Quantification of ergosterol and 3-hydroxy fatty acids in settled house dust by gas chromatography-mass spectrometry: comparison with fungal culture and determination of endotoxin by a Limulus amebocyte lysate assay

Abstract

Ergosterol and 3-hydroxy fatty acids, chemical markers for fungal biomass and the endotoxin of gramnegative bacteria, respectively, may be useful in studies of health effects of organic dusts, including domestic house dust. This paper reports a method for the combined determination of ergosterol and 3-hydroxy fatty acids in a single dust sample and a comparison of these chemical biomarkers determined by gas chromatography-mass spectrometry with results from fungal culture and Limulus assay. Analyses of replicate house dust samples resulted in correlations of 0.91 (ergosterol in six replicates; P < 0.01) and 0.94 (3-hydroxy fatty acids in nine replicates; P < 0.001). The amounts of ergosterol (range, 2 to 16.5 ng/mg of dust) correlated with those of total culturable fungi (range, 6 to 1,400 CFU/mg of dust) in 17 samples, (r ‫؍‬ 0.65; P < 0.005). The amounts of endotoxin (range, 11 to 243 endotoxin units/mg of dust) measured with a modified chromogenic Limulus assay correlated with those of lipopolysaccharide (LPS) determined from 3-hydroxy fatty acid analysis of 15 samples. The correlation coefficient depended on the chain lengths of 3-hydroxy acids used to compute the LPS content. The correlation was high (r ‫؍‬ 0.88 ؎ 0.01; P < 0.001) when fatty acid chains of 10 to 14 carbon atoms were included; the correlation was much lower when hydroxy acids of 16-or 18-carbon chains were included. In conclusion, the results of the described extraction and analysis procedure for ergosterol and 3-hydroxy fatty acids are reproducible, and the results can be correlated with fungal culture and endotoxin activity of organic dust samples.

Characterization and Variability of Endotoxin and 3-Hydroxy Fatty Acids in an Office Building During a Particle Intervention Study

“…The significant negative correlation found for 3-OH-C 16:00 contrasts with Saraf et al (1997) where 3-OH-C 16:00 did not correlate strongly (positively or negatively) with endotoxin activity. Saraf et al (1997) tested the supernatants of several different bacterial suspensions and found that P. cepacia with predominately 3-OH-C 14:00 and 3-OH-C 16:00 fatty acids had greater endotoxin potency than E. coli with predominately 3-OH-C 14:00 , but H. pylori with mostly 3-OH-C 16:00 and 3-OH-C 18:00 had very low potency compared to both P. cepacia and E. coli. If the presence of substantial amounts of 3-OH-C 16:00 and 3-OH-C 18:00 fatty acids is related to reduced endotoxin potency, then this might explain the low endotoxin potency found for office dust in this study where 3-OH-C 16:00 and 3-OH-C 18:00 were the predominant 3-OHFAs in the dust samples.…”

“…The mean endotoxin potency of office dust in this study (0.7 EU/pmole LPS) was approximately half the endotoxin potency reported by Saraf et al (1997) for house dust (∂1.4 EU/pmole LPS), suggesting differences in the LPS composition of dust from these two environments. Differences in the chemical composition of LPS isolated from Enterobacter agglomerans, Citrobacter freundii and Agrobacterium sp.…”

“…Both this study of office dust and the Saraf et al (1997) study of house dust found a significant positive correlation between endotoxin measured by KLARE and 3-OH-C 12:00 measured by GC/MS, although the correlation coefficient for office dust (rΩ0.61) was lower than for house dust (rΩ0.90). The lower correlation coefficient found in this study may be related to the narrower range of endotoxin levels found in office dust samples obtained from one building (24.9-74.9 EU/mg) than in house dust samples obtained from many different homes (11-243 EU/mg).…”

“…Endotoxin activity is typically measured using a Limulus-based bioassay (Milton, 1995a). LPS-associated 3-OHFAs are determined by gas chromatography-mass spectrometry (GC-MS), which provides a quantitative distribution of 3-OHFAs in each sample (Mielniczuk et al, 1993;Gradowska and Larsson, 1994;Saraf et al, 1997).…”

Effects of dog ownership in early childhood on immune development and atopic diseases

“…Ergosterol and muramic acid were measured by gas chromatography-ion trap mass spectrometry. These samples were stored dry in baked glass tubes, and detailed protocols are as described elsewhere [18,19].…”