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Palenzuel and colleagues used enzyme-linked immunosorbent assay (ELISA) to measure matrix metalloproteinase-9 (MMP-9) levels. However, the ELISA method is less sensitive and cannot differentiate the active form of MMP-9 from its inactive proform. Zymography is a more sensitive assay and differentiates both the active and inactive forms of MMP-9.1 By using gelatin zymography, we found a significant increase of proMMP-9 in the fulminant hepatic failure (FHF) group compared to the alcoholic liver disease (ALD), hepatitis C (HCV), and normal controls. Importantly, we observed that the active MMP-9 in 10 patients with FHF was significantly higher (74.0 ± 28.1 pg/mL, P = 0.001) compared to that in others (ALD = 50.6 ± 33.7 pg/mL; HCV = 43.4 ± 13.1 pg/mL; controls = 33.6 ± 6.5 pg/mL) (Fig. 1A). Among patients with FHF, eight had advanced encephalopathy and five clinically had brain edema.

Figure 1.

Expression of α-SMA was detected in (A) Kunming mouse liver and (B) CCl4-induced liver by immunohistochemistry. Sections were incubated with antibody against α-SMA (Abcam, Cambridge, MA; diluted 1:50) followed by visualization with the Histostain-Plus Kit (Invitrogen, Carlsbad, CA) after treatment with 3,3-diaminobenzidine and counterstain with hematoxylin (magnification = ×1000).

We used reverse zymography,1 and observed a significant decrease of tissue inhibitor of metalloproteinase-1 (TIMP-1) levels in the patients with FHF (Fig. 1B). On the other hand, TIMP-1 activities were markedly increased in HCV and did not change in ALD. In all cases, TIMP-2 was not detected. These results further substantiate the presence of active MMP-9.2

Although the blood-brain barrier (BBB) appears structurally intact,3 there is supporting evidence for a vasogenic failure in BBB in FHF.4, 5 Selective permeability can result from subtle alterations without an obvious disruption of the BBB morphology.6, 7 Therefore, unlike brain ischemia or trauma, the vasogenic alterations of BBB in FHF are likely subtle and the permeability is limited to small molecules. Thus, large molecules such as immunoglobulin G and MMP-9 will not penetrate the BBB in FHF. Our data support vasogenic perturbation in the development of brain edema in FHF.8 Further understanding of this vasogenic pathway will likely uncover novel targets of potentially effective therapy.

Justin H. Nguyen M.D.*, * Department of Transplantation, Mayo Clinic Florida, Jacksonville, FL.

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