Evaluation of novel assays in clinical chemistry: quantification of plasma total homocysteine.

BACKGROUND: There is a need for systematic evaluation of methods before their release to the market. We addressed this problem in novel homocysteine assays as part of an European Demonstration Project involving six centers in four countries. METHODS: Two immunological methods for measurement of plasma total homocysteine (P-tHcy), the fluorescence polarization immunoassay (FPIA) and the enzyme immunoassay (EIA), were compared with two comparison methods, HPLC and gas chromatography-mass spectrometry (GC-MS). All laboratories performed the following procedures: (a) familiarization; (b) determination of linearity and precision by analyzing five plasma samples with interrelated concentrations for 20 days; (c) correlation using patients' samples; and (d) assessment of long-term performance. RESULTS: Both immunological methods were linear for P-tHcy between 5 and 45 micromol/L. The intralaboratory imprecision (CV) was <5% for FPIA and <9% for EIA used with a sample processor. The bias was -2% to 3% for FPIA and 2-4% for EIA used with a sample processor. CONCLUSIONS: The immunological methods provide results with little bias compared with HPLC and GC-MS. The imprecision of the assays must be considered in the context of their intended use(s).

Enzyme conversion immunoassay for determining total homocysteine in plasma or serum.

A rapid and precise immunoassay for quantification of total homocysteine in blood samples is presented. The method avoids the use of radioisotopes and chromatographic separations and relies on enzymatic conversion of homocysteine to S-adenosyl-L-homocysteine, followed by quantification of S-adenosyl-L-homocysteine by an enzyme-linked immunoassay in microtiter format. The within- and between-assay imprecision is < 6% and 8%, respectively, and results by the method show good correlation with those by HPLC. Including controls and calibrators in duplicates, 82 samples can be analyzed within 2.5 h. The procedure can be fully automated.

Thermal oxidation of polycyclic aromatic hydrocarbons adsorbed on alumina.

The reevaporation of polycyclic aromatic hydrocarbons (PAH) from secondary alumina (alumina used in a fluidized bed for pot gas cleaning) used in the production of aluminum in aluminum reduction plants has been studied. The secondary alumina contains around 100 ppm of PAH when introduced to the pots. The results of this study indicate that only a minor part of the adsorbed PAH is reevaporated at the pot temperature of 300-400 degrees C and that the use of secondary alumina has little effect on the PAH concentration in the workplace atmosphere of the aluminum reduction plants.

A comparison between different high volume sampling systems for collecting ambient airborne particles for mutagenicity testing and for analysis of organic compounds.

Samples of urban air were collected simultaneously using different sampling systems, including electrostatic precipitation (ESP) and high volume filtration (HVF) on various filters for particle sampling and absorption on activated carbon and organic polymers for sampling of volatiles. Acetone extracts of the samples were analyzed for polycyclic aromatic hydrocarbons (PAH) and tested for mutagenicity with the Ames Salmonella/microsome assay. The results show that the concentrations of PAH found in the various particle-samples were in good agreement, whereas the mutagenic activity of these samples showed large variations. The highest mutagenic activity was found in the samples collected by ESP and on the teflon-coated glassfibre filters, whereas samples collected by high volume filtration with size-fractionation showed the lowest mutagenic activity. We do not know whether the higher activity in samples from the teflon-coated filters compared to those from ordinary glassfibre filters represent filter artifacts or if it represents a more pronounced degradation of mutagenic compounds on the non-coated glassfibre filters. Extracts from filter blanks seemed to interfere with the expression of the mutagenic activity of the positive controls, benzo[a]pyrene and nitropyrene. When sampling volatile compounds, two organic polymers, polyurethane (PUR) and XAD-2, were found suitable for collecting PAH, whereas no PAH could be detected in extracts from the activated carbon. The XAD-2 adsorbent was the most effective for sampling bicyclic PAH. None of the adsorbents yielded extracts well suited for mutagenicity testing, since blank extracts were toxic to the test bacteria. Some extracts of the PUR blanks were weakly mutagenic as well. More emphasis should be placed upon developing more efficient and unreactive adsorbents and on the adaptation of such adsorbents in samplers suited for routine use.

Short-term bioassays of fractionated emission samples from wood combustion.

Extracts of an emission sample from wood burning, consisting of particles and volatiles, have been fractionated on an HPLC silica gel column into five fractions of increasing polarity. Nonfractionated samples and the individual fractions have been tested in three different short-term bioassays: the Ames Salmonella assay, the sister chromatid exchange (SCE) induction-test in Chinese hamster ovary cells (CHO), and the cell transformation test on Syrian hamster embryo (SHE) cells. Most of the total activity was found in the volatile part of the sample with all three bioassays, whereas the particle extract had the highest activity per unit mass extracted. The second most polar fraction contained most of the mass and was also highly active in all assays. The most polar fraction was very potent in the Salmonella assay, but showed only a weak response in the eukaryotic bioassays. Storage of the samples for several months at 0 degrees C revealed that the bacterial mutagens present in the most polar fraction were labile; the mutagenicity was almost totally lost after 1 year's storage.

Contribution of wood combustion to indoor air pollution as measured by mutagenicity in Salmonella and polycyclic aromatic hydrocarbon concentration.

Samples of airborne particles have been collected in the same room when the room was heated by electricity and when heating was done by woodburning. These samples were compared with respect to mutagenic activity and concentration of polycyclic aromatic hydrocarbons (PAH). The effects of the various heating conditions were examined in the presence and absence of tobacco smoking. Whereas wood heating in an "airtight" stove was found to cause only minor changes in the concentration of PAH and no measurable increase of mutagenic activity of the indoor air, both these parameters increased considerably when wood was burned in an open fireplace, yielding PAH concentrations comparable to those of ambient urban air. Relatively high concentrations of moderately polar polycyclic aromatic hydrocarbon derivatives were also found in the indoor air when wood was burned in an open fireplace. Woodburning in the closed stove did, however, result in increased concentrations of mutagenic compounds and PAH on particles sampled in the vicinity of the house. The effects of wood burning in an open fireplace on the mutagenic activity of indoor air could still be considered moderate when compared to those resulting from tobacco smoking in the room. The extracts of particles collected when moderate smoking occurred were several times more mutagenic than samples from urban air collected close to streets with heavy traffic when measured in the Salmonella assay with strain TA98 with metabolic activation.

Mutagenicity testing of high performance liquid chromatography fractions from wood stove emission samples using a modified Salmonella assay requiring smaller sample volumes.

Organic extracts of emissions from wood combustion have been fractionated by high performance liquid chromatography (HPLC) into 25-28 fractions. Each fraction was tested for mutagenic activity in a modified Ames Salmonella/microsome bioassay requiring one-third of the test volumes needed for the ususal test. Direct mutagenic activity was noted predominantly in the most polar fractions, whereas indirect mutagenic activity was associated with the fractions containing polycyclic aromatic hydrocarbons (PAH) and with polar fractions probably consisting of aza-arenes and aromatic amines.

Effect of emissions from residential wood stoves on SCE induction in CHO cells.

The SCE-induction capacity of emissions from an airtight horizontal baffled residential wood stove was investigated in CHO cells. The samples were taken under normal and starved air conditions, from burning birch and spruce separately. Both particle phase and vapour phase were collected. All samples induced a dose-related response in SCE both with and without a metabolic activation system, the rat-liver microsomal fraction. The burning conditions in the stove influenced the mutagenicity of the emissions more than the type of wood; the smoke from wood burning under starved air conditions was more than one order of magnitude more potent in inducing a significant SCE response. With all samples, the response in SCE induction was highest without metabolic activation. The toxicity of the samples, especially those without S9, limited the dose-range tested.

Mutagenicity of extracts from typewriter ribbons and related items.

Extracts of typewriter ribbons and carbon papers were found to be mutagenic in the Salmonella/microsome assay with strain TA98. Fractionation of ribbon extracts indicates that at least 2-3 different classes of mutagenic component are present in these extracts. Nitro-containing compounds may be responsible for the high mutagenicity observed for some of the ribbon extracts in the absence of S9. The results indicate that impurities in the products may be causing part of the mutagenic effect.

Mutagenicity in emissions from coal- and oil-fired boilers.

The mutagenicity of emission samples from three oil-fired and four coal-fired boilers have been compared by using the Salmonella/microsome assay. Very little or no mutagenic activity was observed in samples from five of these boilers. The sample from one oil-fired boiler showed mutagenic activity of about 500 revertants/MJ, and the sample from a coal-fired fluidized bed combustor had an activity of 58,000 revertants/MJ measured with strain TA 98 in the absence of metabolic activation. All samples contained substances that were cytotoxic to the test bacteria, thus making it difficult to obtain linear dose-response curves. Mutagenic activity at low levels may remain undetected due to this toxicity of the samples. Samples with mutagenic activity below the detection limit in the Salmonella test have also been tested for forward mutations at the HGPRT locus in V79 hamster cells. Weak mutagenic effects were detected in two of the samples, whereas the sample from one oil-fired boiler remained negative. In this test, as well as in the Salmonella test, a strong cytotoxic effect could be observed with all samples.

Mutagenicity of air samples from various combustion sources.

The emission of mutagens from various combustion sources was compared. Flue gas samples from power plants and boilers burning coal, oil and wood were studied. Little or no mutagenic activity was observed in samples from big boilers operated under optimal conditions. The mutagenic activity of emission samples from different boiler systems burning the same fuel varied considerably. This variation was larger than the difference obtained from boilers of comparable size utilizing different fuels. The highest mutagenic activity was observed in samples from a small coal combustion unit, utilizing the fluidized-bed technique. In this case the activity was highest without metabolic activation. Extracts from all samples contained toxic compounds that, in high doses, inhibited mutagenicity.

Bioassay of extracts of ambient particulate matter.

Organic extracts from airborne particles collected at various sites in Scandinavia have been tested for mutagenicity in the Ames Salmonella/microsome assay. Extracts from particles in the respirable size fraction (diameter less than 3 microns) were mutagenic with and without metabolic activation. The mutagenic activity varied from day to day, mainly due to variations in meteorological parameters, especially wind speed and atmospheric stability. A seasonal variation could also be observed, with the highest average values in winter time. Samples collected in urban areas were considerably more mutagenic than samples from background areas. The results suggest that exhaust from motor vehicles are the most important source of mutagenic particles in urban areas. Comparison of roof top samples with street level samples indicated that atmospheric reactions cause transformation of nonpolar compounds in the primary emission to more oxygenated mutagenic compounds. It is, however, not known to which degree this causes an overall increase of the mutagenic activity. The mutagenic activity of emissions from stationary combustion sources have also been studied, and residential heating by burning solid fuels in small combustion units have been shown to be a major contributor to mutagens in the environment.

Contribution from motor vehicle exhaust to the mutagenic activity of airborne particles.

Samples of urban airborne particles have been collected simultaneously at street level and at roof top level in a street canyon of Oslo during two summer months. Extracts of the samples were tested for mutagenicity in the Ames Salmonella/microsome assay. The results are discussed in relation to traffic intensity, to meteorological parameters and to results from a similar study performed in the winter. The results indicated that vehicle exhaust was the main source of mutagens at both sampling sites and both seasons. Some additional activity from other sources may be detected in roof level winter samples. The results indicated further that some of the mutagens present at street level had been transformed to more polar compounds before reaching roof level.

Mutagenic activity in photocopies.

Extracts from several different photocopies were mutagenic in the Ames Salmonella assay. The mutagenic behavior was similar for extracts from copies and corresponding toners indicating that toners are directly responsible for the mutagenicity. The mutagenicity is caused by at least two classes of compounds which may be present either alone or in combination in any toner.

Studies on the in vitro uptake of polyamines by Girardi heart cells.

The uptake of spermine and spermidine in human heart cells in culture has been studied. Spermidine was accumulated to a higher degree than spermine whereas more spermine was absorbed to the cell surface. The results indicate that the uptake of spermidine and spermine is mediated by a common carrier. In addition, spermine enters the cells by several other transport mechanisms. The accumulated polyamines were not removed by washing with spermine or spermidine solutions, but they were released by treatment of the cells with butanol.

Effect of fluoride on the activity of ornithine decarboxylase in normal and fluoride resistant LS cells.

Renewal of growth medium caused an induction of ornithine decarboxylase (ODC) activity in LS cells grown in suspension culture. Addition of low concentrations of fluoride ions to the growth medium (up to 1.3 mM) resulted in a further increase in this induction of ODC-activity, whereas addition of 6 mM fluoride caused an inhibition of the induction and resulted in reduced ODC-activity as compared to controls. Since sodium fluoride had no stimulatory or inhibitory effect on the ODC-activity assay, it is likely that the effect is exerted on the regulation of ODC-activity in the cells. The effect of fluoride ions on the induction of ODC-activity upon renewal of the growth medium was markedly less pronounced in fluoride resistant LS cells.

Nickel and strontium distribution in some mouse tissues. Passage through placenta and mammary glands.

Radioactive isotopes of strontium or nickel (89Sr and 63Ni) were injected ip as chlorides on adult female mice and the distribution and retention studied in several soft and hard tissues, including tissues of fetuses and sucklings. The strontium concentration was comparable in liver, kidney and heart and higher than that of brain tissue. The acid soluble part of the mineralized tissues showed a strontium concentration 100-1000 times that of the soft tissues. Progeny receiving strontium through placenta or through mammary glands showed a distribution pattern similar to that of their mother, with retention of strontium in mineralized tissue. The nickel concentration was larger in the kidney than in other organs investigated. No nickel affinity was found for mineralized tissues. Nickel was readily passed through placenta and mammary glands. Contrary to findings for strontium, the nickel concentration in tissues of fetuses was higher than that of their mother.