Like other phospholipid metabolites, N-acyl PEs and NAEs accumulate rapidly in the CNS after the inception of an ischemic injury . This response, likely caused by postischemic rises in the concentration of free Ca21 , may confuse quantitative analysis unless appropriate precautions are taken to prevent it. In our experiments, we have immersed the heads of the animals in liquid nitrogen within 2 sec of decapitation, a procedure that has been used extensively to determine basal levels of free fatty acids and diacylglycerols in brain tissue . Then, by using an analytical approach that combines HPLC purification and GC/MS identification, we have shown that both anandamide and N-arachidonoyl PE are present in brain under these conditions. On average, we measured 11 6 7 pmol of anandamide/gm of wet brain tissue, a value very similar to those obtained very recently by Schmid et al. in pig brain and cow brain , by Sugiura et al. in rat brain , and by Felder et al. in various regions of rat and human brain . Such anandamide levels should be well within the detection limit of the GC/MS isotope dilution method developed by Kempe et al. . These authors did not detect anandamide in their analyses of rat brain tissue, however; differences in the extraction and purification of lipids may account for this discrepancy.The low amounts measured in our study accord better with those obtained with lipid mediators and neuropeptides , than with those obtained with neurotransmitters . Thus, our results reinforce the notion that anandamide may act in the CNS more as a modulatory substance than as a classical neurotransmitter, a possibility that finds further support in its nonvesicular mechanism of release . A comparison of the brain levels of anandamide with those of N-arachidonoyl PE may help clarify the metabolic relationship between these lipid products. The content of N-arachidonoyl PE , twice as large that of anandamide,grow rack systems supports its proposed role as anandamide precursor. Yet, the relatively small proportions of this precursor pool may indicate the existence of an active biochemical mechanism for the resynthesis of N-arachidonoyl PE depleted during neural activity.
In support of this possibility, we have shown previously that biosynthesis of N-acyl PEs in cortical mixed cultures is stimulated by physiologically relevant rises in intracellular Ca21 , elicited by membranedepolarizing agents or by Ca21 ionophore .How may intracellular Ca21 levels regulate N-arachidonoyl PE biosynthesis? Extensive investigations by the laboratory of Schmid and colleagues have shown that formation of other N-acyl PEs is mediated by a Ca21 -dependent N-acyltransferase activity, which transfers a saturated or monounsaturated fatty acyl group from the sn-1 position of phospholipids to the primary amino group of PE . Our results support this general mechanism, and extend it to N-arachidonoyl PE . We have identified and partially characterized a Ca21 – dependent enzyme activity that catalyzes the biosynthesis of N-[14C]arachidonoyl PE, when a phospholipid containing [14C]arachidonate at the sn-1 position is provided as fatty acyl donor. We have also demonstrated that sn-1 arachidonoyl phospholipids are normal, albeit quantitatively minor, components of brain phospholipids. Thus, our results indicate that brain tissue contains the complement of enzymatic activity and lipid substrates necessary for the biosynthesis of N-arachidonoyl PE. While the present study was being drafted, Sugiura et al. reported on the occurrence of a similar enzyme activity and phospholipid substrate in rat brain. Further experiments are necessary to determine whether the N-arachidonoyltransferase activity described in these studies resides in the same enzyme responsible for the biosynthesis of saturated and monounsaturated N-acyl PEs. The discrete tissue localization of N-acyltransferase activity, concentrated in brain and testis, and its relative enrichment in select areas of the CNS, highlight its potential importance in neural signaling. This distribution only partially parallels that of CB1 cannabinoid receptors. In fact, whereas these receptors are densely expressed in cortex, hippocampus, cerebellum, and striatum , they are only sparsely present in the brainstem . A possible explanation for this difference is that anandamide may have additional effects that are not mediated by the activation of any of the known cannabinoid receptor sub-types .
Stimulation of N-acyltransferase activity may account for the Ca21 -dependent increase in N-arachidonoyl PE biosynthesis observed when cortical mixed cultures are exposed to membrane depolarizing agents or Ca21 ionophore . In agreement with this conclusion, we observed that BTNP, a drug that we found to inhibit N-acyltransferase activity in vitro, also prevents ionomycin-induced N-arachidonoyl PE biosynthesis in these cultures.The preceding discussion has focused on the N-acyltransferase pathway as a possible biochemical mechanism for the formation of N-arachidonoyl PE and anandamide, but it is important to note that these lipids represent only a small fraction of the N-acyl PEs and NAEs found in brain. What, if any, are the physiological roles of saturated and monounsaturated N-acyl PEs and NAEs? Although it is not yet possible to provide an adequate answer to this question, several findings indicate that these lipids may be biologically active. For example, N-palmitoylethanolamine was shown to protect cerebellar granule neurons from excitotoxic death and to prevent antigen-induced mast cell activation, possibly by binding to CB2-like cannabinoid receptors . Moreover, N-oleoylethanolamine and N-linoleoylethanolamine were found to inhibit anandamide degradation in vitro, as well as in intact astrocytes in culture , and pharmacological effects of these and other NAEs on cell membrane properties have been reported . These different lines of evidence suggest that the NAEs may constitute a family of lipid signaling molecules, which may serve multiple physiopathological functions in the CNS.Alcohol consumption, particularly heavy use, is prevalent among PWH with rising rates of consumption in older PWH . To date, most studies in older PWH have focused on the combined presence of heavy alcohol use and HIV disease as risk factors for mortality and the development of age-related, multi-system comorbidities . With respect to neurobehavioral health, there is evidence that heavy alcohol use compounds HIV-related neurotoxicity , thereby impairing higher-order neurocognitive abilities critical for daily functioning .
Despite the known adverse neurocognitive effects of heavy alcohol use among older PWH, it is poorly understood whether lower levels of alcohol use similarly increase risk for neurocognitive impairment or, conversely, confer a degree of neuroprotection as has been proposed in prior studies of HIV-seronegative adults . [Note: Definitions of light, moderate and heavy drinking have been arbitrarily characterized across the literature; therefore, we are using “low-risk” henceforth to represent less than heavy alcohol consumption] . The evidence supporting protective effects of alcohol,rolling flood tables suggests an inverted J-shaped association between levels of alcohol consumed and risk for a multitude of diseases , such that there is a higher risk among heavy drinkers and abstainers compared to those with low-risk alcohol consumption . The existing literature examining the association between alcohol consumption and neurocognition among HIV- adults also suggests an inverted Jshaped association, such that low-risk alcohol consumption is associated with better neurocognition than alcohol abstinence, and heavy consumption is associated with the worst neurocognition compared to both no consumption and low-risk consumption . A longitudinal study using the UK Biobank found, among middle and older aged adults, a significant curvilinear association between alcohol consumption and neurocognition. Specifically, neurocognitive performance improved with increased alcohol use, up to one standard drink per day, at which point performance worsened . Results of studies examining this curvilinear association, however, have been inconsistent. Conflicting evidence suggests a positive linear association between neurocognition and alcohol consumption, rather than a curvilinear association . Previous literature suggests that neurocognitive deficits increase with heavier alcohol consumption among older adults . Furthermore, Parsons and Nixon suggest a potential threshold-effect, such that the deleterious effects of alcohol only occur after a specific threshold of consumption ; with heightened effects occurring at heavier levels of alcohol consumption . These inconsistent findings between a curvilinear, linear, and threshold association between alcohol consumption and neurocognition could result from confounding effects of other medical comorbidities, socioeconomic factors, and past alcohol use among current alcohol abstainers, that could contribute more strongly to neurocognitive deficits . The putative multi-system benefits of low-risk alcohol consumption in HIV- individuals have not been systematically investigated among PWH.
Results of earlier studies suggest elevated rates of alcohol use among PWH while more recent reports show similar rates compared to the general population . The majority of alcohol-focused research among PWH has focused on the detrimental effects of heavy drinking or alcohol use disorders . For example, research has consistently shown combined detrimental effects of heavy drinking and HIV disease on neurocognitive function as well as gray and white matter integrity, with the worst outcomes among the heaviest drinkers . Considering that majority of PWH do not report heavier drinking compared to the general population , there is a need for a more comprehensive understanding of the impact of low-risk alcohol consumption among PWH. Examination of whether low-risk drinking exerts differential neurocognitive effects based on HIV serostatus is particularly salient given the increasingly popular recommendations for older adults to follow certain nutritional guidelines . Given that HIV disease can enhance vulnerability to physiological damage from environmental stressors , there may be no level of alcohol associated with better neurocognitive functioning among PWH. Advancing age is independently associated with a higher risk of neurocognitive and neurodegenerative diseases including Alzheimer’s Disease and its precursor mild cognitive impairment . Despite use of combination antiretroviral therapy, older PWH remain particularly vulnerable to HIV-associated neurocognitive impairment and neurodegenerative diseases associated with aging . Considering alcohol consumption is common among PWH, and with advancing age these persons are at a heightened risk for neurocognitive impairment, the present study examined associations between the non-linear effect of recent low-risk alcohol consumption and HIV status on global and domain-specific neurocognitive outcomes. Within the range of low-risk drinking, we hypothesize a curvilinear association between recent alcohol consumption and neurocognition among HIV- individuals, such that intermediate levels of low-risk drinking will be associated with better neurocognitive function compared to non-drinkers and heavier levels; however, we do not expect this curvilinear association among PWH. Participants included 310 PWH and 89 HIV- older adults enrolled in NIH funded research studies at the University of California, San Diego HIV Neurobehavioral Research Program from 2003-2016. Participants were recruited from the greater San Diego area by the HNRP. Regulatory approval was obtained from the University of California San Diego Institutional Review Board prior to the start of protocol implementation. We have previously published several papers using other aspects of these data including medication adherence, age of first alcohol use, and neurocognitive function . The current study represents a secondary analysis of baseline alcohol use and neurobehavioral data from the HNRP. Exclusion criteria for the current analysis included 1) self-reported current or past diagnosis of a psychotic or mood disorder with psychotic features; 2) presence of a neurological condition that could impair neurocognitive function ; 3) positive urine toxicology for illicit drugs or evidence of alcohol intoxication by Breathalyzer test on the day of testing; 4) current diagnosis of AUD; 5) current diagnosis of non-alcohol substance use disorders ; 6) recent “at risk” alcohol consumption as defined per the National Institute on Alcohol Abuse and Alcoholism criteria for “at risk” drinking ; and 7) aged 49 years and younger. The UCSD Institutional Review Board approved this study, and all participants provided written informed consent to participate. Neuromedical Evaluation All participants completed comprehensive medical evaluations and were tested for HIV by enzyme-linked immunosorbent assay . A Western Blot confirmed positive results. Reverse transcriptase-polymerase chain reaction tested levels of HIV viral load in plasma . Psychiatric, Alcohol, and Substance Use Evaluation Current and lifetime mood and substance use disorders were assessed via The Composite International Diagnostic Interview , a fully-structured, computer-based interview . Diagnoses were made in accordance with DSM-IV criteria, as the parent grants from which baseline data were collected were funded before the DSM 5 was published. DSM-IV criteria for alcohol abuse are met when participants report continued alcohol use despite recurring problems . DSM-IV criteria for alcohol dependence are met when participants endorse symptoms of tolerance, withdrawal, and impaired control over drinking . AUD was assigned when DSM-IV criteria for alcohol abuse or dependence was met in order to maintain consistency with DSM 5 criteria and nomenclature.