Alzheimer's Disease: Serum Biological Markers in Relation to Disease Severity

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1 Dorriya A. El-Sayed et al. Alzheimer's Disease: Serum Biological Markers in Relation to Disease Severity Dorriya A. El-Sayed 1, Hanan Salah 1, Mahmoud M. El-Abyary 1, Ashraf M. Zaitoun 1, Ahmed Gaballah 2 Departments of Neurology 1, Clinical Pathology 2, Zagazig University ABSTRACT Background and Purpose: The diagnosis of Alzheimer's disease (AD) is currently based on clinical and neuropsychological examination. To date, there is no blood test available that can discriminate dementia patients from healthy individuals. In the present study, we measured levels of S100B, interleukin-1 (IL-1) and vitamin E in sera of AD patients to find their relation with disease severity in an attempt for intervention of new prophylactic treatment strategies. Patients and Methods: S100B, interleukin-1 (IL-1) and vitamin E were measured in 65 patients (30 males and 35 females mean age years) diagnosed as clinically probable AD. Their levels were compared with those of age and sex matched healthy controls. All patients were evaluated clinically using Minimental State Examination (MMSE), the Global Deterioration Scale and the Clinical Dementia Rating. Patients were divided into three groups according to disease severity on basis of MMSE. Results: There was significant increase of serum S100B, IL-1 levels among patients than controls, while serum vit. E levels were slightly reduced among patients than controls. When comparing serum levels of S100B, IL-1 and vit. E between the three groups of patients: only S100B showed significantly negative correlation with MMSE. Whereas IL-1 and vit. E levels were significantly different in severe cases only in comparison with mild and moderate patients. Conclusion: the definite statement concerning the role of inflammatory biomarkers in AD needs the use of sensitive assays, large patient number and studies showing improvement of cognitive functions after vit. E supplementation are awaited for with interest. (Egypt J. Neurol. Psychiat. Neurosurg., 2009, 46(1): ) INTRODUCTION In recent years, the incidence of dementia as a major public health problem has escalated largely due to an increased life expectancy 1. Alzheimer's disease (AD) is the most common form of senile dementia accounting for 50-60% of all cases, and is considered as the fourth highest cause of disability and death in the elderly 2. Although its occurrence may be age-related, AD is not an inevitable consequence of the aging process 3. Alzheimer's disease is a heterogenous disease with respect to several possible pathophysiological causes and mechanisms contributing to a common stage of cell death 4, but the exact nature of the molecular entity triggering dementia and cognitive deficits is still open to debate 5. In the ideal situation, early diagnosis should be possible with an early and unexpensive blood test, however, no single peripheral biological marker with specificity for AD has been found 6. S100B is predominantly astrocytic protein with a cytokine-like functions. The chronic overdose of S100B in Down's syndrome with its associated progressive neurodogeneration and the resemblance of some clinicopathological features to those of AD have led to the hypothesis that S100B may contribute to disease progression 7. Interleukin-1 (IL- 1), a key molecule in systemic immune responses in health and disease, has analogous roles in the brain, where it may contribute to nerodegeneration 8. Vitamin E (vit. E) is known as a major chain breaking antioxidant in the brain and has been advocated as a modulator of cognitive performance 9. The aim of our study is to measure serum levels of S100B, IL-1 and vit. E. in relation to AD patients, hence early AD stage predictive biomarkers would enable diagnosis of the disease at a premature Correspondence to hsmr2007@yahoo.com. Contact number:

2 Egypt J. Neurol. Psychiat. Neurosurg. phases opening up the prospect for timely application of corrective therapeutic strategies. 178 SUBJECTS AND METHODS A total of 65 patients of both sexes were included in our study, diagnosed as AD (clinically probable)-according to the criteria of National Institute of Neurological and Communication Disorders and AD and Related Disorders Association (NINCDS/AD RDA) 10, with Hachiniski Ischemic Score (HIS)<4 11. Patients were recruited from the outpatient clinic of Neurology Department of Zagazig University Hospitals. Twenty healthy subjects age and sex matched served as controls. All subjects were screened by medical history, general and neurological examination, electrocardiogram and laboratory evaluation of serum electrolytes, glucose, renal and hepatic functions, complete blood count and thyroid function studies. Subjects with past or present major psychiatric disorders, serious head trauma, hypoxia, neurological diseases other than AD, renal or hepatic diseases or chronic obstructive pulmonary diseases were excluded. All subjects were given the Mini Mental State Examination (MMSE) 12 to assess cognitive functions. The assigned scores ranging from 30-for normal function-down to 5 in case of severe dementia. These assessments were further supported by tests of cognitive function in accord with the Global Deterioration Scale (GDS) 13 and the Clinical Dementia Rating (CDR) 14. CDR stages 1 and 2 include persons in the earlier or mild-moderate stages of dementia, stages 3, 4 and 5 include persons in the later advanced stages of dementia. Patients were divided into 3 groups regarding severity of dementia according to the MMSE 15 as follows: * Group I: included mild cases where MMSE ranged from > * Group II: included moderate cases ranging from * Group III: included severe cases with MMSE < Vol. 46 (1) - Jan Magnetic resonance imaging (MRI) scan (0.5- Tesla general Electric SIGMA Contour System) was carried out for all patients to exclude any vascular lesion 16. Serum S100B measurement: Venous blood was withdrawn, sera were separated and stored at 20 C. The assessment was done by the commercially available monoclonal two site immunoluminometric assay and fully automatic LIA-mat system (Byk-Sang Tec diagnostica, Diatzenbach, Germany). The detection limit of this test was 0.02 mg/l. When the S100B level was under the detection limit, the value was recorded as 0.02 mg/l. Serum IL-1B: Venous blood sample was withdrawn and allowed to clot at room temperature for 30 minutes and after being centrifuged for ten minutes, the obtained serum was stored at -80 c. IL-1B levels in serum samples were quantified by ELISA technique, (DIACIONE) research. A monoclonal antibody specific for IL-1B had been coated into wells and microtitre strips.smaples were pippted into those wells and then incubated. During incubation, the IL- 1B antigen and biotinylated monoclonal antibody specific for IL-1B were simultaneously incubated. After washing, the enzyme (streptaridin-peroxylase) is added. After incubated and washing to remove all unbound enzyme, a substrate solution which is acting on the bound enzyme is added to induce a colored reaction product. The intensity of this colored product is directly proportional to the concentration of IL-1B present in the samples. Vit. E measurement: Serum vit. E was estimated by HPLC according to Teissier et al. 17. Fifty ml of standard (SIGMA Aldrichchemico, Germany) and samples of both controls and patients were diluted in 850 ml of methanol mixed twice for ten seconds by vortex shaker. Centrifugation for one minute at rpm then 50 ml of the supernatant were injected directly in HPLC apparatus (pum, Gbc-Lc 1150 Australia) flow rate 1m/min, using fluorescence detector set at 295 nm and 330 nm for excitation and emission respectively. The results were calculated and analyzed by Winchrom software

3 Dorriya A. El-Sayed et al. using calibration curve of vit. E standard which was constructed by using six different concentrations. Statistical analysis: Data were analyzed by a Statistical Package for Social Sciences SPSS for Windows version 11. Data were expressed as mean standard deviation for quantitative variables, number and percentage for qualitative variables. ANOVA, student t-test, chisquare and correlation coefficient were used when appropriate. P-value <0.05 was considered to be statistically significant. RESULTS Sixty five patients diagnosed as clinically probable AD were included in this study. They were 30 males and 35 females, their ages ranged from years (mean SD, ). Twenty healthy subjects served as controls (12 males & 8 females), mean age Table (1) shows demographic data, MMSE and disease duration. MMSE scores were significantly lower among AD patients than controls. These assessments were also supported by tests of cognition in accord with the GDS and the CDR. All clinical evaluations yielded consistent results throughout, therefore only MMSE criteria were used for comparative cognitive and statistical studies. Patients were divided into three groups regarding disease severity in accord with MMSE. - Group I (mild disease): including 34 patients with MMSE ranging from > Group II (moderate disease): including 22 patients with score ranging from Group III (severe cases) including 9 patients scoring less than 10 in MMSE. Serum levels of S100B, IL-1 and vit. E in patients and controls are shown in Table (2) with S100B and IL-1 levels highly significant increase among patients than controls, whereas serum vit. E levels were slightly reduced in patients than controls with no statistical significant difference. When comparing serum levels of the three studied biomarkers between the three groups of patients: Table (3) shows increased serum S100B levels with increasing severity of the disease with statistical significant difference between the three groups. Regarding IL-1 and Vit. E serum levels, a statistically significant difference is only detected between group III in comparison to group I and II, with increased levels of IL-1, and decreased levels of vit. E, where no statistical significant difference were found between group I and II. A significant negative correlation is shown between MMSE and serum S100B in the 3 patients groups. Whereas IL-1 and vit. E did not correlate significantly with disease severity judged by MMSE (Table4). Table 1. Demographic and clinical of patient and control groups. Control (n=20) Patient (n=65) Age in years t P Mean SD Range Sex X 2 P Male 12 (60%) 30 (46.2%) Female 8 (40%) 35(53.8%) MMSE t P Mean SD <0.001 Range <23 Disease duration Mean SD

4 Egypt J. Neurol. Psychiat. Neurosurg Vol. 46 (1) - Jan Range 3-10 Table 2. S100B, IL-1 and vit. E serum levels in patients and controls. Control Patient t P (n=20) (n=65) S100B ** IL <0.001** Vit. E Table 3. Relation of serum S100B, IL-1 and vit. E of AD patients in the three groups. Group I Group II Group III P S100B * <0.01* IL * Vit. E ** Table 4. Correlation between serum biomarkers levels and disease severity according to MMSE in the three patients groups. r P S100B <0.001** IL >0.05 Vit. E 0.12 >0.05 DISCUSSION The leading cause of dementia is AD, which is a progressive neurodegenerative disorder affecting the elderly 18. It is distinguished by a progressive and devasting mental decline that is usually expressed by memory impairment stemming from a disturbance within medial temporal lobe structures such as hippocampus 19. Considering the limited capacity of the central nervous system (CNS) tissue to repair, early intervention in the degenerative processes will be crucial to spare as much tissue as possible 20. Early diagnosis may then help to increase the possibilities for developing and testing new preventive treatment strategies 8. S100B acts as an astrocyte-derived cytokine with a physiologically beneficial role in promoting neuronal survival and development 21, as well as the synthesis of amyloid precursor protein (APP) in neurons and neurites 22. However, pathologically over expressed S100B induces dystrophic changes in neurons and neurites, which correlate with the transformation of benign amyloid deposits into neuritic plaques responsible for cortical atrophy in AD 7. In our study, the serum content of S100B was significantly elevated among AD patients than controls. A finding that is supported by previous two reports 5,23. We were not able to compare our results with most of earlier studies for two reasons: firstly to our knowledge almost all previous studies 7,23,24,25 had measure S100B in cerebro-spinal fluid (CSF) not in serum. Secondly, they had used patients suffering neurological diseases other than dementia as control subjects so the results could have diagnostic value only for discrimination of AD patients from other CNS patients. We have found a significant relation between serum S100B levels and disease severity where S100B levels correlated negatively with the MMSE scores, a finding that lend further support to the suggestion that S100B is an important, early acting pathogenic agent in the 180

5 Dorriya A. El-Sayed et al. progression of the neuropathophysiological changes underlying the clinical manifestation of AD 24. In contrast to our results, previous study 25 did not show significant differences in S100B levels in relation to disease severity. This may be referred to the broad range of clinical dementia severity and relatively larger number of AD patient samples examined in our study. A major inducer of astrocyte activation and S100B expression is the immunomodulatory cytokine IL IL-1 is markedly expressed by activated microglia in AD 27. In the current study, there was a significantly higher levels of serum IL-1 in AD patients in comparison to control subjects, which was also reported by another study 28. Where as most studies that investigated serum showed no difference in serum IL-1 levels between AD patients and controls 29,30,31. This may be regarded to the low level of IL-1 present in serum of normal controls in combination with the low sensitivity of the ELISA systems used. So, differences in mean concentrations can only be measured when a large group of AD patients is included, consequently an increase in IL-1 concentration in serum may have very limited diagnostic value for single patients 23. When comparing serum IL-1 levels between the three groups of our patients, only the severe AD patients (group III) revealed significantly higher levels when compared with the other two groups. This may be explained by the fact that IL-1 is not able to cross the intact blood brain barrier (BBB), and only with progression of the disease to severity stage and disruption of BBB to permit significant peripheral increase of the level of IL-1 to be detected 32. In such case, the altered serum level in the mild and moderate cases may not be due to increased flow of inflammation related proteins from the brain when the BBB is intact. It might be possible that a whole body damage in concentrations of interleukins occurs, which affects the brain tissue specifically. Alternatively, damage to brain tissue may be one of the various effects of an increased peripheral concentration of interleukins 23. Vitamin E is considered as an important antioxidant whose level can be influenced by dietary habits. It is especially important for the brain, considering the high lipid content and a relatively high proportion of polyunsaturated fatty acids of the brain 33. When comparing serum vit. E levels between patients and controls, there was no significant difference with decreased levels among all AD patients under normal dietary circumstances i.e. without any supplementation. Decreased concentrations of vitamins A, C and E in serum of patients with dementia have been reported 34,35,36. Regarding the relation between disease severity and vitamin E concentration, there was a significant decrease of vit. E levels among group III patients compared to group I & II. However, no correlation was found between serum vit. E. and disease severity. A low vitamin status may give an indication of the susceptibility of an individual to oxidative damage. Nevertheless, the causality in the relation between cognitive impairment and vitamin status is not known. As vitamin deficiency is common in older people due to insufficient intake, reduced intestinal absorption 37. Thus dietary intake or supplementation can influence the levels of this vitamin. In summary, S100B plays an important role in initiation and prorogations of AD, though the definite statements concerning inflammation biomarker differences between control and AD patients require the use of sensitive assays, and large patient groups. This is imperative not only for measuring very low concentrations of S100B or IL-1 but all possible AD markers. Studies showing improvement of cognitive functions after vit. E. supplementation is awaited. REFERENCES 1. Ritchie K, and Lovestone S. (2002): The dementias. Lancet; 360 : Blennow K., Leon M. and Zetterberg H, (2006): Alzheimer's disease. Lancet; 368: Bennett D.A., Schneider J.A., Wilson R.S., et al. (2004): Neurofibrillary tangles mediate the association of amyloid load with clinical Alzheimer's disease and level of cognitive function. Arch Neurol; 61: Terry R.D., Masliah E., Salmon D.P., et al. (1991): Physical basis of cognitive alterations in Alzheimer's disease: Synape loss is the major correlate of cognitive impairment. Ann Neurol; 30:

6 Egypt J. Neurol. Psychiat. Neurosurg. 5. Marina A.G., tatyana B.D., mantas M., et al. (2007): Differential neuroimmune markers to the onset of Alzheimers disease neurodegeneration and dementia: Autoantibodies to AB (25-35) oligomers, Sl00B and neurotransmitters.j.neuroimmun;186: Griffin W.S.T., Ling L., Yuckui L., et al. (2006): Interleukin-1 mediates Alzheimer and Lewy body pathologies. J. Neuroimmun; 181: Petzold A., Jenkins R., Watt H.C., et al. (2003): CSF S100B correlates with brain atrophy in AD. Neurosceince Letters; 336: Griffin W.S.T., Robert E.M. (2002): Interleukin-1 in the genesis and progression of and risk for development of neurobal degeneration in Alzheimer's disease. J of Leukocyte Biology; 72: Nourhashemi F., Gillette-Guyonnet S., Andrieu S., et al. (2000): Alzheimer disease: Protective factors. Am J Clin Nutr; 71: Mckhann G., Drachman D., Folstein M., et al. (1984): Clinical diagnosis of AD. Neurology;34: Hachiniski V.C., Liff L.D., Zilhka E., et al. (1975): Cerebral blood flow in dementia. Arch Neuro; 32: Folstein M.F., Folstein S.E., McHugh P.R. (1975): Mini Mnental state practical method for grading the cognitive state of patients for the clinician. J Psychiatr Res; 12: Reisberg B., Ferris S.H., DeLeon M.J., et al. (1982): The global deterioration scale for assessment of primary degenerative dementia. Am J Psychiatr; 139: Morris J.C. (1993): The clinical dementia rating (CDR): current version and scoring rules. Neurology; 43: Molloy D.W., Alemayehu E.,and Roberts R. (1991): Standerdized MMSE. Am J Psychiatry; 148: Wallin A.,and Blennow K. (1994): The clinical diagnosis of vascular dementia. Dementia;5: Teisser E., Walters Laporte E., Duhem C., et al. (1996): Rapid quantification of -tocopherol in plasma and low and high density lipoproteins. Clinical Chemistry, 42(3): Holscher C.(2005): Development of betaamyloid-induced neurodegeneration in Alzheimer's disease and novel neuroprotective strategies Rev.Neurosci.16, Kohler S., Black S.E., Sinden M., et al. (1998): Memory impairments associated with hippocompal versus parahuppocampal-gyrus 2009 Vol. 46 (1) - Jan atrophy:an MR volumetry study in AD. Neurpsychologia;36: Elgh E., Lindqvist Astot A.,and Fagerlund M. (2006): Cognitive dysfunction, hippocampal atrophy and glucocarticoid feedback in AD. Biol Psychiatry; 59: Chan W.Y., Xia C.L, and Dong D.C. (2003): Differential expression of S100 proteins in the developing human hippocampus and temporal cortex. Microsc Res Tech;60: Li Y., Wang J., Sheng J.G., et al. (1998): S100B increases levels of beta-amyloid precursor protein an dits encoding in RNA in rat neuronal cultures. J Neurochem;71: Teunissen C.E., DeVente J.,and Steinbusch H.W.M. (2002): Biochemical markers related to AD in serum and CSF. Neurobiol Aging; 23: Peskin E.R., Griffen WEST, Alama K.T., et al. (2001): CSF S100B is elevated in the earlier stages of AD. Neurochem. International; 39: Green A.J., Harvey R.J., Thompson E.J., et al. (1997): Increased S100B in the CSF of patients with from to temporal dementia. Neuroscience Letters; 235: Sheng J.G., Ito K, Skinner R.D., et al. (1996): In vivo & in vitro evidence supporting an role for the inflammatory cytokine IL-1 is a driving force in AD pathogenesis. Neurobiol Aging; 17: Griffin WEST, Sheng J.G., Royston M.C., et al. (1998): Glial-neuronal interactions in AD: the potential role of a cytokine cycle in disease progression. Brain Pathol; 8: Licastro F., Pedrini S., Caputo L., et al. (2000): Increased plasma levels of IL-1, IL-6 and -1- antichemotrypsin in patients with AD: Peripheral inflammation or signals from the brain. J Neuroimmunol; 103: Lanzrein A.S., Johnston C.M., Perry V.H., et al. (1998): Longitudinal study of inflammatory factors in serum, CSF, and brain tissue in AD. Alzheimer Dis Assoc Disord; 12: Taddei K., Clarnette R., Gandy S.E., et al. (1997): Increased plasma apoe levels in AD. Neuroscience Letters; 223: Tarkowski E., Blennow K., Wallin A., et al. (1999): Intracerbral production of TNF-alpha, a local neuroprotective agent in AD vascular dementia. J Clin Immunol; 19:

7 Dorriya A. El-Sayed et al. 32. Kim S.H., Smith C.J., and VanEldik L.J.(2004): Importance of MAPK pathways for microglial pro-inflammatory cytokine IL-1 beta production. Neurobiol Aging; 25 (14): Grundman M. (2000): Vitamin E and AD: The basis for additional clinical trials. Am J Clin Nutr; 71: Foy C.J., Passmore A.P., Vahidassr M.D., et al. (1999): Plasma chain-breaking antioxidants in AD, vascualr dementia and parkinson's disease. QIM; 92: Sincclair A.J., Bayer A.J., Johnston J., et al. (1998): Altered plasma antioxidant status in subjects with AD and vascular dementia. Int J Geriatr Psychiatry; 13: Zaman Z., Roche S., Fielden P., et al. (1996): Plasma concentrations of vitamin E and A and carotenoids in AD. Age Aging; 21: Ahlskog J.E., Uitti R.J., Low P.A., et al. (1995): No evidence for systemic oxidant stress in parkinson's or AD. Mov Disord;10: الملخص العربى الدالالث البيىكيميائيت في مصل الدم لمرض الزهايمر وعالقتها بشدة المرض 1 S100B S S100 S

8 Egypt J. Neurol. Psychiat. Neurosurg Vol. 46 (1) - Jan S100B 178

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