Lipid mediators and their metabolism in the nucleous: implications for Alzheimer's disease

Lipid mediators are important endogenous regulators derived from enzymatic degradation of glycerophospholipids, sphingolipids, and cholesterol by phospholipases, sphingomyelinases, and cytochrome P450 hydroxylases, respectively. In neural cells, lipid mediators are associated with proliferation, differentiation, oxidative stress, inflammation, and apoptosis. A major group of lipid mediators, which originates from the enzymatic oxidation of arachidonic acid, is called eicosanoids (i.e., prostaglandins, leukotrienes, thromboxanes, and lipoxins). The corresponding lipid mediators of docosahexaenoic acid metabolism are named as docosanoids. They include resolvins, protectins (neuroprotectins), and maresins. Docosanoids produce antioxidant, anti-inflammatory, and antiapoptotic effects in brain tissue. Other glycerophospholipid-derived lipid mediators are platelet activating factor, lysophosphatidic acid, and endocannabinoids. Degradation of sphingolipids also results in the generation of sphingolipid-derived lipid mediators, such as ceramide, ceramide 1-phosphate, sphingosine, and sphingosine 1-phosphate. These mediators are involved in differentiation, growth, cell migration, and apoptosis. Similarly, cholesterol-derived lipid mediators, hydroxycholesterol, produce apoptosis. Abnormal metabolism of lipid mediators may be closely associated with pathogenesis of Alzheimer's disease.     

Emerging roles of resolvins in the resolution of inflammation and pain

Resolvins, including D and E series resolvins, are endogenous lipid mediators generated during the resolution phase of acute inflammation from the omega-3 polyunsaturated fatty acids docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). Resolvins have potent anti-inflammatory and pro-resolution actions in several animal models of inflammation. Recent findings also demonstrate that resolvin E1 and resolvin D1 can each potently dampen inflammatory and postoperative pain. This review focuses on the mechanisms by which resolvins act on their receptors in immune cells and neurons to normalize exaggerated pain via regulation of inflammatory mediators, transient receptor potential (TRP) ion channels, and spinal cord synaptic transmission. Resolvins may offer novel therapeutic approaches for preventing and treating pain conditions associated with inflammation.     

Quinolinic acid reduces the antioxidant defenses in cerebral cortex of young rats

Quinolinic acid (QA), the major metabolite of the kynurenine pathway, is found at increased concentrations in brain of patients affected by various common neurodegenerative diseases, including Huntington's disease and Alzheimer's disease. Recently, a role for QA in the pathophysiology of glutaric acidemia type I (GAI) was postulated. Considering that oxidative stress has been recently involved in the pathophysiology of the brain injury in these neurodegenerative disorders; in the present study, we investigated the in vitro effect of QA on various parameters of oxidative stress, namely total radical-trapping antioxidant potential (TRAP), total antioxidant reactivity (TAR), glutathione (GSH) levels, thiobarbituric acid-reactive substances (TBA-RS) measurement and chemiluminescence in cerebral cortex of 30-day-old rats. QA diminished the brain non-enzymatic antioxidant defenses, as determined by the reduced levels of TRAP, TAR and GSH. We also observed that QA significantly increased TBA-RS and chemiluminescence. Therefore, in vitro QA-treatment of rat cortical supernatants induced oxidative stress by reducing the tissue antioxidant defenses and increasing lipid oxidative damage, probably as a result of free radical generation. In addition, we examined the effect of QA on TBA-RS levels in the presence of glutaric acid (GA) and 3-hydroxyglutaric acid (3HGA), which are accumulated in GAI, as well as in the presence of 3-hydroxykynurenine (3HK), a tryptophan metabolite of the kynurenine pathway with antioxidant properties. It was verified that the single addition of QA or GA plus 3HGA to the incubation medium significantly stimulated in vitro lipid peroxidation. Furthermore, 3HK completely prevented the TBA-RS increase caused by the simultaneous addition of QA, GA and 3HGA. Taken together, it may be presumed that QA induces oxidative stress in the brain, which may be associated, at least in part, with the pathophysiology of central nervous system abnormalities of neurodegenerative diseases in which QA accumulates.     

Cyclooxygenases, lipoxygenases, and epoxygenases in CNS: their role and involvement in neurological disorders

Three enzyme systems, cyclooxygenases that generate prostaglandins, lipoxygenases that form hydroxy derivatives and leukotrienes, and epoxygenases that give rise to epoxyeicosatrienoic products, metabolize arachidonic acid after its release from neural membrane phospholipids by the action of phospholipase A(2). Lysophospholipids, the other products of phospholipase A(2) reactions, are either reacylated or metabolized to platelet-activating factor. Under normal conditions, these metabolites play important roles in synaptic function, cerebral blood flow regulation, apoptosis, angiogenesis, and gene expression. Increased activities of cyclooxygenases, lipoxygenases, and epoxygenases under pathological situations such as ischemia, epilepsy, Alzheimer's disease, Parkinson disease, amyotrophic lateral sclerosis, and Creutzfeldt-Jakob disease produce neuroinflammation involving vasodilation and vasoconstriction, platelet aggregation, leukocyte chemotaxis and release of cytokines, and oxidative stress. These are closely associated with the neural cell injury which occurs in these neurological conditions. The metabolic products of docosahexaenoic acid, through these enzymes, generate a new class of lipid mediators, namely docosatrienes and resolvins. These metabolites antagonize the effect of metabolites derived from arachidonic acid. Recent studies provide insight into how these arachidonic acid metabolites interact with each other and other bioactive mediators such as platelet-activating factor, endocannabinoids, and docosatrienes under normal and pathological conditions. Here, we review present knowledge of the functions of cyclooxygenases, lipoxygenases, and epoxygenases in brain and their association with neurodegenerative diseases.     

Interactions between neural membrane glycerophospholipid and sphingolipid mediators: a recipe for neural cell survival or suicide

The neural membranes contain phospholipids, sphingolipids, cholesterol, and proteins. Glycerophospholipids and sphingolipids are precursors for lipid mediators involved in signal transduction processes. Degradation of glycerophospholipids by phospholipase A(2) (PLA(2)) generates arachidonic acid (AA) and docosahexaenoic acids (DHA). Arachidonic acid is metabolized to eicosanoids and DHA is metabolized to docosanoids. The catabolism of glycosphingolipids generates ceramide, ceramide 1-phosphate, sphingosine, and sphingosine 1-phosphate. These metabolites modulate PLA(2) activity. Arachidonic acid, a product derived from glycerophospholipid catabolism by PLA(2), modulates sphingomyelinase (SMase), the enzyme that generates ceramide and phosphocholine. Furthermore, sphingosine 1-phosphate modulates cyclooxygenase, an enzyme responsible for eicosanoid production in brain. This suggests that an interplay and cross talk occurs between lipid mediators of glycerophospholipid and glycosphingolipid metabolism in brain tissue. This interplay between metabolites of glycerophospholipid and sphingolipid metabolism may play an important role in initiation and maintenance of oxidative stress associated with neurologic disorders as well as in neural cell proliferation, differentiation, and apoptosis. Recent studies indicate that PLA(2) and SMase inhibitors can be used as neuroprotective and anti-apoptotic agents. Development of novel inhibitors of PLA(2) and SMase may be useful for the treatment of oxidative stress, and apoptosis associated with neurologic disorders such as stroke, Alzheimer disease, Parkinson disease, and head and spinal cord injuries.     

Isoprostanes as biomarkers and mediators of oxidative injury in infant and adult central nervous system diseases

Isoprostanes are a family of prostaglandin-like compounds that are generated in vivo by free radical attack of esterified arachidonic acid and then released in free form in biological fluids. Since their discovery in 1990, they have been extensively used as biomarkers of lipid peroxidation and oxidative damage in an increasing number of human diseases. Few members of the isoprostane family are biologically active and could contribute to the functional consequences of oxidant injury. The present review summarises the current knowledge on formation and biological activities of these lipid peroxidation products, focusing on their role as valuable biomarkers to investigate the involvement of oxidative stress in the pathogenesis of infant and adult central nervous system diseases. In addition to isoprostanes, a new class of free radical-mediated peroxidation products, named neuroprostanes, is discussed. Neuroprostanes derive from peroxidation of docosahexaenoic acid, a polyunsatured fatty acid particularly abundant in neurons, and may represent a more selective index of brain oxidant injury than isoprostanes. In spite of some discrepancies in the results reported in different studies, isoprostane and neuroprostane levels in human biological fluids, as well as in experimental models of brain diseases, appear to be valuable indicators not only to monitor the occurrence and the causal role of oxidative stress in brain pathologies, but also for critical selection and evaluation of appropriate antioxidant therapies.     

The effects of the oral administration of fish oil concentrate on the release and the metabolism of [14C]arachidonic acid and [14C]eicosapentaenoic acid by human platelets

It has been suggested by several investigators that eicosapentaenoic acid (C20:5 omega 3, EPA) might have anti-thrombotic effects. In this experiment, the effect of the oral administration of EPA rich fish oil concentrate on platelet aggregation and the release and the metabolism of [1-14C]arachidonic acid and [(U)-14C]eicosapentaenoic acid by human platelets was studied. Eight healthy male subjects ingested 18 capsules of fish oil concentrate (EPA 1.4 g) per day for 4 weeks. Plasma and platelet concentrations of EPA markedly increased, while those of arachidonic acid (C20:4 omega 6, AA) and docosahexaenoic acid (C22:6 omega 3, DHA) did not change. Platelet aggregation induced by collagen and ADP was reduced. Collagen induced [14C]thromboxane B2 (TXB2) formation from [14C]AA prelabeled platelets decreased. There was no detectable formation of [14C]TXB3 from [14C]EPA prelabeled platelets, and the conversion of exogenous [14C]EPA to [14C]TXB3 was lower than that of [14C]AA to [14C]TXB2. The release of [14C]AA from [14C]AA prelabeled platelets by collagen was significantly decreased. These observations raise the possibility that the release of arachidonic acid from platelet lipids might be affected by the alteration of EPA content in platelets.     

Omega-3 fatty acids for the prevention of postpartum depression: negative data from a preliminary, open-label pilot study

Based on the putative relationship between depleted omega-3 fatty acids and postpartum depression, we initiated an open-label pilot study of omega-3 fatty acid supplementation with the aim of preventing postpartum depression. Euthymic pregnant females with a past history of depression in the postpartum period were started on 2960 mg of fish oil (1.4:1 eicosapentaenoic acid:docosahexaenoic acid) per day between the 34th to 36th week of pregnancy and assessed through 12 weeks postpartum. Four of seven participants had a depressive episode during the study period. No participants withdrew from the study due to adverse events. This preliminary, small, open-label pilot study failed to show promising results for the use of omega-3 fatty acid monotherapy beginning at 34 to 36 weeks gestation for the prevention of postpartum depression in patients with a prior postpartum depression history. Controlled studies are lacking.     

Neurological Benefits of Omega-3 Fatty Acids

The central nervous system is highly enriched in long-chain polyunsaturated fatty acid (PUFA) of the omega-6 and omega-3 series. The presence of these fatty acids as structural components of neuronal membranes influences cellular function both directly, through effects on membrane properties, and also by acting as a precursor pool for lipid-derived messengers. An adequate intake of omega-3 PUFA is essential for optimal visual function and neural development. Furthermore, there is increasing evidence that increased intake of the long-chain omega-3 PUFA, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), may confer benefits in a variety of psychiatric and neurological disorders, and in particular neurodegenerative conditions. However, the mechanisms underlying these beneficial effects are still poorly understood. Recent evidence also indicates that in addition to the positive effects seen in chronic neurodegenerative conditions, omega-3 PUFA may also have significant neuroprotective potential in acute neurological injury. Thus, these compounds offer an intriguing prospect as potentially new therapeutic approaches in both chronic and acute conditions. The purpose of this article is to review the current evidence of the neurological benefits of omega-3 PUFA, looking specifically at neurodegenerative conditions and acute neurological injury.     

Plasma fibrinolytic activity after ingestion of omega-3 fatty acids in human subjects

Plasma fibrinolytic activity was measured in human volunteers after 30 day periods of ingestion of a fish oil product (Max Vita) containing eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) and a wheat germ oil product containing alpha linolenic acid. Compliance was confirmed by significant increases in plasma levels of EPA and DHA and by significant falls in serum triglyceride levels. Platelet aggregation in platelet rich plasma and in whole blood was not altered significantly by fish oil or wheat germ oil. Neither fish oil or wheat germ oil caused any significant change in tissue plasminogen activator (tPA) or its inhibitor (PAI) measured enzymatically or in tPA antigen measured by an ELISA method. All these analytes (tPA, PAI, and tPA antigen) were measured before and after venous compression.

There was no evidence of enhanced fibrinolytic activity after ingestion of omega-3 fatty acids in fish oil or in wheat germ oil.     

Red blood cell and adipose tissue fatty acids in mild inactive multiple sclerosis

The fatty acid profiles of phosphatidyl ethanolamine (PE) and phosphatidyl choline (PC) of the red blood cells of 30 patients with mild inactive multiple sclerosis (MS) and 30 healthy controls were studied by gas chromatography. The groups were well matched for factors likely to influence tissue lipid levels, including diet. The MS patients showed a significant reduction in PE eicosapentaenoic acid (p = 0.009) especially in women, and an increase in both PE dihomo-gamma-linolenic acid (p = 0.004) and PC stearic acid (p = 0.04). No reduction in linoleic acid was observed in either the PC or PE fractions of the MS subjects. A similar study of the fatty acid profile in adipose tissue in 26 MS and 35 healthy controls found no detectable eicosapentaenoic acid in either group. However, whereas docosahexaenoic acid was not detectable in any MS patient, 40% of the controls had measurable levels varying from to 0.1 to 0.3% of total estimated fatty acid (p = 0.0003). No reduction in linoleic acid in MS subjects was observed. Supplementation with oral fish body oil demonstrated that n-3 fatty acids were incorporated into red blood cells over 5 weeks and this occurred equally in MS and controls. The effects of oral supplementation on adipose tissue were studied after 1 and 2 years. Whereas many fatty acids such as linoleic acid were raised at 1 year, but did not rise subsequently, eicosapentaenoic acid and docosahexaenoic acid continued to rise through the 2-year period.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ethyl-eicosapentaenoate (E-EPA) attenuates motor impairments and inflammation in the MPTP-probenecid mouse model of Parkinson's disease

Parkinson's disease (PD) is a neurodegenerative disorder, characterized by hypokinesia, but also mood and cognitive disorders. Neuropathologically, PD involves loss of nigrostriatal dopamine (DA) and secondary non-dopaminergic abnormalities. Inflammation may contribute to PD pathogenesis, evident by increased production of pro-inflammatory cytokines. PD onset has been positively associated with dietary intake of omega-(n)-6 polyunsaturated fatty acids (PUFA). On the other hand, omega-(n)-3 PUFA may benefit PD. One of these n-3 PUFA, eicosapentaenoic acid (EPA), is a neuroprotective lipid with anti-inflammatory properties, but its neuroprotective effects in PD are unknown. Thus, we presently tested the hypothesis that EPA can protect against behavioral impairments, neurodegeneration and inflammation in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-probenecid (MPTP-P) mouse model of PD. MPTP-P injections caused hypokinesia in the rotorod and pole test, hyperactivity in the open field, and impaired mice on the cued version (procedural memory) of the Morris water maze. MPTP-P caused a loss of nigrostriatal DA and altered neurochemistry in the frontal cortex and hippocampus. Furthermore, striatal levels of pro-inflammatory cytokines were increased, while the brain n-3/n-6 lipid profile remained unaltered. Feeding mice a 0.8% ethyl-eicosapentaenoate (E-EPA) diet prior to MPTP-P injections increased brain EPA and docosapentaenoic acid (DPA) but not docosahexaenoic acid (DHA) or n-6 PUFA. The diet attenuated the hypokinesia induced by MPTP-P and ameliorated the procedural memory deficit. E-EPA also suppressed the production of pro-inflammatory cytokines. However, E-EPA did not prevent nigrostriatal DA loss. Based on this partial protective effect of E-EPA, further testing may be warranted.     

Omega-3 polyunsaturated fatty acids in Alzheimer's disease: key questions and partial answers

The current rise in the prevalence of Alzheimer's disease (AD) is unfortunately not matched by new treatment options. In the last 10 years, epidemiological, preclinical and clinical data have enlightened the possible preventive action of omega-3 polyunsaturated fatty acids (n-3 PUFA) in AD and other diseases. While the contribution of recent studies to our general knowledge is priceless, many important new questions have been raised. In the present review, we aim at addressing some of these timely interrogations. First, the transport of n-3 PUFA across the blood-brain barrier is underscored based on preclinical data. Second, the relative contribution of two neuroactive n-3 PUFA found in fish oil, docosahexaenoic acid (DHA; 22:6 n-3) and eicosapentaenoic acid (EPA, 20:5 n-3), remains unclear and is reviewed. Third, clinical trials on neurodegenerative diseases consistently remind us that treating early is critical, and this is likely to be the case with n-3 PUFA in AD as well. Fourth, we draw attention to the possibility that the current knowledge translation approach to make health recommendations might have to be adapted to non-patentable endogenous compounds like n-3 PUFA. We propose that answers to these critical questions will be instrumental toward a rational use of n-3 PUFA in AD.     

Functional Vitamin E deficiency in ApoE4 patients with Alzheimer's disease

Oxidative stress has been implicated in the development of Alzheimer's disease (AD). Consequently, antioxidant therapies including Vitamin E (VitE) supplementation for both prevention and treatment of neurodegenerative diseases currently appears to be a promising avenue of research. The aim of the present study was to examine the relationship between AD and the ApoE phenotype, lipid parameters and VitE levels in a large cohort of elderly subjects. No absolute deficit was observed in plasma VitE levels. However in AD, ApoE4 is not associated with an increase in total cholesterol (TC) and VitE levels. Moreover, our results suggest that oxidative stress-induced injury and protection by VitE in AD are related to the ApoE phenotype. Our study strongly supports the hypothesis of an impairment of lipophilic antioxidant delivery to neuronal cells in AD leading to a tissular antioxidant deficiency which could facilitate oxidative stress.     

Neuroprotection mediated by natural products and their chemical derivatives

Neuronal injuries can lead to various diseases such as neurodegenerative diseases, stroke, trauma, ischemia and, more specifically, glaucoma and optic neuritis. The cellular mechanisms that regulate neuronal death include calcium influx and calcium overload, excitatory amino acid release, oxidative stress, inflammation and microglial activation. Much attention has been paid to the effective prevention and treatment of neuroprotective drugs by natural products. This review summarizes the neuroprotective aspects of natural products, extracted from Panax ginseng, Camellia sinensis, soy and some other plants, and some of their chemical derivatives. Their antioxidative and anti-inflammatory action and their inhibition of apoptosis and microglial activation are assessed. This will provide new directions for the development of novel drugs and strategies to treat neurodegenerative diseases.     

Preventive and therapeutic potential of ascorbic acid in neurodegenerative diseases

In this review, we summarize the involvement of ascorbic acid in neurodegenerative diseases by presenting available evidence on the behavioral and biochemical effects of this compound in animal models of neurodegeneration as well as the use of ascorbic acid as a therapeutic approach to alleviate neurodegenerative progression in clinical studies. Ascorbate, a reduced form of vitamin C, has gained interest for its multiple functions and mechanisms of action, contributing to the homeostasis of normal tissues and organs as well as to tissue regeneration. In the brain, ascorbate exerts neuromodulatory functions and scavenges reactive oxygen species generated during synaptic activity and neuronal metabolism. These are important properties as redox imbalance and abnormal protein aggregation constitute central mechanisms implicated in the pathogenesis of neurodegenerative diseases, including Alzheimer's, Parkinson's, and Huntington's diseases, multiple sclerosis, and amyotrophic lateral sclerosis. Indeed, several studies have indicated an association between low serum ascorbate concentrations and neurodegeneration. Moreover, ascorbic acid is a suitable candidate for supplying either antioxidant defense or modulation of neuronal and astrocytic metabolism under neurodegenerative conditions. Ascorbic acid acts mainly by decreasing oxidative stress and reducing the formation of protein aggregates, which may contribute to the reduction of cognitive and/or motor impairments observed in neurodegenerative processes. Although several studies support a possible role of ascorbic acid administration against neurodegeneration, more researches are essential to substantiate the existing results and accelerate the knowledge in this field.     

Association of heat shock proteins and neuronal membrane components with lipid rafts from the rat brain

Lipid rafts are specialized plasma membrane microdomains enriched in cholesterol and sphingolipids that serve as major assembly and sorting platforms for signal transduction complexes. Constitutively expressed heat shock proteins Hsp90, Hsc70, Hsp60, and Hsp40 and a range of neurotransmitter receptors are present in lipid rafts isolated from rat forebrain and cerebellum. Depletion of cholesterol dissociates these proteins from lipid rafts. After hyperthermic stress, flotillin-1, a lipid raft marker protein, does not show major change in levels. Stress-inducible Hsp70 is detected in lipid rafts at 1 hr posthyperthermia, with the peak levels attained at 24 hr, suggesting that Hsp70 may play roles in maintaining the stability of lipid raft-associated signal transduction complexes following neural stress.     

The role of nutrition in Alzheimer's disease: epidemiological evidence

The prevalence of Alzheimer's disease (AD) increases exponentially with age but there is limited knowledge of the modifiable risk factors for AD. However, there is growing evidence for possible dietary risk factors in the development of AD and cognitive decline with age, such as antioxidant nutrients, fish, dietary fats, and B-vitamins. Numerous animal and laboratory studies have shown that antioxidant nutrients can protect the brain from oxidative and inflammatory damage, but there are limited data available from epidemiological studies. There is more substantial epidemiological evidence from a number of recent studies that demonstrate a protective role of omega-3 fatty acids, such as docosahexaenoic acid, in AD and cognitive decline. This review will focus on epidemiological evidence investigating the relationship between nutrition and AD, focusing particularly on the roles of dietary fats and antioxidants.     

Phospholipase A2-generated lipid mediators in the brain: the good, the bad, and the ugly.

Phospholipase A2 (PLA2) generates arachidonic acid, docosahexaenoic acid, and lysophospholipids from neural membrane phospholipids. These metabolites have a variety of physiological effects by themselves and also are substrates for the synthesis of more potent lipid mediators such as eicosanoids, platelet activating factor, and 4-hydroxynonenal (4-HNE). At low concentrations, these mediators act as second messengers. They affect and modulate several cell functions, including signal transduction, gene expression, and cell proliferation, but at high concentrations, these lipid mediators cause neurotoxicity. Among the metabolites generated by PLA2, 4-HNE is the most cytotoxic metabolite and is associated with the apoptotic type of neural cell death. Levels of 4-HNE are markedly increased in neurological disorders such as Alzheimer disease, Parkinson disease, ischemia, spinal cord trauma, and head injury. The purpose of this review is to summarize and integrate the vast literature on metabolites generated by PLA2 for a wider audience. The authors hope that this discussion will jump-start more studies not only on the involvement of PLA2 in neurological disorders but also on the importance of PLA2-generated lipid mediators in physiological and pathological processes.     

Homocysteine induces oxidative stress,inflammatory infiltration,fibrosis and reduces glycogen/glycoprotein content in liver of rats

Hyperhomocysteinemia has been related to various diseases, including homocystinuria, neurodegenerative and hepatic diseases. In the present study we initially investigated the effect of chronic homocysteine administration on some parameters of oxidative stress, named total radical-trapping antioxidant potential, total antioxidant reactivity, catalase activity, chemiluminescence, thiobarbituric acid-reactive substances, and total thiol content in liver of rats. We also performed histological analysis, evaluating steatosis, inflammatory infiltration, fibrosis, and glycogen/glycoprotein content in liver tissue sections from hyperhomocysteinemic rats. Finally, we evaluated the activities of aminotransferases in liver and plasma of hyperhomocysteinemic rats. Wistar rats received daily subcutaneous injection of Hcy from their 6th to their 28th day of life. Twelve hours after the last injection the rats were sacrificed, liver and plasma were collected. Hyperhomocysteinemia decreased antioxidant defenses and total thiol content, and increased lipid peroxidation in liver of rats, characterizing a reliable oxidative stress. Histological analysis indicated the presence of inflammatory infiltrate, fibrosis and reduced content of glycogen/glycoprotein in liver tissue sections from hyperhomocysteinemic rats. Aminotransferases activities were not altered by homocysteine. Our data showed a consistent profile of liver injury elicited by homocysteine, which could contribute to explain, at least in part, the mechanisms involved in human liver diseases associated to hyperhomocysteinemia.