Human dietary δ(15)N intake: representative data for principle food items.

Dietary analysis using δ(15)N values of human remains such as bone and hair is usually based on general principles and limited data sets. Even for modern humans, the direct ascertainment of dietary δ(15)N is difficult and laborious, due to the complexity of metabolism and nitrogen fractionation, differing dietary habits and variation of δ(15)N values of food items. The objective of this study was to summarize contemporary regional experimental and global literature data to ascertain mean representative δ(15)N values for distinct food categories. A comprehensive data set of more than 12,000 analyzed food samples was summarized from the literature. Data originated from studies dealing with (1) authenticity tracing or origin control of food items, and (2) effects of fertilization or nutrition on δ(15)N values of plants or animals. Regional German food δ(15)N values revealed no major differences compared with the mean global values derived from the literature. We found that, in contrast to other food categories, historical faunal remains of pig and poultry are significantly enriched in (15)N compared to modern samples. This difference may be due to modern industrialized breeding practices. In some food categories variations in agricultural and feeding regimens cause significant differences in δ(15)N values that may lead to misinterpretations when only limited information is available.

Rapid determination of urinary di(2-ethylhexyl) phthalate metabolites based on liquid chromatography/tandem mass spectrometry as a marker for blood transfusion in sports drug testing.

Methods of blood doping such as autologous and homologous blood transfusion are one of the main challenging doping practices in competitive sport. Whereas homologous blood transfusion is detectable via minor blood antigens, the detection of autologous blood transfusion is still not feasible. A promising approach to indicate homologous or autologous blood transfusion is the quantification of increased urinary levels of di(2-ethylhexyl) phthalate (DEHP) metabolites found after blood transfusion. The commonly used plasticizer for flexible PVC products, such as blood bags, is DEHP which is known to diffuse into the stored blood. Therefore, a straight forward, rapid and reliable assay is presented for the quantification of the main metabolites mono(2-ethyl-5-oxohexyl) phthalate, mono(2-ethyl-5-hydroxyhexyl) phthalate and mono(2-ethylhexyl) phthalate that can easily be implemented into existing multi-target methods used for sports drug testing. Quantification of the DEHP metabolites was accomplished after enzymatic hydrolysis of urinary glucuronide conjugates and direct injection using isotope-dilution liquid chromatography/tandem mass spectrometry. The method was fully validated for quantitative purposes considering the parameters specificity, linearity (1-250 ng/mL), inter- (2.4%-4.3%) and intra-day precision (0.7%-6.1%), accuracy (85%-105%), limit of detection (0.2-0.3 ng/mL), limit of quantification (1 ng/mL), stability and ion suppression effects. Urinary DEHP metabolites were measured in a control group without special exposure to DEHP (n = 100), in hospitalized patients receiving blood transfusion (n = 10), and in athletes (n = 468) being subject of routine doping controls. The investigation demonstrates that significantly increased levels of secondary DEHP metabolites were found in urine samples of transfused patients, strongly indicating blood transfusion.

Effects of high intensity exercise on isoelectric profiles and SDS-PAGE mobility of erythropoietin.

Exercise induced proteinuria is a common phenomenon in high performance sports. Based on the appearance of so called "effort urines" in routine doping analysis the purpose of this study was to investigate the influence of exercise induced proteinuria on IEF profiles and SDS-PAGE relative mobility values (rMVs) of endogenous human erythropoietin (EPO). Twenty healthy subjects performed cycle-ergometer exercise until exhaustion. VO (2)max, blood lactate, urinary proteins and urinary creatinine were analysed to evaluate the exercise performance and proteinuria. IEF and SDS-PAGE analyses were performed to test for differences in electrophoretic behaviour of the endogenous EPO before and after exercise. All subjects showed increased levels of protein/creatinine ratio after performance (8.8+/-5.2-26.1+/-14.4). IEF analysis demonstrated an elevation of the relative amount of basic band areas (13.9+/-11.3-36.4+/-12.6). Using SDS-PAGE analysis we observed a decrease in rMVs after exercise and no shift in direction of the recombinant human EPO (rhEPO) region (0.543+/-0.013-0.535+/-0.012). Following identification criteria of the World Anti Doping Agency (WADA) all samples were negative. The implementation of the SDS-PAGE method represents a good solution to distinguish between results influenced by so called effort urines and results of rhEPO abuse. Thus this method can be used to confirm adverse analytical findings.

Discrimination of recombinant and endogenous urinary erythropoietin by calculating relative mobility values from SDS gels.

Erythropoietin (EPO) promotes the production of red blood cells, the key factor in the regulation of the oxygen transport, and has been abused by athletes for performance enhancement in endurance sports. Current methods to detect EPO misuse are based on isoelectric focussing (IEF), double blotting, and chemiluminescence detection. A new approach utilizing SDS-PAGE mobilities of target analytes is presented. Employing two internal standards (novel erythropoiesis stimulating protein and recombinant rat EPO), the assay provides a tool which allows the calculation of relative mobility values for endogenous urinary EPO and recombinant epoetins (e.g., Dynepo) and, thus, the distinction of these analytes in doping control samples. A reference group of 53 healthy volunteers and samples originating from a Dynepo (epoetin delta) excretion study conducted with a single person were analyzed and led to a significant discrimination of endogenous urinary and recombinant EPO. A clear differentiation was accomplished over a period of four days post-administration of a single injection of 50 IU/kg body weight. Hence, the method may be useful as a screening procedure in doping control or as complementary confirmation tool to the established IEF assay.

Application of stable carbon isotope analysis to the detection of 17beta-estradiol administration to cattle.

The use of anabolic agents in food producing animals is prohibited within the EU since 1988 (96/22/EC directive). The control of the illegal use of natural steroid hormones in cattle is still an exciting analytical challenge as far as no definitive method and non-ambiguous analytical criteria are available. The ability of gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) to demonstrate the administration of 17beta-estradiol to bovine has been investigated in this paper. By comparison of 13C/12C isotopic ratio of main urinary estradiol metabolite, i.e. 17alpha-estradiol, with two endogenous reference compounds (ERCs), i.e. dehydroepiandrosterone (DHEA) and 5-androstene-3beta,17alpha-diol, the differentiation of estradiol metabolite origin, either endogenous or exogenous, has been proved to be achievable. After treatment, the delta(13)C(VPDB)-values of 17alpha-estradiol reached -27 per thousand to -29 per thousand, whereas delta13CVPDB-values of DHEA remained between -13 per thousand and -20 per thousand depending on the diet, maize and grass, respectively. A significant difference of delta13CVPDB between ERCs and 17alpha-estradiol was measurable over a period of 2 weeks after estradiol ester administration to the animal.