Scaled down method for the reproducible recovery to high purity of human serum albumin from low volume blood samples.

A basic need for a protein-based dosimeter is a purified protein. In this communication we present an isolation protocol and an HPLC-based assay which allows one to determine the purity of the isolated albumin. A total of 168 human blood samples were collected from workers of a benzene processing plant and from nearby countryside at Kohtla-Järve, Estonia. Albumin was isolated from plasma by sequential precipitation and the purity was determined by HPLC. The amount of albumin present in plasma varied between the individuals, being 147 +/- 26 mg/5 ml (n = 168), which is about 59% of plasma albumin. However, the isolated albumin was highly pure (100.9 +/- 8.2%, n = 5). All albumin samples analyzed demonstrate two peaks in HPLC analysis. The two peaks detected were collected and subjected to MS analysis, which demonstrates a difference of 120 mass units between the two albumin products isolated. We have developed an assay, which is easy to carry out and is not too labor intense. The HPLC analysis can be applied to confirm the purity of the isolated albumin as well as to confirm the quantity of the albumin in samples.

Gas chromatographic determination of 3-butene-1,2-diol in urine samples after 1,3-butadiene exposure.

1,3-Butadiene is an important industrial chemical and a common environmental contaminant. Because of its suspected carcinogenicity butadiene-related research has gained high activity. The obvious lack of knowledge so far has been that a biomonitoring method that can detect at least one of the metabolites of butadiene from body fluids or excretas does not exist. In this communication we describe a robust and simple analytical method which can be applied for biomonitoring purposes. We have developed a method that can detect 3-butene-1,2-diol in urine samples of rats inhalation-exposed to various concentrations of 1,3-butadiene. The method is based on liquid-liquid extraction and subsequent gas chromatographic analysis. The extraction efficiency of 3-butene-1,2-diol at a concentration of 2.2 microg/ml was 95% (SD=+/-3%, n=3) and was achieved by using sodium chloride saturation and isopropanol as an extracting solvent. The standard deviation of the gas chromatographic analysis was +/-2% (n=12), the limit of detection was 0.08 microg/ml, the limit of quantitation was 0.11 microg/ml (SD=+/-4.8%, n=3) and the analysis was observed to be linear from 0.11 to 486 microg/ml (R=0.9987). Animals exposed to 1,3-butadiene showed a linear excretion of 3-butene-1,2-diol into urine as a function of butadiene exposure. During the exposure saturation of metabolism or accumulation of 1,3-butadiene or 3-butene-1,2-diol into the body was not observed in any exposure levels used.

Haemoglobin adducts as biomarkers of occupational exposure to 1,3-butadiene.

Adducts of 1,2-epoxy-3-butene (EB) with haemoglobin were monitored in 17 workers from the 1,3-butadiene (BD) production unit of a petrochemical plant and in nine referents employed at the same factory but not exposed to BD. The air concentrations of BD were determined using stationary and personal monitoring. The ambient level of exposure of the plant workers handling butadiene containers (sampling and voiding) was on average 11.2 +/- 18.6 (mean +/- SD) mg/m3. Maintenance and laboratory workers were exposed to levels < or = 1.2 mg/m3. The particular haemoglobin adduct measured was 2-hydroxy-3-butenylvaline, formed by reaction of N-terminal valine with carbon 1 in EB. The adduct levels were increased (0.16 +/- 0.099 pmol/g; n = 10) in plant workers compared with the levels in maintenance and laboratory workers and controls (approximately 0.05 pmol/g; seven laboratory workers and nine controls evaluated). Thus, the method used for adduct determination--derivatization of 200-300 mg globin samples with penta-fluorophenyl isothiocyanate according to the N-alkyl Edman method and detection of the thiohydantoin derivatives by tandem mass spectrometry--is sufficiently sensitive to allow monitoring of exposure to BD down to the p.p.m. level.