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The Dynamics of Circulating Heparin-Binding Protein: Implications for Its Use as a Biomarker

Heparin-binding protein (HBP) is a promising biomarker for the development and severity of sepsis. To guide its use, it is important to understand the factors that could lead to false-positive or negative results, such as inappropriate release and inadequate clearance of HBP. HBP is presumably released only by neutrophils, and the organs responsible for its elimination are unknown. In this study, we aimed to determine whether non-neutrophil cells can be a source of circulating HBP and which organs are responsible for its removal. We found that in two cohorts of neutropenic patients, 12% and 19% of patients in each cohort, respectively, had detectable plasma HBP levels. In vitro, three leukemia-derived monocytic cell lines and healthy CD14+ monocytes constitutively released detectable levels of HBP. When HBP was injected intravenously in rats, we found that plasma levels of HBP decreased rapidly, with a distribution half-life below 10 min and an elimination half-life of 1–2 h. We measured HBP levels in the liver, spleen, kidneys, lungs, and urine using both ELISA and immunofluorescence quantitation, and found that the majority of HBP was present in the liver, and a small amount was present in the spleen. Immunofluorescence imaging indicated that HBP is associated mainly with hepatocytes in the liver and monocytes/macrophages in the spleen. The impact of hematologic malignancies and liver diseases on plasma HBP levels should be explored further in clinical studies.


Supplementary information Contents
Validation of HBP ELISA kit for rat samples Before measurement of HBP in rat samples, the kit was validated for use with rat plasma, urine, and organ lysates by a spike and recovery assay. Plasma, urine and organ homogenate samples from control rats were spiked with 1.25 ng/mL of recombinant HBP, a level near the middle of the detectable range of the kit. HBP level was measured in the spiked and un-spiked sample and the percent recovery of HBP was determined and shown in supplementary table S1. The background level in each sample type is shown in supplementary table S2.
Supplementary table S1. Recovery of spiked HBP in various samples, n=3 of each type, expressed as mean ± standard deviation.

Sample
Recovery ( Recovery was considered acceptable if the mean recovery was between 80-120%. According to these results we analyzed all rat plasma and lung, kidney, and spleen lysates and urine at a minimum dilution of 1:40. Because liver lysate produced a background signal in the ELISA at a 1:40 dilution which was mostly removed at 1:400 dilution, we ran all liver lysates at a dilution of at least 1:400.

Pharmacokinetic analyses
Supplementary figure S1. Plasma HBP levels over time in rats injected intravenously with recombinant human HBP at a dose of 160 µg/kg. Pharmacokinetic graphs for each individual rat with time in minutes vs log HBP. HBP values were log-transformed and the alpha phase (≤10 min) and beta phase (≥10 min) were modelled using linear regression. The blue line represents the estimated mean alpha phase and the red line the mean beta phase. The lines were modelled with the value at 10 minutes for each individual rat as a fixed value. The alpha phase was also modelled not subtracting the predicted beta phase from the values and the green line represents the estimated mean alpha phase from this calculation.

Supplementary figure S2. Plasma HBP levels over time in rats injected intravenously
with recombinant human HBP at a dose of 320 µg/kg. Pharmacokinetic graphs for each individual rat with time in minutes vs log HBP. HBP values were log-transformed and the alpha phase (≤10 min) and beta phase (≥10 min) were modelled using linear regression. The blue line represents the estimated mean alpha phase and the red line the mean beta phase. The lines were modelled with the value at 10 minutes for each individual rat as a fixed value.

Supplementary table S3.
Pharmacokinetic parameters for rats injected with 160 µg/kg HBP were calculated for each individual rat. HBP values were log-transformed and the beta phase (≥10 min) was modelled using linear regression. The fitted values were then subtracted from the alpha phase (0-10 min) and the alpha phase was fitted using linear regression. The alpha phase was also modelled not subtracting the predicted beta phase from the values. Supplementary table S4. Pharmacokinetic parameters for rats injected with 320 µg/kg HBP were calculated for each individual rat. HBP values were log-transformed and the beta phase (≥10 min) was modelled using linear regression. The fitted values were then subtracted from the alpha phase (0-10 min) and the alpha phase was fitted using linear regression.