American Journal of Medical Genetics 35459-467 (1990)

Red Cell Superoxide Dismutase, Glutathione Peroxidase and Catalase in Down Syndrome Patients With and Without Manifestations of Alzheimer Disease Maire E. Percy, Arthur J. Dalton, Vjerica D. Markovic, Donald R. Crapper McLachlan, Jocelyn T. Hummel, Ann C.M. Rusk, and David F. Andrews Departments of Obstetrics and Gynaecology (MEP, J T H , ACMR), Physiology and Medicine (DRCM), Statistics and Preventive Medicine and Biostatistics (DFA), University of Toronto, Mount Sinai Hospital (MEP), and Surrey Place Centre (AJD, VDM), Toronto, Canada. The activities of red blood cell enzymes that scavenge the superoxide radical and hydrogen peroxide were measured in severely to profoundly retarded adult Down syndrome (DS)patients with and without manifestations of Alzheimer disease (AD), and control individuals matched for sex, age, and time of blood sampling. Cu,Zn superoxide dismutase (SOD-1)and glutathione peroxidase (GSHPx) activities were significantly elevated (1.39fold and 1.24-fold,respectively)in DS individuals without AD. When an adjustment was made for the SOD gene dosage effect, DS patients with AD manifestations had significantly lower SOD levels than the matched control individuals. In contrast, DS patients with and without AD had a similar elevation in GSHPx (an adaptive phenomenon). The mean catalase (CAT) activity was no different in DS and control individuals; however, in a paired regression analysis, DS patients without AD had marginally lower CAT activity than control individuals, whereas DS patients with AD had slightly but not significantly higher CAT activity. Thus, AD manifestations in this DS population are associated with changes in the red cell oxygen scavenging processes.

KEY WORDS: Ageing; human erythrocytes; Down syndrome; Alzheimer

Received for publication July 18, 1988;revision received August 22, 1989. Address reprint requests to: Maire E. Percy, Ph.D., Room 423, Surrey Place Centre, 2 Surrey Place, Toronto, Ontario M5S 2C2, Canada. The present address for Arthur J. Dalton is The New York State Institute for Basic Research Developmental Disabilities, Staten Island, New York.

0 1990 Wiley-Liss, Inc.

disease; superoxide dismutase; glutathione perioxidase, catalase INTRODUCTION Alzheimer disease (AD)is a progressive degenerative disease of the brain which may affect more than 11%of all individuals over age 60 years [Terry, 19761. The histopathologic changes which occur in the brain include neurofibrillary tangles and “senile” plaques [Wolstenholme,1970; Katzman et al., 19781.It has been known for more than 50 years that a high proportion of individuals with Down syndrome (DS) who come to autopsy after age 40 years show brain changes that resemble those in AD [Struve, 1929;Jervis, 1948; Wisniewski et al., 19831. These observations suggested a genetic component that predisposes DS subjects to develop AD. Because DS patients have complete or partial trisomy 21, a gene (or genes) on chromosome 21 has been implicated in the development of AD. There is considerable evidence suggesting increased damage from oxidative processes in trisomy 21 [Sinet, 1982; Brooksbank and Balazs, 19841. Because superoxide dismutase plays an important role in the scavenging of oxygen radicals in cells, and because trisomy 21 patients have 3 copies of the Cu,Zn-superoxide dismutase (SOD-1) gene, it has been suggested that the extra SOD-1 gene in DS is responsible for some of the manifestations of DS. In addition to increasing lipid peroxidation in brain in DS [Brooksbank and Balazs, 19841, the unusually rapid elimination of oxygen radicals possibly might affect the oxyradical biosynthesis of neuromediators [Michelson et al., 1977; Sinet, 1982; Jeziorowska et al., 19881. Studies of cells transfected with the cloned human SOD-1 gene [Lieman-Hurwitz et al., 19821have, in fact, shown enhanced lipid peroxidation [Groner et al., 19861. The superoxide dismutases (EC 1.15.1.1, SOD) are enzymes that catalyze the conversion of the superoxide radical (03 to hydrogen peroxide (HzOz) and oxygen

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[Fridovich, 19751.Together with glutathione peroxidase (EC 1.11.1.9, GSHPx) and catalase (CAT) (which catalyze the breakdown of hydrogen peroxide), they provide a crucial defence mechanism in cells against harmful oxygen metabolites. Oxidative damage in cells is thought to be mediated via the iron-catalyzed HaberWeiss reaction (0; + H202-+0 2 + OH) [Halliwell and Gutteridge, 19841. The hydroxyl radical (OH) initiates a chain reaction which leads to lipid breakdown through peroxidation and breakdown of cell membranes [Fee and Teitelbaum, 1972;Brooksbank and Balazs, 19841. Theoretically, any imbalance in the relative levels of SOD, GSHPx and CAT may have deleterious effects on cell membranes. An excess of SOD relative to the peroxidases might result in the buildup of hydrogen peroxide while a relative deficiency of SOD might lead to the buildup of the superoxide radical. Either event might result in the unscheduled oxidation of susceptible molecules, increased membrane permeability and cell death. Three isozymes of SOD have so far been identified. The cytoplasmic form, Cu,Zn-superoxide dismutase (SOD-1), is encoded on chromosome 21 [Tan et al., 1973; Sichitiu et al., 1974; Philip et al., 1978; Levanon et al., 19851. The mitochondria1 form, Mn-SOD, is encoded on chromosome 6 [Creagen et al., 19731. A third isozyme, interstitial or extracellular SOD, is thought to have a separate gene locus [Marklund, 19841. Whether or not these SOD isozymes are always restricted to separate intracellular compartments is a matter that has not yet been unequivocally resolved [Qler, 1975; Rest and Spitznagel, 1977; Crosti et al., 1985a and 1985b1. In some types of cells, the expression of SOD-1 and MnSOD may be coordinately regulated [Crosti et al., 1985a and 1985131.Red cells contain only SOD-1, in addition to glutathione peroxidase which is encoded on chromosome 3 [Wijnen et al., 1978;Johannsmann et al., 19811, and catalase which is encoded on chromosome 11 [Wieacker et al., 1980; Junien et al., 19801, and constitute a useful system for studying interactions among the 3 enzymes. While the neurohistopathology of AD in DS patients has been well documented, considerably less is known about the course of this disease in living persons with DS. Relevant data are difficult to collect because the primary effect of mental retardation can obscure the superimposed changes due to the dementing process. In spite of these difficulties, our group has described shortterm memory deficits, neurological symptoms, and other characteristics which are thought to reflect the development of AD in adult DS patients over age 44 years [Dalton et al., 1974; Crapper et al., 1975; Wisniewski et al., 19851. Because it is possible to identify some DS patients with AD manifestations from those without by the application of these clinical procedures, we asked whether there were differences in red cell oxidative processes in DS patients with and without clinical manifestations of AD relative to matched control individuals.

PROCEDURES Patients The study was carried out in accordance with a University of Toronto approved protocol. Thirty-four DS pa-

tients from group homes or institutions in Ontario ranging in age from 31 to 70 years, and healthy control individuals matched for sex, age (IT 2 years) and time of blood sampling ( 21 hr) were included in the present study. Most of the DS patients were severely to profoundly mentally retarded. They are a subset of a larger group whose cognitive function is being monitored in a longitudinal fashion. Five of the 34 (2 without AD manifestations and 3 with) were carriers of hepatitis B. The control individuals were healthy volunteers, usually from the same residences as the DS patients. Blood samples were taken from the DS patients a t the time of administration of the cognitive tests and neurological examination. Biochemical and genetic studies were carried out independently without knowledge of the clinical status of the patients. The information was analyzed and correlated when the laboratory work was completed. Since most DS patients over age 40 years have neuropathological findings of AD a t autopsy, the DS patients with documented behaviourlneurological deterioration have been described as having “manifestations” of AD rather than as “having” AD throughout the text.

Clinical Methods Cognitive functions, defined as learning capacities and short-term memory retention, were assessed quantitatively in the DS patients a s described previously [Dalton et al., 19741. In most cases, these cognitive assessments had been conducted between 2-6 times at 1-to 2-year intervals. DS patients were designated as significantly impaired in cognitive function if their test scores were more than 2 SD below the mean for their peer group (institution or group home residents) on a t least 2 occasions, and the results could not be attributed to other causes, such a s poor eyesight, poor hearing, ill health, and distractibility. Some patients never learned to do the tests, and were designated as “untestable.” The documentation of cognitive impairment is considered to be a sensitive clinical indicator of moderately advanced AD in severely to profoundly retarded DS patients. Scores from tests for cognitive function, results from neurological and clinical assessments, and other clinical data obtained from reports by staff and medical records, were used to classify the DS patients. Those with apparent manifestations of AD were examined in detail for medical and neurological diseases other than DS or AD which might account for behaviourallcognitive deterioration [McLachlan et al., 1984;Wisniewski et al., 19851. In keeping with previously published diagnostic criteria, a diagnosis of probable AD or possible AD was assigned to the DS patients thought to have AD [McKhann et al., 19841. Of the 34 DS patients, 9 (4 males and 5 females) showed signs consistent with the diagnosis of probable AD; 20 (11 males and 9 females) appeared to be unaffected. In the other 5 cases, it was not possible to make a classification even on the basis of repeated clinical examination; data from these DS patients and the corresponding control individuals were not included in the present analyses. Three of the 5 DS patients who were hepatitis carriers had manifestations of AD, Four of the 9 DS patients with probable AD died subsequent to

Alzheimer Disease in Down Syndrome completion of the study; in all 4 cases, the diagnosis of AD was supported by autopsy findings. (Refer to Table I for a summary of the characteristics of the DS patients with manifestations of AD.)

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Blood samples from the pairs of subjects (a DS patient and a control individual matched for age, sex and time of venipuncture) were collected over a period of 2 % years. Hemoglobin Determination Following receipt of each pair of specimens, plasma was Hemoglobin concentrations of heparinized whole separated by centrifugation. The red cells were then blood and red cell lysates were determined using Drab- washed 3 times in 2 ~010.9%NaCl a t room temperature; kin’s method with methemoglobin as a standard (Kit 0.5-ml aliquots of packed red cells were flash-frozen in liquid nitrogen and stored a t - 80°C for subsequent 525-A, Sigma Chemical Co.). analysis. Following collection of the test samples, 250 ml Cytogenetic Studies of T Lymphocytes of blood was obtained in a single venipuncture from a Small aliquots of heparinized blood (5-10 drops) were healthy normal individual to be used a s a working stanincubated in 10 ml of alpha medium containing 15% dard; washed red cells from this working standard were fetal calf serum, 50 Fgiml reconstituted phytohemag- aliquotted and frozen in the same way as the test samglutinin, 5 USP unitsiml heparin, 100 p,g of penicillin ples. On the day that lysates were prepared from frozen and 100 kg of streptomyciniml, for 72 hr at 37°C. After cells of the “paired” DS and control individuals, a lysate 71 hr of incubation, ethidium bromide (10 pg/ml of cul- was also prepared in parallel from the working stanture) was added to one half the culture tubes in order to dard, and handled in parallel with the test samples (see obtain cells with longer chromosomes for high-resolu- below). Enzymatic activity in the test samples and worktion banding. Colcemid (0.2 kg/ml final conc) was added ing standard was calculated as apparent units per mg to stop cell division 20 min before harvest. KC1 (0.075 M, Hb; activities in the test samples were then calibrated 20 min) was used as hypotonic treatment and meth- against those of the working standard. The enzyme asano1:glacial acetic acid (3:l)was used as a fixative. Chro- says for SOD-1, GSHPx and CAT were conducted, remosome spreading was achieved by dropping cell sus- spectively, over successive intervals of about 2 months. pension on cold wet slides, blow-drying, and heating on a As the red cell enzymes are not completely stable in hot plate for 3 min at 75°C. G-banding was routinely heparinized blood or during storage in harvested red applied to each specimen using a modified method of cells or lysates even at - 8O”C, the paired design minimized intrapair and intergroup variability. Calibration Seabright 119711. Chromosome studies were carried out on 25 of the 29 against the red cell working standard minimized interDS patients. With one exception, chromosomes were an- assay variability and compensated for storage effects on alyzed from at least 30 cells. Ofthe 25 who were studied, GSHPx and CAT activities in the frozen lysates. More17 had triscimy 21; one was an unusual mosaic with a over, a pilot study indicated that the red cell working balanced trimslocation (45,XX,t(14;21)/46,XX,t(14;21), standard was more effective in controlling for interassay t-21) not pi*eviously described, 30% of the cells being variability in the SOD-1 assays than a purified enzyme nontrisomic; 7 had trisomy 21 with 2-4% normal cells standard. Red cell Cu,Zn-superoxide dismutase (SOD-1) activ(designated as “occult” mosaicism). Three of 7 DS patients in the group with manifestations of AD, and 4 of ity was measured at room temperature using the pro18 in the group with none who were analyzed cyto- cedure of Winterbourn et al. [19741. Detection of SOD in genetically had occult mosaicism. Chromosome studies this procedure is based on its ability to inhibit the reduction of nitrobluetetrazolium by superoxide which is genwere not carried out on the control individuals. erated by the reaction of photoreduced riboflavin and oxygen. Lysates were prepared from the frozen aliquots TABLE I. Characteristics of the Down Syndrome Patients of washed, packed red cells by the addition of a n equal Considered t o Show Manifestations of Alzheimer Disease* volume of ice-cold, sterile distilled water, and the hemoglobin concentration of each was adjusted t o 10 g/dl. Patient number 1. 2a 3 8 4 5 6 7 8 9 Aliquots of these lysates were flash-frozen for subseSexb m f f m m f f f m quent measurement of glutathione peroxidase and catas s m sip ? ? Level of m e x a l rem p p lase activities. Chloroform-ethanol extracts were pretardationc Memory test,$$ = I I I = I I u t u t pared from the lysates for measurement of SOD activity. Neurological/clinica1 pr pr pr pr pr pr pr pr pr For the SOD assays, we determined by interpolation the assessmente volume (in ~ 1 of) extract which produced the same perAutopsyf AD AD AD L L AD I> L L centage inhibition as 130 p1 of the control extract used *Refer to the “Clinical Methods” section of PROCEDURES for further as working standard. The reciprocal of this volume was details. designated a s the enzyme activity of the extract. For *Carrier of hepatitis H each individual, we analyzed 10 different volumes of bAbbreviations: m, male; f, female. Level of mental retardation: m, moderate; s, severe; p, profound; ?, chloroform-ethanol extract (10-500 ~ 1 in) duplicate, unknown. relative to their own blanks. In one experiment, SOD dMemory tests: I, significant impairment; ut, untestable; = , unable to activity was assessed for a DS patient and the paired assess. NeurologicaUclinical assessment: pr, probable AD, in keeping with control individual; in a second experiment, SOD activity published diagnostic criteria [McKhann et al, 19841. was assessed for the DS patient and the working stan‘Autopsy: AD, patient died 1-2 years after participation in study, the dard. The advantage of the procedure of Winterbourn et histopathological features of the brain being consistent with the clinical manifestations of AD; L, patient still living. al. is that the red cell SOD activity for 2 subjects can be

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tested simultaneously. Also, the reaction is carried out near physiological pH. Glutathione peroxidase (GSHPx) activity was measured in aliquots of the flash frozen red cell lysates that had been subjected to SOD analysis and stored at - 80°C at a hemoglobin concentration of 10 gidl. Upon thawing, the lysates were adjusted to a hemoglobin concentration of 5gid1, and the GSHPx activity was then measured using a modification of the method of Giinzler [19741as described by Sinet et al. [1975]. This assay is based on the ability of the enzyme to convert reduced glutathione in the presence of peroxide to oxidized glutathione. In our case, t-butyl hydroperoxide was used as substrate, and oxidized glutathione was converted to reduced glutathione by glutathione reductase and nicotinamide adenine dinucleotide phosphate (NADPH). The oxidation of NADPH was recorded at 340 nm. Prior to assay, hemoglobin and catalase were inactivated in lysates by treatment with potassium ferricyanide and KCN in phosphate buffer at pH 7.0. Assays were carried out sequentially on lysates from a DS patient, the paired control individual and the working standard. The activity of the first 2 lysates was calculated relative to the standard, and each set of assays was repeated 4 times. Catalase (CAT) activity was measured by monitoring the formation of oxygen from hydrogen peroxide using the oxygen electrode as described by Percy et al. [19841. For these assays, lysates frozen at a hemoglobin concentration of 10 gidl were thawed and diluted 100-fold. Oxygen formation was measured using 6 different volumes of lysate (10-60 p1). Assays were carried out sequentially for a DS patient, the paired control individual, and the working standard using the smallest volume of lysate. The set of assays was then repeated using larger volumes of lysate. Catalase activity was calculated per unit of extract from a plot of reaction rate versus volume of red cell lysate assayed. The spectrophotometric assays for SOD-1and GSHPx on samples from hepatitis carriers were carried out in sealed, disposable cuvettes. Because of potential aerosol formation, samples from these persons were not assayed for CAT activity using the oxygen electrode.

Statistical Analyses Means of the red cell parameters and subject age were compared using Student’s 2-tailed t-test. Because of their skewed distribution, red cell data were first transformed logarithmically. To eliminate variation due to reagents, season and other factors, the analysis was based on data from caseicontrol pairs obtained simultaneously. Differences between pairs of DS patients and control individuals were thought possibly to depend on age and sex. To study these relations and to allow for such effects in assessing the differences between the groups of DS patients with and without manifestations of AD, multiple linear regression methods were also used [Snedecor and Cochran, 19811. A linear model for caseicontrol differences as a function of age, sex and manifestations of AD was fitted and the significance of the last term was assessed. These calculations were completed using GLIM, a statistical package distributed by the Numerical Analysis Group, Oxford, England.

RESULTS The results of the comparisons are given in Tables I1 and 111. Table I1 presents a comparison of mean (log)red cell parameters. The significance of caseicontrol differences were assessed using a simple t-test and after correcting for age and sex. Table I11 summarizes the case/ control differences corrected for age and sex. 1. Within the DS group, those with manifestations of AD were significantly older than those without (Table 11).Although the DS patients in this study were not selected randomly, this observation is consistent with our previous conclusion that cognitive impairment, thought to be an indicator of moderately advanced AD, is significantly associated with increasing age in DS [Percy et al., 19851. In practice, it was not possible to select large enough groups of DS patients with or without AD manifestations who were matched in age. For this reason, statistical procedures had to be applied to examine the effect of age and the Alzheimer process on enzyme activities. 2. The comparison of means (Table 11)indicated that DS is associated with significant increases in SOD-1 and GSHPx but no apparent alteration in CAT activity. Overall, the 29 DS patients had a 1.31-fold elevation in SOD-1 and a 1.25-foldelevation in GSHPx. 3. The elevation in mean SOD-1that was apparent in DS patients overall was more pronounced in DS patients with no clinical manifestations of AD (1.39-fold vs. 1.31-fold).By contrast, DS patients with manifestations of AD had no significant elevation in SOD-1, their mean level being only 1.16 times that for the matched control individuals. There were no significant differences in the relative GSHPx activities (1.25 and 1.24, respectively), or CAT activities (238 and 1.18, respectively), between the 2 DS groups (Table 11).The reduction in SOD-1 activity in the DS patients with AD is further demonstrated by dividing the SOD activities for the DS patients by 1.5 to correct for the triple gene dosage effect in DS. In this case, the mean SOD-1for the DS patients with AD manifestations was significantly lower than that of the matched control individuals. The paired analysis (Table 111) in which corrections were applied for sex and age effects, although minor, corroborated the above conclusions regarding SOD-1 and GSHPx but suggested that CAT activity differed marginally in the 2 categories of DS patients, those with AD having slightly higher, and those who were unaffected having lower, CAT activity than the matched control individuals. Although a tendency for the SOD-1 activity to decrease with subject age has been noted previously LPercy et al., 19851, the application of multiple linear regression in the present study indicated that these age effects were not significantly different in the DS patients and the matched control individuals. 4. Hemoglobin (giml) of blood decreased with age in the DS and control groups (Prowdon J, Cupples LA, Nee L, Myers RH, O’Sullivan D, Watkins PC, Amos JA, Deutsch CK, Bodfish .JW, Kinsbourne M, Feldman RG, Bruni A, Amaducci L, Foncin J-F, Gusella JF (1987b): Absence of duplication of chromosome 21 genes in familial and sporadic Alzheimer’s disease. Science 238564-668. Sichitiu S, E inet PM, Lejeune J , Frezal J (1974):Surdosage de la form dimerique de l’indophenoloxydase dans la trisomie 21, secondaire au surdc sage genetique. Humangenetik 23:65-72. Sinet PM (..982):Metabolism of oxygen derivatives in Down’s syndrome. Ann NY Acad Sci 396:83-94. Sinet PM, 1,ejeune H (1979): Glutathione peroxidase, hexose monophosphate shunt and I.Q. Life Sci 24:29-34. Sinet PM, Michelson AM, Bazin A, Lejeune J , Jerome N (1975): Increase irl glutathione peroxidase activity in erythrocytes from trisomy 21 subjects. Biochem Biophys Res Commun 67:910-915. Scrnerville .MJ: Bergeron C, Grima EA, McLachlan DR, Yoong LKK, Percy N:E (1989): Selective expression of the superoxide disI

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Red cell superoxide dismutase, glutathione peroxidase and catalase in Down syndrome patients with and without manifestations of Alzheimer disease.

The activities of red blood cell enzymes that scavenge the superoxide radical and hydrogen peroxide were measured in severely to profoundly retarded a...
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