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Clinical Chemistry 48: 1305-1313, 2002;
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(Clinical Chemistry. 2002;48:1305-1313.)
© 2002 American Association for Clinical Chemistry, Inc.

Measurement of the Noncomplexed Free Fraction of Tissue Inhibitor of Metalloproteinases 1 in Plasma by Immunoassay

Mads N. Holten-Andersen1a, Ib Jarle Christensen1, Hans Jørgen Nielsen2, Hans Lilja3, Gillian Murphy4, Vibeke Jensen1, Nils Brünner1 and Timo Piironen1

1 The Finsen Laboratory, Rigshopitalet, Strandboulevarden 49, 2100 Copenhagen, Denmark.

2 Department of Surgical Gastroenterology, Hvidovre University Hospital, 2650 Hvidovre, Denmark.

3 Department of Clinical Chemistry, Malmö University Hospital, 20502 Malmö, Sweden.

4 School of Biological Sciences, University of East Anglia Norwich, NR4 7TJ, United Kingdom.

aAuthor for correspondence. Fax 45-35-25-11-17; e-mail mads{at}finsenlab.dk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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) µg/L] and EDTA plasma [58.9 (13.3) µg/L] from 76 donors (r2 = 0.82). In 154 donors, the ratio of free TIMP-1 [mean (SD), 64.5 (18.0) µg/L] to total TIMP-1 [83.8 (19.8) µg/L] in EDTA plasma was 0.77. Plasma concentrations of free and total TIMP-1 correlated significantly to age (free, r2 = 0.19; total, r2 = 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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Tissue inhibitor of metalloproteinases 1 (TIMP-1)1 is a 28-kDa glycoprotein that has been demonstrated to bind and form noncovalent 1:1 stoichiometric complexes with the matrix metalloproteinases (MMPs) (1), thereby inhibiting the matrix-degrading properties of this family of zinc-dependent enzymes (2), which play a pivotal role in the growth and spread of cancer. However, distinct from this antiproteolytic property of TIMP-1, recent reports have described functions that are different from or even opposite its role as an enzyme inhibitor, such as stimulation of cell growth (3)(4)(5), and inhibition of apoptosis (6)(7), indicating that the role of TIMP-1 in cancer progression may be dual. In keeping with the growth-promoting role of TIMP-1, high tumor tissue concentrations of TIMP-1 mRNA and protein have been reported in various cancers (8)(9)(10)(11), with high TIMP-1 concentrations being associated with poor prognosis of cancer patients (8)(10)(12). Because the TIMP-1 concentrations in cancer tissue extracts are predictive of patient outcome, attempts have been made to establish immunoassays for measurement of TIMP-1 in plasma, as blood in many ways is a more attractive sample material than tumor tissue (13). Several different ELISAs have been described for the measurement of TIMP-1 in either tissue extracts or blood (14)(15)(16)(17). However, large discrepancies in measured TIMP-1 concentrations have been reported among the various assays (16)(17)(18)(19)(20). These inconsistencies may in part be explained by differences in sample material (serum vs plasma), assay calibration differences, or different specificities and affinities of the assay reagents to the various molecular forms of TIMP-1, i.e., noncomplexed (free) TIMP-1 and TIMP-1 in complex with MMPs.

Using an assay for the measurement of total TIMP-1 (18), we previously reported on the total TIMP-1 concentrations in plasma samples from healthy donors and patients with inflammatory bowel disease, breast cancer, and colorectal cancer (21). No significant difference in total TIMP-1 concentrations was found in plasma samples from healthy donors, patients with inflammatory bowel disease, or patients with primary breast cancer, whereas patients with colorectal cancer had significantly increased plasma total TIMP-1 (21). In addition, we have reported on the prognostic value of preoperative plasma concentrations of total TIMP-1 in patients with colorectal cancer and demonstrated that high total TIMP-1 concentrations in plasma strongly predict short survival independently of other clinicopathologic variables, such age, gender, and Dukes stage (22). Other investigators have also demonstrated that plasma or serum concentrations of TIMP-1 are increased in colorectal cancer patients (16)(17) and that measurement of TIMP-1 in blood has prognostic value (23). Finally, we recently reported that measurement of total plasma TIMP-1 may be of diagnostic use in the detection of early-stage colon cancer (21). We hypothesize that, as for other potential diagnostic markers of cancer, such as prostate-specific antigen, new specific immunoassays for the detection of specific fractions of the antigen may provide significant additional clinical information (24). Because TIMP-1 can exist both in the free form and in complex with MMPs, we now report on the development and validation of an immunoassay for the measurement of free TIMP-1 with which we have collected data on the concentrations of free TIMP-1 in plasma from healthy donors and related these to the corresponding total TIMP-1 concentrations in samples from the same donors. In addition, by applying the immunoassays to plasma samples collected preoperatively from colorectal cancer patients, we could determine the free and total TIMP-1 concentrations in those patients and compare them with the results obtained for healthy blood donors.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
antibody characterization
Two monoclonal anti-TIMP-1 antibodies (MAC15 and MAC19) (18) were characterized for their ability to recognize and specifically bind free TIMP-1 and TIMP-1 complexed with MMP-9. Purified recombinant human TIMP-1 (18) was incubated with affinity-purified recombinant human MMP-9 (gelatinase B; purchased from Diabor) in a 1:2 molar ratio for 1 h at 30 °C, generating TIMP-1/MMP-9 complexes as reported previously (25). To determine the ability of the two monoclonal anti-TIMP-1 antibodies to bind free TIMP-1 or TIMP-1/MMP-9 complex, 96-well microtiter plates (Maxisorp; Nunc) were coated overnight at 4 °C with a polyclonal anti-MMP-9 antibody (diluted 1:1000; kindly supplied by Professor Niels Borregaard, Rigshopitalet, Copenhagen, Denmark). After blocking and washing as described previously (18), TIMP-1 (16 and 8 µg/L), MMP-9 (16 and 8 µg/L), or TIMP-1/MMP-9 complex (16 and 8 µg/L) was added to the plates (100 µL/well) and incubated for 1 h at 30 °C. After additional washes, the plates were incubated for 1 h at 30 °C with MAC15 or MAC19 (500 and 375 µg/L, respectively; 100 µL/well). After another wash step, an alkaline phosphatase-conjugated rabbit anti-mouse antibody (Dako), diluted 1:1000, was added to the plates and incubated for 1 h at 30 °C. After final washes, p-nitrophenyl phosphate substrate solution (Sigma) was added to each well, and the plate was immediately placed in a Ceres 900TM plate reader (Bio-Tek Instruments) for kinetic rate measurements [milliabsorbance units (mAU)/min] over 60 min at 405 nm. All samples were assayed in duplicate.

free timp-1 elisa
By exchanging the monoclonal anti-TIMP-1 detection antibody MAC15 with MAC19, the established ELISA for measurement of total TIMP-1 (18) was changed to an immunoassay that specifically detected free TIMP-1. In brief, the ELISA for the detection of free TIMP-1 was constructed as follows: 96-well microtiter plates (Maxisorp) were coated overnight at 4 °C with an affinity-purified sheep polyclonal anti-TIMP-1 antiserum (4 mg/L; 100 µL/well). After blocking and washing as described previously (18), we constructed a calibration curve for recombinant free TIMP-1 by adding serial dilutions containing 12, 6, 3, 1.5, 0.75, 0.375, and 0.1875 µg/L (100 µL/well) to the plates; we also included a 1:26 dilution of a citrate plasma pool as a control and blank wells containing only sample dilution buffer. Plasma samples diluted 1:26 in sample dilution buffer (20 µL of sample plus 500 µL of sample buffer; 100 µL/well) were added to the plates, which were then incubated for 1 h at 30 °C. After the binding of TIMP-1, plates were washed and incubated with 100 µL/well MAC19 (375 µg/L) for 1 h at 30 °C. After another round of washes, plates were incubated for 1 h at 30 °C with the alkaline phosphatase-conjugated rabbit anti-mouse antibody. After the final wash step, p-nitrophenyl phosphate substrate solution was added (100 µL/well), and the plate was immediately read using kinetic rate measurements (mAU/min) in a plate reader at 405 nm for every 10 min over 60 min.

All calibration curves were constructed and samples assayed in duplicate. Details on the buffers, wash steps, blocking solutions, and other reagents have been published previously (18). The intraassay imprecision was determined from 24 duplicate measurements of a control citrate plasma pool diluted 1:26 on one plate, whereas interassay imprecision was calculated from the measurements of the same plasma pool run as an internal control on 24 plates on different days. Because the immunoassays for the detection of free and total TIMP-1 differed only in detection antibody and because both assays were calibrated using the same recombinant free TIMP-1 calibrator, determinations of free and total TIMP-1 from the same individual could readily be compared.

recovery and dilution experiments
Recombinant TIMP-1 was added to 1:26 dilutions of citrate- and EDTA-plasma pools, and recovery of the TIMP-1 signal was measured with the free TIMP-1 ELISA. Recombinant TIMP-1 was added to the plasma pool solutions to give final concentrations of 0.1875–12 µg/L as described above, and the recovery for each plasma pool was calculated from the slope of the line representing TIMP-1 signal as a function of concentration, where 100% recovery was defined as the slope obtained when recombinant TIMP-1 was diluted in the sample dilution buffer. For dilution experiments, EDTA- and citrate-plasma pools were diluted serially (1:10, 1:20, 1:40, 1:80, 1:160, 1:320, 1:640, and 1:1280; for the 1:10 dilution, 100 µL of sample was mixed with 900 µL of sample buffer; subsequent dilutions were made by mixing 500 µL of the previous dilution with 500 µL of sample buffer) in sample buffer and tested for a linear reduction in the free TIMP-1 signal until linearity was no longer fulfilled.

cross-reactivity
The described ELISA was tested for cross-reactivity to recombinant TIMP-2, -3, and -4 (TIMP-2 from Amersham; TIMP-3 and -4 from Chemicon International). The four recombinant TIMPs were added in duplicate at 50, 30, 20, 10, 5, 2.5, 1, and 0.5 µg/L (100 µL/well) to a plate coated with the polyclonal sheep anti-TIMP-1 antiserum as described above and incubated for 1 h at 30 °C. In an analogous experiment, the immunoassay for the detection of total TIMP-1 was tested for cross-reactivity to TIMP-2, -3, and -4.

healthy blood donors
Through collaboration with the blood bank of the University Hospital of Hvidovre (Copenhagen, Denmark), we acquired corresponding EDTA- and citrate-plasma samples from one set of 76 healthy blood donors for analysis of free TIMP-1. This set of blood donors comprised 43 males and 33 females [median (range) ages, 42 (19–57) and 37 (20–58) years, respectively]. A small difference in sampling procedure when collecting EDTA and citrate plasma from the donors made a systematic correction factor necessary for the citrate-plasma samples: EDTA-plasma tubes contained dry anticoagulant material, whereas citrate-plasma tubes contained a small amount of liquid citrate buffer, which gave a small and systematic dilution error (9/10). Therefore, all measured citrate-plasma values were multiplied by a factor of 10/9.

A second set of EDTA-plasma samples was available from 78 healthy blood donors, comprising 33 males and 45 females [median (range) ages, 58 (50–68) and 60 (51–79) years, respectively]. These volunteers were all healthy, were not taking any medicine, and had no clinical signs or symptoms of cancer or joint, liver, metabolic, or hormonal disease (26). All plasma samples were drawn as peripheral blood with minimal stasis, according to a previously described protocol (18), and stored at -80 °C until the day of analysis, when samples were thawed quickly in a water bath at 37 °C and placed on ice until the 1:26 dilutions were prepared. Total TIMP-1 had been measured previously in all plasma samples from the healthy donors (18)(21).

colorectal cancer patients
Preoperative EDTA-plasma samples from a set of 65 colorectal cancer patients were also included in the study. Twenty-one patients had rectal cancer, and 44 had colon cancer. Eleven patients had Dukes stage A disease, 27 had Dukes stage B, 14 had Dukes stage C, and 13 had Dukes stage D. The median age was 73 years (range, 40–94 years), and the patient group comprised 33 females and 32 males. Blood samples were collected, stored, and analyzed according to the procedure described above for the blood donors. Blood samples were obtained with informed consent from all donors and cancer patients in accordance with the Helsinki Declaration, and the study was approved by the Central National Ethical Committee.

stastistical analyses
The SAS® software package (Ver. 8; SAS Institute) was used to manage patient data and to perform statistical analyses. Descriptive statistics for free and total TIMP-1 included the median, range, mean, and SD as well as the 95% confidence intervals for mean differences. Paired t-tests were used when appropriate. The association between the TIMP-1 concentration and age was estimated by linear regression analysis and included gender and donor groups. Correlation coefficients were calculated between free and total TIMP-1. Calculations were done on log-transformed TIMP-1 values. Significance was set at 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
antibody characterization
TIMP-1/MMP-9 complexes captured by the polyclonal anti-MMP-9 antibody were detected by MAC15 (59.7 and 30.4 mAU/min at antigen concentrations of 16 and 8 µg/L, respectively), whereas MMP-9 bound by the polyclonal anti-MMP-9 antibody was not (generated signal at or below background). Free TIMP-1, which would not be bound by the polyclonal anti-MMP-9 antibody, also was not detected. When antigens were detected with MAC19, TIMP-1/MMP-9 complexes, MMP-9, and free TIMP-1 captured by the polyclonal anti-MMP-9 antibody were not detected above background. From these results it was evident that MAC15 binds both free TIMP-1 and TIMP-1 bound in complex to MMP, whereas MAC19 binds only free TIMP-1, as we reported recently (25). In support of the specificity of MAC19 for free TIMP-1, Cooksley et al. (27) have previously demonstrated that MAC19 does not bind other TIMP-1/MMP complexes, such as TIMP-1/MMP-1 and TIMP-1/MMP-3.

free timp-1 elisa
The design of the ELISA for the detection of free TIMP-1 is depicted in Fig. 1 . The epitope on TIMP-1 recognized by MAC19 is covered when a complex is formed with MMP-9 (Fig. 1B ). Thus, the described TIMP-1 ELISA measures only the free form of the MMP inhibitor. Calibration curves for the free TIMP-1 ELISA were constructed by plotting the rate of color development (mAU/min) against the concentration of recombinant TIMP-1 over the range 0.1875–12 µg/L (Fig. 2 ). The background rate in blank wells was deducted from all measurements. The calibration curve was fitted using a four-parameter fit, and the correlation coefficient for the fitted curve was typically >0.99. The median (SD) rate in the blank wells, which contained no recombinant TIMP-1, was 0.49 (0.14) mAU/min. The limit of detection for the assay, defined as the concentration of TIMP-1 corresponding to a signal 3 SD above the mean for the TIMP-1 blank, was 0.2 µg/L, or 9.7% of the mean of the measured concentration of free TIMP-1 in citrate-plasma samples from healthy donors, diluted 1:26. Because the plasma samples measured with the described immunoassay were diluted 1:26 in sample buffer, the resulting ELISA signal for any given sample fell within the linear range of the calibration curve, depicted in Fig. 2 as the interval between the two dashed lines. The intraassay CV for 24 replicates of a control citrate-plasma pool measured on the same plate was 6.4%, and the interassay CV for 24 successive assays of the plasma pool was 7.9%. This plasma pool had a free TIMP-1 concentration of 53.6 µg/L.



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  		Figure 1. Principle of the free TIMP-1 ELISA.

I, polyclonal sheep anti-TIMP-1 antiserum; II, monoclonal anti-TIMP-1 antibody (MAC19); III, alkaline phosphatase-conjugated rabbit anti-mouse antibody. The epitope to which MAC19 binds is readily accessible when TIMP-1 is not in complex (A), but when TIMP-1 is complexed with MMP, the epitope is covered and MAC19 will not bind (B).



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  		Figure 2. Calibration curve for the free TIMP-1 ELISA.

Rate measurements (mAU/min) for duplicate recombinant free TIMP-1 calibrators in the range 0.1875–12 µg/L were collected automatically every 10 min over 60 min at 405 nm. The background rate in wells with no calibrator added (blank signal) has been deducted. The calibration curve is calculated from a four-parameter fit.

recovery and dilution experiments
Recovery of the specific signal for free TIMP-1 was determined by addition of increasing concentrations of recombinant TIMP-1 to diluted plasma pools (dilution factor, 1:26) and subsequent measurement of the ELISA signal (Fig. 3 ). In citrate plasma, the recovery was 105%, and in EDTA plasma, the recovery was 96% when compared with the slope of the corresponding curve for recombinant TIMP-1 diluted in sample dilution buffer (defined as 100% recovery). Thus, the recovery of TIMP-1 signal from an internal standard was acceptable for both citrate and EDTA plasma. The signals for both the EDTA and the citrate plasma were linear down to a 1:320 dilution in sample buffer (data not shown). The chosen dilution of 1:26 for plasma samples measured for free TIMP-1 was thus well within the linear dilution range of the assay.



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  		Figure 3. Recovery of ELISA signal from recombinant TIMP-1 (µg/L) added in increasing concentrations to assay dilution buffer ({circ}), a 1:26 dilution of EDTA-plasma pool ({triangledown}), and a 1:26 dilution of citrate plasma pool ({triangleup}).

cross-reactivity
Because no signal above background was generated even at the highest added concentration of TIMP-2, -3, or -4 (50 µg/L), the cross-reactivity was determined to be <0.4% based on calculation of the ratio between the detection limit of the free TIMP-1 assay (0.2 µg/L) and the highest concentrations of recombinant free TIMP-2, -3, and -4 tested. Similarly, the cross-reactivity for the immunoassay for the detection of total TIMP-1 was <0.2%.

free timp-1 in edta and citrate plasma from healthy blood donors
In the set of 76 healthy blood donors, the mean (SD) free TIMP-1 concentrations in corresponding citrate- and EDTA-plasma samples were 55.5 (11.5) and 58.9 (13.3) µg/L, respectively, as can be seen in Table 1 . The calculated differences in free TIMP-1 in EDTA and citrate plasma are shown in Fig. 4A as a function of the mean concentration of free TIMP-1 in EDTA and citrate plasma (28). The difference in the TIMP-1 concentration was not dependent on the mean concentration of free TIMP-1 in EDTA and citrate plasma. A paired-means comparison revealed that the free TIMP-1 concentrations in EDTA-plasma samples were, on average, slightly higher [mean (SD), 3.5 (14.3) µg/L] than in the corresponding citrate-plasma samples (P <0.0001), which is seen in Fig. 4A . The corresponding free TIMP-1 concentrations in citrate and EDTA plasma are shown in Fig. 4B . Clearly, a strong correlation between EDTA- and citrate-plasma concentrations of free TIMP-1 from individual donors exists (r2 = 0.82; P <0.0001). The orthogonal regression line for the relationship between free TIMP-1 in EDTA and citrate plasma is shown in Fig. 4B .


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  		Table 1. Results for two donor sets.



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  		Figure 4. Scatter plots for free TIMP-1 in corresponding EDTA- and citrate-plasma samples from healthy donors.

(A), the difference between free TIMP-1 (µg/L) concentrations in corresponding EDTA- and citrate-plasma samples (EDTA - citrate) from healthy donors is plotted as a function of the mean free TIMP-1 concentration (µg/L) in EDTA and citrate plasma [(EDTA + citrate)/2]. (B), free TIMP-1 (µg/L) concentrations in corresponding EDTA- and citrate-plasma samples from healthy donors. The orthogonal regression line is shown in B. The equation for the line is: loge(free TIMP-1 EDTA) = 1.139 x loge(free TIMP-1 citrate) - 0.381.

free and total timp-1 in healthy blood donors
In addition to the 76 blood donors described above, we analyzed free TIMP-1 in EDTA plasma from a second set of 78 donors. Thus, a total of 154 EDTA plasma samples from healthy donors were analyzed for free TIMP-1. The mean (SD) free TIMP-1 concentration for the 154 donors was 64.5 (18.0) µg/L, which was close to the median (range) for free TIMP-1: 63.7 (29.8–134.0) µg/L. All data on total and free TIMP-1 concentrations in donors are given in Table 1Up . Fig. 5A illustrates the free TIMP-1 concentrations in EDTA plasma in the two sets of blood donors as a function of the age of the donors. The free TIMP-1 concentrations were lower in the first set of donors, who were substantially younger than the second donor set, but there was no difference in free TIMP-1 plasma concentrations adjusted for age between the two donor groups. Thus, a significant association between free TIMP-1 and age in both sets of blood donors was demonstrated (linear regression analysis, P <0.0001). A slightly larger variance was found in free TIMP-1 concentrations in the second set of donors (P = 0.02), but there were no statistically significant differences between the concentrations (P = 0.14) or the slopes (P = 0.2). Therefore, a regression analysis was performed for all 154 healthy blood donors (r2 = 0.19; P <0.0001). The regression line for free TIMP-1 and age for all donors is shown in Fig. 5A . This association predicted a mean increase of 50% in free TIMP-1 over an age span of 45 years. In the adjusted model, gender had no influence on the concentration of free TIMP-1 in plasma (P = 0.30).



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  		Figure 5. Scatter plots for free TIMP-1 (µg/L) in EDTA-plasma samples from two sets of healthy donors.

•, first set of 76 donors; {triangleup}, second set of 78 donors. (A), TIMP-1 concentration plotted as a function of donor age (years). The regression line depicting the association between free TIMP-1 and age of the donors is: loge(free TIMP-1) = 0.01 x age + 3.67. (B), corresponding concentrations of free and total TIMP-1 in EDTA-plasma samples from the two individual sets of donors. Orthogonal regression lines are shown for each set of donors: solid line, first donor set; dashed line, second donor set. The equations for the first and second donor sets are, respectively: loge(free TIMP-1) = 1.287 x loge(total TIMP-1) - 1.474; and loge(free TIMP-1) = 1.383 x loge(total TIMP-1) - 2.035.

From our previous studies (18), corresponding total TIMP-1 concentrations were available for the same 154 donors (Table 1Up ). Total TIMP-1 was significantly higher than free, and when we calculated the ratio of free and total TIMP-1, the mean ratio was 0.79 for the first (younger) set of donors; the second (older) set had a slightly lower mean ratio of 0.74 (P = 0.04). Thus, a very small (r2 = 0.07) but significant inverse relationship between the ratio of free to total TIMP-1 and donor age (P = 0.007) could be demonstrated. When we disregarded this slight age dependency, we obtained a mean ratio of 0.77 for all 154 blood donors, indicating that approximately three-fourths of the total TIMP-1 in a given donor EDTA-plasma sample was free TIMP-1. This is illustrated Fig. 5BUp , in which free TIMP-1 values are plotted against corresponding total TIMP-1 concentrations. We found a significant correlation between free and total TIMP-1 (r2 = 0.72; P <0.0001), which can also be seen from the orthogonal regression lines for this relationship in the two sets of donors (Fig. 5BUp ). As for free TIMP-1, a moderate but significant correlation to donor age was found for total TIMP-1 (r2 = 0.27; P <0.0001). The regression line for the model for total TIMP-1 and age was similar to that for free TIMP-1, with a mean increase of 50% in total TIMP-1 over an age span of 46 years. Again, gender was excluded from the final regression model (P = 0.28).

free and total timp-1 in colorectal cancer patients
The mean (SD) and median (range) concentrations of free and total TIMP-1 in the preoperative EDTA-plasma samples from the patients were as follows: free TIMP-1, 109.3 (82.7) and 84.9 (44.7–424.0); total TIMP-1, 187.2 (140.5) and 141.0 (80.7–790.6). Shown in Fig. 6A are the free TIMP-1 concentrations in the blood donors and colorectal cancer patients, including the regression line (solid line) and the 95% upper reference limit (dashed line) for free TIMP-1 and age in the blood donors. As seen in Fig. 6 , of the 154 blood donors, 5 had free TIMP-1 values above the upper reference limit, whereas 15 of the 65 colorectal cancer patients had free TIMP-1 values above the limit, reflecting that colorectal cancer patients have significantly increased free TIMP-1 in plasma (P <0.0001). Neither free nor total TIMP-1 was associated with age, and no effect of gender was demonstrated for TIMP-1 concentrations in the colorectal cancer patients. There was a significant difference in free and total TIMP-1, with a mean ratio of 0.58. This ratio was significantly lower than the ratio in the healthy blood donors (P <0.0001) as can be seen from Fig. 6B , which shows the ratio of free to total TIMP-1 plotted vs age of the blood donors or colorectal cancer patients. No effect of gender or age on the ratio was found in the colorectal cancer patients.



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  		Figure 6. Scatter plots for free TIMP-1 concentrations in EDTA plasma from blood donors and colorectal cancer patients.

•, 154 blood donors; {triangleup}, 65 colorectal cancer patients. (A), free TIMP-1 plotted as a function of donor age (years). The regression line [loge(free TIMP-1) = 0.01 xage + 3.67] depicting the association between free TIMP-1 and donor age is plotted (solid line) along with the calculated 95% upper reference limit (dashed line; loge(free TIMP-1) = 0.01 x age + 4.08. (B), association between the ratio of free and total TIMP-1 (µg/L) and donor and patient age. The mean ratios for colorectal cancer patients (0.58; solid line) and healthy blood donors (0.77; dashed line) are plotted.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study describes the establishment and validation of a new immunoassay for the detection of free TIMP-1 in plasma samples. Using this assay, we report on the concentrations of free TIMP-1 in both EDTA- and citrate-plasma samples from healthy donors and compare these with concentrations in EDTA-plasma samples from colorectal cancer patients. In the blood donors, free TIMP-1 concentrations fell within a narrow range and were, on average, slightly higher in EDTA- than in citrate-plasma samples from the same donor; however, there was a strong correlation between the concentrations in EDTA- and citrate-plasma samples. Regression analyses demonstrated that free TIMP-1 in plasma from healthy donors was significantly correlated to donor age, whereas no effect of gender was found. A mean increase of 50% in free TIMP-1 in plasma over an age span of 45 years was found.

Total TIMP-1 was measured by our previously described ELISA (18), and we found that colorectal cancer patients had significantly increased concentrations of free and total TIMP-1 compared with the blood donors. By comparing free to total TIMP-1 in blood donor plasma, we found that TIMP-1 in plasma from healthy blood donors consists predominantly of TIMP-1 in its free form: a mean ratio of 0.77 of the total TIMP-1 in plasma was free TIMP-1. This ratio of free to total TIMP-1 was significantly lower in plasma from colorectal cancer patients, who on average had a ratio of 0.58, meaning that less TIMP-1 was in its free form in the colorectal cancer patients. This finding indicates that relatively more of the TIMP-1 present in the blood of cancer patients is complexed with MMPs, perhaps reflecting generally higher concentrations of MMPs in these patients.

An immunoassay for the measurement of free TIMP-1 has previously been reported by Clark et al. (29). The assay was based on two monoclonal antibodies, of which the detection antibody was specific for free TIMP-1. The mean (SD) free TIMP-1 concentration in seven EDTA-plasma samples from healthy volunteers was 109 (35) µg/L. Although this study was limited, free TIMP-1 concentrations were higher than those we report here. Although EDTA plasma was measured in both studies and the immunoassays were both specific for free TIMP-1, the differences in measured concentrations of free TIMP-1 in blood from healthy donors may be explained simply by differences in assay calibration.

We recently reported that total TIMP-1 concentrations in preoperative plasma samples from colorectal cancer patients were significantly increased compared with plasma concentrations in healthy blood donors, patients with inflammatory bowel disease, or patients with primary breast cancer (21). On the basis of these results, it was suggested that measurement of total TIMP-1 in plasma could be used to detect early-stage colon cancer with high specificity and sensitivity (21). The data from the present study demonstrate that the relationship between free and total TIMP-1 in plasma samples from patients with colorectal cancer may carry significant clinical information regarding identification of patients. However, because the present study included a limited number of patients, we plan to determine TIMP-1 concentrations in larger collections of plasma from colorectal cancer patients to establish the potential diagnostic and prognostic value of free and total TIMP-1.

Recently, it was demonstrated that several MMPs measured by immunoassays are increased in the blood of cancer patients compared with blood donors. Zucker et al. (19) reported that MMP-9 concentrations are increased in plasma from patients with gastrointestinal or breast cancer and that the concentrations of MMP-9/TIMP-1 complexes are increased in plasma from patients with metastatic gastrointestinal or gynecologic cancer. Similarly, Jung et al. (30) have shown that plasma concentrations of MMP-3 are increased in patients with prostate cancer. We therefore suggest that the observed increase in total TIMP-1 in colon cancer patients reflects an increased amount of MMP/TIMP-1 complexes. In analogy to prostate-specific antigen, algorithms the include free and total TIMP-1 will be established that are aimed at increasing the diagnostic specificity and sensitivity of TIMP-1 measurements in the diagnosis of early-stage colon cancer.

In conclusion, we have established and validated an immunoassay for the detection of plasma free TIMP-1. Thus, measurements of both free and total TIMP-1 are feasible. Relating these two variables, the present study indicates that the ratio of free to total TIMP-1 may carry information regarding identification of cancer patients. However, further investigations in larger sample sets are warranted to confirm these results.


   Acknowledgments
 
This work was supported by the Danish Cancer Society, the Danish Cancer Research Fund, the Else and Mogens Wedell-Wedellsborgs Foundation, Manufacturer Einar Willumsens Foundation, Novartis, and the Mrs. Ingeborg Albinus Larsens Foundation.


   Footnotes
 
1 Nonstandard abbreviations: TIMP-1, tissue inhibitor of metalloproteinases 1; MMP, matrix metalloproteinase; and mAU, milliabsorbance units.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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