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New perspectives in metal contamination of health food supplements and antacid tablets and Alzheimer's disease
NEUROBIOLOGY OF AGING, VOLUME l J, 1990 ABSTRACTS OF' SECOND INTERNATIONAL CONFERENCE ON ALZHEIMER'S DISEASE ENVIRONMENTAl. FACT()RS
316
the basis of electrophoretic mobility, coeluted with alpha-2-macroglobulln as a minor component of the first peak and was routinely detected in eletrophoresis. While contamination of peaks 2, 3 and 4 by proteins other than immunoglobulin, albumin and transferrin is likely, no other proteins were apparent in electrophoretic analyses. After DFO treatment, there was an evident redistribution of aluminum with a marked reduction in the amount associated with alpha-2-macroglobulin immunoglobulin and to a lesser extent albumin and transferrin. This decrease was accompanied by the formation of a new aluminum peak in a region of the chromatogram devoid of absorbance at 280 nm. However, employing detection at 214 nm and gel electrophoresis this new peak was found to be associated with a protein of approximate 60-70 Kd with an apparently high affinity for aluminum. Amino acid analysis showed a high content of acidic residues which is consistent with metal ion binding properties. (Supported by NSERC and the Alshelmer Society of Ontario of Peterborough and Hallburton)
261 EVIDENCE THAT LIPID PEROXIDATIONAND TOTAL IRON ARE INCREASED IN ALZHEIMER'S BRAIN. * Anne C. Andom, Robert S, Bdtton, Bruce R. Bacon. D e ~ of Psychiatryand Medicine,LSU MedicalCenter,Shreveport,LA 71130 Lipid peroxidationmay cause, or be a consequenceof tissue damage in a variety of disease states (HeJllw~, B. and Gutterldge, J.M., Arch. Biochem. and Blophy&, 246:501-514,1986). We havepreviouslydemonstratedthat the produotlon of tl'dobmbltur~ acid reactive products (TBAR) can be used as a measureof lipid pero~ddmionIn particulatemembranefragmentsof human brain (Andom, et aL MOf. PharmacoL, 33:155-162, 1988). To test the hypothesisthat .&Jzheimer'sdbeased (AD) brain had Increasedlipid peroxldation,we stimulated TBAR production with ascorbate(1.0 raM) In pertlculatemembranefragmentsof Young (< 45), aged (> 65), AD and aged matched controls for the AD. Our results were: CONDITION YOUNG
AGED AGEMATCH TO AD AD
TBAR (nmoles MDA/mg protein)
N
25.5 + 6.7 12.7 + 3.4*
4 7
10.8 + 2.3* 4.7 ± 1.4"*
4 5
M ~ O (MDA) was determInedas previouslydescrihed(Andom,et aL IBID) in p~liouk~e memlxane fragmentsderivedfrom young (<45 y.o.), aged >65 y.o.), agematchedto AD sample (ave. age 71.5--+5.0years), and AD s e m ~ (ave. age 71:4+4.4 years). The * denotes statisticallysignificantdifferencesas coml~red to young, as detennined by Student's t-test at p <0.05. The ** denotes stattstlcally slgnlflcentdifferencesbetweenthe AD group and Its age matchedcontrof at p <0.01.
These findings suggest that AD brain has increased lipid peroxldaUonaslivlty ant~, henceTBARI:~:x:luctioncannothe stimulatedas effec~aly poatmortem This finding b not reproduced in cases of young Indlvldualssuffedng acute anc0da (N=3; nmob MDA/MG protein = 20.3+6.4). However,we have ~ that AD brain has 3.87 + 1.25 up total iron/rng protein (N = 5) as comparedto aged matched nomADcontrolswhich have 1.94-+0.7ug/mg protein (N= 4), a statistically difference at p <0.05. Whether the postmortem reduction In stimulated TBAR productionin AD Is due to increasesof di- and td-valentmetals,or ~ anoxla is under Investigation. The results of these studies will also be presented. 262 ACCUMULATION OF FERRITIN IN ALZHEIMER DISEASE (AD) BRAIN. JiG. JOSHI', J. FLEMING', H. LASS=~M~IN=, K. IQBAL" AND I. GRUNDKE-IQBAL'. 'DEPT OF BIOCHEM, UNIV OF TN, KNOXVILLE, T N USA, 37996-0840, =INST BRAIN RES, UNIV VIENNA, AUSTRIA AND =INST B A S I C RES, STATEN ISLAND, NY 10314 USA. Brain iron levels increase with age. Some areas of the brain reach a maximal level and maintain it while others continue iron accumulation throughout life. About 30% of this iron is stored in ferritin (FER). This major iron ~ protein is composed of heavy, H (60%) and light, L (40%) chains. We ~ FER from normal and AD brain. The amounts of FER obtained from AD brain were ~ then ~ from normal brain and c o n f i n e d increased levels of Fe. The two native proteins were carbohydrate-free, similar in MW, IEF pattern and resolved by S D S P A G E into only two subunits in the expected relative proportions and reacted with H and L chain antibodies on Western blots. Up0n RPLC on a C18 column, the H and L fractions resolved into a t least ~ and three peaks respectively. The total amino acid c o m ~ of the major H peaks was similar to each other and to H chain of human heart; the L chain corresponded to the L chain of liver FER; I m m ~ studies carded out with liver FER antibodies reveled a marked ~ in FER immunoreectJvity in the neuritic (senile) plaques inAD ~ p u s . The increased FER accumulation was almost exclusively ~ w i t h the microglia which appeared to be greatly proliferated. In contrast, in the
diffuse pre-amyloid plaques without neuritic changes the microglia appeared to be normal. No correlation between increased FER/microglia activity and blood brain barrier damage as examined by IgG diffusion was detected. FER might participate in the formation of amyloid through the action of free radicals generated during the release of iron from FER. Alternatively the FER microglia system might be secondarily involved in the removal and processing of the amyloid. (Supported by the Council for Tobacco Research and Cole Neuroscience Foundation to JGJ, Ministry for Sci and Res, Austria to H,L and NIH grants NS 18105, AG04220 and AG05892 to IGI and KI). 263 NEW PERSPECTIVES IN METAL CONTAMINATION OF HEALTH FOOD SUPPLEMENTS AND ANTACID TABLETS AND ALZHEIMER'S DISEASE. *B.M. Boulos, A.W. yon Smolinski. School of Public Health, and College of Pharmacy, University of Illinois at Chicago, Chicago, Illinois 60680 USA. Tablets of the mineral dolomite, a calcium magnesium carbonate, and antacid tablets containing mainly calcium carbonate were found to contain traces of toxic metals such as lead, cadmium, copper, manganese and other. Since there is some indication for an etiologic role of aluminum in dementing illness of the Ahheimer's type calcium supplement tablets were analyzed for aluminum: Ten tablets of the same brand, randomly selected, were weighed, dried, pulverized and low temperature plasma ashed. An aliquot of the ash was dissolved in 35% metal free nitric acid, diluted and analyzed for aluminum using a Perkin-Elmer Model 5000 atomic absorption spectrophotometer equipped with HGA 500 graphite furnace, AS-1 Autosampler and PRS-10 printer sequencer. The aluminum content of dolomite tablets ranged from 0.4-1.3 mg/g powder (n=10, ,'~=0.9+0.3 rag/g, p=0.05). The aluminum level of antacid tablets was lower. It ranged from 8.7-14.1 ug/g powder (n=10, 'X=10.9+1.4 ug/g, p=0.05). The toxicity of aluminum has only recently been recognized. Research suggests that aluminum is implicated in Alzheimer's disease and dialysis dementia. Indications are that aluminum binds to DNA, deposits in abnormal neurofibrillary tangles in the brain and inhibits the enzyme hexokinase. We do not claim that these levels of aluminum are high enough to cause hazards in the general population, yet they may cause adverse effects in high risk groups. The need exists to determine such metal levels in other brands and initiate some regulation to limit the maximal content of traces of toxic metals in such supplements, as a large sector of the public uses mega-doses of such products. 264 CALCIUM AND PHOSPHORUS IN DEMENTIA *M.N. Subhash, T.S. Padmashree, K.N. Srinivas, S.K. Shankar. Department of Neurochemistry, Psychiatry & Neuropatt~31ogy, Institute of Mental Health and NeuroSciances, Beng~dore-560 029, India Altered calcium homeostasis appears to be pathologically important in age related decline in brain function and various neurodegenerative disorders. Since the relationship between caldum and phosphorus in cerebrospinai fluid has not been studied so extensively =n aging, total and diffusible calcium and phosphorus in cerebrospinal fluid was estimated (by atomic absorption spectrophotomet~) in patients with dementia (65) and compared with geriatric controls (20) in India. A significant decrease in both calcium and phosphorus in C,SF was observed in Alzheimer's type dementia (13<0.01) and multiple infarct dementia (p<0.01) when compared to adult controls. A 60% dem,eaas in diffusible CSF Ca we8 observed both in patients andgartatdccontrols when compared to adult controls (p<0.001). Diffusible Pi was also decreased in rheas groups (p<0.05). However, a marginal decrulse in serum CA and slight increase in Pi was observed in both ~ and geriatric controls. A stgnifw,ant decrease in Ca and Pi i n ~ when compared to aged controls suggests that this lowering of Ca and Pi is not solely due to aging process and may indicate a role in the pathology of these disorders. This decrease might be due to cellular
316
the basis of electrophoretic mobility, coeluted with alpha-2-macroglobulln as a minor component of the first peak and was routinely detected in eletrophoresis. While contamination of peaks 2, 3 and 4 by proteins other than immunoglobulin, albumin and transferrin is likely, no other proteins were apparent in electrophoretic analyses. After DFO treatment, there was an evident redistribution of aluminum with a marked reduction in the amount associated with alpha-2-macroglobulin immunoglobulin and to a lesser extent albumin and transferrin. This decrease was accompanied by the formation of a new aluminum peak in a region of the chromatogram devoid of absorbance at 280 nm. However, employing detection at 214 nm and gel electrophoresis this new peak was found to be associated with a protein of approximate 60-70 Kd with an apparently high affinity for aluminum. Amino acid analysis showed a high content of acidic residues which is consistent with metal ion binding properties. (Supported by NSERC and the Alshelmer Society of Ontario of Peterborough and Hallburton)
261 EVIDENCE THAT LIPID PEROXIDATIONAND TOTAL IRON ARE INCREASED IN ALZHEIMER'S BRAIN. * Anne C. Andom, Robert S, Bdtton, Bruce R. Bacon. D e ~ of Psychiatryand Medicine,LSU MedicalCenter,Shreveport,LA 71130 Lipid peroxidationmay cause, or be a consequenceof tissue damage in a variety of disease states (HeJllw~, B. and Gutterldge, J.M., Arch. Biochem. and Blophy&, 246:501-514,1986). We havepreviouslydemonstratedthat the produotlon of tl'dobmbltur~ acid reactive products (TBAR) can be used as a measureof lipid pero~ddmionIn particulatemembranefragmentsof human brain (Andom, et aL MOf. PharmacoL, 33:155-162, 1988). To test the hypothesisthat .&Jzheimer'sdbeased (AD) brain had Increasedlipid peroxldation,we stimulated TBAR production with ascorbate(1.0 raM) In pertlculatemembranefragmentsof Young (< 45), aged (> 65), AD and aged matched controls for the AD. Our results were: CONDITION YOUNG
AGED AGEMATCH TO AD AD
TBAR (nmoles MDA/mg protein)
N
25.5 + 6.7 12.7 + 3.4*
4 7
10.8 + 2.3* 4.7 ± 1.4"*
4 5
M ~ O (MDA) was determInedas previouslydescrihed(Andom,et aL IBID) in p~liouk~e memlxane fragmentsderivedfrom young (<45 y.o.), aged >65 y.o.), agematchedto AD sample (ave. age 71.5--+5.0years), and AD s e m ~ (ave. age 71:4+4.4 years). The * denotes statisticallysignificantdifferencesas coml~red to young, as detennined by Student's t-test at p <0.05. The ** denotes stattstlcally slgnlflcentdifferencesbetweenthe AD group and Its age matchedcontrof at p <0.01.
These findings suggest that AD brain has increased lipid peroxldaUonaslivlty ant~, henceTBARI:~:x:luctioncannothe stimulatedas effec~aly poatmortem This finding b not reproduced in cases of young Indlvldualssuffedng acute anc0da (N=3; nmob MDA/MG protein = 20.3+6.4). However,we have ~ that AD brain has 3.87 + 1.25 up total iron/rng protein (N = 5) as comparedto aged matched nomADcontrolswhich have 1.94-+0.7ug/mg protein (N= 4), a statistically difference at p <0.05. Whether the postmortem reduction In stimulated TBAR productionin AD Is due to increasesof di- and td-valentmetals,or ~ anoxla is under Investigation. The results of these studies will also be presented. 262 ACCUMULATION OF FERRITIN IN ALZHEIMER DISEASE (AD) BRAIN. JiG. JOSHI', J. FLEMING', H. LASS=~M~IN=, K. IQBAL" AND I. GRUNDKE-IQBAL'. 'DEPT OF BIOCHEM, UNIV OF TN, KNOXVILLE, T N USA, 37996-0840, =INST BRAIN RES, UNIV VIENNA, AUSTRIA AND =INST B A S I C RES, STATEN ISLAND, NY 10314 USA. Brain iron levels increase with age. Some areas of the brain reach a maximal level and maintain it while others continue iron accumulation throughout life. About 30% of this iron is stored in ferritin (FER). This major iron ~ protein is composed of heavy, H (60%) and light, L (40%) chains. We ~ FER from normal and AD brain. The amounts of FER obtained from AD brain were ~ then ~ from normal brain and c o n f i n e d increased levels of Fe. The two native proteins were carbohydrate-free, similar in MW, IEF pattern and resolved by S D S P A G E into only two subunits in the expected relative proportions and reacted with H and L chain antibodies on Western blots. Up0n RPLC on a C18 column, the H and L fractions resolved into a t least ~ and three peaks respectively. The total amino acid c o m ~ of the major H peaks was similar to each other and to H chain of human heart; the L chain corresponded to the L chain of liver FER; I m m ~ studies carded out with liver FER antibodies reveled a marked ~ in FER immunoreectJvity in the neuritic (senile) plaques inAD ~ p u s . The increased FER accumulation was almost exclusively ~ w i t h the microglia which appeared to be greatly proliferated. In contrast, in the
diffuse pre-amyloid plaques without neuritic changes the microglia appeared to be normal. No correlation between increased FER/microglia activity and blood brain barrier damage as examined by IgG diffusion was detected. FER might participate in the formation of amyloid through the action of free radicals generated during the release of iron from FER. Alternatively the FER microglia system might be secondarily involved in the removal and processing of the amyloid. (Supported by the Council for Tobacco Research and Cole Neuroscience Foundation to JGJ, Ministry for Sci and Res, Austria to H,L and NIH grants NS 18105, AG04220 and AG05892 to IGI and KI). 263 NEW PERSPECTIVES IN METAL CONTAMINATION OF HEALTH FOOD SUPPLEMENTS AND ANTACID TABLETS AND ALZHEIMER'S DISEASE. *B.M. Boulos, A.W. yon Smolinski. School of Public Health, and College of Pharmacy, University of Illinois at Chicago, Chicago, Illinois 60680 USA. Tablets of the mineral dolomite, a calcium magnesium carbonate, and antacid tablets containing mainly calcium carbonate were found to contain traces of toxic metals such as lead, cadmium, copper, manganese and other. Since there is some indication for an etiologic role of aluminum in dementing illness of the Ahheimer's type calcium supplement tablets were analyzed for aluminum: Ten tablets of the same brand, randomly selected, were weighed, dried, pulverized and low temperature plasma ashed. An aliquot of the ash was dissolved in 35% metal free nitric acid, diluted and analyzed for aluminum using a Perkin-Elmer Model 5000 atomic absorption spectrophotometer equipped with HGA 500 graphite furnace, AS-1 Autosampler and PRS-10 printer sequencer. The aluminum content of dolomite tablets ranged from 0.4-1.3 mg/g powder (n=10, ,'~=0.9+0.3 rag/g, p=0.05). The aluminum level of antacid tablets was lower. It ranged from 8.7-14.1 ug/g powder (n=10, 'X=10.9+1.4 ug/g, p=0.05). The toxicity of aluminum has only recently been recognized. Research suggests that aluminum is implicated in Alzheimer's disease and dialysis dementia. Indications are that aluminum binds to DNA, deposits in abnormal neurofibrillary tangles in the brain and inhibits the enzyme hexokinase. We do not claim that these levels of aluminum are high enough to cause hazards in the general population, yet they may cause adverse effects in high risk groups. The need exists to determine such metal levels in other brands and initiate some regulation to limit the maximal content of traces of toxic metals in such supplements, as a large sector of the public uses mega-doses of such products. 264 CALCIUM AND PHOSPHORUS IN DEMENTIA *M.N. Subhash, T.S. Padmashree, K.N. Srinivas, S.K. Shankar. Department of Neurochemistry, Psychiatry & Neuropatt~31ogy, Institute of Mental Health and NeuroSciances, Beng~dore-560 029, India Altered calcium homeostasis appears to be pathologically important in age related decline in brain function and various neurodegenerative disorders. Since the relationship between caldum and phosphorus in cerebrospinai fluid has not been studied so extensively =n aging, total and diffusible calcium and phosphorus in cerebrospinal fluid was estimated (by atomic absorption spectrophotomet~) in patients with dementia (65) and compared with geriatric controls (20) in India. A significant decrease in both calcium and phosphorus in C,SF was observed in Alzheimer's type dementia (13<0.01) and multiple infarct dementia (p<0.01) when compared to adult controls. A 60% dem,eaas in diffusible CSF Ca we8 observed both in patients andgartatdccontrols when compared to adult controls (p<0.001). Diffusible Pi was also decreased in rheas groups (p<0.05). However, a marginal decrulse in serum CA and slight increase in Pi was observed in both ~ and geriatric controls. A stgnifw,ant decrease in Ca and Pi i n ~ when compared to aged controls suggests that this lowering of Ca and Pi is not solely due to aging process and may indicate a role in the pathology of these disorders. This decrease might be due to cellular