Whole Blood Assay for Elastase, Chymotrypsin, Matrix Metalloproteinase-2, and Matrix Metalloproteinase-9 Activity

Abstract

The ability to measure protease activity in the blood is important for the development of future diagnostics and for biomedical research. Presently, protease assays require sample preparation, making them time-consuming, costly, less accurate, and unsuitable for point-of-care (POC) diagnostics. Recently, we demonstrated a unique method for measuring clinically relevant levels of trypsin activity in only a few microliters of whole blood. This assay utilizes a charge-changing fluorescent peptide substrate that produces a positively charged fluorescent product fragment upon cleavage by the target protease. Using a simple electrophoretic format, the fragments could be rapidly separated, concentrated, and detected directly from a whole blood sample. We now report on the development of new protease substrates for the measurement of elastase, chymotrypsin, matrix metalloproteinase (MMP)-2, and MMP-9 activity in whole blood. In these studies, detection limits ranging from 1 to 40 pg in 6 μL of 1× phosphate-buffered saline (PBS) (0.2-6 ng/mL) were achieved after a only 1 h reaction of enzyme and substrate. In subsequent experiments measuring spiked protease in whole blood (with endogenous protease present), detection limits ranging from 100 to 200 ng/mL were achieved after a 1 h reaction. Thus, these new substrates demonstrate broad applicability toward clinically relevant detection of important disease-relevant proteases.

The Autodigestion Hypothesis for Shock and Multi-organ Failure

“…This information will serve as the basis for understanding the mechanisms that lead to the escape of digestive enzymes from the lumen of the intestine in pathophysiological conditions (9). More sensitive and specific diagnostic tools to detect degrading enzyme activity in patients are required (32). To minimize autodigestion, more specific digestive enzyme inhibitors and optimal delivery methods need to be developed while at the same time the nutritional needs of the patients may have to be met by alternative pathways, for example by intravenous delivery.…”

Proof of Concept: Matrix metalloproteinase inhibitor decreases inflammation and improves muscle insulin sensitivity in people with type 2 diabetes

“…[21] A global assay for MMP activity levels was also performed on plasma samples using the SensoLyte®520 Generic MMP assay kit according to manufacturer’s instructions (#71158; AnaSpec, San Jose, CA. ).…”

An Emerging Role of Degrading Proteinases in Hypertension and the Metabolic Syndrome: Autodigestion and Receptor Cleavage

“…In contrast to inflammatory markers such as the acute phase protein c-reactive protein or fibrinogen, which have minimal diurnal variations, proteinase protein levels and especially proteinase activities may fluctuate, so the time of day, meals, medication, or medical conditions other than hypertension must be considered. Resolution of this issue will require systematic in vivo measurements using improved zymographic techniques that can read nanomolar concentrations of fresh active enzymes with minimal sample preparation and storage [87]. Techniques to measure MMP activity in blood vessels of humans in vivo are also required.…”