Detection of in vivo matrix metalloproteinase activity using microdialysis sampling and liquid chromatography/mass spectrometry.
Matrix metalloproteinases (MMPs) are a family of endoproteases that break down extracellular matrix and whose upregulation contributes to several diseases. A liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed to quantify MMP-1 and MMP-9 substrates and their N-terminal peptide products in samples obtained from implanted microdialysis sampling probes. In vitro studies with purified human MMP-1 and MMP-9 were used to optimize the assay and determine the effectiveness of the local delivery of a broad-spectrum MMP inhibitor, GM 6001. Localized delivery of GM 6001 at 10 microM was sufficient to completely inhibit product formation in vitro. In vivo studies in male Sprague-Dawley rats were performed with microdialysis probes implanted into the subcutaneous tissue. Directly after microdialysis probe implantation, infusions of the MMP-1 and MMP-9 substrates (50 microM each) resulted in recovered product concentrations of approximately 2 microM. During a 50 microM GM 6001 coinfusion with the substrates, a 30% and 25% reduction in product formation for the MMP-1 and MMP-9 substrates was obtained, respectively. Blank dialysates were negative for enzymatic activity that could cleave the MMP substrates. This method allowed for the activity of different MMPs surrounding the microdialysis probe to be observed during in vivo sampling.
Wang
Ying
Y
Department of Chemistry and Chemical Biology, Rensselaer Polytechnic Institute, 110 Eighth Street, Troy, New York 12180, USA.
Zagorevski
Dmitri V
DV
Lennartz
Michelle R
MR
Loegering
Daniel J
DJ
Stenken
Julie A
JA
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D004151:Dipeptides / Q000032:analysis* / Q000494:pharmacology; D006801:Humans; D008297:Male; D020781:Matrix Metalloproteinase 1 / Q000737:chemistry / Q000378:metabolism*; D020780:Matrix Metalloproteinase 9 / Q000737:chemistry / Q000378:metabolism*; D061965:Matrix Metalloproteinase Inhibitors; D017551:Microdialysis; D051381:Rats; D017207:Rats, Sprague-Dawley; D053719:Tandem Mass Spectrometry
D004151:Dipeptides; D061965:Matrix Metalloproteinase Inhibitors; C078131:N-(2(R)-2-(hydroxamidocarbonylmethyl)-4-methylpentanoyl)-L-tryptophan methylamide; D020780:Matrix Metalloproteinase 9; D020781:Matrix Metalloproteinase 1