Measurement of the noncomplexed free fraction of tissue inhibitor of metalloproteinases 1 in plasma by immunoassay.
BACKGROUND
We previously found differences in total concentrations of tissue inhibitor of metalloproteinases 1 (TIMP-1) in plasma from donors and cancer patients. Because TIMP-1 can exist in more than one molecular form, a new immunoassay to specifically detect free TIMP-1 was developed and concentrations were determined in plasma from healthy donors and colorectal cancer (CRC) patients.


METHODS
We established and validated an immunoassay for the specific measurement of free TIMP-1 that uses a polyclonal anti-TIMP-1 antibody for capture and a monoclonal anti-TIMP-1 antibody that binds only free TIMP-1 for detection of antigen. Plasma samples from healthy donors and CRC patients were assayed for free TIMP-1. Total TIMP-1 was measured by our previously published assay.


RESULTS
The mean (SD) concentrations of free TIMP-1 were similar in citrate [55.5 (11.5) microg/L] and EDTA plasma [58.9 (13.3) microg/L] from 76 donors (r(2) = 0.82). In 154 donors, the ratio of free TIMP-1 [mean (SD), 64.5 (18.0) microg/L] to total TIMP-1 [83.8 (19.8) microg/L] in EDTA plasma was 0.77. Plasma concentrations of free and total TIMP-1 correlated significantly to age (free, r(2) = 0.19; total, r(2) = 0.27; P <0.0001), increasing 50% over an age span of 45 years. Free and total TIMP-1 were significantly increased in CRC patients (P <0.0001), whereas the ratio of free to total TIMP-1 (mean, 0.58) was significantly lower than in donors.


CONCLUSIONS
Most of the TIMP-1 in donor plasma is present in its free form, and free TIMP-1 increases with age. Free and total TIMP-1 are increased in CRC patient plasma, but the ratio of free to total TIMP-1 is significantly lower in these patients than in donors.
Holten-Andersen
Mads N
MN
The Finsen Laboratory, Rigshopitalet, Strandboulevarden 49, 2100 Copenhagen, Denmark. mads@finsenlab.dk
Christensen
Ib Jarle
IJ
Nielsen
Hans Jørgen
HJ
Lilja
Hans
H
Murphy
Gillian
G
Jensen
Vibeke
V
Brünner
Nils
N
Piironen
Timo
T
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000925:Anticoagulants; D019343:Citric Acid; D015179:Colorectal Neoplasms / Q000097:blood; D004492:Edetic Acid; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D008666:Metalloendopeptidases / Q000378:metabolism; D008875:Middle Aged; D012016:Reference Values; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000097:blood* / Q000378:metabolism
D000925:Anticoagulants; D019715:Tissue Inhibitor of Metalloproteinase-1; D019343:Citric Acid; D004492:Edetic Acid; D008666:Metalloendopeptidases