Apolipoprotein E genotyping and questionnaire-based assessment of lifestyle risk factors in dyslipidemic patients with a family history of Alzheimer's disease: test development for clinical application.

The cholesterol-raising properties of the apolipoprotein E (APOE) epsilon-4 (ε-4) allele has been validated in the South African population. Mounting evidence supports the added value of APOE genotyping for the evaluation of cardiovascular risk in dyslipidemic patients beyond its established role in the diagnosis of late-onset Alzheimer's disease (AD). The aim of this study was to determine the potential benefits of combining AD family history with questionnaire-based lifestyle assessment to facilitate the clinical interpretation of APOE genotyping results. A total of 580 unrelated South African individuals prospectively enrolled in a chronic disease screening program incorporating a genetic component (2010-2015) was selected for inclusion in this study based on the presence (75) or absence (505) of AD family history. Biochemical assessment of their lipid profiles was performed according to standard laboratory protocols. All study participants were genotyped for the APOE ε-2/ε-3/ε-4 alleles using allele-specific TaqMan real-time polymerase chain reaction technology. In patients without a family history of AD, APOE genotype modified the relationship between alcohol intake and body mass index (p = 0.026), with a significant positive correlation noted between these parameters being limited to ε-4 allele carriers. APOE genotype also modified the association between alcohol intake and total serum cholesterol in patients with a positive family history of AD (p = 0.026). We demonstrated the benefits of a questionnaire-based approach for assessment of lifestyle risk factors to facilitate clinical interpretation of APOE genotyping results for targeted intervention in a genetic subgroup of dyslipidemic patients at increased risk for AD.

Apolipoprotein E ε-4 as a genetic determinant of Alzheimer's disease heterogeneity.

Alzheimer's disease (AD) displays a high degree of heterogeneity in terms of its etiology, presentation, prognosis, and treatment response. This can partly be explained by high-penetrance mutations in the amyloid precursor protein, presenilin 1 and presenilin 2 genes causing amyloid beta aggregation, which is a major pathogenic mechanism in the development of early-onset AD in a small subgroup of patients. Late-onset AD is considered a polygenic disorder in which cumulative risk resulting from interaction with modifiable environmental risk factors may be responsible for the majority of cases. The ε-4 allele of the apolipoprotein E (APOE) gene has emerged as the most significant genetic risk factor for late-onset AD, influencing nearly every pathogenic domain affected in AD. It is a major risk factor for cerebral amyloid angiopathy, recognized as a common pathological finding in an AD subtype associated with white matter dysfunction. The APOE ε-4 allele is also a known risk factor for ischemic stroke, which can result in vascular dementia or contribute to subcortical vascular dysfunction. In this review, we evaluate the clinical relevance of APOE genotyping in relation to cholesterol metabolism and available evidence on risk reduction strategies applicable to AD.

Association of cocaine- and amphetamine-related transcript, leptin and leptin receptor gene polymorphisms with anthropometric obesity phenotype indicators in South African learners.

BACKGROUND/AIMS: Obesity has increased rapidly in South African children and adolescents. Genes involved in appetite regulation have been extensively studied worldwide, but their role in the obesity phenotype in South African Black and mixed-ancestry school adolescents is unknown. METHODS: Seven common polymorphisms in LEP, GHRL, CART and LEPR were analysed for genotype and haplotype association with anthropometric obesity phenotype indicators in South African Black and mixed-ancestry adolescent school learners. RESULTS: The CART c.517A→G polymorphism was significantly associated with obesity susceptibility. The LEPR Lys(109)Arg G allele was associated with an average reduction of 2.36 kg/m(2) in body mass index (BMI), 5.66 cm in waist circumference (WC) and 1.61 cm in mid-upper-arm circumference (MUAC). This was confirmed by haplotype analysis. Additionally, a haplotype of the LEP polymorphisms significantly increased BMI, MUAC and hip circumference, while LEPR haplotypes were associated with differences in MUAC. CONCLUSION: Our findings suggest that c.517A→G and Lys(109)Arg contribute to the variation in anthropometric obesity phenotype indicators observed among Black African and mixed-ancestry South African learners. Furthermore, haplotypes of LEP, LEPR and GHRL polymorphisms were associated with varying measurements of weight, BMI and WC. Further studies are required to confirm our results in a larger and homogeneous study population group.

Membrane saturated fatty acids and disease progression in multiple sclerosis patients.

The risk of developing multiple sclerosis is associated with increased dietary intake of saturated fatty acids. We determined the fatty acid composition within the different phospholipid fractions of red blood and peripheral blood mononuclear cell membranes of 31 patients diagnosed with multiple sclerosis and 30 healthy control subjects using gas chromatography. Individual saturated fatty acids were correlated with the severity of neurological outcome as measured by the Kurtzke Expanded Disability Status Scale. Significant increases were found in multiple sclerosis peripheral blood mononuclear cell membrane sphingomyelin C14:0 and phosphatidylinositol C22:0. In the peripheral blood mononuclear cell membranes, C22:0 and C24:0 showed positive correlations, while C14:0, C16:0 and C20:0 showed inverse correlations with the Functional System Scores. In conclusion, this study is in accordance with previous studies that have shown an increase in shorter long-chain SATS in MS patients. In addition, this study also showed that higher C14:0 and C16:0 reflected better disease outcome as demonstrated by the inverse correlation with the EDSS and FSS. We have also characterized the specific SATS, that is, long-chain SATS that may increase the risk of developing MS.

Erythrocyte membrane fatty acids in patients with multiple sclerosis.

BACKGROUND: Reports on fatty acids levels in multiple sclerosis remain inconclusive. OBJECTIVE: To determine the erythrocyte membrane fatty acid levels in multiple sclerosis patients and correlate with Kurtzke Expanded Disability Status Scale. METHODS: Fatty acid composition of 31 multiple sclerosis and 30 control individuals were measured by gas chromatography. RESULTS: The membrane phosphatidylcholine C20:4n - 6 concentration was lower in the multiple sclerosis patients when compared to that of the control group, P = 0.04 and it correlated inversely with the EDSS and FSS. CONCLUSION: Decrease in C20:4n - 6 in the erythrocyte membrane could be an indication of depleted plasma stores, and a reflection of disease severity.

Pathogenic Mechanisms Underlying Iron Deficiency and Iron Overload: New Insights for Clinical Application.

Iron uptake, utilisation, release and storage occur at the gene level. Individuals with variant forms of genes involved in iron metabolism may have different requirements for iron and are likely to respond differently to the same amount of iron in the diet, a concept termed nutrigenetics. Iron deficiency, iron overload and the anemia of inflammation are the commonest iron-related disorders. While at least four types of hereditary iron overload have been identified to date, our knowledge of the genetic basis and consequences of inherited iron deficiency remain limited. The importance of genetic risk factors in relation to iron overload was highlighted with the identification of the HFE gene in 1996. Deleterious mutations in this gene account for 80-90% of inherited iron overload and are associated with loss of iron homeostasis, alterations in inflammatory responses, oxidative stress and in its most severe form, the disorder hereditary haemochromatosis (HH). Elucidation of the genetic basis of HH has led to rapid clinical benefit through drastic reduction in liver biopsies performed as part of the diagnostic work-up of affected patients. Today, detection of a genetic predisposition in the presence of high serum ferritin and transferrin saturation levels is usually sufficient to diagnose HH, thereby addressing the potential danger of inherited iron overload which starts with the same symptoms as iron deficiency, namely chronic fatigue. This review provides the scientific back-up for application of pathology supported genetic testing, a new test concept that is well placed for optimizing clinical benefit to patients with regard to iron status.

Oral zinc augmentation with vitamins A and D increases plasma zinc concentration: implications for burden of disease.

A study evaluating zinc supplementation in patients with Alzheimer's disease yielded variable zinc plasma levels in spite of positive cognitive and physiological results. In an attempt to raise and sustain plasma zinc levels, a single patient was given 15 mg zinc/day with various combinations of vitamins. A sustained raise in plasma zinc concentration (and therefore its potential bioavailability) was obtained only when the zinc was augmented with both vitamins A and D (in RDA concentrations). In order to verify these results, a follow-up study was conducted in 70 volunteers. Seven groups of 10 healthy subjects received various combinations of zinc and the two vitamins A and D, namely: zinc, vitamin A, vitamin D, zinc plus vitamin A, zinc plus vitamin D, vitamins A and D, and zinc plus vitamins A and D. Plasma zinc levels were determined at baseline, 3 weeks and 6 weeks. Plasma zinc levels increased significantly (p < 0.02) from 11.82 (+/-2.60) to 13.32 (+/-3.04) mum/L only in the group receiving the combination of zinc and vitamins A and D. This novel method of increasing plasma zinc levels by the augmentation of vitamins A and D may have implications for the reduction of burden of disease.

The effect of stress on the antioxidative potential of serum: implications for Alzheimer's disease.

There is growing consensus in the literature that oxidation status is increased in Alzheimer's disease (AD), and that antioxidant supplementation as prevention or treatment strategy should be investigated further. In the present study the total antioxidant status (TAS) was found to be highly significantly lower in 22 AD patients (p < 0.0001) than in 22 age- and gender matched non-demented controls. The TAS was also lower than controls in 22 patients with vascular dementia, but not significantly. The increased oxidation status in AD was verified using the benzoate hydroxylation method. The origin of the enhanced oxidation status in AD has not been elucidated. To determine whether a causal effect between stress and oxidative status of serum can be demonstrated, a rat model was used with two different kinds of stressors, swim stress (exercise) and restraint stress (non-exercise stress). Following swim stress the maximum oxidative effect was observed at one hour post stress (p < 0.001). At 24 h the oxidative status had recovered significantly to below control values. Restraint stress, however, showed progressively increased oxidation which attained significance after 24 h (p < 0.005). It is postulated that stress may contribute to the higher oxidation status in AD patients.

Iron and the folate-vitamin B12-methylation pathway in multiple sclerosis.

UNLABELLED: Some subjects with multiple sclerosis (MS) present with low blood iron parameters. Anecdotal reports and a single patient study suggest that iron supplementation may be beneficial in these subjects. Myelin is regenerated continually, but prerequisites for this process are iron and a functional folate-vitamin B12-methylation pathway. The aim of this study was to determine iron status, folate and homocysteine in MS subjects, and to evaluate the effect on MS symptoms if deficiencies were addressed. RESULTS: In relapsing-remitting MS subjects, serum iron concentration correlated significantly with age at diagnosis (r=0.49; p=0.008). In Caucasian female MS subjects, serum iron and ferritin concentrations were significantly lower than in matched controls. In a 6-month pilot study, 12 subjects taking a regimen of nutritional supplements designed to promote myelin regeneration, improved significantly neurologically as measured by the Kurzke EDSS (Total Score means 3.50 to 2.45, 29.9%; p=0.021). These were significantly improved (p=0.002) compared to 6 control group patients taking multivitamins (Kurzke Score increased by 13.9% from 4.83 to 5.50). Both groups had significantly reduced homocysteine concentrations at 6 months, suggesting that methylation is necessary but not sufficient for myelin regeneration.

Serum concentrations of some metals and steroids in patients with chronic fatigue syndrome with reference to neurological and cognitive abnormalities.

Chronic fatigue syndrome is defined by the Atlanta Centers for Disease Control (Atlanta, GA, USA) as debilitating fatigue lasting for longer than 6 months. Symptoms include disturbances of cognition. Certain factors have in the past been shown to influence cognition, including metals such as aluminum, iron, and zinc; and steroids such as dehydroepiandrosterone. In the present study, concentrations of these factors were determined in the serum and plasma of patients and their age- and gender-matched healthy controls (10 women and 5 men in each group). In addition, copper, dehydroepiandrosterone sulphate, cortisol, cholesterol, hemoglobin, ferritin and transferrin concentrations, as well as transferrin genetic subtypes were determined in both groups. The results indicate that patients had significantly increased serum aluminum and decreased iron compared to controls. In the females, serum iron and dehydroepiandrosterone sulphate were significantly decreased and correlated. Total cholesterol was significantly increased, and significantly negatively correlated with dehydroepiandrosterone sulphate. There were no differences in zinc, copper, cortisol, hemoglobin, transferrin and ferritin concentrations, or in transferrin genetic subtypes.

Earlier age of onset of Alzheimer's disease in patients with both the transferrin C2 and apolipoprotein E-epsilon 4 alleles.

The etiology of Alzheimer's disease is now known to be multifactorial. The genetic factors transferrin C2 (TfC2) and apolipoprotein E epsilon 4 (ApoE-epsilon 4) have both been associated with Alzheimer's disease (AD). Transferrin is the carrier protein for iron in the blood, while ApoE is involved with the transport and redistribution of lipids. In the present study, the polymerase chain reaction (PCR) method was used to determine the frequency of both TfC2 and ApoE-epsilon 4 in 27 AD patients, 9 vascular dementia (VaD) patients, and 27 controls. Patients were diagnosed according to the criteria as set out in the 4th edition of the Diagnostic and Statistical Manual of the American Psychiatric Association (DSM-IV). The frequency of the TfC2 allele for the AD patients was 24%, while for the VaD patients it was 12.5%, which was not significantly different from the controls at 13%. The frequency of ApoE-epsilon 4 for the AD patients was 44%, for the VaD patients 22%, and controls 17%. Of the 27 AD patients, 8 had both TfC2 and ApoE-epsilon 4. The age of onset of the disease in these 8 patients (51-67 years, mean 60.25) was significantly earlier (p < 0.02) than in the remaining AD patients (49-76 years, mean 66.9). None of the VaD patients had both the TfC2 and the ApoE-epsilon 4 alleles.

Glycosylation of transferrin in Alzheimer's disease and alcohol-induced dementia.

Transferrin is a glycosylated metal-binding serum protein. Carbohydrate-deficient transferrin (CDT) is a marker of recent and heavy alcohol intake. A genetic variant of transferrin, TfC2, occurs with increased frequency in patients with Alzheimer's disease (AD). Hence the question arose whether, in addition to an altered amino acid sequence, there could also be a difference in the glycosylation state of transferrin in patients with dementia. Serum samples of 37 AD and 13 Alcohol-induced dementia patients as well as 10 healthy controls were analyzed for abnormal Tf variants, using isoelectric focusing followed by blotting with anti-Tf antibodies. This allowed the direct visualization of glycosylation variants of transferrin, and assessment of any increase in underglycosylated forms (di-, mono- and asialo transferrin).

A comparative study of the effects of cholesterol, beta-sitosterol, beta-sitosterol glucoside, dehydroepiandrosterone sulphate and melatonin on in vitro lipid peroxidation.

The free radical scavenging abilities of the structurally related steroids beta-sitosterol, beta-sitosterol glucoside (plant sterols and sterolins), cholesterol, and dehydroepiandrosterone sulphate (DHEAS) were compared with melatonin (an efficient free radical scavenger) in an in vitro system which measures lipid peroxidation of platelet membranes in the presence of iron (Fe2+). Lipid peroxidation is a process whereby cellular membranes are damaged due to the oxidative deterioration of polyunsaturated lipids, which may lead to cell death and disease in living organisms. Substances such as vitamin E protect cellular membranes against oxidative damage due to their chemical structures. The steroids cholesterol, beta-sitosterol, beta-sitosterol glucoside and dehydroepiandrosterone (DHEA) are structurally related to each other. During aging, serum concentrations of DHEA, DHEAS and melatonin decrease, while the concentration of cholesterol tends to increase. The aim of the present study was to compare the role these substances play in lipid peroxidation over a wide concentration range. At concentrations lower than the free iron in the reaction mixture, all the steroids investigated decreased lipid peroxidation. At higher concentrations, cholesterol and beta-sitosterol increased lipid peroxidation, while DHEAS and melatonin continued to decrease lipid peroxidation.

Melatonin prevents beta-amyloid-induced lipid peroxidation.

Beta-amyloid is a major constituent of senile plaques that occur in the brains of Alzheimer's disease (AD) patients. Cell culture studies have shown that high concentrations of beta-amyloid are toxic and damage biological macromolecules. A number of experiments have shown that melatonin is a potent antioxidant. Melatonin not only neutralizes oxygen-derived free radicals but can also scavenge species of other types such as carbon-centered free radicals. The present study was designed to determine whether beta-amyloid toxicity would cause lipid peroxidation of human platelet membranes. Since aluminum has been implicated in the etiology of AD, we investigated the effects of aluminum on lipid peroxidation and whether the harmful effects of beta-amyloid are aggravated by aluminum. We also investigated whether melatonin had the ability to protect against beta-amyloid toxicity. Our results indicate that both beta-amyloid and aluminum dose-dependently increased lipid peroxidation in platelet membranes. Aluminum was more potent than beta-amyloid. Incubation of platelet membranes with increasing concentrations of aluminum in the presence of 100 microM beta-amyloid (fragment 25-35) resulted in lipid peroxidation levels of similar magnitude as the two substances, respectively. Prior administration of melatonin dose-dependently inhibited this effect. These results confirm the toxic effects of beta-amyloid to biological membranes. While aluminum itself damages membranes, its presence did not exacerbate the toxic effects of beta-amyloid. Melatonin effectively reduced the lipid peroxidation induced by beta-amyloid and aluminum, suggesting that its supplementation to AD patients may be beneficial.

A new model for the pathophysiology of Alzheimer's disease. Aluminium toxicity is exacerbated by hydrogen peroxide and attenuated by an amyloid protein fragment and melatonin.

OBJECTIVES: Although Alzheimer's disease (AD) is the leading cause of dementia in developed countries, there is an as yet unexplained lower prevalence of the disease in parts of Africa. AD is characterised by a catastrophic loss of neurons; free radicals (oxidative toxins) have been implicated in the destruction of the cells through the process of lipid peroxidative damage of cell membranes. Previously aluminium (Al) and a fragment of beta amyloid (A beta 25-35) were shown to exacerbate free-radical damage, while melatonin reduced this effect. The aim of the present study was: (i) to investigate the conditions determining the toxicity of Al and A beta 25-35; and (ii) to assess whether melatonin could attenuate the damage done by both aluminium and the amyloid fragment, thus suggesting a pathway for the aetiology of AD. DESIGN: An in vitro model system was used in which free radicals were generated, causing lipid peroxidation of platelet membranes, thus simulating the disease process found in the brain. RESULTS: 1. Al and A beta 25-35 caused lipid peroxidation in the presence of the iron (II) ion (Pe2+), Al being more toxic than A beta 25-35. 2. A beta 25-35 attenuated the lipid peroxidation promoted by Al. 3. Hydrogen peroxide (H2O2) greatly exacerbated the toxicity of Al and A beta 25-35. 4. Melatonin prevented lipid peroxidation by Al and A beta 25-35 in the absence of H2O2, but only reduced the process when H2O2 was present. CONCLUSIONS: In the light of the results obtained from the present study, the following hypotheses are formulated. 1. In AD, excessive quantities of Al are taken up into the brain, where the Al exacerbates iron-induced lipid peroxidation in the lysosomes. 2. In response, the normal synthetic pathway of amyloid protein is altered to produce A beta fragments which attenuate the toxicity of Al. In the process of sequestering the Al and iron, immature plaques are formed in the brain. 3. Microglia are activated, in an attempt to destroy the plaques by secreting reactive oxygen species such as H2O2. At this point in the disease process, lipid peroxidation causes a catastrophic loss of brain cells. 4. Melatonin, together with other free radical scavengers in the brain, reduces the free-radical damage caused by Al and A beta, except in the latter stages of the disease process. Since melatonin is produced by the pineal gland only in the dark, the excess of electric light in developed countries may help explain why AD is more prevalent in these countries than in rural Africa.

Zinc and platelet membrane microviscosity in Alzheimer's disease. The in vivo effect of zinc on platelet membranes and cognition.

OBJECTIVES: To investigate the effects of oral zinc supplementation on: (i) plasma zinc concentrations; (ii) platelet membrane microviscosity in vivo; and (iii) cognitive function of Alzheimer's disease (AD) patients. DESIGN: An open-labelled pilot study. SETTING: University of Stellenbosch Medical School and Stikland Hospital. SUBJECTS: Six volunteer AD patients. OUTCOME MEASURES: Plasma zinc levels, platelet membrane microviscosity and cognition (MMSE and ADAS-cog tests). RESULTS: Oral zinc supplementation (30 mg/day) did not increase plasma zinc levels significantly, but significantly increased platelet membrane microviscosity (P = 0.02; 6 patients). Four patients, who underwent 12 months of evaluation, showed modest cognitive improvement on psychometric testing (Mini-Mental State Examination and the cognitive portion of the Alzheimer's Disease Assessment scale scores). CONCLUSIONS: While earlier literature promoted the use of zinc in AD patients, a recent study has contradicted this and implicated zinc in the aetiology of Alzheimer's disease. On the basis of the above results, it may be premature to single out zinc as a causal agent in AD.

Free radical scavenging effects of melatonin and serotonin: possible mechanism.

Melatonin has been reported to be a potent free radical scavenger, but the mechanism by which it protects membranes from lipid peroxidation is poorly understood. The present study addresses this problem by comparing the free radical scavenging properties of melatonin and serotonin, two indoles with similar structure, but differing solubilities. Both serotonin and melatonin significantly prevented lipid peroxidation of platelet membranes. Additionally, melatonin significantly decreased the microviscosity (increased the fluidity) of platelet membranes, while serotonin had the opposite effect. These data led us to postulate that serotonin exerts its free radical scavenging action in the aqueous phase, or at the water-membrane interface, while melatonin positions itself within the lipid bilayer where it protects membrane phospholipids against free radical attack.

Transferrin C2 and Alzheimer's disease: another piece of the puzzle found?

A significant increase in the occurrence of the transferrin C2 genetic subtype has been found in patients with Alzheimer's disease. This variant has previously been linked to diseases thought to be associated with free radical damage. We hypothesize that Alzheimer's disease is caused by free radical damage to membranes of endocytic vesicles due to defective binding of iron and aluminium by Tf C2. The aluminium binds to the membranes, creating pores, while the iron reacts with H2O2 and superoxide radicals produced by activated microglia (brain phagocytes), to produce hydroxyl radicals (oxidative toxins), which attack the fatty acids in the membranes through these pores. In order to treat the disease successfully, it would be necessary to alleviate the multiple deficiencies caused by these toxins by constantly providing the cells with antioxidants and other essential nutrients. In addition, a drug that would stimulate the regrowth of neurons is needed.

Lipid peroxidation and platelet membrane fluidity--implications for Alzheimer's disease?

In humans, the fluidity of cell membranes generally decreases with age. Unexpectedly, several laboratories have found increased fluidity of platelet membranes (mainly endoplasmic reticulum) in patients with Alzheimer's disease (AD) compared with controls. In the present study, free radical induced lipid peroxidation was found to increase the fluidity of platelet membranes. Hydroxyl radicals were generated in the presence of Fe2+ and EDTA at low concentrations of ascorbate. It is hypothesised that platelet membranes are unable to restore their microviscosity by incorporating cholesterol. There may be a link between the result obtained in this study, the recently discovered decreased cholesterol content of affected AD neuronal membranes, and the increased frequency of epsilon 4 apolipoprotein E (a cholesterol carrier) found in AD patients.