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Studies to specify various metalloproteins in cell cytosol of tissues using chromatographic separation methods and plasma mass spectrometry for element detection require a careful step by step sample preparation. These steps involve (i) bioptic sample removal from tissue, (ii) extraction and preparation of the tissue supernatant, (iii) the chromatographic separation of the proteins, and finally (iv) the on-line transfer of the column eluate into the plasma mass spectrometer. Each of the analytical steps has to be carefully monitored to avoid undesired changes in the sample composition which could be caused by enzymatic and/or oxidative processes as well as by external element contamination. This presentation introduces a concept to ensure that environmental element contamination does not occur to bias the analytical results.

Our first project aimed to determine the average values of Fe and Zn in normal German human brain (5 individuals, 10 brain parts). Determinations were carried out by instrumental neutron activation analysis in Berlin. Quality control measurements were performed using National Institute of Standard Technology standard reference materials. The present results show non-homogeneous distribution of Fe and Zn in normal human brain. Our second goal was to study the possible elemental concentration changes in German patients with Alzheimer disease (5 subjects, 10 brain regions). Fe and Zn values are found to be significantly changed in some AD brain regions compared to the controls. Another object of this work was to extend the method for the determination of elemental concentration not only in whole brain samples (high fat content) but - applying two types of solvent extraction - in lipid fraction and in brain tissue without lipid.

A method for the identification of selenocysteine and selenomethionine in protein hydrolysates was developed. The proteins were subjected to acid hydrolysis after they had been carboxymethylated to prevent decomposition of selenocysteine during this process. After precolumn derivatization of the amino acids with o-phthaldialdehyde, the hydrolysate was chromatographed on C18 columns. The selenoamino acids were detected either by the fluorescence of their o-phthaldialdehyde derivatives (detection limit 30 pmol for selenomethionine and 170 pmol for selenocysteine) or by selenium determination in the eluate using atomic absorption spectrometry (detection limit 0.3 pmol) or, with 75Se-labelled compounds, the measurement of the tracer activity. With the latter procedure the detection limit, which depends on the specific activity of the Se tracer, could be decreased to the femtomole range. The method was successfully applied to the identification of selenocysteine in several newly found mammalian selenium-containing proteins.

Commercially available calf serum did not supply the cultured murine fibroblast cell line L929 with amounts of selenium and alpha-tocopherol sufficient to protect against peroxide damage. Supplementation of the culture medium with 30 microM alpha-tocopherol or 50 nM sodium selenite led to a substantial increase of cellular alpha-tocopherol concentrations from 18 +/- 3.0 to 3179 +/- 93.0 pmol/10(6) cells or cellular selenium concentrations from 0.17 +/- 0.02 to 1.75 +/- 0.16 ng/10(6) cells, respectively. L929 fibroblasts grown in selenite-containing medium also had markedly raised activities of both cytosolic glutathione peroxidase (from 11 +/- 0.9 to 67.2 +/- 4.2 mU/10(7) cells) and phospholipid hydroperoxide glutathione peroxidase (from 0.2 to 9.5 +/- 0.9 mU/10(7)cells). Supplementation with alpha-tocopherol inhibited single-strand breaks induced by low concentrations of H2O2 only, whereas an adequate selenium supply almost completely inhibited single-strand breaks induced by up to 30 microM H2O2 and also significantly reduced H2O2-induced cell death. An inadequate selenium supply and corresponding increase of GPx activity upon selenite supplementation was also observed with other cell lines, for instance, D10N, ECV-304, HepG2, and THP-1. Our data strengthen the relevance of standardized and adequate supplementation of tissue culture media with antioxidants to improve viability and genetic stability of cultured cells in general and in particular, if they are oxidatively challenged.

Objective: Evaluation of the iatrogenic aluminum load by aluminum-contaminated nutritive infusion solutions in long-term total parenteral nutrition (TPN)., Patients: 16 consecutive patients (6 children, 10 adults) who had to undergo total parenteral nutrition for more than one month. Three of them were from a home parenteral nutrition program. The duration of TPN was up to 68 months. The parenteral aluminum load was calculated on the basis of the individual TPN programs. Six patients were exposed to an additional parenteral aluminum load in the course of intensive care. Comparative group: To establish a reference range, the serum aluminum concentrations were determined in 71 unloaded patients who had to undergo minor surgical procedures. ALUMINUM ANALYSIS: Strict adherence to a contamination-free sampling and processing technique. The aluminum determination was performed at the Hahn-Meitner-Institute Berlin by means of graphite-furnace atomic absorption spectrometry (GFAAS)., Results: The TPN-associated daily aluminum load was 3.5 +/- 0.4 micrograms/kg body weight (bw) in children and 2.2 +/- 1.8 micrograms/kg bw in adults. 59 +/- 6% of the intravenous aluminum load in children and 42 +/- 16% in adults was due to the highly contaminated small-volume calcium, inorganic phosphate, trace element and vitamin parenterals. The median serum aluminum concentration under TPN was 10.9 micrograms/l (range: 5.0-26.9 micrograms/l) and was thus 7.3 times higher than in the preoperative control group (median: 1.5 micrograms/l, 95% confidence interval: < 0.6-3.5 micrograms/l). Individual values ranged up to 36.8 micrograms/l., Conclusions: The aluminum intake of patients on parenteral nutrition in Germany is thus on occasion considerably above the ASCN/ASPEN recommendations for the limitation of intravenous aluminum loading (ASCN: American Society for Clinical Nutrition; ASPEN: American Society for Parenteral and Enteral Nutrition). The toxicological significance of parenteral aluminum loading is discussed. The results suggest that limits should be established for the Aluminum contamination of infusion solutions.

Background: For patients with disturbed aluminum (Al) excretion, a high Al intake is not without risk. As main aluminum sources infusion solutions and solutions for parenteral nutrition have been identified. This study will give current survey of aluminum loading of the above-mentioned preparations., Material and Methods: The aluminum loading of 139 different infusion solutions and solutions for parenteral nutrition was determined. The solutions were from the clinical pharmacy of the Klinikum Steglitz of the Free University Berlin or were bought in a public pharmacy. The aluminum content was determined by means of two different, independent analytical methods: a) graphite furnace atomic absorption spectroscopy (GFAAS) and b) inductively coupled plasma atomic emission spectroscopy (ICP-AES). The agreement of the measured values was good except for five samples, where different values were found. Mistakes due to contamination were excluded on the basis of the results of measuring standard reference materials., Results: Small-volume additives of TPN (total parenteral nutrition) formulations were highly contaminated with aluminum, e.g. Ca and phosphate solutions (29-12,000 micrograms/l), vitamin C solutions (700-1,200 micrograms/l) and trace element solutions (67-6,200 micrograms/l). Furthermore about 44% of the crystalline amino acid solutions and lipid emulsions had an aluminum content of 25 to 55 micrograms/l. Low aluminum levels were found in carbohydrate solutions, NaCl and KCl solutions and in distilled water (aqua ad injectabilia)., Conclusions: Many of the solutions for parenteral nutritional support have an aluminum content which exceeds, in part considerably, the suggested threshold concentration of 25 micrograms/l (0.93 mumol/l), recommended by the American Society for Clinical Nutrition (ASCN) and the American Society for Parenteral and Enteral Nutrition (ASPEN). The pharmaceutical industry should be required to check the manufacturing process for avoidable sources of contamination, and threshold values for aluminum loading by intravenously applied pharmaceuticals should be laid down in the German and European pharmacopoeia. In cases where contaminations cannot be eliminated during the manufacturing process after careful checking, the aluminum content of the infusion solution should be declared for the user.

The Enschi district in Hubei province, Peoples Republic of China is geochemically one of the two seleniferous regions, producing both selenium (Se) black tea and the Se green tea. Three samples of green tea with different Se contents and one non-Se tea were analysed. The following mineral and trace elements were determined by inductively coupled plasma-atomic emission spectrometry (ICP-AES): K, Ca, Mg, Na, P, S, Al, Mn, Fe, Ba, Sr, Co, Ni, Cu, Zn, Mo, and Cr. Except for Mo, Co, and Cr, all other elements in infusions of the samples analysed were also measured, since their concentrations are lying over their detection limits. The Se content in the tea samples and in the infusions were measured with graphite furnace atomic absorption spectrometry (GFAAS). The accuracy of Se determination was tested by measuring untreated tea samples with instrumental neutron activation analysis (INAA). The Se content in the measured samples was 1 to 8.5 micrograms/g. In addition to Se, 17 other elements were measured in the tea samples and 14 others in the infusions. With this data the extractable part of this elements in the infusion were calculated. Up to 10% of the Se was found in a first infusion.

Serum concentration and urine excretion of aluminium were examined during intensive care in 26 premature infants receiving long-time parenteral nutrition. In addition, aluminium content was analysed in all fluids and medicaments used for intravenous therapy and oral feeding. Several fluids for parenteral nutrition and milk formulae were highly contaminated with aluminium, especially serum conserves, human albumin, calcium gluconicum, and special diets for enteral disorders. Small premature infants have a predisposition to aluminium loading during intensive care. In 22 premature infants serum aluminium levels exceeded 10 micrograms/L. There was a significantly negative correlation between serum aluminium levels and gestational ages. Altogether, infants with high aluminium levels showed significantly more complications, especially bronchopulmonary dysplasia, necrotizing enterocolitis, cholestasis, and osteopenia.

Neutron activation analysis via the short-lived radionuclide 66Cu was applied in the determination of the level of copper in tissues. With this procedure the samples are analysed non-destructively and can therefore be used for further histological tests. The applicability of the method in the analysis of biopsy samples is discussed.

Taking the necessary precautions essential for accurate trace element analysis, the zinc concentrations of 227 samples of amniotic fluid taken at term were determined by means of neutron activation analysis. Half of the zinc was found to be bound to the particles present in amniotic fluid. In the control group (123 cases) the zinc concentrations ranged from 25-261 micrograms/l for untreated samples and from 14-143 micrograms/l when the samples were centrifuged for 10 min at 22 000 g prior to analysis. The values showed log-normal frequency distributions and were lower than any of the values published in the literature to date. No statistically significant difference could be found, when zinc concentrations of various risk groups (mothers suffering from gestosis or diabetes mellitus, newborns hypo- or hypertrophic, twin births) were compared with the zinc concentration of the control group. The amniotic fluid zinc concentration is not, therefore, a suitable indicator for the diagnosis of disturbances of the embryonic development.