced reduction of the AChE-staining density was compensated for by septal grafting. In conclusion, both histochemical and biochemical methods showed that cholinergic hippocampal parameters were drastically impaired by 192 IgG-saporin lesions, but were almost completely restored by septal grafting. The graft responded to intrinsic serotonergic regulation.

Potential active-site residues in polyneuridine aldehyde esterase, a central enzyme of indole alkaloid biosynthesis, by modelling and site-directed mutagenesis.

In the biosynthesis of the antiarrhythmic alkaloid ajmaline, polyneuridine aldehyde esterase (PNAE) catalyses a central reaction by transforming polyneuridine aldehyde into epi-vellosimine, which is the immediate precursor for the synthesis of the ajmalane skeleton. The PNAE cDNA was previously heterologously expressed in E. coli. Sequence alignments indicated that PNAE has a 43% identity to a hydroxynitrile lyase from Hevea brasiliensis, which is a member of the alpha/beta hydrolase superfamily. The catalytic triad, which is typical for this family, is conserved. By site-directed mutagenesis, the members of the catalytic triad were identified. For further detection of the active residues, a model of PNAE was constructed based on the X-ray crystallographic structure of hydroxynitrile lyase. The potential active site residues were selected on this model, and were mutated in order to better understand the relationship of PNAE with the alpha/beta hydrolases, and as well its mechanism of action. The results showed that PNAE is a novel member of the alpha/beta hydrolase enzyme superfamily.