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Reply to J Rood and SR Smith

Strathclyde Institute of Pharmacy and Biomedical Sciences
University of Strathclyde
204 George Street
Glasgow G1 1XW
United Kingdom
E-mail: clett.erridge{at}strath.ac.uk

Teresa Attina and David J Webb

The Queen's Medical Research Institute
University of Edinburgh
Edinburgh EH16 4TJ
United Kingdom

Dear Sir:

The results presented by Rood and Smith are interesting and a useful reminder that the limulus amoebocyte lysate (LAL) assay for endotoxin may be confounded by a number of established inhibitors or enhancers (1). However, the suggestion that triacylglycerol may act as a nonspecific enhancer of endotoxin detection by the LAL assay has been examined previously. Specifically, Emancipator et al (2) reported that coincubation of endotoxin-containing samples with increasing concentrations of Intralipid (Fresenius-Kabi, Clayton, NY) led to an overestimation of endotoxin concentrations when spectrophotometric measurement of the LAL assay was used.

However, because Intralipid itself was determined by Egberts et al (3) to have a high absorbance at the wavelength used to measure the LAL assay (405 nm), those researchers aimed to correct their LAL assay absorbance values for the inherent turbidity of Intralipid. This was achieved by subtracting the absorbance values obtained for Intralipid in phosphate-buffered saline without LAL reagent from the absorbance values obtained from corresponding LAL assay measurements of the same samples. When this correction was applied, Intralipid was found to have no effect on the measurement of endotoxin concentrations by the LAL assay, when it was included in the reactions at dilutions from 1:1000 to 1:20 (2). Excess turbidity is therefore the mechanism by which Intralipid increases absorbance in the LAL assay.

This observation leads to the obvious concern that the elevated endotoxin concentrations we and others have observed in postprandial plasma could have been confounded by the increased turbidity of lipemic postprandial plasma samples. To quantify the maximum extent to which this may have contributed to our results, we selected several of the most lipemic plasma samples from our previous study (with triacylglycerol concentrations ranging from 2.4 to 3.4 mmol/L); we retrospectively measured the absorbance of these samples and matched preprandial plasma samples (0.9–1.3 mmol triacylglycerol/L) at 405 nm after 1:20 dilution in water, as is usually performed in the standard LAL assay. We found that the mean increase in absorbance between preprandial and postprandial (lipemic) samples in water alone was 0.037 absorbance units. This finding compares with the mean absorbance value obtained from the original LAL assays of the postprandial samples, ie, 0.207. However, the final step in our limulus assay protocol is to add 0.25 volumes of 50% acetic acid. This treatment immediately clears any visible turbidity from lipemic plasma samples and reduces the difference in nonspecific absorbance between preprandial and postprandial samples to just 0.003 absorbance units. Because this protocol was applied to all of the samples tested in our study, it is likely that nonspecific effects of turbidity arising from lipemia accounted for only 1.5% of the increase in endotoxin readings that we observed. Because endotoxin has a high affinity for lipoproteins (4), it is likely that gut-derived endotoxin is associated with chylomicrons after a fatty meal, which may explain why a stronger correlation appears to exist between elevated triacylglycerol concentrations and endotoxin concentrations than may be explained by this result alone.

Nevertheless, we acknowledge that direct measurement of endotoxin concentrations in plasma samples will remain challenging, given the fluctuating nature of circulating endotoxin concentrations and the fact that they are close to the limit of sensitivity of the LAL assay. We therefore support the suggestion of Rood and Smith that further progress in this area will likely demand a novel approach to the detection of fat-induced endotoxin translocation. They rightly pointed out that the gel-clot method of endotoxin determination is not affected by issues of turbidity or triacylglycerol concentrations and that it may provide a clearer picture of the effects of diet on endotoxemia. Accordingly, it is interesting to note that the gel-clot method of endotoxin detection was used by Yoshimatsu et al (5) to show that apolipoprotein E–deficient mice develop portal endotoxemia when fed a high-fat diet but not when fed normal chow. Clearly, further studies are warranted to investigate the relevance of this novel potential pathway of endotoxin exposure in the context of inflammatory diseases.

ACKNOWLEDGMENTS

None of the authors had a personal or financial conflict of interest.

REFERENCES

1. Sullivan JD, Watson SW. Factors affecting the sensitivity of Limulus lysate. Appl Microbiol 1974;28:1023–6.[Medline]
2. Emancipator K, Csako G, Elin RJ. In vitro inactivation of bacterial endotoxin by human lipoproteins and apolipoproteins. Infect Immun 1992;60:596–601.[Abstract/Free Full Text]
3. Egberts J, van den Heuvel B, Duiser HJ, van Dam W, Lentjes EGWM, Kanhai HH. Iterative, spectrophotometric method for determination of amniotic fluid bilirubin concentrations: comparison with the Liley method. Clin Chem 2002;48:2045–7.[Free Full Text]
4. Harris HW, Grunfeld C, Feingold KR, et al. Chylomicrons alter the fate of endotoxin, decreasing tumor necrosis factor release and preventing death. J Clin Invest 1993;91:1028–34.[Medline]
5. Yoshimatsu M, Terasaki Y, Sakashita N, et al. Induction of macrophage scavenger receptor MARCO in nonalcoholic steatohepatitis indicates possible involvement of endotoxin in its pathogenic process. J Exp Pathol 2004;85:335–43.

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