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Triglyceride concentrations and endotoxemia

Department of Clinical Chemistry
Pennington Biomedical Research Center
6400 Perkins Road
Baton Rouge, LA 70808
E-mail: jennifer.rood{at}pbrc.edu

Steven R Smith

Department of Molecular Endocrinology
Pennington Biomedical Research Center
Baton Rouge, LA

Dear Sir:

In a recent article in the Journal, Erridge et al (1) provided data to suggest that circulating endotoxin concentrations increase after a high-fat meal. One other report in the literature described elevated endotoxin concentrations after 4 wk of a high-fat diet (2).

Given the scientific interest in gut permeability to lipopolysaccharide (LPS; endotoxin) and to inflammatory disease processes and metabolism (3, 4), we pursued the measurement of LPS by using methods similar to those described by Erridge et al. Using the chromogenic limulus amebocyte lysate (LAL) QCL-1000 assay (Cambrex Bioscience, Walkersville, MD), we analyzed 40 clinical serum samples. The assay is reported as a quantitative test for gram-negative endotoxin (ie, LPS). Samples are measured spectrophotometrically at 405–410 nm after the addition of LAL. The absorbance of the sample is in direct proportion to the amount of endotoxin present in the sample, and it can be calculated by using a standard curve. We intentionally selected samples with a range of triglyceride values (mean: 287 mg/dL; range: 22–1067 mg/dL). Triglycerides and endotoxin values were highly correlated (r² = 0.73). Endotoxin concentrations ranged from 0.198 to 1.22 endotoxin units/mL. This association could be explained by the affinity of endotoxin for the VLDL particle as a carrier, or it could represent cross-reactivity of the assay for another lipid, such as triglyceride or phospholipid.

To test the hypothesis that triglycerides may bias the endotoxin assay, we performed the assay with reaction buffer and Intralipid (Fresenius-Kabi, Mississauga, Canada), with or without plasma. Intralipid is a sterile, nonpyrogenic fat emulsion containing phospholipids and triglycerides, and it is approved for clinical use via intravenous administration. Briefly, we performed the assay with both sterile pyrogen-free water and serum samples spiked with Intralipid; dilutions of Intralipid varied from 1: 30 to 1:10 000. The manufacturer recommends heat inactivation of the samples before assay; we varied the duration at 37 °C from 5 to 60 min. In all cases, as the Intralipid concentration increased, so did the endotoxin concentration (r² ranged from 0.909 to 0.995). This was true for both spiked serum and sterile water samples.

These experiments suggest that the chromogenic assay results are confounded by elevated triglyceride. It was previously shown that intravenous fat emulsions interfere with the total bilirubin assay, which is also a chromogenic procedure (5). Because triglyceride values increase, endotoxin values increase, and this increase is not driven by endotoxin. This hypothesis assumes that Intralipid is endotoxin free; that assumption was confirmed by conversations with the manufacturer. Intralipid is certified as pyrogen-free by using the LAL gel clot method. These data are important to the clinical researcher, the basic (animal) researcher, and, potentially, medical laboratories or clinicians. Caution should be used in interpreting data from biological samples by using this assay. Both high-fat meals and high-fat diets are known to increase circulating triglycerides and could provide a false-positive result. It is possible that the newer fluorescence endotoxin assays (Cambrex Bioscience) are less affected by the triglycerides in biological materials, such as plasma and serum. This possibility should be considered in future investigations.

ACKNOWLEDGMENTS

Neither author had a personal or financial conflict of interest.

REFERENCES

1. Erridge C, Attina T, Spickett CM, Webb DJ. A high-fat meal induces low-grade endotoxemia: evidence of a novel mechanism of prostprandial inflammation. Am J Clin Nutr 2007; 86(5):1286–92.[Abstract/Free Full Text]
2. Cani PD, Amar J, Iglesias MA, et al. Metabolic endotoxemia initiates obesity and resistance. Diabetes 2007;56(7):1761–72.[Medline]
3. Suliburk J, Helmer K, Moore F, Mercer D. The gut in systemic inflammatory response syndrome and sepsis. Enzyme systems fighting multiple organ failure. Eur Surg Res 2008;40(2):184–9 (Epub 2007 Nov 12).[Medline]
4. Jergensen VL, Ibsen M, Andresen L, Schulzke JD, Perner A. Effects of endotoxaemia on markers of permeability, metabolism and inflammation in the large bowel of healthy subjects. Acta Anaesthesiol Scand 2007;51(8):1085–92.[Medline]
5. McKiel RR, Barron N, Needham CJ, Wilkins TA. Liposyn interference with neonatal billrubin measurements. Clin Chem 1982;28(11):2334–5.[Free Full Text]

This Article Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Google Scholar Articles by Rood, J. Articles by Smith, S. R PubMed PubMed Citation Articles by Rood, J. Articles by Smith, S. R Agricola Articles by Rood, J. Articles by Smith, S. R