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Faster diagnosis? Chlorinated lipids in sepsis

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Amy Carpenter Aquino

October 2019—Chlorinated lipids have been shown to be new potential biomarkers for sepsis, and continuing research into their role could lead to faster diagnosis, said David A. Ford, PhD, of Saint Louis University School of Medicine, at this year’s AACC annual meeting. Dr. Ford, a professor of biochemistry and molecular biology, discovered chlorinated lipids in 2002, and at the AACC meeting he shared recent research on the association between chlorinated lipids and lung injury and death in sepsis patients.

“Can we define new biomarkers?” he asked at the start of his talk. “And do they have a functional role in mediating some of the injury—the multiorgan failure—that we see during sepsis?”

Multiorgan failure during sepsis is the result of dysregulated blood and endothelial interactions that lead to microcirculatory collapse. The National Institutes of Health in 2014 expanded the number of investigators invited to research microcirculatory dysfunction, Dr. Ford said. He and co-principal investigators Jane McHowat, PhD, professor of pathology at Saint Louis University School of Medicine, and Ronald Korthuis, PhD, professor of medical pharmacology and physiology at the University of Missouri School of Medicine, have been studying the role of chlorinated lipids in sepsis, and their work has been funded by $1.78 million in NIH grants.

Dr. Ford

“In particular, there are bloodborne elements interacting with the endothelium that cause injury. That’s where we got involved in this problem—investigating the microcirculatory dysfunction that leads to organ failure and ultimately death in these patients.”

In respect to the microcirculatory collapse, Dr. Ford and colleagues have been exploring “whether the polymorphonuclear cells—the neutrophils—had a role in generating novel compounds, whether these novel compounds could be biomarkers, and whether they could also participate in an injury,” he said.

Dr. Ford’s laboratory first zeroed in on the role of myeloperoxidase, the predominant protein in neutrophils, which is unleashed when a neutrophil is activated resulting in the production of hypochlorous acid or bleach. “Your white blood cells are making bleach” to destroy bacteria that are taken up by the neutrophil, Dr. Ford said. “It engulfs and destroys them with the strong oxidant hypochlorous acid.”

His laboratory was further interested in whether there were biomolecules in the neutrophil and surrounding tissue that could be targeted by hypochlorous acid to lead to other novel compounds. “We are mining for new biomarkers and mediators,” with chlorinated lipids as a targeted approach and then also performing unbiased analyses.

“We’re doing experiments in our lab that are untargeted,” he said, using high-resolution mass spectrometry to find molecules. “Once you find those, you can make them your new targets. Then you can plug them into biological assays and see whether they have an effect.” Computational modeling then can be performed with machine learning. “The whole process is considered a systems biology approach.”

Dr. Ford and colleagues discovered early on that bleach created by the neutrophils can target the vinyl ether bond of a phospholipid called plasmalogen. Plasmalogens, a molecular subclass of phospholipids, are the most abundant phospholipids in the plasma membrane of many cells, including those of the heart, endothelium, and brain. “The brain, heart, and vascular system are highly enriched with plasmalogens, which have a vinyl ether linkage at the sn-1 position of the glycerol backbone of the phospholipids,” he said.

The vinyl-ether bond of the plasmalogen is responsible for generating chlorinated lipids.

“The molecules that we found in my laboratory are chlorinated fatty aldehydes [2-CLFALD] and chlorinated fatty acids [2-CLFA] that get liberated from the vinyl ether linkage of the plasmalogens,” Dr. Ford said. These chlorinated lipids, which are usually 16 or 18 carbons in length, are a deleterious side product of the inflammatory response of the neutrophil.

In a separate study, Dr. Ford and Nuala Meyer, MD, MS(MSTR), and Jason Christie, MD, MSCE, of the Division of Pulmonary and Critical Care Medicine, University of Pennsylvania Perelman School of Medicine, showed a link between 2-CLFA and acute respiratory distress syndrome in adult ICU patients who were suspected of having sepsis. The study was conducted as part of a larger University of Pennsylvania study called the Molecular Epidemiology of Sepsis in the ICU (MESSI) cohort.

“We found that the 2-chlorofatty acids in the plasma of these patients were elevated in septic patients who developed acute respiratory distress syndrome,” Dr. Ford said. “They were also elevated in patients who did not survive.”

Plasma was collected at day zero from patients who were brought into the ICU and verified to have sepsis based on blood culture test results (Meyer NJ, et al. JCI Insight. 2017;2[23]:e96432).

“We then wanted to find out whether they [chlorinated lipids] had a role in causing injury at the endothelial layer in the microcirculation,” Dr. Ford said. Dr. Korthuis, of the University of Missouri, performed a technique called intravital microscopy, which makes it possible to visualize white blood cells rolling across the vasculature. “You can measure whether white blood cells are rolling and adhering to the microcirculation. Dr. Korthuis superfused the chlorinated fatty acid on the mesenteric microcirculation and saw increased leukocyte rolling as well as leukocyte adhesion” to the endothelium.

“The aldehyde also had an effect,” Dr. Ford said. However, “The fatty acid that doesn’t have chlorine, palmitic acid, had no effect on leukocyte rolling or on adhesion.”

After determining that chlorinated lipids affect endothelial cell surface adhesion, they wanted to see whether they could look at adhesion molecule expression and whether in cell models that would have an effect on neutrophil, platelet, and endothelial function, he said. “We also looked at whether these chlorinated lipids cause netosis.”

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