**3. Results**

#### **3.1. Elemental profiles of erythrocytes**

The main transport of erythrocytes is oxygen from the lungs to cells and carbon dioxide back to lungs serving different organs including brain. The elemental profile of the erythrocytes reflect metabolism of oxygen supplied cells but also the metabolism of the erythrocyte itself. 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 of Pb, Cd, Ag and U were significant higher than those of controls.

**2. Material and methods**

participated voluntarily according to Helsinki declaration.

406 Pharmacology and Nutritional Intervention in the Treatment of Disease

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

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‐

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

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

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

The main transport of erythrocytes is oxygen from the lungs to cells and carbon dioxide back to lungs serving different organs including brain. The elemental profile of the erythrocytes reflect metabolism of oxygen supplied cells but also the metabolism of the erythrocyte itself.

for 114Sn. The analytical technique used is described in more details [1, 2, 3].

isotopes were validated against Oyster tissue 1566a, Bovine liver 1577a.

**2.1. Patients**

**2.2. Samples**

propylene bottles.

**2.3. ICP-MS instrumentation**

**2.4. Reagents and standards**

**3.1. Elemental profiles of erythrocytes**

test was used.

**3. Results**

**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) higher accumulation.

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

in cells including brain cells. Accumulation of elements in the human erythrocytes are a result

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

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

409

At present there is lack of methods to describe the accumulated effects of metal ions in erythrocytes in terms of stability constants or conditional constants. There is lacking informa‐ tion of e.g. solvent properties, pH, temperature, redox properties, lack of knowledge of interacting compounds. Elemental profiles of erythrocytes may be an alternative to estimate changes of trace elements and metal ions at the cellular level. The discussion below will be focused on some effects on membrane integrity by lead, cadmium, silver and uranium because

**4.2. Initiation of eryptosis by accumulated lead, cadmium silver and uranium in erythrocytes** It has been suggested that mature erythrocytes may enter programmed death without the aid of caspase systems [15,16]. Heavy metal ions and organic compounds may be involved in early senescence [17, 18, 19]. When erythrocytes are growing old they will change gradually, fragments will be cleaned up [20, 21], mainly in spleen, liver, bone marrow by the macro‐ phages. Most material of captured senescent erythrocytes e.g. iron and metabolized organic

Silver, cadmium ions induce suicidal erythrocyte death [24, 25] and it is likely that erythro‐ cytes and cells dependent on erythrocytes of patients with Alzheimer's disease with accumulated silver and also lead, cadmium and uranium may show early eryptosis. Eryptosis may also be initiated via formation of phosphatidylserine by lead, cadmium, hemin as

**4.3. Erythrocyte channels exposed to accumulated ions of lead, cadmium, silver and uranium** The accumulated lead, cadmium, silver and uranium may interfere the activity in the eryth‐ rocyte channels for cations, anions and water. Cadmium, lead, silver may interfere with potassium, calcium [29, 30] and other ions in erythrocyte membranes [31, 32]. Binding in vitro of cadmium to β-amyloid channels was reported by [33]. Exposure to accumulated lead, cadmium, silver, uranium ions to β-amyloid or other compounds is likely to support the

Anions e.g. chlorides, carbonates ions are transported in anion channels [34, 35, 36] may react with silver, lead, cadmium and uranium disturbing channel transport and oxygen handling. Aquaporin channels in the erythrocyte membrane are important for flow of water and shape [37, 38, 39]. Accumulated cadmium, lead, silver, uranium ions associated to erythrocyte aquaporin channels may decrease the capacity to shrink and expand. Water exchange may also be important for osmotic regulation when the erythrocytes enter capillaries. It cannot be excluded that lead, cadmium, silver, uranium may release important elements. e.g. calcium from their association sites. Released calcium may form non-selective channels in

of low exposure and not of a sudden insult which calls for sensitive biomarkers.

it is not fully known how these ions are associated to other compounds.

pathophysiological process by association to available binding sites.

material will be reused [22, 23].

stimulator [26, 27, 28].

membranes [40].

**4.1. Effects of accumulated lead, cadmium, silver and uranium in the erythrocytes**

**Figure 3.** Distribution of silver in erythrocytes of 13 patients with Alzheimer's disease. Patient 1-9 men, 10-13 women. Error bar 10%. The mean value of Ag in Alzheimer's patients was 7.4 μg/kg, standard error 1.5 μg/kg, mean of con‐ trols 1.3 μg/kg, The concentration of silver in the erythrocytes of patients was significant higher (Wilcoxon, p< 0.005) than that of controls.

**Figure 4.** Concentration of uranium in erythrocytes of 13 patients with Alzheimers disease. Patient 1-9 men, 10-13 women. Error bar 10 %. The mean value of U in Alzheimer's patients was 0.55 μg/kg, standard error 0.16 μg/kg, mean of controls 0.11 μg/kg, The concentration of uranium in the erythrocytes of patients with Alzheimer's disease was sig‐ nificant higher (Wilcoxon, p<0.01) than that of controls
