Shedding Light on your Blood Endotoxin Level

A drop of your blood is all that is needed to see if you have a dangerous level of circulating Endotoxin. With a measurement time of 30 minutes it detects Picogram amounts.

My Chemistry students favourite laboratory practical was the joy of making Luminol Chemiluminescence.

I had to take them into a rather cramped storeroom a few at a time to see something like this:

Photo credit.¹

So I was thrilled to find that a fast, sensitive and specific test for Gram-negative bacterial Endotoxin Lipid A was published in 1998 by Canadian researchers that relies on Luminol Chemiluminescence and the CR₁ and CR₃ receptor-induced oxidant production of polymorphonuclear leucocytes as a detection platform.²

The predominant oxidant reacting with the lumiphor Luminol in whole blood is HOCl (Hypochlorous Acid).

The US FDA approved this rapid whole blood Endotoxin assay, now called EAA or Endotoxin Activity Assay (EAA) in 2002. It is a semi-quantitative test where levels are reported as low, EAA level < 0.40 units, intermediate, 0.40–0.59 and high, ≥ 0.60 EA units and uses each patients own blood as a control.

The EAA test employs murine monoclonal IgM antibody against the Endotoxin Lipid A epitope in the the presence of Zymosan from Saccharomyces cerevisiae (brewer's yeast or baker's yeast, extracts of which I like in Vegemite and Guiness).³

Oxidant released in response to Complement Opsonized⁴ Zymosan⁵ results in Luminol oxidation and subsequent light emission.

Sensitivity of the EA assay is dependent upon optimal antigen–antibody equivalence and duration of pre-incubation with the whole blood sample. A positive control 800 Picogram/ml Escherichia coli 055:B5 Endotoxin at an antibody concentration of 0.8 μg per assay. EAA works over a wide range of Endotoxin concentrations 0.017–1.6 EU/ml or 20–2000 picogram/ml. The researchers equate 1 Endotoxin Unit with 1,200 picogram (1.2 nanogram) of the supertoxin.

Endotoxin causes Organ Failure in Covid19 Patients

I recently came across a paper⁶ reporting on the Cytokine Storm caused by Covid19 infection from March to June 2020 which stated:

High levels of Endotoxin activity were found in a majority of critically-ill COVID-19 patients admitted to the ICU and were associated with greater risk for Cardiovascular and Renal Failure. Further investigation is needed to determine if endotoxin reducing strategies are useful in treating severe COVID-19 infection.

The researchers used EAA to correlate Endotoxin with Length of Stay in ICU as well Organ Failure and to make decisions regarding the utility of attempting to reduce Endotoxin by application of an Endotoxin removal device by Hemoperfusion, such as Toraymyxin.

Toraymyxin uses a cartridge containing fibres made of polystyrene derivatives (alphachloro-aceto-amidomethylated polystyrene). Polymyxin B is immobilized on the surface of the fibres which adsorbs and removes Endotoxin from the patient’s circulating blood.⁷

1. Tavo Romann. 2013. https://en.m.wikipedia.org/wiki/File:Chemiluminescence.jpg

2. Alexander D. Romaschin, David M. Harris, Melanie B. Ribeiro, Jeffrey Paice, Debra M. Foster, Paul M. Walker, John C. Marshall. 1998. A rapid assay of endotoxin in whole blood using autologous neutrophil dependent chemiluminescence. https://www.sciencedirect.com/science/article/abs/pii/S0022175998000039

3. https://en.wikipedia.org/wiki/Saccharomyces_cerevisiae

4. https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/opsonization

5. https://en.wikipedia.org/wiki/Zymosan

6. Philip T.H. Dela Cruz, Danielle Davison, David P. Yamane, Everett Chu and Michael Seneff. 2023. Increased Endotoxin Activity in COVID-19 Patients Admitted to the Intensive Care Unit. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9676678/

7. https://www.toraymyxin.toray/en/toraymyxin/