We investigated whether circulating ascorbic acid in humans is protein bound or free and whether ascorbic acid exists in its reduced form alone as ascorbic acid or in its reduced and oxidized forms as ascorbic acid and dehydroascorbic acid, respectively. Ascorbic acid and dehydroascorbic acid were determined by using HPLC with coulometric electrochemical detection, and protein binding was determined by centrifugal ultrafiltration. Ascorbic acid was free in plasma and serum of normal, healthy volunteers, 10 men and 10 women. Ascorbic acid was detectable only in its reduced form. However, dehydroascorbic acid could be made to appear in samples processed under oxidizing conditions. Because circulating ascorbic acid is free and is detected only as reduced vitamin, ascorbic acid may be available without intermediates for peripheral utilization. Dehydroascorbic acid may not be present in plasma and serum of normal humans unless assay conditions permit ascorbic acid oxidation.

Macrophages are aptly positioned to function as the primary line of defence against invading pathogens in many organs, including the lung and peritoneum. Their ability to phagocytose and clear microorganisms has been well documented1 , 2. Macrophages possess several substances with which they can kill bacteria, including reactive oxygen species, nitric oxide, and antimicrobial proteins3 -9 . We proposed that macrophage-derived proteinases may contribute to the antimicrobial properties of macrophages. Macrophage elastase (also known as matrix metalloproteinase 12 or MMP12) is an enzyme predominantly expressed in mature tissue macrophages 10 and is implicated in several disease processes, including emphysema11. Physiological functions for MMP12 have not been described. Here we show that Mmp12 −/− mice exhibit impaired bacterial clearance and increased mortality when challenged with both Gram-negative and Gram-positive bacteria at macrophage-rich portals of entry, such as the peritoneum and lung. Intracellular stores of MMP12 are mobilized to macrophage phagolysosomes after the ingestion of bacterial pathogens. Once inside phagolysosomes, MMP12 adheres to bacterial cell walls where it disrupts cellular membranes resulting in bacterial death. The antimicrobial properties of MMP12 do not reside within its catalytic domain, but rather within the carboxy-terminal domain. This domain contains a unique four amino acid sequence on an exposed β loop of the protein that is required for the observed antimicrobial activity. The present study represents, to our knowledge, the first report of direct antimicrobial activity by a matrix metallopeptidase, and describes a new antimicrobial peptide that is sequentially and structurally unique in nature.To determine whether MMP12 has a role in host defence against bacteria, we subjected Mmp12 −/− mice and wild-type littermates to intraperitoneal (i.p.) injection of either Staphylococcus aureus or Escherichia coli. In each case, there was a significant survival advantage for wild-type mice as compared to Mmp12 −/− mice over a 3-day period (Fig. 1a-c, g). Lower titres of i.p. S. aureus injection (1 ×10 7 colony-forming units (c.f.u.)), which did not result in mortality in wild-type mice, still proved >50% lethal in Mmp12 −/− mice. Soon afterCorrespondence and requests for materials should be addressed to A.M.H. (houghtonm@dom.pitt.edu). * These authors contributed equally to this work.Author Information Reprints and permissions information is available at www.nature.com/reprints. Author Contributions A.M.H. performed in vivo and in vitro studies, contributed to data interpretation and mechanistic advance, prepared the manuscript and figures, and performed all revisions. W.O.H. performed in vivo and in vitro studies and contributed to study design, data analysis, and mechanistic advance. C.S.R. performed CTD processing studies. F.X.G.-R. constructed the three-dimensional homology model of MMP12 CTD. S.D.S. generated Mmp12 −/− mice and all recombinant proteins, was responsible fo...

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