Ethanol, Vitamins, and Brain Dysfunction

Chapter 44

Ethanol, Vitamins, and Brain Dysfunction Emilio González-Reimers1, Camino Fernández-Rodríguez1, Emilio González-Arnay2, Francisco SantolariaFernández1 1Servicio

de Medicina Interna, Hospital Universitario de Canarias, Universidad de La Laguna, La Laguna, Tenerife, Canary Islands, Spain; de Anatomía, Anatomía Patológica e Histología, Hospital Universitario de Canarias, Universidad de La Laguna, La Laguna, Tenerife, Canary Islands, Spain 2Departamento

Abbreviations cAMP  Cyclic adenosine monophosphate CD  Cluster determinant CREB  cAMP responsive element binding protein CRMP-2  Collapsin response mediator protein 2 DNA  Deoxyribonucleic acid EGF  Epidermal growth factor GABA  Gamma-amino-butyric acid HMBG-1  High mobility group box 1 IL  Interleukin MAP-LC3  Microtubule-associated protein-light chain 3 MCP  Monocyte chemoattractant protein MRI  Magnetic resonance imaging NFκB  Nuclear factor kappa B NADP  Nicotinamide adenine dinucleotide phosphate NADPH  Reduced nicotinamide adenine dinucleotide phosphate NGF  Nerve growth factor NMDA  N-Methyl-d-aspartate NOX  Nicotinamide adenine dinucleotide phosphate oxidase ROS  Reactive oxygen species TLR  Toll-like receptor TNF  Tumor necrosis factor

Brain alterations are frequent among alcoholics and include several distinct clinicopathological pictures (Table 1). Some of them are uncommon, such as central pontine myelinolysis or Marchiafava-Bignami disease. Prevalence of Wernicke encephalopathy associated with thiamine deficiency shows important geographic variations (Harper, Fornes, Duyckaerts, Lecomte, & Hauw, 1995), but the most frequent central nervous system alterations are brain atrophy, accompanied by cognitive dysfunction and several neuropsychological alterations, and cerebellar atrophy, related to motor alterations and gait disturbance, and possibly also with cognitive impairment. The true prevalence of brain atrophy is not well known. In a study by Torvik and Torp (1986), cerebellar atrophy was found in 42% of alcoholics under 70 years—a prevalence even higher than that observed among nonalcoholic 478

individuals over 70 years. This figure roughly parallels that of cognitive dysfunction, which affects 50–80% of alcoholics. A link exists between atrophy and altered cognition, although deranged blood flow, reversible with prolonged abstinence (Gansler et al., 2000), may contribute. In addition, several associated conditions, such as liver dysfunction, protein–calorie malnutrition, and deficiency in some antioxidant vitamins and trace elements, also damage the brain. This chapter reviews the mechanisms leading to brain atrophy in adolescent or adult alcoholics, and the potential role of vitamin alterations on these changes.

BRAIN ATROPHY AFFECTS BOTH WHITE MATTER AND GRAY MATTER Gray Matter Atrophy Gray matter atrophy can be viewed as a result of an imbalance between altered neuronogenesis and increased neuron degeneration. Brain tissue, including cortical neurons, derives from the neuroepithelium of the neural tube. This neuroepithelium is formed by pluripotent stem cells that are able to differentiate both into neurons and glia; it is located in an area known as the ventricular zone. In this area, stem cells evolve to form immature neurons and the radial glia, which, in turn, generate cortical astrocytes but also preserve the ability to transform into new neurons. After the seventh week, both neurons and glia from the ventricular zone migrate to the subventricular zone—an area that progressively evolves into the main neurogenetic area (Meyer, Perez-Garcia, Abraham, & Caput, 2002). Cells of this area give raise to the six-layered neocortex of the mammalian brain through radial migration—a process disrupted by in utero ethanol exposure. Ethanol also affects dendritic arborization of developing hippocampal neurons (Kumada, Jiang, Cameron, & Komuro, 2007). The Cajal-Retzius neurons produce reelin, an essential matrix protein that organizes the normal structure of the cortex (Lambert de Rouvroit & Goffinet, 1998). This protein becomes altered by ethanol in vitro (Mooney, Siegenthaler, & Miller, 2004). These changes, of undisputed importance in the development of the so-called fetal alcohol syndrome, may be also relevant in the

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TABLE 1  Brain Alterations in Alcoholic Patients Brain atrophy (frontal lobes, hippocampi) Cerebellar atrophy Wernicke–Korsakoff syndrome Centropontine myelinolisis Marchiafava-Bignami disease (necrosis of corpus callosum) Ethanol intoxication Witdrawal syndrome Trauma (subdural + parenchymatous hematoma) Stroke Optic neuritis Frontal atrophy and/or cerebellar atrophy are observed in 50–70% of heavy alcoholics.

adulthood. Indeed, hippocampus is an extraordinarily important neurogenetic area, with activity persisting until late adulthood. A normal hippocampus increases in size and myelination from childhood to adulthood (Suzuki et al., 2005). Studies of these authors performed on 23 younger adolescents, aged 13–14 years, compared with 30 older ones (19–21 years) revealed a marked volume increase among the latter, especially among males. Progressive myelination was also observed by Benes, Turtle, Khan, and Farol (1994) in 164 individuals; the myelination process was especially intense in the first two to three decades, especially among women, although it persisted during the lifetime. Indeed, it is currently accepted that, in humans, neuronogenesis continues in the subventricular zone of the wall of the lateral ventricles and in the hippocampal subgranular zone of the dentate gyrus throughout life (Knoth et al., 2010). These authors found doublecortin expression—a marker of neuronogenesis—in the granular zone of the dentate gyrus in brains of 54 individuals whose age at death ranged from birth to 100 years. Maximal expression was observed, however, during adolescence and young adulthood, until 30–40 years of age. In addition to these active neurogenetic areas, specialized forms of astrocytes from the subventricular zone migrate to the olfactory bulb, where they evolve to mature neurons (Lim & Alvarez-Buylla, 2014). Therefore, adolescence and young adulthood are important age periods for the full maturation and development of the hippocampus. However, in this life period, binge drinking is most common. Ethanol heavily affects hippocampal neuronogenesis both during embryonic development (possibly underlying the cognitive deficits observed in the fetal alcohol syndrome, as commented) and in adulthood (as discussed below). Hippocampus affectation is especially important among adolescent binge drinkers, who frequently show a striking hippocampal volume shrinkage, especially in the right hippocampus. Agartz, Momenan, Rawlings, Kerich, and Hommer (1999) in a study of 52 excessive drinkers (age range 27–53 years) compared with 36 controls, found a decrease in both hippocampi volume among alcoholic women; however, no differences were observed in left hippocampi among men but were found in right ones. De Bellis et al. (2000) reported that both right and left hippocampi were atrophied in 12 alcoholics compared

with 24 controls. Nagel, Schweinsburg, Phan, and Tapert (2005) found decreased left hippocampal volumes in 14 alcoholics aged 15–17 years compared to 17 controls, although hippocampal volume did not correlate with alcohol consumption. From these and other studies, it can be concluded that the adolescent hippocampus is especially sensitive to the deleterious effects of ethanol. These changes are in a certain way similar to those observed in degenerative situations. In patients with Alzheimer disease, a reduction in the left hippocampal volume was related to brain dysfunction. Although it is clear that hippocampal atrophy is typical in heavy alcoholics, there is controversy about the relative importance of blunted neuronogenesis or increased neuron loss in its pathogenesis. Several authors, using animal models, have shown decreased neuronogenesis both in the hippocampus (Crews & Nixon, 2009; He, Nixon, Shetty, & Crews, 2005) and subventricular zone, but others have challenged this view. A study by Sutherland et al. (2013) of the brains of 15 chronic alcoholics and 16 age-matched controls failed to find differences in the number of proliferative cells in the olfactory bulb (however, mean age was around 55 years, far from the adolescent period). This result is challenging because, in line with the active migration of neuron precursor cells from the subventricular zone to the olfactory bulb, hyposmia has been reported as an early predictor of dementia in degenerative brain diseases and it has been proposed as a manifestation of brain atrophy in alcoholics (Rupp et al., 2003). Ethanol also affects cerebellar development. Cerebellar neurons are especially sensitive to the noxious effects of ethanol, partly dependent on the altered signaling mechanisms mediated by the retinoic acid receptors and NMDA receptors and partly dependent on altered growth factors (Kumar, LaVoie, DiPette, & Singh, 2013).

White Matter Atrophy White matter atrophy is also striking among alcoholics. The prefrontal white matter, corpus callosum, and cerebellum (Harper, 2009) are especially affected areas. However, some controversy exists: postmortem examination revealed that Purkinje cell volume was reduced among 10 alcoholics aged 45.5 years compared with 10 age-matched controls, but no changes were observed regarding white matter cerebellar atrophy (Andersen, 2004). In part, white matter atrophy results from axonal injury secondary to neuronal death, but alterations in myelination have been also described (Harper, 2009). Repeated binge-drinking episodes lead to demyelination in the prefrontal cortex of adolescent rats but not in adult ones (Pascual, Pla, Miñarro, & Guerri, 2014), reinforcing the greater vulnerability of the brain during adolescence and early youth. Also, a single binge-drinking episode by a mother may lead to oligodendrocyte apoptosis in the fetus, and, consequently, decreased myelin production. This was shown in an experimental model in which macaque mothers were exposed to ethanol (Creeley, Dikranian, Johnson, Farber, & Olney, 2013) in an amount necessary to achieve a blood ethanol concentration in the range of 300–400 mg/dl (similar to those achieved during a binge-drinking episode) during 8 h. Also, in rat models, chronic ethanol exposure reduces the dendritic growth of newborn neurons (He et al., 2005). The functional nature of white matter atrophy was shown by Bartsch et al. (2007), who used magnetic resonance imaging (MRI) to study 15 alcoholic patients who completed abstinence,

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and repeated the exploration 6–7 weeks after sobriety. This population was compared with 10 controls. Overall, brain mass showed a 2% increase after abstinence, especially in the superior vermis, perimesencephalic, supratentorial and infratentorial periventricular borders, and frontomesial and frontoorbital edges. These morphologic changes were accompanied by biochemical ones, suggesting a recovery from white matter lesions consistent with remyelination, and neuropsychological improvement (attention and concentration). Therefore, white matter damage seems to occur in relation to alcohol intake and is reversible after abstinence. White matter lesions are also heavily dependent on vascular disease. Brain MRI studies allow the assessment of white matter hyperintensity, a parameter related to reduced blood flow. However, MRI studies using automated methods to assess white matter signal hyperintensity or diffusion tensor imaging (which allows examination of random mobility of tissue water, informing about the integrity of white matter fiber bundles) have yielded conflicting results in alcoholics (Cardenas et al., 2013).

FUNCTIONAL CONSEQUENCES Brain morphologic alterations in alcoholics lead to functional changes. For instance, repeated withdrawal syndromes are associated with impaired cognitive function, but it seems that also acute ethanol ingestion deranges cognitive function (Duka, Townshend, Collier, & Stephens, 2003). Binge drinking impairs learning; interestingly, memory impairment is more intense when alcohol is consumed during adolescence than when it is consumed in adulthood, in parallel with anatomic and biochemical changes, which are also more intense when alcohol is consumed during adolescence. Moreover, one of the neuropsychological effects of ethanol-mediated organic brain damage may be the altered ability to abandon drinking habitus, which may be associated with a higher risk for resumption of ethanol drinking. In a study of 75 alcoholics treated against alcohol dependence, future relapsers showed smaller brain volumes in the mesocorticolimbic system than nonrelapsers (Cardenas et al., 2011). Cerebellar alteration may lead to ataxia and gait disturbance, although it has been also shown that it may also lead to cognitive dysfunction (Fitzpatrick, Jackson, & Crowe, 2008).

PATHOGENESIS OF NEUROTOXICITY IN ALCOHOLICS Many data support the hypothesis that proinflammatory cytokines and oxidative stress contribute to brain damage. Ethanol promotes activation of the transcription factor nuclear factor kappa B (NFκB). Enhanced transcription of this factor is associated with increases in proinflammatory cytokines, especially tumor necrosis factor (TNF)-α and interleukin (IL)-1β and chimiokines, such as monocyte chemoattractant protein 1 (MCP-1). These cytokines are produced by activated glia, and numerous conditions, including ischemia, Alzheimer disease, brain injury, infection, or toxic exposure are potent inductors of microglia activation. In addition, proinflammatory cytokines produced elsewhere can reach the central nervous system, cross the blood–brain barrier, and secondarily activate microglia. Therefore, systemic inflammatory response due to ethanol consumption is involved in brain atrophy (Crews & Nixon, 2009).

Moreover, the duration of raised TNF values in brain is long lasting. A single intraperitoneal lipopolysaccharide injection provokes raised cytokine levels during 10 months, and activation of toll-like receptors (TLR) in microglia leads to a more prolonged secretion of cytokines in brain than in peripheral tissues. TLR-4 plays a pivotal role in this process. Alfonso-Loeches, Pascual-Lucas, Blanco, Sanchez-Vera, and Guerri (2010) have shown that neuroinflammation is blunted in mice lacking TLR-4 receptors. Ethanol also increases brain expression of TLR-3 and high mobility group box 1 (HMGB-1), a cytokine-like protein that functions as a TLR coagonist. Microglial activation, by multiple pathways, ultimately leads to microglia synthesis of proinflammatory cytokines, induction of NFκB, induction of NOX, and increased reactive oxygen species (ROS) production. ROS can activate neurons and cause neuronal induction of NOX leading to neuronal death (Qin & Crews, 2012). Therefore, microglial activation occurs by several different pathways, including ethanol itself, a local production of proinflammatory cytokines, and an induction of inflammation by cytokines produced in distant organs that reach microglia via a saturable blood to brain transport system. Microglial activation of NFκB constitutes a key step in neuronal damage. It opposes to transcription of the cAMP responsive element binding protein (CREB). This transcription factor can be viewed as a neuron survival factor, because it protects neurons from apoptosis and excitotoxicity, and its expression keeps an inverse relation with the presence of ethanol. In fact, ethanol reduces levels of DNA binding to CREB in hippocampal entorhinal cortex slice cultures (Crews & Nixon, 2009), coincident with the enhancement in oxidative enzymatic pathways and NFκB transcription. Because NFκB induces NOX and enhances ROS production, ultimately, oxidative stress is an important mediator of the effects of ethanol on neurodegeneration. This fact raises questions about the role played by antioxidants in brain alterations. In other settings, Patel, Rogers, and Huang (2008) showed that flavonoids protect patients with Alzheimer disease, and Mehlig et al. (2008) showed that ingestion of wine in moderate amounts may protect against dementia. In addition, several studies suggest that antioxidants could protect the brain from binge ethanol-induced damage (Crews & Nixon, 2009). These findings support the importance of analyzing the role of antioxidant vitamins on brain alterations in the alcoholic patient.

LIPOSOLUBLE VITAMINS As discussed below, the role of vitamin A or vitamin E deficiency on brain alterations and altered cognition has been well described for decades; more recently, the knowledge about vitamin D deficiency and brain alteration has suffered an explosive expansion. We comment on some relevant features regarding the effects of deficiency of these vitamins in alcoholics in the following sections. Recent research has pointed out that vitamin K deficiency seems to play a role in brain alterations, participating in sphingolipid metabolism and also as a brain antioxidant (Ferland, 2012).

Vitamin E Deficiency Vitamin E (tocopherol) was discovered about 90 years ago, as a product contained in lettuce that prevented fetal loss in animals

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fed a rancid lard diet. Its function was previously thought to be restricted to the reproductive area. Later, it became evident that its principal effect was the protection of polyunsaturated fatty acids from oxidation. This effect was also evident in membrane phospholipids; therefore, there are theoretical basis that support a protective role of vitamin E on damaged myelin and neuronal cells. This has been experimentally demonstrated in murine models in which vitamin E deficiency was associated with cognitive dysfunction (Nishida et al., 2006). In vitamin E deficient animals, there is apoptosis and deposition of β-amyloid proteins, but before these histological changes occur, there is an alteration of collapsin response mediator protein 2 (CRMP-2), a cytoplasmatic protein involved in normal axonal function. These changes take place in the face of an increased expression of microtubule-associated protein-light chain 3 (MAP-LC3), an autophagy-related protein, possibly enhanced by increased oxidative damage (Fukui et al., 2012). Axonal dysfunction took place before any changes were detectable in the cell bodies of the hippocampal neurons. Vitamin E deficiency also causes ataxia, mainly due to its deleterious action on Purkinje cells. Chronic ethanol feeding results in decreased serum vitamin E levels, partly due to an increased vitamin E demand by the liver due to increased transformation into α-tocopheryl quinone after scavenging of ROS (although in a murine model, ethanol-treated rats showed increased liver α-tocopherol concentration; Reilly, Patel, Peters, & Preedy, 2000). In alcoholics, poor nutrition and malabsorption probably contribute to decreased vitamin E levels. Therefore, less vitamin E remains available for performing antioxidant functions in tissues other than the liver, and possibly, vitamin E deficiency contributes to brain damage. Bondy, Guo, and Adams (1996) found that rats treated with α-tocopherol (200 mg/kg body weight by daily intraperitoneal injection for 15 days) showed raised levels of glutathione in both brain and liver. Simultaneous treatment with α-tocopherol prevented ethanol-induced decrease in liver and brain glutathione levels. Ethanol-treated rats also showed hyperhomocysteinemia and DNA damage, which were reverted by vitamin E supplementation (Shirpoor, Salami, Khadem-Ansari, Minassian, & Yegiazarian, 2009). Vitamin E is a liposoluble vitamin whose absorption may be impaired in liver diseases. Therefore, theoretically, the development of liver cirrhosis should aggravate vitamin E deficiency in alcoholics—a result confirmed in a recent study (Figure 1). However, relationships among vitamin E levels and brain alterations are poor.

Vitamin A Deficiency Vitamin A is usually ingested as beta carotene, which is transformed in the intestinal mucosa and (especially) in the liver into vitamin A. Carotenoids, in a similar way to α-tocopherol, protect unsaturated fatty acids from oxidative damage. Malnutrition and associated malabsorption contribute to vitamin A deficiency in alcoholics (Halsted, 2004). The situation becomes aggravated by ethanol-mediated microsomal cytochrome P-450 induction, which leads to vitamin A depletion. Indeed, liver vitamin A levels are reduced in alcoholics, and increased liver vitamin A breakdown has been reported in these patients (Clugston & Blaner, 2012). In the brain, retinoic acid is involved in cognition and neuronal development. As with other vitamins, experimental data support a role for decreased vitamin A levels on brain affectation. In adult

FIGURE 1  Serum vitamin E levels in cirrhotics and noncirrhotics. Lower values are observed in cirrhotics. Circles represent outliers; asterisks represent extreme values.

rats, it has been clearly shown that memory and spatial learning become severely impaired with vitamin A deprivation. Vitamin A deficiency reduces hippocampal neuronogenesis and leads also to a marked reduction in retinoic acid receptors, leading to memory impairment (Etchamendy et al., 2003). The decrease in the number of hippocampal neurons and impaired brain function associated with vitamin A deprivation can be reversed by vitamin A supplementation (Bonnet et al., 2008). However, vitamin A supplementation may be also harmful, as it has been associated with increased oxidant capacity (Behr et al., 2012). In a study performed in Kuala Lumpur on 333 individuals aged 60 or more years, the prevalence of mild cognitive impairment was 21.1%. Binary logistic regression indicated that the predictors of cognitive impairment were being married, being overweight or obese, and having vitamin A deficiency (Shahar et al., 2013). However, in a study on centenarians, the relationship between vitamin A levels (in brain or serum) and cognitive performance was weak (Johnson et al., 2013). Several authors have reported reduced levels of vitamin A (or its metabolites) in serum and liver of alcoholics (Halsted, 2004). Being a liposoluble compound, the same reasons argued for a more intense depletion of vitamin E in cirrhotics are also valid for vitamin A. In accordance, we found that vitamin A levels were markedly decreased in cirrhotics (Figure 2), keeping a relationship with deranged liver function. Also, vitamin A levels kept an independent relationship with cerebellar atrophy.

Vitamin D Deficiency It has been shown that systemic effects of vitamin D lay far beyond bone and calcium homeostasis. Vitamin D exerts positive effects on neuronal differentiation, migration, and proliferation, and it inhibits apoptosis. Individuals older than 60 with cognitive impairment show lower vitamin D levels than individuals without cognitive impairment (Chei et al., 2014). The results of the study by Chei et al. confirm those reported by many others, with few exceptions. Meta-analysis of five cross-sectional and two longitudinal studies including 7688 participants showed an increased risk of cognitive impairment in those with low vitamin D compared

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FIGURE 2  Serum vitamin A in cirrhotics, noncirrhotics, and controls. Lower values are observed in cirrhotics. Circles represent outliers.

with normal vitamin D (OR 2.39, 95% CI 1.91–3.00; p < 0.0001, Etgen, Sander, Bickel, Sander, & Förstl, 2012). Although heterogeneity of these population-based studies and the many confounding variables inherent to these studies oblige one to interpret these results with caution, several brain functions are clearly influenced by vitamin D. Vitamin D receptor is present in the brain, especially in the hippocampus. Vitamin D is involved in brain development and may be considered as a potent antioxidant in the adult brain (Briones & Darwish, 2014). Vitamin D deficiency is common among alcoholics. It is usual to define vitamin D deficiency as serum vitamin D values below 20 ng/ml, and vitamin D insufficiency when vitamin D levels are below 30 ng/ml. Most studies in alcoholics show that the prevalence of vitamin D deficiency is about 40–60%, and that of vitamin D insufficiency reaches 60–90%, although normal values have been reported by other authors (e.g., Santori et al., 2008). We recently found vitamin D insufficiency in 73 out of 128 alcoholics, and vitamin D deficiency in 36 of them (Figure 3). Several factors may contribute to vitamin D alterations, including dietary habits, latitude, sun exposure, malabsorption due to pancreatic insufficiency, or portal hypertension. In addition, chronic ethanol consumption may alter renal metabolism of 25 OH D3, inducing the synthesis of the inactive metabolite 24–25 (OH)2 D3 (Shankar et al., 2008). The deficient vitamin D levels observed in alcoholics may not only impair the antioxidant defense in brain, leading to increased neurodegeneration. Vitamin D deficiency is also related to increased blood pressure both by renin-dependent and reninindependent mechanisms, leading to the development of lacunar infarcts and vascular dementia. In addition, vitamin D deficiency is associated with insulin resistance.

HYDROSOLUBLE VITAMINS Vitamin B12, Folic Acid, and Homocysteine Cobalamin deficiency leads to several neurological syndromes, including myelopathy with ataxia, neuropsychiatric syndromes, neuropathy, optic neuritis, and cognitive deficits. Although in several studies vitamin B12 levels are related to dementia and

FIGURE 3  Prevalence of normal vitamin D, vitamin D insufficiency (below 30 ng/ml), and vitamin D deficiency (below 20 ng/ml) in cirrhotics and noncirrhotics. Vitamin D insufficiency was equally frequent among cirrhotics and noncirrhotics.

judgment impairment, and the decrease of B12 levels over time is also related to incident dementia, it is also clear that cognitive changes already occur, even when B12 levels are in the lowest quartile of normality (Hin et al., 2006). Equally important, it has been also disentangled that serum B12 levels may be not a good marker to assess the status of the vitamin in tissues; plasma holotranscobalamin, methylmalonic acid, or homocysteine are better indicators, and variations of the serum levels of these parameters are associated with significant changes in cognitive performance. In parallel with the functional changes, a decrease in B12 is also related to the intensity of brain atrophy. These alterations are due in part to the defective myelin synthesis associated with B12 deficiency. Indeed, it was shown that B12 in the form of adenosylcobalamine is an essential cofactor of myelin synthesis (Thakkar & Billa, 2014). However, demyelination associated with B12 deficiency is also heavily influenced by increased cytokine levels, such as nerve growth factor (NGF), TNF-α, and the soluble complex CD40:CD40 ligand, in the face of decreased neurotrophic molecules, such as epidermal growth factor (EGF) and IL-6 (Scalabrino, Veber, & Mutti, 2008). These findings establish a link between B12 deficiency and neuroinflammation. S-adenosylmethionine is a key product in central nervous system metabolism because it acts as a methyl donor. Its levels are dramatically dependent both on B12 and folate, because these substances are required in the methylation of homocysteine to methionine, with the latter being the precursor of S-adenosylmethionine. Therefore, it is not surprising that both folate and vitamin B12 deficiency may cause similar brain alterations, including dementia, and a demyelinating myelopathy. Cobalamin and/or folate deficiency lead to increased homocysteine levels. Homocysteine can be degraded to cystathionine and cysteine by vitamin B6 (Figure 4).

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FIGURE 4  Role of B12, B6, and folate in homocysteine metabolism. B12 or folate deficiency blocks the transformation of homocysteine into methionine, whereas B6 deficiency blocks the catabolism of homocysteine into cystathionine.

Hyperhomocysteinemia is a classic factor associated with vascular risk and cognitive deficiency, with the hippocampus being especially sensitive to hyperhomocysteinemia. Possibly, oxidative damage is the underlying mechanism of the deleterious effect of hyperhomocysteinemia. Homocysteine downregulates glutathione peroxidase, therefore impairing the antioxidant machinery. There are several reports showing increased levels of homocysteine in alcoholics. In a study on 52 individuals who showed high levels of homocysteine, low levels of folate and B6 but normal B12 levels were observed. Hyperhomocysteinemia was related to hippocampal atrophy (Bleich et al., 2003). Heese et al. (2012), in another study on 168 alcoholics, found high homocysteine levels (median = 15.3 μmol/l), which decreased after abstinence (median 10.7 μmol/l at the 11th day), keeping a relation with riboflavin and folate, but not with cobalamin levels. Results of these studies are in accordance with observations performed on otherwise healthy elderly subjects and in cases of dementia. In a study on 1092 subjects aged 76 years without dementia at inclusion, 111 developed dementia (83 of them due to Alzheimer disease) over an 8-year study period. The adjusted relative risk of dementia was 1.4 (95% confidence interval, 1.1–1.9) for each increase of one standard deviation of the log-transformed homocysteine levels. Plasma homocysteine levels higher than 14 μmol/l were associated with a double risk for developing Alzheimer disease (Seshadri et al., 2002). In another prospective study of 107 individuals aged 61–87 years without cognitive impairment at enrollment, vitamin B12 in the lowest tertile (<308 pmol/l) was associated with increased rate of brain volume loss along a 5-year study period (Vogiatzoglou et al., 2008) and more rapid cognitive decline. However, precise pathogenetic mechanisms, as well as the role of vitamin B12 in brain atrophy of alcoholics, are largely unknown. Moreover, although folate deficiency is a common finding in alcoholics, there is a trend to raised serum B12 levels, especially in cirrhotics. Preliminary data on 114 alcoholic patients showed that B12 levels were significantly higher among Child C patients compared with Child A and Child B ones (KW = 27.9; p < 0.001; Figure 5). However, neither folic acid, homocysteine, nor B12 were related with the intensity of frontal atrophy.

Vitamin C Deficiency Vitamin C acts as a scavenger of ROS: ascorbic acid oxidizes to monodehydroascorbic acid, which is later deoxidized by the glutathione reductase activity, linking the beneficial effect of vitamin C to selenium stores. Several studies have reported decreased vitamin C in patients with dementia, supporting the importance of oxidative stress on brain damage. However, in the review performed by Crichton, Bryan, and Murphy (2013), there is no conclusive evidence that dietary antioxidants protect against the development of dementia. One year of treatment with vitamin C and E did not modify the course of Alzheimer disease, although it was accompanied by an antioxidant effect, as assessed by cerebrospinal fluid analysis (Aarlt et al., 2012). In contrast, several studies point out a beneficial effect for ethanolinduced hippocampal neurodegeneration, both in the developing rat fetus and in adult animals. Among alcoholics, vitamin C deprivation has been described, especially among smokers (Guequen et al., 2003).

Vitamin B3 Deficiency Deficiency of some vitamins may provoke more or less acute clinical neurologic syndromes and response to specific therapy, but it complicates the clinical course of the alcoholics. Niacin deficiency is an example. Both reduced intake and impaired absorption may lead to vitamin B3 deficiency, which, in severe cases, may present with the full-blown picture of diarrhea, dermatitis, and dementia; the latter is more a delirium/confusion state than a true dementia. Given the lack of stores in the body, this clinical picture may ensue as soon as 60 days of dietary deprivation. Niacin plays a role in the redox reaction involving reduced nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate (NADPH). The possibility exists that niacin is derived from tryptophan metabolism, but the conversion from tryptophan to niacin requires riboflavin, thiamine, and pyridoxine, probably equally deficient in alcoholics deprived

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includes impaired oxidative metabolism, alteration of mitochondrial function, and reduction of thiamine-dependent enzyme activity, such as transketolase, α-ketoglutarate dehydrogenase, and pyruvate dehydrogenase—all leading to neuronal death. Studies searching for a genetic predisposition for Wernicke– Korsakoff encephalopathy have yielded inconclusive results (Guerrini, Thomson, & Gurling, 2009). Likely, thiamine and ethanol exert synergistic effects, at least regarding white matter shrinkage: brain atrophy and atrophy of corpus callosum are observed in relation with Wernicke’s encephalopathy in cases related to ethanol misuse. Cerebellar shrinkage is another feature observed both in thiamine deficiency conditions and ethanol.

CONCLUSIONS FIGURE 5  Serum vitamin B12 levels according to Child’s classification. Vitamin B12 increases among Child C patients. Circles represent outliers.

from niacin. Therefore, malnourished alcoholics may develop the clinical-pathological features of niacin deficiency. In a study of 20 necropsies, widespread central neuronal chromatolysis affecting cranial nerve and pontine nuclei and Betz neurons (Ishii & Nishihara, 1981) was observed in patients with niacin deficiency who had presented with confusion, hallucination, tremor, and extrapyramidal rigidity. Extrapyramidal signs, oppositional hypertonus, confusion, hallucinations, and insomnia are cardinal, although nonspecific, features of the so-called alcoholic pellagra encephalopathy. Niacin deficiency may provoke acute delirium in the alcoholic patient, mimicking withdrawal syndrome (Oldham & Ivkovic, 2012). Often, associated vitamin deficiencies usually complicate the clinical picture of pellagra (López, Olivares, & Berrios, 2014).

Thiamine Deficiency and Wernicke Encephalopathy In many alcoholics, multiple vitamin deficiencies coexist, obscuring differential diagnosis of the neurologic disturbance that prompted admission to a hospital. Previously mentioned cases (López et al., 2014; Oldham & Ivkovic, 2012) and others (e.g., Ishii and Nishihara, 1981) constitute paradigmatic examples of a chronic alcoholic patient, like many attended in our unit, in whom severe impairment of protein–calorie nutritional status, which confers a poor prognosis, is associated with deficits of several micronutrients and vitamins. The Wernicke–Korsakoff syndrome is due to thiamine deficiency, which may occur in the alcoholic patient from one or more of the following mechanisms: inadequate intake, impaired absorption, a reduced liver storage, and decreased transformation of thiamine in its active form. It consists mainly of a constellation of focal neurological symptoms, including ophthalmoplegia, stupor or coma, and cerebellar dysfunction. These symptoms typically improve after appropriate treatment with thiamine; however, in many patients (nearly 80% in some series) the Korsakoff psychosis ensues, characterized by a more or less permanent cognitive impairment, confabulation, anterograde amnesia, visuospatial alteration, reduced affect, and impaired problem-solving capacity. Lesions are more prominent in mammillary bodies and the dorsomedial and anterior nuclei of the thalamus. Pathogenesis

In conclusion, ethanol exerts several effects on brain function; the most commonly observed are brain atrophy, cerebellar atrophy, ventricular enlargement, and white matter shrinkage. Neuronogenesis is severely impaired, and neuronal death is increased. Underlying mechanisms include cytokine-derived inflammatory response. These cytokines are either locally produced or have a systemic origin, but in either case they generate inflammation accompanied by increased ROS production. The increased lipid peroxidation is aggravated by the fact that antioxidant defense is impaired, particularly those mechanisms that depend on some antioxidant vitamins, such as vitamin A, vitamin E, vitamin D, and vitamin C, among others, whose levels are usually depressed in alcoholics. Despite this, and despite the abundance of experimental studies apparently confirming this hypothesis, results in human beings are more controversial. In general, trials adding vitamin supplements have yielded, in the best of cases, only modest results, with the exceptions of the well-known therapeutic effect of thiamine on Wernicke encephalopathy and niacin on pellagra-­associated dementia. However, ethanol withdrawal leads to a marked improvement of brain performance and reversal of atrophy, thus remaining as the only effective therapy for this situation.

APPLICATIONS TO OTHER ADDICTIONS AND SUBSTANCE MISUSE In addition to ethanol, other drugs damage the brain, although the underlying pathogenic mechanisms differ. Many studies support the role of ethanol in neuroinflammation, oxidative stress, and ultimately neuronal death. These features are also observed in polydrug abusers. Findings include neurodegenerative alterations, neuronal loss, axonal damage, and microglial activation. Oxidative stress may underlie the toxic effects of opiates, heroin, or morphine. Consumption of these drugs leads to depletion of antioxidant mechanisms, including cellular antioxidant systems and antioxidant vitamins; however, antioxidant therapy, although efficacious in some experimental models, fails to exert protection in humans. Cocaine, amphetamine, and synthetic derivatives severely impair brain blood perfusion. Stroke is a relatively common consequence. Opiates may also cause hypoxic brain damage due to respiratory depression. Studies have also shown depletion of the total antioxidant capacity among cocaine and/or methamphetamine consumers. Cocaine consumption is also associated with extensive white

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matter damage. Interestingly, chronic cocaine use alters the response to avoid punishment, enhances perseverative responding, and possibly, by increasing ROS production, contributes to the reinforcing effect of the drug; at the same time, oxidative stress impairs learning and memory. Consumption of marijuana is associated with smaller hippocampi and altered frontal white matter, and a greater impulsivity. Therefore, most drugs damage the brain by mechanisms related to neuroinflammation and excessive ROS production. Although there are theoretical bases for antioxidant vitamins supplementation and some experimental data support their efficacy, clinical studies have yielded poor results.

DEFINITION OF TERMS Hippocampus  This gyrated region of the temporal lobe is mainly composed of two interrelated regions of the archicortex called dentate gyrus and Cornu Ammonis. It is heavily involved in learning, behavior, and memory acquisition. Archicortex  This part of brain cortical tissue is organized in three layers (instead of six, as most of the remaining cortex). In the human brain, it includes the hippocampus and olfactory cortex. Microglia  The phagocytes of the central nervous system share many properties with other cells of the reticuloendothelial system, including cytokine production and oxygen free radical generation. Oxidative stress  This alteration of the structure of diverse molecules occurs by reaction with highly reactive oxygen species. Antioxidants  These enzymatic pathways or cofactors transform reactive oxidant species into less reactive ones. Excitotoxicity  The mechanism of neuronal cell death is derived from intense and prolonged activation of excitatory neurotransmitter receptors. This activation alters intracellular enzymatic pathways, ultimately leading to cell death. This mechanism is especially important during ethanol withdrawal. Toll-like receptors  These cell surface receptors are usually activated by-products derived from microorganisms, such as membrane structures from gram-negative bacteria. In the brain, toll-like receptors are present in microglia cells. Apoptosis  In this process, cells suffer a series of modifications affecting membranes, cytoplasma, and the nucleus, which ultimately lead to cell death, typically without accompanying inflammatory reaction. Cytokines  These small protein molecules are of importance in the immune response. They usually act on transcription factors that activate genes. Their functions are varied, mainly inducing the synthesis of inflammatory mediators and activating several kinds of cells, especially endothelial cells, immune cells, and phagocytes, among others. Transcription factors  These intracellular proteins bind to DNA sequences in the nucleus and modify the transcription of the DNA genome into messenger RNA, therefore orchestrating the synthesis of certain proteins.

KEY FACTS Key Facts of Ethanol-Mediated Brain Damage among Adolescents l  Binge

drinking is defined by the ingestion of at least five drinks (for men) or four (for women) during the same drinking episode.

l Binge

drinking is more common during adolescence. episodic pattern of heavy drinking in a short time is associated with episodic increases in glutamate release, which can cause excitotoxicity and neuronal death. l  During youth, the hippocampus is more sensitive to the noxious effects of alcohol-derived neuroinflammation and oxidative damage. This explains why this pattern of consumption is more damaging to the brain than regular excessive consumption. l  Possibly, hippocampal damage during binge drinking may predispose an individual to alcohol addiction. l This

Key Facts of Gray Matter l Gray

matter is composed of neuronal bodies. neuronogenesis takes place during infancy, adolescence, and youth. l Ethanol inhibits neuronogenesis. l Ethanol increases neuronal death. l The deficiency of some vitamins, such as thiamine, also leads to neuronal death and the effects are potentiated by ethanol. l Significant

SUMMARY POINTS l  Brain

atrophy, especially affecting the frontal lobes and hippocampi, and cerebellar atrophy are major manifestations of heavy alcoholism. l Both gray matter and white matter are affected. l The secretion of proinflammatory cytokines and oxidative damage constitute the main pathogenic mechanisms involved. Therefore, there is a theoretical basis for the therapeutic use of antioxidants. l Decreased levels of vitamin E, vitamin A, vitamin D, and vitamin C have been reported in patients or experimental models of ethanol-mediated brain atrophy. l  Experimental data are promising regarding the therapeutic effect of vitamins supplementation, but clinical trials show no definite benefits. The best therapy is alcohol abstention. l  Clinical syndromes due to specific vitamins deficiency may coexist in the same patient, and with a superimposed ethanol withdrawal syndrome, obscuring the diagnosis and making difficult the implementation of adequate therapy.

REFERENCES Agartz, I., Momenan, R., Rawlings, R. R., Kerich, M. J., & Hommer, D. W. (1999). Hippocampal volume in patients with alcohol dependence. Archives of General Psychiatry, 56, 356–363. Alfonso-Loeches, S., Pascual-Lucas, M., Blanco, A. M., Sanchez-Vera, I., & Guerri, C. (2010). Pivotal role of TLR4 receptors in alcohol-induced neuroinflammation and brain damage. The Journal of Neuroscience, 30, 8285–8295. Andersen, B. B. (2004). Reduction of Purkinje cell volume in cerebellum of alcoholics. Brain Research, 1007, 10–18. Arlt, S., Müller-Thomsen, T., Beisiegel, U., & Kontush, A. (2012). Effect of one-year vitamin C- and E-supplementation on cerebrospinal fluid oxidation parameters and clinical course in Alzheimer’s disease. Neurochemical Research, 37, 2706–2714.

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Bartsch, A. J., Homola, G., Biller, A., Smith, S. M., Weijers, H. G., Wiesbeck, G. A., … Bendszus, M. (2007). Manifestations of early brain recovery associated with abstinence from alcoholism. Brain, 130 (Pt 1), 36–47. Behr, G. A., Schnorr, C. E., Simões-Pires, A., da Motta, L. L., Frey, B. N., & Moreira, J. C. (2012). Increased cerebral oxidative damage and decreased antioxidant defenses in ovariectomized and sham-operated rats supplemented with vitamin A. Cell Biology and Toxicology, 28, 317–330. Benes, F. M., Turtle, M., Khan, Y., & Farol, P. (1994). Myelination of a key relay zone in the hippocampal formation occurs in the human brain during childhood, adolescence, and adulthood. Archives of General Psychiatry, 51, 477–484. Bleich, S., Bandelow, B., Javaheripour, K., Müller, A., Degner, D., Wilhelm, J., … Kornhuber, J. (2003). Hyperhomocysteinemia as a new risk factor for brain shrinkage in patients with alcoholism. Neuroscience Letters, 335, 179–182. Bondy, S. C., Guo, S. X., & Adams, J. D. (1996). Prevention of ethanolinduced changes in reactive oxygen parameters by alpha-tocopherol. Alcohol and Alcoholism, 31, 403–410. Bonnet, E., Touyarot, K., Alfos, S., Pallet, V., Higueret, P., & Abrous, D. N. (2008). Retinoic acid restores adult hippocampal neurogenesis and reverses spatial memory deficit in vitamin A deprived rats. PLoS One, 3, e3487. Briones, T. L., & Darwish, H. (2014). Decrease in age-related tau hyperphosphorylation and cognitive improvement following vitamin D supplementation are associated with modulation of brain energy metabolism and redox state. Neuroscience, 262, 143–155. Cardenas, V. A., Durazzo, T. C., Gazdzinski, S., Mon, A., Studholme, C., & Meyerhoff, D. J. (2011). Brain morphology at entry into treatment for alcohol dependence is related to relapse propensity. Biological Psychiatry, 70, 561–567. Cardenas, V. A., Greenstein, D., Fouche, J. P., Ferrett, H., Cuzen, N., Stein, D. J., & Fein, G. (2013). Not lesser but greater fractional anisotropy in adolescents with alcohol use disorders. NeuroImage. Clinical, 2, 804–809. Chei, C. L., Raman, P., Yin, Z. X., Shi, X. M., Zeng, Y., & Matchar, D. B. (October 3, 2014). Vitamin D levels and cognition in elderly adults in China. Journal of the American Geriatrics Society, 62(11), 2125–2129. Clugston, R. D., & Blaner, W. S. (2012). The adverse effects of alcohol on vitamin A metabolism. Nutrients, 4, 356–371. Creeley, C. E., Dikranian, K. T., Johnson, S. A., Farber, N. B., & Olney, J. W. (2013). Alcohol-induced apoptosis of oligodendrocytes in the fetal macaque brain. Acta Neuropathologica Communications, 1, 23. Crews, F. T., & Nixon, K. (2009). Mechanisms of neurodegeneration and regeneration in Alcoholism. Alcohol and Alcoholism, 44, 115–127. Crichton, G. E., Bryan, J., & Murphy, K. J. (2013). Dietary antioxidants, cognitive function and dementia–a systematic review. Plant Foods for Human Nutrition, 68, 279–292. De Bellis, M. D., Clark, D. B., Beers, S. R., Soloff, P. H., Boring, A. M., Hall, J., … Keshavan, M. S. (2000). Hippocampal volume in adolescent-onset alcohol use disorders. American Journal of Psychiatry, 157, 737–744. Duka, T., Townshend, J. M., Collier, K., & Stephens, D. N. (2003). Impairment in cognitive functions after multiple detoxifications in alcoholic inpatients. Alcoholism Clinical and Experimental Research, 27, 1563–1572. Etchamendy, N., Enderlin, V., Marighetto, A., Pallet, V., Higueret, P., & Jaffard, R. (2003). Vitamin A deficiency and relational memory deficit in adult mice: relationships with changes in brain retinoid signalling. Behavioural Brain Research, 145, 37–49.

Etgen, T., Sander, D., Bickel, H., Sander, K., & Förstl, H. (2012). Vitamin D deficiency, cognitive impairment and dementia: a systematic review and meta-analysis. Dementia and Geriatric Cognitive Disorders, 33, 297–305. Ferland, G. (2012). Vitamin K, an emerging nutrient in brain function. Biofactors, 38, 151–157. Fitzpatrick, L. E., Jackson, M., & Crowe, S. F. (2008). The relationship between alcoholic cerebellar degeneration and cognitive and emotional functioning. Neuroscience and Biobehavioral Reviews, 32, 466–485. Fukui, K., Kawakami, H., Honjo, T., Ogasawara, R., Takatsu, H., Shinkai, T., … Urano, S. (2012). Vitamin E deficiency induces axonal degeneration in mouse hippocampal neurons. Journal of Nutritional Science and Vitaminology, 58, 377–383. Gansler, D. A., Harris, G. J., Oscar-Berman, M., Streeter, C., Lewis, R. F., Ahmed, I., & Achong, D. (2000). Hypoperfusion of inferior frontal brain regions in abstinent alcoholics: a pilot SPECT study. Journal of Studies on Alcohol, 61, 32–37. Gueguen, S., Pirollet, P., Leroy, P., Guilland, J. C., Arnaud, J., Paille, F., … Herbeth, B. (2003). Changes in serum retinol, alpha-tocopherol, vitamin C, carotenoids, zinc and selenium after micronutrient supplementation during alcohol rehabilitation. Journal of the American College of Nutrition, 22, 303–310. Guerrini, I., Thomson, A. D., & Gurling, H. M. (2009). Molecular genetics of alcohol-related brain damage. Alcohol and Alcoholism, 44, 166–170. Halsted, C. H. (2004). Nutrition and alcoholic liver disease. Seminars in Liver Disease, 24, 289–304. Harper, C. (2009). The neuropathology of alcohol-related brain damage. Alcohol and Alcoholism, 44, 136–140. Harper, C., Fornes, P., Duyckaerts, C., Lecomte, D., & Hauw, J. J. (1995). An international perspective on the prevalence of the Wernicke-­ Korsakoff syndrome. Metabolic Brain Disease, 10, 17–24. Heese, P., Linnebank, M., Semmler, A., Muschler, M. A., Heberlein, A., Frieling, H., … Hillemacher, T. (2012). Alterations of homocysteine serum levels during alcohol withdrawal are influenced by folate and riboflavin: results from the German Investigation on Neurobiology in Alcoholism (GINA). Alcohol and Alcoholism, 47, 497–500. He, J., Nixon, K., Shetty, A. K., & Crews, F. T. (2005). Chronic alcohol exposure reduces hippocampal neurogenesis and dendritic growth of newborn neurons. European Journal of Neuroscience, 21, 2711–2720. Hin, H., Clarke, R., Sherliker, P., Atoyebi, W., Emmens, K., Birks, J., … Evans, J. G. (2006). Clinical relevance of low serum vitamin B12 concentrations in older people: the Banbury B12 study. Age and Ageing, 35, 416–422. Ishii, N., & Nishihara, Y. (1981). Pellagra among chronic alcoholics: clinical and pathologic study of 20 necropsy cases. Journal of Neurology, Neurosurgery, and Psychiatry, 44, 209–215. Johnson, E. J., Vishwanathan, R., Johnson, M. A., Hausman, D. B., Davey, A., Scott, T. M., … Poon, L. W. (2013). Relationship between serum and brain carotenoids, α-tocopherol, and retinol concentrations and cognitive performance in the oldest old from the Georgia Centenarian Study. Journal of Aging Research, 2013, 951786. Knoth, R., Singec, I., Ditter, M., Pantazis, G., Capetian, P., Meyer, R. P., … Kempermann, G. (2010). Murine features of neurogenesis in the human hippocampus across the lifespan from 0 to 100 years. PLoS One, 5(1), e8809. Kumada, T., Jiang, Y., Cameron, D. B., & Komuro, H. (2007). How does alcohol impair neuronal migration? Journal of Neuroscience Research, 85, 465–470.

Ethanol, Vitamins, and Brain Dysfunction Chapter | 44  487

Kumar, A., LaVoie, H. A., DiPette, D. J., & Singh, U. S. (2013). Ethanol neurotoxicity in the developing cerebellum: underlying mechanisms and implications. Brain Sciences, 3, 941–963. Lambert de Rouvroit, C., & Goffinet, A. M. (1998). The reeler mouse as a model of brain development. Advances in Anatomy Embryology and Cell Biology, 150, 1–106. Lim, D. A., & Alvarez-Buylla, A. (September 12, 2014). Adult neural stem cells stake their ground. Trends in Neurosciences, 37(10), 563–571 pii:S0166-2236(14) 00149–0. López, M., Olivares, J. M., & Berrios, G. E. (2014). Pellagra encephalopathy in the context of alcoholism: review and case report. Alcohol and Alcoholism, 49, 38–41. Mehlig, K., Skoog, I., Guo, X., Schütze, M., Gustafson, D., Waern, M., … Lissner, L. (2008). Alcoholic beverages and incidence of dementia: 34-year follow-up of the prospective population study of women in Goteborg. American Journal of Epidemiology, 167, 684–691. Meyer, G., Perez-Garcia, C. G., Abraham, H., & Caput, D. (2002). Expression of p73 and reelin in the developing human cortex. The Journal of Neuroscience, 22, 4973–4986. Mooney, S. M., Siegenthaler, J. A., & Miller, M. W. (2004). Ethanol induces heterotopias in organotypic cultures of rat cerebral cortex. Cerebral Cortex, 14, 1071–1080. Nagel, B. J., Schweinsburg, A. D., Phan, V., & Tapert, S. F. (2005). Reduced hippocampal volume among adolescents with alcohol use disorders without psychiatric comorbidity. Psychiatry Research, 139, 181–190. Nishida, Y., Yokota, T., Takahashi, T., Uchihara, T., Jishage, K., & Mizusawa, H. (2006). Deletion of vitamin E enhances phenotype of Alzheimer disease model mouse. Biochemical and Biophysical Research Communications, 350, 530–536. Oldham, M. A., & Ivkovic, A. (2012). Pellagrous encephalopathy presenting as alcohol withdrawal delirium: a case series and literature review. Addiction Science and Clinical Practice, 7, 12. Pascual, M., Pla, A., Miñarro, J., & Guerri, C. (2014). Neuroimmune activation and myelin changes in adolescent rats exposed to highdose alcohol and associated cognitive dysfunction: a review with reference to human adolescent drinking. Alcohol and Alcoholism, 49, 187–192. Patel, A. K., Rogers, J. T., & Huang, X. (2008). Flavanols, mild cognitive impairment, and Alzheimer’s dementia. International Journal of Clinical and Experimental Medicine, 1, 181–191. Qin, L., & Crews, F. T. (2012). Chronic ethanol increases systemic TLR3 agonist-induced neuroinflammation and neurodegeneration. Journal of Neuroinflammation, 9, 130. Reilly, M. E., Patel, V. B., Peters, T. J., & Preedy, V. R. (2000). In vivo rates of skeletal muscle protein synthesis in rats are decreased by acute ethanol treatment but are not ameliorated by supplemental alphatocopherol. Journal of Nutrition, 130, 3045–3049.

Rupp, C. I., Kurz, M., Kemmler, G., Mair, D., Hausmann, A., Hinterhuber, H., & Fleischhacker, W. W. (2003). Reduced olfactory sensitivity, discrimination, and identification in patients with alcohol dependence. Alcoholism Clinical and Experimental Research, 27, 432–439. Santori, C., Ceccanti, M., Diacinti, D., Attilia, M. L., Toppo, L., D’Erasmo, E., … Minisola, S. (2008). Skeletal turnover, bone mineral density, and fractures in male chronic absusers of alcohol. Journal of Endocrinological Investigation, 31, 321–326. Scalabrino, G., Veber, D., & Mutti, E. (2008). Experimental and clinical evidence of the role of cytokines and growth factors in the pathogenesis of acquired cobalamin-deficient leukoneuropathy. Brain Research Reviews, 59, 42–54. Seshadri, S., Beiser, A., Selhub, J., Jacques, P. F., Rosenberg, I. H., D’Agostino, R. B., … Wolf, P. A. (2002). Plasma homocysteine as a risk factor for dementia and Alzheimer’s disease. The New England Journal of Medicine, 346, 476–483. Shahar, S., Lee, L. K., Rajab, N., Lim, C. L., Harun, N. A., Noh, M. F., … Jamal, R. (2013). Association between vitamin A, vitamin E and apolipoprotein E status with mild cognitive impairment among elderly people in low-cost residential areas. Nutritional Neuroscience, 16, 6–12. Shankar, K., Liu, X., Singhal, R., Chen, J. R., Nagarajan, S., Badger, T. M., & Ronis, M. J. (2008). Chronic ethanol consumption leads to disruption of vitamin D homeostasis associated with induction of renal 1,25 dihydroxyvitamin D3 24 hydroxylase (CYP24A1). Endocrinology, 149, 1748–1756. Shirpoor, A., Salami, S., Khadem-Ansari, M. H., Minassian, S., & Yegiazarian, M. (2009). Protective effect of vitamin E against ethanol-­ induced hyperhomocysteinemia, DNA damage, and atrophy in the developing male rat brain. Alcoholism Clinical and Experimental Research, 33, 1181–1186. Sutherland, G. T., Sheahan, P. J., Matthews, J., Dennis, C. V., Sheedy, D. S., McCrossin, T., … Kril, J. J. (2013). The effects of chronic alcoholism on cell proliferation in the human brain. Experimental Neurology, 247, 9–18. Suzuki, M., Hagino, H., Nohara, S., Zhou, S. Y., Kawasaki, Y., Takahashi, T., … Kurachi, M. (2005). Male-specific volume expansion of the human hippocampus during adolescence. Cerebral Cortex, 15, 187–193. Thakkar, K., & Billa, G. (August 13, 2014). Treatment of vitamin B12 deficiency-­Methylcobalamine? Cyancobalamine? Hydroxocobalamin?clearing the confusion. European Journal of Clinical Nutrition, 69(1), 1–2. Torvik, A., & Torp, S. (1986). The prevalence of alcoholic cerebellar atrophy. A morphometric and histological study of an autopsy material. Journal of Neurological Sciences, 75, 43–51. Vogiatzoglou, A., Refsum, H., Johnston, C., Smith, S. M., Bradley, K. M., de Jager, C., … Smith, A. D. (2008). Vitamin B12 status and rate of brain volume loss in community-dwelling elderly. Neurology, 71, 826–832.