U. Amstutz¹, S. Farese², S. Aebi², C.R. Largiadèr¹

¹Institute of Clinical Chemistry, Inselspital University Hospital, University of Bern, Bern, Switzerland, ²Department of Medical Oncology (DOLS), Inselspital University Hospital, Universtiy of Bern, Bern, Switzerland

The activity of dihydropyrimidine dehydrogenase (DPD), the key enzyme of pyrimidine catabolism, is thought to be an important determinant for the occurrence of severe toxic reactions to 5-fluorouracil (5-FU), one of the most commonly prescribed chemotherapeutic agents for the treatment of solid cancers. Genetic variation in the DPD gene (DPYD) has been proposed as a main factor for variation in DPD activity in the population. However, only a small proportion of severe toxicities in 5-FU based chemotherapy can be explained with such rare deleterious DPYD mutations resulting in severe enzyme deficiencies. Recently, hypermethylation of the DPYD promoter region has been proposed as an alternative mechanism for DPD deficiency and thus as a major cause of severe 5-FU toxicity. Here, the prognostic significance of this epigenetic marker with respect to severe 5-FU toxicity was assessed in 22 cancer patients receiving 5-FU based chemotherapy. None of the investigated patients were carriers of a known deleterious DPYD mutation. Twelve of these 22 patients experienced severe toxic side effects following drug administration, while all others did not show any or only a mild toxic reaction to 5-FU. The methylation status of the DPYD promoter region in peripheral blood mononuclear cells was evaluated by analyzing for each patient at least 20 different clones of a PCR-amplified 209 base pair fragment of the bisulfite-modified DPYD promoter region. The fragments were sequenced to detect bisulfite-induced, methylation-dependent sequence differences. No sign of DPYD promoter methylation was observed in any of the investigated patient samples, whereas in a control experiment, as little as 10% methylated genomic DNA could be detected. This indicates that DYPD promoter hypermethylation is, if at all, only of minor importance as a prognostic factor for severe toxicity in 5-FU based chemotherapy.

Dr. Andres Hanspeter¹, Dr. Wunderli Samuel¹

¹Federal Office of Metrology METAS, Lindenweg 50, CH-3003 Bern-Wabern, Switzerland

The national regulations and European directive for in-vitro diagnostics (98/79/EG from 27. October 1998) require that traceability of values assigned to calibrators and/or control materials must be assured through available reference measurement procedures and/or available reference materials of a higher order. METAS' contribution is to provide traceable reference values for chemical activity measurements of ions in complex electrolytes and of glucose in solutions. These two types of analyte measurements are most common tasks of a clinical chemical laboratory and of high relevance to the physicians. The chemical activity of dissolved ions and of glucose in physiological liquids corresponds to the freely available part of these components of a mixture. It determines their biochemical effects. In physiological solutions, such as blood serum, chemical activity clearly differs from ion or substrate concentration. Potentiometric and chronoamperometric measurements using ion-selective electrodes and biosensors, respectively, allow to directly determine chemical activity. However, many measurement instruments and methods are currently in use which do not clearly differentiate between total substance concentration and its biologically active component. At equilibrium, the membrane potential of an ion-selective membrane is mainly dependent on the chemical activity of the target ion outside the membrane and is best described by a modified Nikolskij-Eisenman equation. Thus the potentiometric signal of the ion-selective electrode is a direct measure for the chemical activity for a particular electrolyte ion. The uncertainty of the activity is dominated by the uncertainties of the diffusion potential and the response behaviour of the potentiometric measurement chain. In the physiological range the expanded uncertainty equals to 4 % rel. for the monovalent ions of sodium, potassium and chloride, whereas for divalent ions Calcium and Magnesium the uncertainty doubles. Glucose oxidase is often used to determine glucose enzymatically, a process during which the chemically active form of β-D-glucose is transformed highly selectively into 5-gluconolactone. Two electrons are released for each converted glucose molecule. This electron current is the evaluable phenomenon in chronoamperometry. The uncertainty of this activity determination is dominated by the uncertainties in the response behaviour of the asymptotic current and the amount of substance content in the gravimetrically prepared reference solutions. For aqueous glucose solutions in the physiological range the expanded uncertainty is below 4 % rel. Dissemination of chemical activity reference values is either done by the determination of the chemical activity of working standards or by direct calibration of measuring instruments. Traceability of electrolyte and glucose measurement results on a national standard ensures international comparability and avoids laboratory dependent measurement results.

U. Amstutz¹, S. Farese², S. Aebi², C.R. Largiadèr¹

¹Institute of Clinical Chemistry, Inselspital University Hospital, University of Bern, Bern, Switzerland, ²Department of Medical Oncology (DOLS), Inselspital University Hospital, Universtiy of Bern, Bern, Switzerland

5-fluorouracil (5-FU) is among the most commonly prescribed chemotherapeutic agents for the treatment of solid cancers. A high variation is observed in both tumor response and side effects to this drug, for which a genetic basis is suggested. Various rare deleterious mutations in the dihydropyrimidine dehydrogenase gene (DPYD), the key enzyme of 5-FU catabolism, have repeatedly been reported to be associated with severe toxicity in 5-FU based chemotherapy. However, the importance of these gene variants for the prediction of 5-FU toxicity is still unclear. Furthermore, most research was focused on rare coding variants, whereas a comprehensive analysis at the haplotype level, also covering potentially regulatory gene variants, is missing. Therefore, the aim of the study presented here was to sequence the entire coding region and flanking intronic sequences of DPYD in 111 cancer patients receiving 5-FU based chemotherapy in order to infer recombination patterns and haplotype structures, and correlate them with the occurrence of severe toxic side effects to 5-FU. Thirty-six variable sites in the DPYD sequence were identified, however, none of the known deleterious DPYD mutations were observed in 24 patients experiencing severe 5-FU toxicity. On the other hand, various novel intronic polymorphisms were detected, some of which showed associations with severe adverse side effects to 5-FU. Linkage analysis revealed that the observed associations of four of these polymorphisms were attributable to a single haplotype overrepresented in patients with severe 5-FU toxicity in a linkage block ranging from intron 3 to exon 11, comprised of three novel intronic polymorphisms (IVS5+18G>A, IVS6+139G>A, IVS9-51T>G) and one synonymous mutation (1236G>A). Out of eight observations of this haplotype, five were found in patients with severe adverse effects, and the only homozygous patient experienced lethal 5-FU toxicity. This observed association of a haplotype comprised of intronic and synonymous polymorphisms thus confirms the usefulness of the haplotype-based approach to detect new genetic variation located outside the coding regions, which may be useful for predicting severe 5-FU toxicity due to a potential effect on mRNA splicing or gene regulation. Furthermore, a comparison with other studies suggests potential geographic differences in the prognostic significance of previously described deleterious DPYD mutations in relation to severe 5-FU toxicity.

S. Ayaz¹, A. Ayaz²

¹Hematology Department, Turkey High Speciality Hospital, Ankara, Turkey, ²Department of Nutrition and Dietetics of Hacettepe University, Ankara/Turkey

Background: This study was carried out to determine the nutritional status and the relationship between cognitive functions and Food consumption of patients with Alzheimer type senile dementia. Metod: The study was conducted on 20 elderly patients (12 males, 8 females) with new diagnosis of Alzheimer type senile dementia who were admitted to the Dementia Clinic of Gülhane Military Medical Academy Neurology Department and 20 healthy alderly individuals (7 male,13 female).A questionnaire and an evaluation of the cognitive functions using a mini mental status examination (MMSE) test were applied to all participants. Anthropometric measurements, body compositionwith bioelectric impedance analysis (BIA) and biochemical analysis were performed and consecutive 3 day food consumption with physical activity records were obtained for all participants. Biochemical parameters and homosystein level are measured with autoanalyzer( Abbott System, USA). Results: MMSE test values were 18.3 + 0.5 points in Alzheimer disease patients,but were 27.3 + 0.3 points in the control group (p<0.05).According to food consumption only the differences between carbohydrate,heme iron and sodium consumption of Alzheimer disease patients and control group were found to be statistically significant (p<0.05).There were no significant differences between the two groups in physical activity, basal metabolic rate (BMR) and total energy expenditure (p>0.05). In addition, no significant correlations were found between MMSE test values and energy and nutrient consumptions in these two groups. However,it was found that waist and hip circumference (cm), triceps skinfold thickness (mm), body fat mass (kg) and body water (%) (p<0.05) were different between the two groups of males and this was statistically important. According to biochemical analysis, serum alkaline phosphatase, total protein, albumin, transferin levels of women and serum vitamin B12 levels of men were significantly different in both groups (p<0.05). In general, it was found that serum transferin, prealbumin and serum vitamin B12 levels in Alzheimer disease patients were significantly higher than the control group (p<0.05). There were negative correlations between serum homocysteine and serum vitamin B12 levels in controls (r = -0.642, p< 0.01) and between MMSE test values and serum homocysteine levels in the Alzheimer disease patients group (r = -0.606 , p<0.01). Conclusion: According to the findings of this study , it was concluded that monitoring serum vitamin B12 and folic acid levels and management of balanced nutrition at all stages of the Alzheimer disease would be beneficial.