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ORIGINAL ARTICLE
Year : 2017  |  Volume : 30  |  Issue : 4  |  Page : 1065-1071

The value of osteopontin and matrix metalloproteinase-3 in the assessment of bone mineral density in postmenopausal women


1 Department of Biochemistry, Faculty of Medicine, Menoufia University, Menoufia, Egypt
2 Department of Biochemistry and Molecular Medicine, Faculty of Medicine, Zagazig University, Zagazig, Egypt
3 Department of Clinical Pathology, Faculty of Medicine, Menoufia University, Menoufia, Egypt
4 Department of Internal Medicine, Faculty of Medicine, Menoufia University, Menoufia, Egypt

Date of Submission10-Jan-2017
Date of Acceptance29-Jan-2017
Date of Web Publication04-Apr-2018

Correspondence Address:
Seham A Khodeer
Department of Clinical Pathology, Faculty of Medicine, Menoufia University, Menoufia
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/mmj.mmj_41_17

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  Abstract 


Objective
The aim of this study was to assess the value of osteopontin (OPN) and matrix metalloproteinase-3 (MMP-3) in the assessment of bone mineral density (BMD) in postmenopausal women.
Background
Osteoporosis is a widespread age-related skeletal disorder characterized by bone loss that increases skeletal fragility.
Patients and methods
This study was conducted on 172 postmenopausal female individuals. OPN and MMP-3 were assayed by enzyme-linked immunosorbent assay. BMD assessment was carried out by dual-energy X-ray absorptiometry. The patients were divided into three groups according to BMD: osteopenic, osteoporotic and control groups.
Results
OPN and MMP-3 levels were significantly greater in the osteoporotic and osteopenic groups than in the control group (P < 0.001). In the osteoporotic group, negative correlations between OPN, calcium, and BMD were found as well as positive correlations between OPN and MMP-3. By using the receiver operating characteristic curve, the sensitivities of both OPN and MMP-3 were found to be equal (93%), and the specificities were 100 and 84%, respectively.
Conclusion
OPN and MMP-3 were significantly increased in postmenopausal women with osteoporosis, suggesting their role in bone turnover. The preserving of normal bone mass density is a challenge for postmenopausal women to prevent bone disabilities. Hence, further studies are recommended to use the presence of OPN and MMP-3 as a regular monitoring system – being sensitive and easy to measure – for the early detection of osteoporosis in postmenopausal women.

Keywords: C-terminal telopeptide type I, matrix metalloproteinase-3, osteopontin


How to cite this article:
Abdu Allah AM, El Tarhouny SA, Khodeer SA, Shehab-Eldin WA. The value of osteopontin and matrix metalloproteinase-3 in the assessment of bone mineral density in postmenopausal women. Menoufia Med J 2017;30:1065-71

How to cite this URL:
Abdu Allah AM, El Tarhouny SA, Khodeer SA, Shehab-Eldin WA. The value of osteopontin and matrix metalloproteinase-3 in the assessment of bone mineral density in postmenopausal women. Menoufia Med J [serial online] 2017 [cited 2018 May 21];30:1065-71. Available from: http://www.mmj.eg.net/text.asp?2017/30/4/1065/229221




  Introduction Top


Osteoporosis is a major health problem, as dealing with it is very deficient concerning its diagnosis and treatment[1]. Women are more susceptible to osteoporosis than men, representing ~80% of all patients with osteoporosis. Generally, women have smaller and thinner bones than men, as there is a sharp decline in the production of estrogen after menopause[2]. The method by which the decreased level of estrogen causing bone loss can be detected is not clear yet[3],[4]. Bone radiological studies are of little predictive utility, because biochemical changes precede radiological changes. Dual-energy X-ray absorptiometry is the standard method used to determine bone mineral density (BMD). It provides information on changes in bone mineral contents and is useful for follow-up of bone mass or for the study of bone mass changes in the same patient[5].

Matrix metalloproteinase-3 (MMP-3), also called stromlysin-1, is released by osteoblasts. As estrogen deficiency occurs, osteoblasts are activated by osteoclasts. Osteoclasts resorb bone over a period of 3 weeks, creating cavities that are termed as remodeling space. The resorption is followed by osteoblast activation and osteoid formation, filling the cavities over a period of 3 months. Hence, MMP-3 is released in extra concentrations in the matrix around the osteocyte and its lacuna to play a role in collagen and cartilage metabolism[3],[4]. In premature bone tissue, noncollagen proteins are altered by osteopontin (OPN)/MMP-3 complexity before mineralization of bone matrix so as to maintain local microcircumstance by protein-lyses of complexity. It occurs in osteoporosis enhanced by osteoclasts as osteoblasts are creeping and persisting in states with increasing mineralization and high bone turnover[6],[7].

Approximately 90% of the organic matrix of bone is type I collagen, a helical protein that is cross-linked at the N-terminal and C-terminal ends of the molecule. During bone resorption, osteoclasts secrete a mixture of acid and neutral proteases that degrade the collagen fibrils into molecular fragments including C-terminal telopeptide (CTX). As the bone ages, the α form of aspartic acid present in CTX converts to the β form (β-CTX). β-CTX is released into the blood stream during bone resorption and serves as a specific marker for the degradation of mature type I collagen. Elevated serum concentrations of β-CTX have been reported in patients with increased bone resorption. The major drawback of CTX is its circadian difference; hence, the samples required should be taken in the morning while fasting, for accurate results[8].

The aim of the study was to evaluate OPN and MMP-3 in the assessment of BMD in postmenopausal women. The study may support the identification of female individuals at high risk of osteoporosis to implement early prevention.


  Patients and Methods Top


This study was carried out at Clinical Pathology, Biochemistry and Internal Medicine Departments, Menoufia University Hospitals, Faculty of Medicine, in the period between March 2013 and June 2015.

The study included 116 postmenopausal women from those attending the Outpatient Clinic of Internal Medicine, Menoufia University Hospitals, in addition to 56 apparently healthy postmenopausal volunteers. All women were subjected to the following: complete history taking, detailed physical examination, blood pressure, and BMI as calculated by dividing the individual's weight by the square height [BMI = weight (kg)/height (m)2]. Laboratory investigation of fasting blood glucose, calcium, phosphorus, lipid profile, and kidney function tests, and assay of OPN, CTX-I, and MMP-3 were also carried out. BMD was measured using 'Lunar Achilles Express' QUS examination. BMD values were given as g/cm 2, and the results were reported as T scores (SD above or below values for a young healthy population). Participants sharing in this study were classified into three groups according to the WHO classification (1994) using the T score (more than −1: normal, −1 to −2.49: osteopenia, and −2.5 or less: osteoporosis). Low BMD (osteopenia and osteoporosis) was considered if the T score was of −1 or less as follows: group I consisted of 66 postmenopausal women suffering from osteopenia; group II consisted of 67 postmenopausal women suffering from osteoporosis; and group III consisted of 40 apparently healthy women as a control group.

Exclusion criteria were as follows: patients suffering from oligomenorrhea, patients who had experienced the stopping of menstruation before the age of 40 years, patients who were suffering from bone diseases due to renal affection, known hepatic patients, patients suffering from parathyroid and thyroid problems, diabetes mellitus, hyperprolactinemia, rheumatoid arthritis, malabsorption syndromes, and hypertension, and those who had undergone the removal of their ovaries. Female patients on treatment with glucocorticoids, estrogens, Ltroxin, parathyroid hormone, vitamin D and calcium supplementations, and antiosteoporotic drugs were also excluded from the study.

The study design was accepted by the Local Ethics Committee. Informed consents were obtained from the women who participated in the study.

Methods

Sampling

Samples were taken under complete aseptic conditions, after an overnight fasting; 5 ml venous blood samples were collected in sterile plain tubes and allowed to clot at 37°C. The serum was separated by centrifugation and put into two tubes: one for the immediate assay of fasting blood glucose, calcium, phosphorus, lipid profile, and kidney function tests, and the other tube was divided into three aliquots and kept immediately at −80°C until assay of MMP-3, OPN, and CTX-I.

Laboratory methods

Biochemical tests for detection of fasting blood glucose, kidney function, total cholesterol, triglycerides, and high-density lipoprotein cholesterol were performed on Synchron C × 9 autoanalyzer using the kit supplied by Beckman Instrument Inc. (Fullerton, California, USA). Low-density lipoprotein cholesterol was calculated in consistency with Friedewald equation.

Measurements of serum OPN: human OPN ELISA (Aviva Systems Biology Corporation, San Diego, California, USA) was carried out using a quantitative technique called 'sandwich' ELISA in which the target protein (antigen) is bound in a 'sandwich' format by the primary capture antibodies coated to each well-bottom, and the secondary detection antibodies were added subsequently. The absorbance was read by the spectrophotometer. Concentrations were calculated according to the standard curve.

Measurements of serum MMP-3: measurement of serum MMP-3 was carried out using MMP-3 Human ELISA kit (ab100607MMP-3; Cambridge, UK). This assay utilizes an antibody specific for human MMP-3 coated on a 96-well plate. The absorbance was read by the spectrophotometer.

Measurements of serum CTX-I: measurements of serum CTX-I was carried out using Abnova Human ELISA kit (http://www.abnova.com). The microtiter plate provided in this kit had been precoated with an antibody specific to CTX-I. The concentration of CTX-I in the samples is calculated by comparing the optical density of the samples to the standard curve.

Bone mineral density

BMD measurement was carried out on one leg of all study participants, with the individual in the sitting position, using a water-based Achilles Express ultrasonometer 'Lunar Achilles Express' (NY 10980 USA) QUS examination. The patient's result was expressed as a T score and Z score as a percentage compared with the reference population. The diagnostic criteria for osteopenia/osteoporosis in the studied individuals was characterized according to the WHO classification (1994) using a T score (more than − 1: normal, −1 to − 2.49: osteopenia, and − 2.5 or less: osteoporosis).

Statistical analysis

The statistical analysis was undertaken using SPSS software (version 17; SPSS Inc., Chicago, Illinois, USA). One-way analysis of variance (F test) and Kruskal–Wallis test were used to collectively indicate the presence of any significant difference between several groups for quantitative variables. The post-hoc test was used after one-way analysis of variance (F test) or Kruskal–Wallis test to show any significant difference between the individual groups. χ2-Test was used for qualitative variables. P value less than 0.05 was considered significant. Receiver operating characteristic curve was plotted.


  Results Top


Serum osteopontin in osteopenic, osteoporotic, and control groups

There were high statistically significant differences between the three studied groups (P< 0.001 for all). The highest level was observed in the osteoporotic group (56.05 ± 13.01 ng/ml), followed by the osteopenic group (31.70 ± 9.82 ng/ml) and finally by the control group (12.35 ± 5.55 ng/ml) [Table 1].
Table 1: Characteristics and laboratory results of the studied groups

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Serum matrix metalloproteinase-3 in osteopenic, osteoporotic, and control groups

There were high statistically significant differences between the studied groups (P< 0.001 for all). The highest level was observed in the osteoporotic group (187.16 ± 70.14 ng/ml), followed by the osteopenic group (136.39 ± 50.74 ng/ml) and finally by the control group (78.01 ± 33.61 ng/ml) [Table 1].

Serum C-terminal telopeptide type I in osteopenic, osteoporotic, and control groups

There were high statistically significant differences between the studied groups (P< 0.001 for controls vs. osteoporotic and controls vs. osteopenic, and P < 0.05 for osteopenic and osteoporotic groups); the highest level was observed in the osteoporotic group (1.74 ± 0.39 ng/ml), followed by the osteopenic group (0.69 ± 0.32 ng/ml) and finally the control group (0.52 ± 0.29 ng/ml) [Table 1].

Osteopontin/matrix metalloproteinase-3 in osteopenic, osteoporotic and control groups

No statistically significant difference was found between the osteoporotic (0.25 ± 0.18 ng/ml) and the osteopenic (0.23 ± 0.23 ng/ml) groups (P = 0.827). Meanwhile, the osteoporotic group showed higher statistically significant differences than both the osteopenic and the control groups. Moreover, the osteopenic group had a higher statistically significant difference than the control group [Table 1].

Correlation between osteopontin, matrix metalloproteinase-3, and other parameters in the postmenopausal women with osteoporosis group

OPN was positively correlated with MMP-3 and CTX-I, and was found to have negative correlations with both calcium and BMD. Meanwhile, no correlations were found between OPN and both Alkaline phosphatase (ALP) and BMI [Table 2].
Table 2: Correlation between osteopontin and other parameters in the postmenopausal women with osteoporosis group

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Receiver operating characteristic curve for osteopontin and matrix metalloproteinase-3 osteopontin/matrix metalloproteinase-3 ratio between osteoporotic and control groups

As regards OPN, the cutoff value for osteoporosis was 36.5 ng/ml. The area under the curve was 99.9%, with a sensitivity of 93%, specificity of 100%, positive predictive value (PPV) of 100%, and negative predictive value (NPV) of 96% [Figure 1].
Figure 1: Receiver operating characteristic (ROC) curve for studied variables between osteoporosis and controls.

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With regard to MMP-3, the cutoff value for osteoporosis was 99 ng/ml. The area under the curve was 95.2%, with a sensitivity of 93%, specificity of 84%, PPV of 85%, and NPV of 92%.

As regards the OPN/MMP-3 ratio, the cutoff value for osteoporosis was 0.18 ng/ml. The area under the curve was 77.1%, with a sensitivity of 86%, specificity of 64%, PPV of 71%, and NPV of 82%.

Receiver operating characteristic curve for osteopontin and matrix metalloproteinase-3 osteopontin/matrix metalloproteinase-3 ratio to differentiate between osteopenic and the control groups

With respect to OPN, the cutoff value for osteoporosis was 19.5 ng/ml. The area under the curve was 97.0%, with a sensitivity of 93%, specificity of 91%, PPV of 92%, and NPV of 93% [Figure 2].
Figure 2: Receiver operating characteristic (ROC) curve for studied variables between osteopenia and controls.

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As regards MMP-3, the cutoff value for osteoporosis was 87.0 ng/ml. The area under the curve was 79.3%, with a sensitivity of 81%, specificity of 64%, PPV of 71%, and NPV of 77%.

With regard to the OPN/MMP-3 ratio, the cutoff value for osteoporosis was 0.18 ng/ml. The area under the curve was 75.6%, with a sensitivity of 76%, specificity of 62.5%, PPV of 68%, and NPV of 71%.


  Discussion Top


Our study revealed that the highest level of OPN was in favor of postmenopausal women with osteoporosis. Group I of postmenopausal women with osteoporosis had higher levels of serum OPN compared with both group II of postmenopausal women with osteopenia and group III of normal postmenopausal women. OPN has several functions that involve bone regeneration, control of inflammation, and cell survival[9]. OPN enhances the expression of CD44 that causes an increase in osteoclast motility and bone resorption[10]. It also has an important role in the sympathetic control of bone mass by acting through β2 adrenergic receptors/cAMP signaling[11]. OPN −/− mice express resistance to bone resorption that results from ovariectomy compared with wild-type mice[12], which explains the crucial role of OPN in postmenopausal osteoporosis. Our study was in accordance with a study carried out by Chang et al.[13], who reveal the risk of increased serum levels of OPN that cause osteoporosis in menopause. Our results support the results obtained by Dai[3] who studied OPN in 120 postmenopausal Chinese women volunteers. He divided them also into three groups: normal, osteopenia, and osteoporosis groups. OPN levels were significantly increased in the osteoporosis than in the osteopenic and normal volunteers. Moreover, Fodor et al.[5] stated that OPN levels were significantly higher in the osteoporosis group versus the osteopenic and normal groups.

In the present study, the highest level of MMP-3 was recorded by the osteoporosis group, followed by the osteopenia group and lastly by the normal group. Regarding the OPN/MMP ratio, there were greater statistically significant differences between both the osteopenia and normal groups as well as the osteoporosis and the normal group. Meanwhile, no statistically significant variance was detected between the osteopenia and osteoporosis groups. Our results were partially in accordance with Dai[3]. He studied MMP and the OPN/MMP ratio in the osteoporosis, osteopenia and normal groups and found statistically significant differences in the studied groups regarding both parameters. MMP has a family of zinc-dependent proteinases that take part in many pathological and physiological functions such as embryonic development, formation of new blood vessels, wound repair, periodontal disease, autoimmune arthritis, and cancer occurrence and spread. MMP that is secreted by osteoblasts has a role in bone metabolism by degradation of the bone matrix. MMP-3 affects bone resorption by degrading denatured type-II collagen and other constituents of the bone organic matrix. Much evidence has been found to confirm the critical role of MMPs that are secreted by osteoblasts in the initiation of bone resorption together with bone formation through degradation of the mineralized osteoid layer of the bone surface to permit osteoclasts to fix themselves to the mineralized matrix. In vitro, normal osteoblasts secrete copious amounts of MMP-3 under the influence of OPN, thereafter OPN/MMP-3 complexes enhance bone matrix ossification before bone mineralization[4],[14]. MMP-3 could destroy the extracellular matrix of the bone, especially in postmenopausal osteoporosis. In low estrogen concentrations, MMP could enhance osteoclasts maturation, immigration, and sticking along with degrading the matrix[3].

Our results revealed that the concentrations of CTX-I in both groups of osteopenia and osteoporosis were significantly increased than in the controls. These results are in line with a study carried out by Im et al.[15] and Al-Daghri et al.[2]. Bone is made of a calcified organic matrix that contains 90% type I collagen. During bone resorption, type I collagen is degraded, and small fragments are liberated into the blood stream. Higher CTX-I levels are associated with lower BMD values in osteoporosis[16]. This is consistent with our findings, indicating a high rate of bone resorption in osteoporotic female individuals.

Our study addressed the relationship between serum OPN concentrations and BMI, calcium, ALP, CTX-I, MMP-3, and BMD in postmenopausal female individuals with osteoporosis. Our data showed no statistically significant correlations between OPN and BMI. The results revealed by Cho et al.[17] were not able to show a relation between serum OPN levels and BMI. Meanwhile, Chang et al.[13] found a negative correlation with body weight and height. Moreover, OPN levels were positively correlated with MMP-3 and OPN/MMP-3 ratio. Dai[3] stated that MMP-3 was probably the initiating factor of postmenopausal osteoporosis, and the OPN-MMP-3 complex was probably the connection to discover the start of postmenopausal osteoporosis. OPN concentrations were negatively correlated with BMD as well as the level of serum calcium, and positively with CTX. Moreover, there was a study that involved 124 postmenopausal female individuals, and this study confirms that OPN levels were a risk factor for the occurrence of osteoporosis in menopause. Serum OPN concentrations were positively correlated with bone resorption markers such as carboxy-terminal collagen cross-links as reported by Chang et al.[13]. Moreover, our results were in accordance with the results obtained by Fodor et al.[5] and Yang et al.[18].

By using the receiver operating characteristic curve between the control and osteoporotic groups, the cutoff value of OPN for osteoporosis was found to be 36.5 ng/ml. In a study carried out by Fodor et al.[5], the cutoff value of OPN for osteoporosis diagnosis was 9.47 ng/ml. The cutoff value of MMP-3 was 99 ng/ml. The cause of discrepancies between human studies might be due to the type of samples, age, race, and changes in types of detection kits[19]. MMP-3 and OPN were equal in sensitivity (93%); meanwhile, OPN was higher in specificity than MMP-3 (100 and 84%, respectively). We concluded that OPN is a more specific marker to osteoporosis; hence, it could help in its diagnosis. Meanwhile, combined measuring of OPN, CTX, and MMP could help in the early detection of osteoporosis, as there is positive correlation between CTX and OPN.


  Conclusion Top


OPN and MMP-3 levels were significantly increased in both postmenopausal women with osteopenia and those with osteoporosis compared with those with normal BMD, suggesting their role in bone turnover. The preserving of normal BMD is a challenge for postmenopausal women to prevent bone disabilities. Hence, further studies are recommended for the capability of using OPN and MMP-3 as a regular monitoring system – being sensitive and easy to measure – for the early detection of osteoporosis in postmenopausal women.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

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Min Y. Update on denosumab treatment in postmenopausal women with osteoporosis. Endocrinol Metab 2015; 30:19–26.  Back to cited text no. 1
    
2.
Al-Daghri N, Yakout S, Al-Shehri E, Al-Fawaz H, Aljohani N, Al-Saleh E. Inflammatory and bone turnover markers in relation to PTH and vitamin D status among Saudi postmenopausal women with and without osteoporosis. Int J Clin Exp Med 2014; 7:3528–3535.  Back to cited text no. 2
    
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7.
Fedarko NS, Jain A, Karadag A, Fisher LW. Three small integrin binding ligand N-linked glycoproteins (SIBLINGs) bind and activate specific matrix metalloproteinases. FASEB J 2004; 18:734–736.  Back to cited text no. 7
    
8.
Garnero P, Borel O, Delmas PD. Evaluation of a fully automated serum assay for C-terminal cross-linking telopeptide of type I collagen in osteoporosis. Clin Chem 2001; 47:694–702.  Back to cited text no. 8
    
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11.
Nagao M, Feinstein TN, Ezura Y, Hayata T, Notomi T, Saita Y, et al. Sympathetic control of bone mass regulated by osteopontin. Proc Natl Acad Sci USA 2011; 108:17767–17772.  Back to cited text no. 11
    
12.
Yoshitake H, Rittling SR, Denhardt DT, Noda M. Osteopontin-deficient mice are resistant to ovariectomy-induced bone resorption. Proc Natl Acad Sci USA 1999; 96:8156–8160.  Back to cited text no. 12
    
13.
Chang IC, Chiang TI, Yeh KT, Lee H, Cheng YW. Increased serum osteopontin is a risk factor for osteoporosis in menopausal women. Osteoporos Int 2010; 21:1401–1409.  Back to cited text no. 13
    
14.
Huang W, Carlsen B, Rudkin G, Berry M, Ishida K, Yamaguchi DT, et al. Osteopontin is a negative regulator of proliferation and differentiation in MC3T3-E1 preosteoblastic cells. Bone 2004; 34:799–808.  Back to cited text no. 14
    
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18.
Yang DH, Chiang TI, Chang IC, Lin FH, Wei CC Cheng YW. Increased levels of circulating advanced glycation end-products in menopausal women with osteoporosis. Int J Med Sci 2014; 11:453–460.  Back to cited text no. 18
    
19.
Constantinescu D, Vornicu M, Grigoriu C, Cozmei C, Grigoriu BD. Assaying for circulating osteopontin in practice: a technical note. Eur Respir J 2010; 35:1187–1188.  Back to cited text no. 19
    


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