Email updates

Keep up to date with the latest news and content from Critical Care and BioMed Central.

This article is part of the supplement: Sepsis 2012

Poster presentation

Preliminary results for the use of proteinase K to achieve release of LPS from the Alteco LPS Adsorber® after perfusion with LPS containing blood

E Hansen1*, L Pierre2 and S Blomqvist12

  • * Corresponding author: E Hansen

Author Affiliations

1 Alteco Medical AB, Lund, Sweden

2 Faculty of Medicine, Lund University, Sweden

For all author emails, please log on.

Critical Care 2012, 16(Suppl 3):P95  doi:10.1186/cc11782


The electronic version of this article is the complete one and can be found online at: http://ccforum.com/content/16/S3/P95


Published:14 November 2012

© 2012 Hansen et al.; licensee BioMed Central Ltd.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Background

The effect of Alteco LPS Adsorber® to remove LPS from the circulation is based on the incorporation of a synthetic peptide that binds to the lipid A moiety of LPS, the binding capacity in one adsorber exceeding 7.5 μg LPS. Positive clinical effects of the adsorber when used in patients with Gram-negative sepsis have been reported [1]. Measurement of LPS in human blood, however, is hampered by difficulties such as contamination and interference. The aim of this pilot study was to evaluate a method to release LPS from the LPS adsorber after perfusion with LPS containing blood.

Methods

Two adsorbers (A1, A2) containing the active peptide and one dummy adsorber (D) with no peptide were used. Whole blood (500 ml) from healthy pigs was collected in a bag containing heparin, 37 μg LPS was added. A roller pump was used to recirculate the blood through the adsorber. The pump flow was set at 150 ml/minute and the duration of the perfusion was 2 hours. After the end of perfusion the adsorbers were rinsed with 500 ml Krebs solution. A solution of 20 mg proteinase K in 50 ml Tris buffer with pH 7.4 was prepared and 2.5 ml CaCl2 was added. The adsorbers were kept at 37°C and the proteinase K solution [2] was perfused through the adsorbers at 5 ml/minute for 6 hours. Samples for LPS analysis (chromogenic LAL) [3] were drawn before the perfusion and then after 30, 240 and 360 minutes. The enzyme activity was checked using a synthetic substrate [4].

Results

The concentrations of LPS before and during perfusion with proteinase K are shown in Table 1.

Conclusion

The LPS values found before the start of the perfusion indicate contamination of the solution. The increase in LPS seen in all adsorbers after 30 minutes is probably due to traces of blood components. The later increase in LPS after treatment with proteinase K in the active adsorbers indicates that the adsorber is effective in capturing LPS from whole blood and that proteinase K is able to dislodge LPS bound to the adsorber.

References

  1. Ala-Kokko TI, Laurila J, Koskenkari J: A new endotoxin adsorber in septic shock: observational case series.

    Blood Purif 2011, 32:303-309. PubMed Abstract | Publisher Full Text OpenURL

  2. Petsch D, Deckwer WD, Anspach FB: Proteinase K digestion of proteins improves detection of bacterial endotoxin by the Limulus amebocyte lysate assay: application for endotoxin removal from cationic proteins.

    Anal Biochem 1998, 259:42-47. PubMed Abstract | Publisher Full Text OpenURL

  3. European Pharmacopeia. Volume 1. 6th edition. EDQM; 2008. OpenURL

  4. Bajorath J, Hinrichs W, Saenger W: The enzymatic activity of proteinase K is controlled by Calcium.

    Eur J Biochem 1988, 176:441-447. PubMed Abstract | Publisher Full Text OpenURL