Effects of prenatal nutrient supplementation and early life exposures on neurodevelopment at age 10: a randomised controlled trial - the COPSYCH study protocol.

INTRODUCTION: Nutrient deficiency and immune and inflammatory disturbances in early life may compromise neurodevelopment and be implicated in the aetiology of psychiatric disorders. However, current evidence is limited by its predominantly observational nature. COpenhagen Prospective Study on Neuro-PSYCHiatric Development (COPSYCH) is a research alliance between Copenhagen Prospective Studies on Asthma in Childhood (COPSAC) and Center for Clinical Intervention and Neuropsychiatric Schizophrenia Research with the overall aim to investigate effects of prenatal and early life exposures on neurodevelopment at 10 years. COPSYCH will investigate the impact of prenatal n-3 long-chain polyunsaturated fatty acids (n-3 LCPUFA) and high-dose vitamin D supplementation on neurodevelopment reflected by brain development, neurocognition and psychopathology. Moreover, the neurodevelopmental impact of early life exposures such as infections, low grade inflammation and the gut microbiome will be scrutinised. METHODS AND ANALYSIS: COPSYCH is based on the prospective and ongoing COPSAC2010 birth cohort of 700 mother-child pairs. Randomised controlled trials of supplementation with n-3 LCPUFA and/or high-dose vitamin D or placebo in the third trimester were embedded in a factorial 2×2 design (ClinicalTrials.gov: NCT01233297 and NCT00856947). This unique cohort provides deep phenotyping data from 14 previous clinical follow-up visits and exposure assessments since birth. The ongoing 10-year visit is a 2-day visit. Day 1 includes a comprehensive neurocognitive examination, and assessment of psychopathological dimensions, and assessment of categorical psychopathology. Day 2 includes acquisition of brain structural, diffusion and functional sequences using 3 Tesla MRI. Study outcomes are neurocognitive, psychopathological and MRI measures. ETHICS AND DISSEMINATION: This study has been approved by the Danish National Committee on Health Research Ethics and The Danish Data Protection Agency. The study is conducted in accordance with the guiding principles of the Declaration of Helsinki. Parents gave written informed consent before enrolment.

Differential effect of quetiapine and lithium on functional connectivity of the striatum in first episode mania.

Mood disturbances seen in first-episode mania (FEM) are linked to disturbed functional connectivity of the striatum. Lithium and quetiapine are effective treatments for mania but their neurobiological effects remain largely unknown. We conducted a single-blinded randomized controlled maintenance trial in 61 FEM patients and 30 healthy controls. Patients were stabilized for a minimum of 2 weeks on lithium plus quetiapine then randomly assigned to either lithium (serum level 0.6 mmol/L) or quetiapine (dosed up to 800 mg/day) treatment for 12 months. Resting-state fMRI was acquired at baseline, 3 months (patient only) and 12 months. The effects of treatment group, time and their interaction, on striatal functional connectivity were assessed using voxel-wise general linear modelling. At baseline, FEM patients showed reduced connectivity in the dorsal (p = 0.05) and caudal (p = 0.008) cortico-striatal systems when compared to healthy controls at baseline. FEM patients also showed increased connectivity in a circuit linking the ventral striatum with the medial orbitofrontal cortex, cerebellum and thalamus (p = 0.02). Longitudinally, we found a significant interaction between time and treatment group, such that lithium was more rapid, compared to quetiapine, in normalizing abnormally increased functional connectivity, as assessed at 3-month and 12-month follow-ups. The results suggest that FEM is associated with reduced connectivity in dorsal and caudal corticostriatal systems, as well as increased functional connectivity of ventral striatal systems. Lithium appears to act more rapidly than quetiapine in normalizing hyperconnectivity of the ventral striatum with the cerebellum. The study was registered on the Australian and New Zealand Clinical Trials Registry (ACTRN12607000639426). http://www.anzctr.org.au.

White Matter Disruptions in Schizophrenia Are Spatially Widespread and Topologically Converge on Brain Network Hubs.

White matter abnormalities associated with schizophrenia have been widely reported, although the consistency of findings across studies is moderate. In this study, neuroimaging was used to investigate white matter pathology and its impact on whole-brain white matter connectivity in one of the largest samples of patients with schizophrenia. Fractional anisotropy (FA) and mean diffusivity (MD) were compared between patients with schizophrenia or schizoaffective disorder (n = 326) and age-matched healthy controls (n = 197). Between-group differences in FA and MD were assessed using voxel-based analysis and permutation testing. Automated whole-brain white matter fiber tracking and the network-based statistic were used to characterize the impact of white matter pathology on the connectome and its rich club. Significant reductions in FA associated with schizophrenia were widespread, encompassing more than 40% (234ml) of cerebral white matter by volume and involving all cerebral lobes. Significant increases in MD were also widespread and distributed similarly. The corpus callosum, cingulum, and thalamic radiations exhibited the most extensive pathology according to effect size. More than 50% of cortico-cortical and cortico-subcortical white matter fiber bundles comprising the connectome were disrupted in schizophrenia. Connections between hub regions comprising the rich club were disproportionately affected. Pathology did not differ between patients with schizophrenia and schizoaffective disorder and was not mediated by medication. In conclusion, although connectivity between cerebral hubs is most extensively disturbed in schizophrenia, white matter pathology is widespread, affecting all cerebral lobes and the cerebellum, leading to disruptions in the majority of the brain's fiber bundles.

Dopamine, fronto-striato-thalamic circuits and risk for psychosis.

A series of parallel, integrated circuits link distinct regions of prefrontal cortex with specific nuclei of the striatum and thalamus. Dysfunction of these fronto-striato-thalamic systems is thought to play a major role in the pathogenesis of psychosis. In this review, we examine evidence from human and animal investigations that dysfunction of a specific dorsal fronto-striato-thalamic circuit, linking the dorsolateral prefrontal cortex, dorsal (associative) striatum, and mediodorsal nucleus of the thalamus, is apparent across different stages of psychosis, including prior to the onset of a first episode, suggesting that it represents a candidate risk biomarker. We consider how abnormalities at distinct points in the circuit may give rise to the pattern of findings seen in patient populations, and how these changes relate to disruptions in dopamine, glutamate and GABA signaling.

Altered striatal functional connectivity in subjects with an at-risk mental state for psychosis.

Recent functional imaging work in individuals experiencing an at-risk mental state (ARMS) for psychosis has implicated dorsal striatal abnormalities in the emergence of psychotic symptoms, contrasting with earlier findings implicating the ventral striatum. Our aims here were to characterize putative dorsal and ventral striatal circuit-level abnormalities in ARMS individuals using resting-state functional magnetic resonance imaging (fMRI) and to investigate their relationship to positive psychotic symptoms. Resting-state fMRI was acquired in 74 ARMS subjects and 35 matched healthy controls. An established method for mapping ventral and dorsal striatal functional connectivity was used to examine corticostriatal functional integrity. Positive psychotic symptoms were assessed using the Comprehensive Assessment of At-Risk Mental State and the Positive and Negative Syndrome Scale. Compared with healthy controls, ARMS subjects showed reductions in functional connectivity between the dorsal caudate and right dorsolateral prefrontal cortex, left rostral medial prefrontal cortex, and thalamus, and between the dorsal putamen and left thalamic and lenticular nuclei. ARMS subjects also showed increased functional connectivity between the ventral putamen and the insula, frontal operculum, and superior temporal gyrus bilaterally. No differences in ventral striatal (ie, nucleus accumbens) functional connectivity were found. Altered functional connectivity in corticostriatal circuits were significantly correlated with positive psychotic symptoms. Together, these results suggest that risk for psychosis is mediated by a complex interplay of alterations in both dorsal and ventral corticostriatal systems.

Neurological soft signs are not "soft" in brain structure and functional networks: evidence from ALE meta-analysis.

BACKGROUND: Neurological soft signs (NSS) are associated with schizophrenia and related psychotic disorders. NSS have been conventionally considered as clinical neurological signs without localized brain regions. However, recent brain imaging studies suggest that NSS are partly localizable and may be associated with deficits in specific brain areas. METHOD: We conducted an activation likelihood estimation meta-analysis to quantitatively review structural and functional imaging studies that evaluated the brain correlates of NSS in patients with schizophrenia and other psychotic disorders. Six structural magnetic resonance imaging (sMRI) and 15 functional magnetic resonance imaging (fMRI) studies were included. RESULTS: The results from meta-analysis of the sMRI studies indicated that NSS were associated with atrophy of the precentral gyrus, the cerebellum, the inferior frontal gyrus, and the thalamus. The results from meta-analysis of the fMRI studies demonstrated that the NSS-related task was significantly associated with altered brain activation in the inferior frontal gyrus, bilateral putamen, the cerebellum, and the superior temporal gyrus. CONCLUSIONS: Our findings from both sMRI and fMRI meta-analyses further support the conceptualization of NSS as a manifestation of the "cerebello-thalamo-prefrontal" brain network model of schizophrenia and related psychotic disorders.

Frontal delta power associated with negative symptoms in ultra-high risk individuals who transitioned to psychosis.

It has recently been shown that treatment with long-chain omega-3 polyunsaturated fatty acids (PUFAs) could decrease the rate of transition to psychosis, and improve psychiatric symptoms and global functioning in people at ultra-high risk (UHR) for psychosis. Previous studies have suggested that resting state brain activity measured with electroencephalography (EEG) may represent an objective biomarker of changes in neural function associated with supplementation with omega-3 PUFAs. It has also been proposed that although resting state EEG cannot, by itself, predict transition to psychosis in UHR individuals, the combination of resting state EEG with negative symptoms may be a valid predictor of transition. The present study investigated whether treatment with omega-3 PUFAs influenced resting state EEG in UHR participants, and whether or not the association of the participants' resting state EEG with their levels of negative symptoms was dependent on their transition status. The brain activity of 73 UHR participants was recorded in the context of a randomized, placebo-controlled trial of the effects of supplementation with omega-3 PUFAs. The UHR participants who subsequently transitioned to psychosis (UHR+) did not differ from those who did not transition (UHR-) in terms of resting state EEG power in any frequency band. However, negative symptom scores were associated with increased delta activity in the frontal region of the UHR+ participants, but not in the UHR- participants. Treatment with omega-3 PUFAs did not induce changes in resting state EEG in either group. The results suggest that decreased frontal delta activity, in combination with high levels of negative symptoms, may be a risk factor for subsequent transition to psychosis in UHR individuals.

Midline brain abnormalities in established bipolar affective disorder.

BACKGROUND: Morphologic changes of cortico-limbic regions have been reported in bipolar disorder, but it remains unclear whether midline brain abnormalities relevant to cortico-limbic connectivity are also present. METHODS: We used magnetic resonance imaging to investigate the size of the adhesio interthalamica (AI) and cavum septi pellucidi (CSP), as well as third ventricular volume, in 26 patients with bipolar I disorder and 24 matched controls. RESULTS: CSP length and prevalence of a large CSP did not differ between the groups, but bipolar patients had significantly shorter AI and larger third ventricles compared to controls. LIMITATIONS: A comprehensive investigation of medication effects was not possible due to incomplete medication data. CONCLUSIONS: These findings implicate a role for the AI and connected brain regions in the neurobiology of bipolar disorder.

Midline brain structures in teenagers with first-presentation borderline personality disorder.

Brain morphologic changes have been reported in borderline personality disorder (BPD), but it remains largely unknown whether BPD is associated with midline brain abnormalities. We used magnetic resonance imaging to investigate the length of the adhesio interthalamica (AI) and cavum septum pellucidum (CSP) as well as third ventricular volume in 20 teenagers with first-presentation BPD and 20 healthy controls. While the CSP length did not differ between the groups, the AI was significantly shorter in BPD patients than in controls. Furthermore, the BPD patients had a significantly larger third ventricle than controls. These preliminary findings suggest that ongoing neuroimaging studies should further evaluate a potential involvement of midline brain structures in the pathogenesis of BPD.

Reconciling neuroimaging and neuropathological findings in schizophrenia and bipolar disorder.

PURPOSE OF REVIEW: Although structural magnetic resonance imaging (sMRI) and neuropathological investigations offer complementary information that can be used to formulate and test hypotheses about pathophysiological mechanisms in psychiatric disorders, the findings from these two fields are seldom integrated in a systematic manner. In this study, we overview recent sMRI findings in schizophrenia and bipolar disorder and consider how they relate to neuropathological data. RECENT FINDINGS: sMRI research indicates that schizophrenia is associated with volumetric reductions in a network of frontal, temporal, limbic, striatal, and thalamic regions. Some of these abnormalities are apparent prior to psychosis onset and may progress with ongoing illness. sMRI findings in bipolar disorder have been more variable, with both volumetric increases and decreases being reported across several brain regions at different illness stages. Neuropathological studies of both patient groups suggest the cellular changes associated with these volumetric differences affect diverse tissue compartments in a regionally heterogeneous way. SUMMARY: These findings suggest that any putative pathophysiological mechanism in schizophrenia or bipolar disorder should account for the dynamic, complex, and regionally heterogeneous brain abnormalities seen in these patients. We contend that greater integration of the findings from these two fields will facilitate more targeted and hypothesis-driven research in the future.

Adhesio interthalamica in individuals at high-risk for developing psychosis and patients with psychotic disorders.

Abnormal neurodevelopment in midline structures such as the adhesio interthalamica (AI) has been reported in psychotic disorders, but it is unknown whether individuals at risk for the disorder share the AI findings observed in patients with florid psychosis. Magnetic resonance imaging of 162 patients with first-episode psychosis (FEP), 89 patients with chronic schizophrenia, 135 individuals at ultra high-risk (UHR) of psychosis (of whom 39 later developed psychosis), and 87 healthy controls were used to investigate the length and prevalence of the AI. The relation of the AI length to lateral ventricular enlargement was also explored. The patients with FEP and chronic schizophrenia as well as UHR individuals had a shorter AI than the controls, but there was no difference in the AI findings between the UHR individuals who did and did not subsequently develop psychosis. There was a negative correlation between the AI length and lateral ventricular volume in all the diagnostic groups. The absence of the AI was more common in the chronic schizophrenia patients when compared with all other groups. These results support the notion that the AI absence or shorter length could be a neurodevelopmental marker related to vulnerability to psychopathology, but also suggest that schizophrenia patients may manifest progressive brain changes related to ongoing atrophy of the AI after the onset.

The positive effect of integrated care on depressive symptoms in stroke survivors.

BACKGROUND: Depressive symptoms occur in approximately one-third of stroke patients. We sought to evaluate whether an integrated model of stroke care and secondary prevention reduced depressive symptomatology in stroke survivors. METHODS: The integrated care (IC) model is a multifaceted program that provides ongoing collaboration between a specialist stroke service and primary care physicians, using telephone tracking, a bi-directional information feedback loop, management of vascular risk factors, and regular screening for depressive symptoms. RESULTS: Patients exposed to the IC model exhibited significantly fewer depressive symptoms than controls at 12 months post stroke (as measured by the PHQ-9 screening tool; p = 0.006). At 12 months, 30/91 (33%) of the treatment group had depressive symptoms, compared to 52/95 (55%) of the control group (p = 0.003). With other variables adjusted for, the major associates of being depressed at 12 months were group allocation and physical disability. CONCLUSION: The integrated care approach provides a framework for detecting and monitoring depressive symptoms, and appears to be protective against post-stroke depression.

A 1H-MRS investigation of the medial temporal lobe in antipsychotic-naïve and early-treated first episode psychosis.

Schizophrenia is associated with significant brain abnormalities, including changes in brain metabolites as measured by proton magnetic resonance spectroscopy (MRS). What remains unclear is the extent to which these changes are a consequence of the emergence of psychotic disorders or the result of treatment with antipsychotic medication. We assessed 34 patients with first episode psychosis (15 antipsychotic naïve) and 19 age- and gender-matched controls using short-echo MRS in the medial temporal lobe bilaterally. Overall, there were no differences in any metabolite, regardless of treatment status. However, when the analysis was limited to patients with a diagnosis of schizophrenia, schizophreniform or schizoaffective disorder, significant elevations of creatine/phosphocreatine (Cr/PCr) and myo-inositol (mI) were found in the treated group. These data indicate a relative absence of temporal lobe metabolic abnormalities in first episode psychosis, but suggest that some treatment-related changes in mI might be apparent in patients with schizophrenia-spectrum diagnoses. Seemingly illness-related Cr/PCr elevations were also specific to the diagnosis of schizophrenia-spectrum disorder and seem worthy of future study.

Ethyl-eicosapentaenoic acid in first-episode psychosis. A 1H-MRS study.

Ethyl-eicosapentaenoic acid (E-EPA) is an omega-3 fatty acid that has been used in a range of neuropsychiatric conditions with some benefits. However, its mechanism of action is unknown. Here, we investigate its effects on in vivo brain metabolism in first-episode psychosis (FEP). Proton magnetic resonance spectroscopy at 3 T was performed in the temporal lobes of 24 FEP patients before and after 12 weeks of treatment in the context of a larger double-blind, placebo-controlled E-EPA augmentation study. Treatment group effects for glutathione (F1,12=6.1, p=0.03), and a hemisphere-by-group interaction for glutamine/glutamate (F1,20=4.4, p=0.049) were found. Glutathione increased bilaterally and glutamate/glutamine increased in the left hemisphere following E-EPA administration. Improvement in negative symptoms correlated with metabolic brain changes, particularly glutathione (r=-0.57). These results suggest that E-EPA augmentation alters glutathione availability and modulates the glutamine/glutamate cycle in early psychosis, with some of the metabolic brain changes being correlated with negative symptom improvement. Larger confirmatory studies of these postulated metabolic brain effects of E-EPA are warranted.

Early maternal deprivation reduces prepulse inhibition and impairs spatial learning ability in adulthood: no further effect of post-pubertal chronic corticosterone treatment.

Prolonged maternal deprivation leads to long-term alterations in hypothalamic-pituitary-adrenal (HPA) axis activity, disturbances of auditory information processing and neurochemical changes in the adult brain, some of which are similar to that observed in schizophrenia. Here we report the adult behavioural effects of maternal deprivation (12h on postnatal days 9 and 11) in Wistar rats on paradigms of auditory information processing (prepulse inhibition), sensitivity to dopamimetics (amphetamine-induced hyper-locomotion) and cognition (T-maze delayed alternation and Morris water-maze). In addition, we examined the long-lasting effect of chronic 21-day corticosterone treatment during the post-pubertal period (i.e., postnatal days 56-76) on each of these behavioural paradigms in maternally deprived and control rats. Behavioural testing commenced 2 weeks after the termination of corticosterone treatment. Maternal deprivation led to a significant reduction in PPI and impaired spatial learning ability in adulthood, but did not affect the behavioural response to amphetamine. Post-pubertal chronic corticosterone treatment did not have any major long-lasting effects on any of the behavioural measures in either maternally deprived or control rats. Our findings further support maternal deprivation as an animal model of specific aspects of schizophrenia.

Evidence of altered prefrontal-thalamic circuitry in schizophrenia: an optimized diffusion MRI study.

MRI diffusion tensor imaging (DTI), optimized for measuring the trace of the diffusion tensor, was used to investigate microstructural changes in the brains of 12 individuals with schizophrenia compared with 12 matched control subjects. To control for the effects of anatomic variation between subject groups, all participants' diffusion images were nonlinearly registered to standard anatomical space. Significant statistical differences in mean diffusivity (MD) measures between the two groups were determined on a pixel-by-pixel basis, using Gaussian random field theory. We found significantly elevated MD measures within temporal, parietal and prefrontal cortical regions in the schizophrenia group (P > 0.001), especially within the medial frontal gyrus and anterior cingulate. The dorsal medial and anterior nucleus of the thalamus, including the caudate, also exhibited significantly increased MD in the schizophrenia group (P > 0.001). This study has shown for the first time that MD measures offer an alternative strategy for investigating altered prefrontal-thalamic circuitry in schizophrenia.

Increased pituitary volume in antipsychotic-free and antipsychotic-treated patients of the Æsop First-Onset Psychosis Study

Subjects at their first psychotic episode show an enlarged volume of the pituitary gland, but whether this is due to hypothalamic–pituitary–adrenal (HPA) axis hyperactivity, or to stimulation of the prolactin-secreting cells by antipsychotic treatment, is unclear. We measured pituitary volume, using 1.5-mm, coronal, 1.5 T, high-resolution MRI images, in 78 patients at the first psychotic episode and 78age- and gender-matched healthy controls. In all, 18 patients were antipsychotic-free (12 of these were antipsychotic-naý¨ve), 26 werereceiving atypical antipsychotics, and 33 were receiving typical antipsychotics. As hypothesized, patients had a larger pituitary volume than controls (+22percent , p=0.001). When divided by antipsychotic treatment, and compared to controls, the pituitary volume was 15 percent larger in antipsychotic-free patients (p¼0.028), 17 percent larger in patients receiving atypicals (p¼0.01), and 30 percent larger in patients receiving typicals (p=0.001). Patients receiving typicals not only had the largest pituitary volume compared to controls but also showed a trend for a larger pituitary volume compared to the other patients grouped together (11 percent, p¼0.08). When divided by diagnosis, and compared to controls, the pituitary volume was 24 percent larger in patients with schizophrenia/schizophreniform disorder (n¼40, p=0.001), 19 percent larger in depressed patients (n¼13, p¼0.022), 16 percent larger in bipolar patients (n¼16, p¼0.037), and 12 percent larger in those with other psychoses (n¼9, p¼0.2). In conclusion, the first-episode of a psychotic disorder is associated with a larger pituitary independently of the presenceof antipsychotic treatment, and this could be due to activation of the HPA axis. Typical antipsychotics exert an additional enlarging effecton pituitary volume, likely to be related to activation of prolactin-secreting cells...

Thalamic and caudate volumes in monozygotic twins discordantfor schizophrenia.

OBJECTIVE: The thalamus and caudate nucleus are key subcortical structures in the fronto-striato-thalamic pathways that have been implicated in the pathogenesis of schizophrenia. Previous studies have been inconsistent in identifying structural and functional abnormalities in these structures. However, methodologies in these studies have been unreliable and some have not adequately matched patients and controls. METHODS: Using algebraically-manipulated double-echomagnetic resonance (MR) images, we developed a reliable method to estimate caudate and thalamic volumes in a group of 13 monozygotic(MZ) twins; eight discordant for schizophrenia and five normal.Initially, volumes were measured on four image types: proton density(PD), T2-weighted, summed (PD + T2) and subtracted (PD-T2) to determine the most reliable method. RESULTS: There was a significant method by region interaction, where caudate volumes measured on PD images were significantly larger than those measured on T2-weighted images, while the opposite was found for thalamic volumes. However, there was no interaction of method by diagnosis. Test-retest reliability was highest for the summed images. Using summed images to measure the volumes of the caudate and thalamus in each twin, we found significantly increased caudate volumes in affected twins compared to their unaffected cotwins,but no significant difference in thalamic volume. CONCLUSIONS: Our results in a small sample of MZ twins discordant for schizophrenia do not support the presence of structural abnormalities in the thalamus. The findings in the caudate are consistent with previously reported effects of antipsychotic medication. We also report a reliable method for assessing the volumes of subcortical structures. However, volumetric estimates of brain structures may be dependent on which method is used and the structure being assessed. Such interactions need to be considered in future studies investigating brain structural abnormalities in schizophrenia and other disorders.

Implications of lipid biology for the pathogenesis of schizophrenia.

OBJECTIVE: Preclinical and clinical data suggest that lipid biology is integral to brain development and neurodegeneration. Both aspects are proposed as being important in the pathogenesis of schizophrenia. The purpose of this paper is to examine the implications of lipid biology, in particular the role of essential fatty acids (EFA), for schizophrenia. METHODS: Medline databases were searched from 1966 to 2001 followed by the cross-checking of references. RESULTS: Most studies investigating lipids in schizophrenia described reduced EFA, altered glycerophospholipids and an increased activity of a calcium-independent phospholipase A2 in blood cells and in post-mortem brain tissue. Additionally, in vivo brain phosphorus-31 Magnetic Resonance Spectroscopy (31P-MRS) demonstrated lower phosphomonoesters (implying reduced membrane precursors) in first- and multi-episode patients. In contrast, phosphodiesters were elevated mainly in first-episode patients (implying increased membrane breakdown products), whereas inconclusive results were found in chronic patients. EFA supplementation trials in chronic patient populations with residual symptoms have demonstrated conflicting results. More consistent results were observed in the early and symptomatic stages of illness, especially if EFA with a high proportion of eicosapentaenoic acid was used. CONCLUSION: Peripheral blood cell, brain necropsy and 31P-MRS analysis reveal a disturbed lipid biology, suggesting generalized membrane alterations in schizophrenia. 31P-MRS data suggest increased membrane turnover at illness onset and persisting membrane abnormalities in established schizophrenia. Cellular processes regulating membrane lipid metabolism are potential new targets for antipsychotic drugs and might explain the mechanism of action of treatments such as eicosapentaenoic acid.