Quantitative two-dimensional electrophoretic detection of possible urinary protein biomarkers of occupational exposure to cadmium.

To search for new urinary protein biomarkers of cadmium toxicity, we used quantitative two-dimensional electrophoresis (2DE) and analysed urine samples from 18 male cadmium recovery plant employees whose mean age was 47 +/- 15.6 years (+/- 1 SD) and whose urine cadmium levels ranged from 0.14 microgram l-1 to 20.4 micrograms l-1 (0.06-37.1 micrograms g-1 creatinine). Image analysis of the silver-stained gels yielded intensity (concentration) values for a mean number, per person, of 825 +/- 184 urinary proteins (spots) and found 596 +/- 218 matched proteins (the same proteins in two or more gels) per person. Total urinary protein and the sum of all spot intensities were positively correlated (P = 0.0447 and P = 0.0616, respectively) with urinary cadmium (UCD), as measured by atomic absorption spectroscopy. The combined sum of the intensities of all acidic proteins with a relative molecular weight (M(r)) below 40 kDa was correlated with UCD (P = 0.0461), revealing a low M(r), acidic proteinuria as UCD increased. Multiple hypothesis testing by regression analysis of the intensities of matched proteins with UCD revealed 14 unidentified proteins that were considered candidates for biomarkers of cadmium exposure. The best two candidate proteins--those having M(r)s of less than 13.9 kDa and relative glyceraldehyde-3-phosphate dehydrogenase (G3PDHr) coordinates of -19.7 and -27.2--were excellently resolved in the 2DE gels, and their intensities increased by 323% and 857%, respectively, over the UCD range that was tested. Two other proteins with M(r)s of 23.9 kDa and 29.2 kDa and with acidic net charges were not as well resolved. Six very acidic proteins, with M(r)s ranging from 88.8 to 90.7 kDa and with intensities highly correlated with UCD, appeared to be related and were resolved as a 'charge train' (a group of related proteins, or isoforms, differing only by small changes in net charge). Four proteins appeared to increase only when the UCD concentration was above a threshold of 16 micrograms l-1.

Identification of haptoglobin alpha-2FF variants in mid-trimester maternal serum as potential markers for Down syndrome.

Exploratory protein analysis in mid-trimester maternal sera by 2-dimensional electrophoresis and image analysis was performed to determine the differences between mothers carrying fetuses with Down syndrome (DS) and control mothers. Nine haptoglobin alpha-1 and alpha-2 (Hp1 and Hp2) variants were detected. Three apparent isoforms of the Hp2FF protein having the same relative charge but different Mr's were detected, with the two smaller variants--Hp2FF (-0.7k) and Hp2FF (-1.3k)--believed to be first reported here. Ninety-two percent of the cases (n = 83) had the Hp2FF (-0.7k) protein, and 61% had Hp2FF (-1.3k). The geometric mean concentration of Hp2FF (Mr = 18.0 kd) was found to be significantly increased (P less than 0.01) in mid-trimester sera from cases (n = 27) compared to controls (n = 56). When the concentrations of the three Hp2FF isoforms were added, the cases had larger sums on average than controls (P less than 0.01).

Factors affecting T3 and T4 proficiency testing.

Problem areas within a proficiency testing (PT) program are performance evaluation and sample stability. The different units used in the various T3 uptake methodologies make performance evaluation complex. To facilitate this evaluation, a normalization method for T3 uptake performance evaluation has been developed. Sample stability studies for T3 uptake indicate that, at room temperature, sample values increase after storage for about seven days. Room temperature sample stability studies for T4 using a competitive protein binding (CPB) method indicate that the apparent T4 content of pooled serum increases after about one week. Fatty acids are shown to be an interfering substance in the T4 CPB method as well as the T4 radioimmunoassay (RIA) method. This interference increases with a decrease in carbon chain length from C18 to C12 and with an increase in unsaturation of fatty acids. The B/B0 ration for arachidonic acid at a concentration of 0.48 micronMoles per tube is 17.4 in a CPB method and 87.1 in a radioimmunoassay method indicating that the greater effect is in the CPB method. The increase in T3 uptake values are probably also due to the interfering effect of fatty acids.

Optimization of T assay: a model study.

Factors involved in the optimization of competitive binding radioassays have been analyzed using a thyroxine (T4) radioassay as a model system. The effects of kinetic errors are minimized by the use of the lowest possible incubation temperature for the separation step and by the use of labeled T4 of high purity (which is not consistently available from commerical sources). The use of labeled T4 containing significant quantities of labeled triiodothyronine (T3) can lead to a marked increase in bias and a decrease in precision with relatively small errors in the separation step due to the relatively short half-life of the T3-binder complex. The determination of the dissociation rate constant (not to be confused with the affinity constant) allows one to make useful estimates of the tolerances that can be allowed in the separation step in order to achieve a desired level of accuracy and precison.

Interference of fatty acids in the competitive protein-binding assay for serum thyroxine.

An increase in apparent thyroxine values obtained by competitive protein-binding assay on storage of sera is well documented. We find that the major source of this positive bias is probably the unsaturated nonesterified fatty acids. Nonesterified fatty acids cause a positive basis in the competitive protein-binding assay for serum thyroxine because they inhibit the binding of radioactive thyroxine by the serum-binding reagent (probably thyroxine-binding globulin). This inhibition by fatty acids may be due to the formation of a fatty acid/thyroxine complex. The degree of inhibition caused by the fatty acids depends on the length of the carbon chain of the fatty acid and the degree of saturation. Short-chain fatty acids are more potent inhibitors of thyroxine binding than those with a longer chain, and unsaturated fatty acids are more potent inhibitors of thyroxine binding than are saturated fatty acids. The polyunsaturated fatty acid, arachidonic acid, was the most potent inhibitor of all the fatty acids tested. Triglycerides (triacylglycerols) insignificantly inhibit thyroxine binding in this assay.