MRDR-HPLC method is more reliable and faster. With these modifications, the MRDR may find wider application for the detection of VAD. References 1. Tanumihardjo SA, Cheng JC, Permaesih D, Muherdiyantiningsih, Rustan E, Muhilal, et al. Refinement of the modified-relative-dose-response test as a method for assessing vitamin A status in a field setting: experience with Indonesian children. Am J Clin Nutr 1996;64: 966 -71. 2. de Pee S, Yuniar Y, West CE, Muhilal. Evaluation of biochemical

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... of vitamin A status in breast-feeding and non-breast-feeding Indonesian women. Am J Clin Nutr 1997;66: 190 -1. Matrix metalloproteinases (MMPs), zinc-dependent proteinases belonging to the family of matrixins, are involved in the processes of normal development and growth as well as in several pathologic states (1 ). MMP-2 (gelatinase A; EC 3.4.24.24) and MMP-9 (gelatinase B; EC 3.4.24.35) play crucial roles in the proteolytic cascade leading to extracellular matrix degradation (2 ) . To evaluate the usefulness of MMPs as tumor markers, several authors have measured MMP-2 and MMP-9 in the blood of cancer patients, using serum or plasma samples, and reached contradictory conclusions (3, 4 ). The preanalytical steps of blood sampling and processing influence the concentrations of MMPs and their inhibitors (5, 6 ), but no data are available concerning the effect of blood specimen collection methods on the appearance of the full spectrum of MMP isoforms on gelatin zymograms. We simultaneously collected blood samples from 20 healthy volunteers (median age, 36 years; range, 23-55 years) into plastic tubes with no additive, plastic tubes with a silica gelcoated surface as coagulation accelerator, lithium heparin-coated plastic tubes, and potassium EDTA-coated plastic tubes (Monovette Systems; Sarstedt). The tubes were kept at room temperature and centrifuged immediately after venipuncture (1600g for 15 min at 4°C), and the supernatants were stored at Ϫ80°C until analysis. Human gelatinases were prepared as described previously (7 ). Gelatin zymography was performed under nonreducing conditions on 7.5% polyacrylamide mini slab gels (Bio-Rad), copolymerized with 1.5 g/L 90 Bloom gelatin (Sigma) (8 ). Aliquots containing 50 g of total protein (Bio-Rad) were used for each zymographic test. To assay gelatin lysis, scaled aliquots of proteins were run in triplicate and submitted to computer-assisted densitometric scans using Image Pro-Plus software (Cybernetics); the semiquantitative results were expressed as a percentage vs control or calibrator. The gelatin zymography technique allows detection of all MMPs in circulating blood, including the largest isoform (225 kDa; Fig. 1 ). The four bands showing gelatinase proteolytic activity were fibroblast-derived pro-MMP-2 (72 kDa) and neutrophilderived pro-MMP-9 (92, 130, and 225 kDa) (7, 8 ). As shown in Fig. 1A , pro-MMP-2 is commonly present in both plasma and sera from healthy humans, although in most samples other major proteolytic activities were seen at M r 92, 130, and 225 kDa. All gelatino-Fig. 1. Gelatin zymograms of plasma and serum MMPs. Samples (50 g of total protein) were analyzed on 7.5% gels containing 1.5 g/L 90 Bloom gelatin. The calibrators (lane Marker) were capillary whole-blood gelatinases; molecular masses (kDa) are indicated. (A), lanes 1 and 2, gelatinases from plasma samples collected into potassium EDTA-coated and lithium heparin-coated plastic tubes, respectively; lanes 3 and 4, MMPs from serum collected in tubes with clot activator and with no additive, respectively. (B), blood MMP zymograms incubated in the presence of 2 g/L potassium EDTA (lanes 1 and 2) , 30 kIU/L lithium heparin (lanes 3 and 4) , or the incubation buffer (50 mmol/L Tris-HCl, pH 7.5, 100 mmol/L NaCl, 5 mmol/L CaCl 2 , 1 mmol/L ZnCl 2 , 0.2 mL/L Brij-35, 2.5 mL/L Triton X-100, and 0.2 g/L NaN 3 ) without additives (lanes 5 and 6).