**2. Material and methods**

### **2.1. Patients**

Thirteen diagnosed Alzheimer patients, 9 men and 4 women (mean age 61 y) and age matched controls (mean age 58 y) were selected for the study living in the same area. All patients participated voluntarily according to Helsinki declaration.

As erythrocytes carry ferro ions in hemoglobin and other divalent elements e.g. zinc, magne‐ sium, calcium they will also reflect external influence of metabolism. The elemental profile of erythrocytes will be a status report of cell metabolism translated in terms of metal ions and trace elements. In this study we report accumulated ions: silver, cadmium, lead and uranium in the erythrocytes of patients with Alzheimer's disease. The concentration of lead (wet weight) in the erythrocytes of patients with Alzheimer's disease is demonstrated in Figure 1. The result of cadmium analysis (wet weight) of the erythrocytes is shown in Figure 2. The concentration of silver in the erythrocytes is indicated in Figure 3. The concentration of uranium in the erythrocytes was significant higher that of controls Figure 4. In summary the concentrations

Metabolism Changes as Indicated by the Erythrocytes of Patients with Alzheimer's Disease

http://dx.doi.org/10.5772/57511

407

**Figure 1.** Lead concentration in erythrocytes of 13 patients with Alzheimer's disease. Patient 1-9 men, 10-13 women. Error bar 10%. The mean value of Pb in Alzheimer's patients was 157 μg/kg, mean of controls 31 μg/kg, standard error 52.8 μg/kg. Pb increases normally with age but the Alzheimer group indicate significant (Wilcoxon, p< 0.005)

**Figure 2.** Distribution of cadmium in erythrocytes of 13 patients with Alzheimer's disease. Patient 1-9 men, 10-13 women. Error bar 10%. The mean value of Cd in Alzheimer's patients was 11.5 μg/kg, standard error 3.9 μg/kg, mean of controls 1.3 μg/kg, The concentration of Cd in the erythrocytes of patients with Alzheimer's disease was significant

of Pb, Cd, Ag and U were significant higher than those of controls.

higher accumulation.

higher (Wilcoxon, p< 0.05) than that of controls.

### **2.2. Samples**

Whole blood (2x7 ml) was drawn into Vacutainer tubes for trace element analysis (BDH, with sodium heparin as anticoagulant). Centrifugation was started half an hour after venopuncture. The erythrocytes were separated by centrifugation at 120xg at 4 C for 15 minutes. After removing the buffy coat the erythrocytes were washed twice with 0.9 % NaCl at 1000xg for 5 minutes. The erythrocytes were transferred to cryo vials (Nunc) weighed and freezed at-18 C. The samples (0.6-0.8g, wet weight) were digested with nitric acid and hydrogen peroxide (both ultra pure) in microwave oven. The samples were diluted with Milli-Q water in 25 ml poly‐ propylene bottles.

### **2.3. ICP-MS instrumentation**

For the elemental analysis of the erythrocytes ICP-MS, Elan 6100 DRC was used in peak hopping mode. The isotopes monitored: 107Ag, 114Cd, 208Pb and 238U. Integration time: 5 sweep, 100 msek. Internal standard 10 ppb Rh was supplied by externally feeding. 114Cd was corrected for 114Sn. The analytical technique used is described in more details [1, 2, 3].

#### **2.4. Reagents and standards**

Ultra pure nitric acid and hydrogen peroxide was obtained from Merck. ICP-MS standards were obtained from Johnson & Matthey, SRM 1566a and 1577a from NIST. The different isotopes were validated against Oyster tissue 1566a, Bovine liver 1577a.

The estimated accumulated mean error in the analysis of samples was ± 10 % or less generated in the sampling procedure, preparation, digestion, volumetric and weighing errors and error in the ICP-MS analysis. For statistical calculations of ICP-MS results Wilcoxon's nonparametric test was used.
