Distribution and kinetics of lipoprotein-bound endotoxin

Abstract

Lipopolysaccharide (LPS), the major glycolipid component of gram-negative bacterial outer membranes, is a potent endotoxin responsible for pathophysiological symptoms characteristic of infection. The observation that the majority of LPS is found in association with plasma lipoproteins has prompted the suggestion that sequestering of LPS by lipid particles may form an integral part of a humoral detoxification mechanism. Previous studies on the biological properties of isolated lipoproteins used differential ultracentrifugation to separate the major subclasses. To preserve the integrity of the lipoproteins, we have analyzed the LPS distribution, specificity, binding capacity, and kinetics of binding to lipoproteins in human whole blood or plasma by using high-performance gel permeation chromatography and fluorescent LPS of three different chemotypes. The average distribution of O111:B4, J5, or Re595 LPS in whole blood from 10 human volunteers was 60% (+/-8%) high-density lipoprotein (HDL), 25% (+/-7%) low-density lipoprotein, and 12% (+/-5%) very low density lipoprotein. The saturation capacity of lipoproteins for all three LPS chemotypes was in excess of 200 microg/ml. Kinetic analysis however, revealed a strict chemotype dependence. The binding of Re595 or J5 LPS was essentially complete within 10 min, and subsequent redistribution among the lipoprotein subclasses occurred to attain similar distributions as O111:B4 LPS at 40 min. We conclude that under simulated physiological conditions, the binding of LPS to lipoproteins is highly specific, HDL has the highest binding capacity for LPS, the saturation capacity of lipoproteins for endotoxin far exceeds the LPS concentrations measured in clinical situations, and the kinetics of LPS association with lipoproteins display chemotype-dependent differences.

Figures

FIG. 1

Comparison of the TNF-α-inducing capacity of labeled or unlabeled O111:B4 (A), J5 (B), or Re595 (C) in whole blood from three healthy volunteers. LPS at concentrations of 1, 10, 100, and 1,000 ng/ml of whole blood was incubated for 4 h at 37°C, and TNF-α levels were measured in duplicate. The values represent the average ± 1 standard deviations.

FIG. 2

Competition between labeled and unlabeled LPS for binding to plasma lipoproteins. Graphs for BODIPY-O111:B4 (A), NBD-J5 (B), and NBD-Re595 (C) show the distribution of labeled LPS lipoprotein in association with VLDL, LDL, or HDL in plasma in the presence of unlabeled LPS for plasma concentrations approaching saturation (200, 300, and 200 μg/ml) for the three chemotypes. The LPS mixtures were added to the plasma samples and incubated for 20 min at 37°C. Correction was done for the natural fluorescent background of the plasma components at the excitation and emission wavelengths used. Linear regression was used to generate the curves.

FIG. 3

(A) Chromatographic profile of the major plasma lipoprotein classes separated by HPGC with online cholesterol detection. The average molecular masses calculated from retention characteristics were 5 MDa for VLDL, 1 MDa for LDL, and 300 kDa for HDL. Horizontal bars indicate the lipoprotein fractions pooled for further analysis. (B) SDS-PAGE analysis of these lipoprotein fractions with subsequent silver staining, showing the protein composition of the lipoproteins. M, molecular weight in thousands; lane A, VLDL; lane B, LDL; lane C, HDL. Each well was loaded with 20 μl containing a total of approximately 2 μg of protein.

FIG. 4

Chromatographic LPS cholesterol (A) and fluorescence (B) profiles of normal (continuous lines) and delipidated (dotted lines) plasma. Both normal plasma and delipidated plasma were incubated with NBD-J5 LPS for 60 min at 37°C, and the fluorescence and cholesterol lipoprotein profiles were determined as described in Materials and Methods.

FIG. 5

Chromatographic profiles showing dose response of LPS-lipoprotein association with three LPS chemotypes, O111:B4, (A), J5 (B), and Re595 (C). All main lipoprotein classes indicated in panel A apply also to panels B and C. LPS at the concentrations indicated was incubated for 1 h at 37°C. All chromatograms were corrected for the inherent fluorescence of plasma components. The data are representative of a number of independent experiments.

FIG. 6

LPS binding capacities of plasma lipoproteins. Diagrams of lipoprotein-associated BODIPY-O111:B4 (A), NBD-J5 (B), and NBD-Re595 (C) LPS show a dose-dependent distribution of LPS on VLDL, LDL, and HDL in plasma obtained from healthy volunteers. The values represent the mean ± 1 standard deviation of duplicate results after correction of the natural fluorescent background of the plasma components at the excitation and emission wavelengths used. Nonlinear regression data fit was used to generate the curves.

FIG. 7

LPS binding kinetics of plasma lipoproteins after incubation of 24 μg of BODIPY-O111:B4 (A), 35 μg of NBD-J5 (B), and 40 μg of NBD-Re595 (C) per ml. After incubation of individual plasma samples for 10, 20, 40, 60, and 120 min at 37°C, 60 μl of plasma diluted 1:1 with TBST elution buffer was analyzed by HPGC as described in the text. All points represent peak areas corrected for inherent background fluorescence of plasma components at the excitation and emission wavelengths used. Nonlinear regression data fit was used to generate the curves. The graphs are representative of one of the four LPS concentrations used in these experiments which all produced similar results.