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Letters to the Editor |
1 Institute of Histology and Laboratory Analysis, Faculty of Sciences and Technologies University of Urbino Carlo Bo Urbino, Italy
2 Department of Pharmacology Faculty of Medicine University of Sao Paulo Ribeirao Preto, Brazil
3 Department of Morphology Estomatology and Physiology Dental School University of Sao Paulo Ribeirao Preto, Brazil
aAddress correspondence to this author at: Institute of Histology and Laboratory Analysis, Faculty of Sciences and Technologies, University Carlo Bo, Via O. Ubaldini 7, 61029 Urbino (PU), Italy. Fax 39-0722-322370; e-mail f.mannello{at}uniurb.it.
To the Editor:
Whether serum or plasma is the best specimen for determination of matrix metalloproteinases (MMPs) is a matter of debate, as are the influences of sample collection and processing on MMP concentrations (1)(2)(3). MMP-2 concentrations do not differ significantly in plasma and serum, whereas MMP-9 concentrations are significantly higher in serum than in plasma, and in plasma differently affected by anticoagulants(2)(3). Furthermore, both unexpected protein peaks and increased MMP-9 concentrations occur in serum samples collected in tubes coated with clot accelerators such as kaolin or silica gel(2)(4).
To find an explanation of the differences in MMP-9 concentrations among citrate plasma, serum, and serum with clot accelerators (Sca), we analyzed the effects of silicate on MMP in whole blood (WB), plasma, serum, and buffy coat (BC), as well as in culture media of U-937 myelomonocytic leukemia cells (SF U937).
We obtained peripheral blood (PB) and BC samples from 30 healthy volunteers age 23–55 years (median 36 years). Samples were collected into plastic tubes (Vacutainer® from Becton Dickinson). After centrifugation at 500g for 15 min at 4 °C, the supernatants of cell culture, BCs, plasma, and serum with and without silicate were analyzed by use of Western blot (75-7F7 and GE-213 monoclonal antibodies against MMP-2 and -9, respectively; Calbiochem) and gelatin zymography (7.5% polyacrylamide gels containing 2 g/L gelatin 90 Bloom type A from porcine skin; Sigma) (2). U-937 cells were cultured in serum-free conditions (to avoid endogenous bovine serum gelatinases) and treated for 3 h with silicate (54 mg/L). Calibrators were prepared from capillary WB(2). The accuracy/precision of gelatinolytic activities were evaluated by zymogram densitometry with Image Pro-Plus software (Cybernetics)(4). Differences were compared using the Mann–Whitney U-test; P values <0.05 were considered statistically significant. All study participants gave informed consent, and the work was carried out in accordance with the ethics standards of the Helsinki Declaration of 1975, as revised in 1983.
Western blots identified pro-MMP-2 (Gelatinase A, EC 3.4.24.24, 72 kDa) and pro- and complexed forms of MMP-9 (Gelatinases B, EC 3.4.24.35, of 92, 130, and 225 kDa) in whole PB (Fig. 1
, lanes 2 and 3). Citrate plasma results showed that pro-MMP-2 expression did not differ significantly between serum and plasma, nor did it change with silicate treatment in any of the paired sample types. MMP-9 forms were found in significantly higher amounts in serum (mean 5-fold higher; P <0.001) than in citrate samples (Fig. 1
, lanes 6 vs 4 serum). However, addition of silicate to previously separated citrate plasma and serum did not noticeably change the zymographic profile of MMP-9 with respect to untreated samples (Fig. 1
, lane 4 vs 5, and lanes 6 vs 7).
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Addition of silicate to citrate plasma tubes before PB collection increased MMP-9 (Fig. 1
, lanes 8 and 9) a mean of 4-fold (P <0.001). Addition of silicate into empty plastic tubes for serum collection before PB addition also significantly increased MMP-9 concentrations (P <0.001) (data not shown). Addition of silicate to citrate and serum tubes before PB addition resulted in similar trends of MMP increase vs silicate concentration: MMP-9 activity (µg/L) = 28.0 x silicate (mg/L) – 9.4, r2 = 0.94, and MMP-9 activity (µg/L) = 30.6 x silicate (mg/L) + 91.6, r2 = 0.88, respectively.
When samples were collected into empty serum tubes to which buffered silicate (silicate dissolved in PBS containing 137 mmol/L NaCl, 10 mmol/L phosphate, 2.7 mmol/L KCl, pH 7.4) was added before PB collection, all MMP-9 forms were increased, and the zymographic profile was similar to that of Sca (Fig. 1
, lanes 10 and 11). Samples collected in the presence of nonbuffered silica (nonsoluble silica particles sprayed into plastic tubes or with silica-gel) (http://catalog.bd.com/ecat/msds/d01/vs60313.pdf) showed MMP-9 release 1.5-fold higher than for buffered silicate (data not shown). Serum collected in plastic tubes with clot accelerators showed the highest MMP-9 activities (Fig. 1
, lane 11). The addition of silicate to BCs isolated from citrate PB significantly enhanced MMP-9 release in buffered solution (Fig. 1
, lanes 12 and 13). Similarly, silicate addition to U-937 cells cultured in serum-free media significantly increased MMP secretion (Fig. 1
, lanes 14 and 15). Thus silicates increase in vitro release of MMP-9 forms from leukocytes. Our observations are consistent with the findings that during silicosis both macrophages and lymphocytes secrete enhanced amounts of MMP-9 forms (5).
To optimize the diagnostic accuracy of PB MMPs as biomarkers, we strongly recommend avoiding the use of serum samples, particularly in serum with clot activators containing silica/silicate. We believe the increased MMP-9 observed in these specimens reflects both the interfering effects of the coagulation/fibrinolysis processes (4) and the induction by silicates of MMP-9 release from leukocytes.
Acknowledgments
Grant/funding support: None declared.
Financial disclosures: None declared.
References
The following articles in journals at HighWire Press have cited this article:
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  F. Mannello Serum or Plasma Samples?: The "Cinderella" Role of Blood Collection Procedures Preanalytical Methodological Issues Influence the Release and Activity of Circulating Matrix Metalloproteinases and Their Tissue Inhibitors, Hampering Diagnostic Trueness and Leading to Misinterpretation Arterioscler Thromb Vasc Biol, April 1, 2008; 28(4): 611 - 614. [Full Text] [PDF]
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 K. Jung, S. Klotzek, C. Stephan, F. Mannello, and M. Lein Impact of Blood Sampling on the Circulating Matrix Metalloproteinases 1, 2, 3, 7, 8, and 9 Clin. Chem., April 1, 2008; 54(4): 772 - 773. [Full Text] [PDF]
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K. Jung Matrix Metalloproteinase-8 and Tissue Inhibitor of Metalloproteinase-1 in Serum Do Not Reflect the Analytes Circulating in Blood Arterioscler Thromb Vasc Biol, March 1, 2008; 28(3): e15 - e16. [Full Text] [PDF]
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