INTRODUCTION:

Osteoporosis is an illness associated with increased bone turnover and reduced bone mass¹^,². It is well known that osteoporosis is a significant public health issue. The most common chronic metabolic bone disease, which is characterized by increased fragility of the bone, is osteoporosis³. More than 200 million people are currently estimated to have osteoporosis⁴. In the world, 1 in 3 women over the age of 50 and 1 in 5 men will suffer from osteoporotic fractions during their lifetime, according to recent statistics from the International Osteoporosis Foundation⁵. Each fracture is an impending sign of another. Until there is a fracture, Osteoporosis has no clinical manifestations.

Fractures cause significant morbidity; they can cause mortality in men in particular. Moreover, it is a common, age-dependent, silent illness that affects a large number of women, causing serious problems^6,7.

The resorption and formation of bone tissues is a continual process; when the resorption rate is higher than the formation rate, bone loss occurs. Bone mass increases between birth and adulthood; at puberty, the density of the bone is at its highest, i.e. reaching peak bone mass (PBM), and then the bone mass loss begins. Several factors such as nutrition, endocrine status, health, growth, sex, and activity are considered as the major determinants of PBM. Bone remodeling, which involves extracting the aged bone and replacing it with a newer bone, is used to repair and prevent micro fractures, thereby helping maintain a healthy skeleton⁸. There are other determining factors involved that are a cause of or result from osteoporosis. Generally, MMPs are the leading enzymes responsible for all ECM components for degradation or resorption^9,10.

MMPs are strictly homologated and can degrade a wide range of components from the extracellular or sub-membranous materials of the enzymatically activated extracellular, zinc, and calcium-dependent endopeptidases[5]^11,12. MMP targets include proteases, protease inhibitors, latent growth factors, blood clotting factors, chemical molecules, growth factor binding proteins, cells, and cell adhesion molecules¹³^,¹⁴.

Human matrix metallopeptidase 12 (MMP12), also known as metalloelastase macrophage, was first described in human alveolar macrophages¹⁵. Macrophages and neutrophils are both recruited at the injury site; they express a variety of proteinases and release them into the extracellular wound space¹⁶. MMP12 has also been described in terms of its elastase activity, secreted from macrophages. Since then, the list of MMP12 substrates has expanded to include other ECM and non-ECM components, with increased functions in different cellular processes^17,18.

Lysyl oxidase (LOX) (EC 1.4.3.13) is also known as protein-lysine 6-oxidase¹⁹. This enzyme is necessary if a healthy extracellular matrix is to be biosynthesised and morphogenesis and connective tissue repairs are to be achieved. Four similar lysyl oxidase isoforms (LOXL1, LOXL2, LOXL3, and LOXL4) of the protein are found in the same gene family. In recent years, LOX has been involved in several biological processes, such as cell motility and migration, cell and transcription regulation, altered condensation of chromatin, suppression of channels, and metastases^20,21. Besides the activation of the covalent interconnector and transcription regulations of ECM, LOX is involved in tissue growth, repair and reshaping as well as glaucoma²², fibrosis²³, cancer, and metastases^22,24.

Several biochemical parameters and markers are altered in osteoporosis patients. Therefore, we suggest conducting more longitudinal trials to assess the role of these parameters in both female and male patients. In the present work, we aimed to investigate the relations between serum LOX, MMP-12, and some other measured parameters among osteoporosis patients.

MATERIALS AND METHODS:

Study Population:

The research was carried out at the Chemistry Department, College of Science for Women, University of Baghdad. A total of 110 participants in the study were divided into two groups: Group 1 comprised 60 osteoporotic women aged 30–65 years, and group 2 consisted of 50 healthy women aged 35–52 years. The samples were obtained from the individuals who entered the outdoor patient department at Baghdad Teaching Hospital, Iraq. Patients with rheumatoid, arthritis, and liver diseases as well as smokers were excluded from the study.

Samples collection and processing:

10ml of blood was collected in a gel tube for each patient. After coagulation, the blood was centrifuged up to 3000rpm at room temperature and then separated into two aliquots and stored at -20°C. It was subsequently was used for measuring the parameters under study.

Determination of biochemical parameters:

Serum LOX was measured using a quantitative enzyme immunoassay (sandwich method). The kit was supplied by the Chinese company Cusabio. MMP-12 serum was measured in the same way as LOX using the kit supplied by other Chinese company biological Shanghai. phosphorus and the ALP kit were supplied by Agappe, India. Serum Mg was determined using the kit supplied by HUMAN GmBH, Germany, serum Ca using the kit supplied by Linear Chemicals, Spain, and serum vitamin D using the Elecsys cobas kit supplied by Roche, Germany.

Statistical analysis:

Statistical analysis was performed using SPSS version 26. Mean and standard errors were used for numerical variables, and log transform was applied for skewed data. The significance of the difference between the mean values was measured using an independent t-test. Dendrogram, correlation, multiple regression, and receiver operating characteristic (ROC) curves were used to further understand the relationship between the variables and calculation of sensitivity with specificity.

RESULTS:

Demography results:

The research population’s anthropometric and biochemical characteristics are shown in Table1. The results regarding the anthropometric parameters showed a significant difference (p = 0.02) between the mean age of the patient group (47±1.616) and that of the control group (39±1.30). While osteoporosis is known as an age-related disease, this aspect could not be researched due to the lack of comparable age ranges between the two groups.

There was no significant difference (p = 0.651) between the BMI of the patient group (30.40±1.02) and that of the control group (31.99±1.03). A significant increase (p < 0.001) in ALP was noted in both the patient (104.33± 7.36) and control (64.58±3.68) groups. No significant decrease in Ca level (p > 0.05) was observed in the patient (8.67±0.10) and control (8.71±0.11) groups. Mg result showed a highly significant decrease (p < 0.001) in the patient group as compared to the control group. Meanwhile, no significant difference (p = 0.25) was noticed in the P level of the studied groups. Vit D did not show any significant difference either. The T-score appeared significantly different (p < 0.001) between the patient group (26.68 ±1.02) and the control group (7.02 ±0.70), as shown in Table 1.

Table 1:Age, BMI, ALP, Ca, Mg, T-score %, Vit D, and P levels for the patient and control groups.

P-value	Control group [Mean ± SE] (n = 50)	Osteoporosis group [Mean ± SE] (n = 60)	Parameter
0.02*	39 ± 1.30	47 ± 1.61	Age (year)
0.65	31.99 ± 1.03	30.40 ± 1.02	BMI (Kg/m²)
0.00*	64.58 ± 3.68	104.33 ± 7.36	ALP (U/L)
0.37	8.71 ± 0.11	8.67 ± 0.10	Ca (mg/dl)
0.00*	2.11 ± 0.04	1.40 ± 0.06	Mg (mg/dl)
0.00*	7.02 ± 0.70	26.68 ± 1.02	T-score %
0.120	22.97 ± 0.04	15.79 ± 0.03	Vit D (ng/ml)
0.25	3.64 ± 0.09	3.40 ± 0.10	P (mg/dl)

The data obtained were presented as Mean ± SE.

*Independent t-test was performed; significant level at P-value < 0.05.

BMI: body mass index, ALP: Alkaline Phosphatase, Ca: calcium, Mg: magnesium, Vit D: vitamin D, P: Phosphorus.

New biochemical parameters’ results:

LOX and MMP-12 results are presented in Table 2. MMP-12 did not appear to have significant results when the patient and control groups were compared. Meanwhile, the LOX results showed interesting findings; the mean LOX in the patient group was 7.96 ± 0.45 compared to 12.78 ±0.04 in the control group. The results showed a significant decrease in the levels of LOX (P < 0.05) in osteoporosis patients compared to the healthy control group.

Table 2: MMP-12 and LOX levels in blood serum from study participants.

p-value

Control group

[Mean ± SE]

(n = 50)

Osteoporosis group

[Mean ± SE]

(n = 60)

Parameter

0.14

5.89 ± 0.07

6.47 ± 0.05

MMP-12 (ng/ml)

0.001*

12.78 ± 0.04

7.96 ± 0.45

LOX (ng/ml)

The data obtained were evaluated by Mean ± SE.

*Independent -t-test was performed; significant level at P-value < 0.05.

MMP-12: Human matrix metallopeptidase 12; LOX: lysyl oxidase.

Cluster analysis of multivariate for all parameters in studied subjects:

Cluster analysis groups variables by searching for related or interdependent variables, combining them in clusters or segments, and separating them from other, dissimilar, variables. Ward’s approach is one of the most common type of cluster analyses²⁵. This method is typically displayed using a dendrogram. In this test, no prior assumption of the clustering is made.

In the present study, cluster analysis was used to discover the similarities in the studied variables. According to the coefficients, the variables in all the studied groups were distributed into two clusters with mild shifting in some groups. Fig. 1: a shows the multivariable cluster analysis results using patient data. The variables were identified and classified into two clusters. The first cluster included the most well-known diagnostic tests as well as LOX, MMP-12, and T-score. Cluster two included ALP. A fusion of the first cluster with the second took place to form a single group. Fig. 1 :b shows the multivariable cluster analysis results using control data. The variables were identified and classified into two clusters. The first cluster included Vit D, LOX, MMP-12, Mg, T-score, P, and Ca. Cluster two included ALP. The results showed that LOX and MMP-12 were close to each other in the patient group.

Fig. 1: The cluster analysis of multivariate for all parameters among patients in the group[a] and control [b].

The relationship between the studied parameters:

A correlation study was carried out using the Pearson test, and the results are presented in Table 3. The results show a positive correlation of MMP-12 with T-score (p < 0.05) and Vit D (p < 0.05). Moreover, a positive correlation between Vit D and T-score (p < 0.05) as well as ALP and BMI (p < 0.05) were found. In contrast, a negative correlation between P and BMI (p < 0.001) was found in the osteoporosis group. The presented correlations appeared even though there was no significant difference in those parameters when a t-test was conducted with the osteoporosis and control groups. Further, no significant correlation was found between the T-score result and LOX, Mg, Ca, and P, which indicates that the relation was not linear because the change in the variables neither increased nor decreased systematically compared to the T-score.

Table 3 :Correlation between MMP-12, LOX and other parameters in female osteoporosis patients.

Parameters		MMP-12	LOX	Vit D	Mg	Ca	P	ALP	BMI
T_score	Pearson Correlation	0.304^*	-0.070	0.273^*	-0.201	-0.080	0.162	0.055	0.022
BMI	Pearson Correlation	0.020	0.190	-0.002	-0.129	-0.053	-0.333^**	0.319^*
ALP	Pearson Correlation	-0.230	0.022	-0.042	0.178	0.153	-0.110
P	Pearson Correlation	0.038	-0.065	-0.091	-0.082	0.045
Ca	Pearson Correlation	0.021	0.092	0.083	-0.065
Mg	Pearson Correlation	-0.112	-0.068	0.065
vit_D	Pearson Correlation	0.269^*	-0.073
LOX	Pearson Correlation	-0.113

*Significant difference between parameters at 0.05 level.

**Significant difference between parameters at 0.001 level.

BMI: body mass index, ALP: Alkaline Phosphatase, Ca: calcium, Mg: magnesium, Vit D: vitamin D, P: Phosphorus. MMP-12: human matrix metallopeptidase 12, LOX: lysyl oxidase.

Table 4: Multiple regression analysis of ALP, phosphorus, Ca, Vit D, Mg, LOX, and MMP-12 with dependent variables T- score, LOX, and MMP-12 for the patients and controls.

Control			Osteoporosis patients			Parameters
Dependent Variable			Dependent Variable
MMP-12	LOX	T-score	MMP-12	LOX	T-score
No dep.	No dep.	No dep.	No dep.	No dep.	No dep.	ALP
No dep.	No dep.	No dep.	No dep.	No dep.	No dep.	Ca
No dep.	No dep.	No dep.	No dep.	No dep.	No dep.	Mg
No dep.	↓↓	No dep.	No dep.	No dep.	No dep.	P
No dep.	_↑	No dep.	No dep.	No dep.	↑	Vit -D
No dep.		No dep.	No dep.		No dep.	LOX
	No dep.	No dep.		No dep.	No dep.	MMP-12
No dep.	No dep.		No dep.	No dep.		T-score

↑↑↑, 3 ≦ t < 3.5 and p < 0.05 ↑↑, 2.5 ≦ t < 3.0 and p < 0.05 ↑, 2 ≦ t < 2.5 and p < 0.05 ↓, − 2.5 ≦ t < − 2 and p < 0.05

↓↓, − 3 ≦ t < − 2.5 and p < 0.05 ↓↓↓, − 3.5 ≦ t < − 3 and p < 0.05 Not dep: Not dependent

Multiple regression:

The multivariate regression method was used to identify the degree of association of the independent variables to the dependent variable. The analysis revealed that the significant predictor of T-score in osteoporosis patients was Vit-D, as shown in Table 4. The dependence between Vit D and T-score (t < 2.028 and p < 0.05) in the patient group, absence of dependence between Vit D and T-score in the control group. High dependence in opposite direction of P and LOX variables (t < -3.403 and p < 0.001) was observed in control subject, and dependence ( t < 2.077 and p < 0.05) in Vit D with LOX in the control group were noticed.

Receiver operating characteristic (ROC):

According to the ROC curve, the area under the curve (AUC) for LOX was 0.650, and 95% CI AUC was 0.548–0.752, while the best cut–off point was found to be 10.3 ng/ml. This meant that the test value higher than 10.3 ng/ml represented healthy conditions whereas the value less than (10.3 ng/ml) represented an unusual case, as shown in Table 5 and Fig 2. According to the ROC curve, the AUC for MMP-12 was 0.579, and the 95% CI AUC was 0.459–0.679. The MMP-12 result, which is shown in Table 5 and Fig 3, did not have a clear cut-off value.

Table 5: ROC curve for the MMP-12 and LOX of the patient and control groups.

Marker	AUC	95%CI AUC	Cut-off value	P-value
MMP-12	0.579	0.459–0.679	6.63 ng/ml	0.212
LOX	0.650	0.548–0.752	10.3 ng/ml	0.007

AUC: Area under the curve.

Fig. 2: The ROC curve for MMP-12

Fig.3: The ROC curve for LOX

DISCUSSION:

No previous studies have indicated the LOX levels of fragile patients; however, there have been numerous studies of LOX levels in relation to other diseases such as chronic kidney disease²⁶. Collagens, which are synthesised by fibroblasts, facilitate a healing process cascade in skin, bone, and cardiovascular tissues after a LOX-catalysed connection reaction. The above-presented phenomena may explain the decrease in LOX level in osteoporosis patients, as observed in this study²⁷.

Previous researchers have reported that a decrease in the LOX concentration could be induced by TNF-a in endothelial cells, osteoblasts, and fibroblasts²⁸. Further, some researchers have concluded that the level of LOXs released by damaged tissues could further be modulated by some chemical mediators such as IL-1-1 VEGF, BMP-1, IGF-1 and MMP-2²⁷. LOX improves osteoclasts’ association with osteoblasts to maintain a healthy and regular bone rotation²⁹. The reduction in LOX activity can, therefore, affect the balance of osteoclasts and osteoblast; the same was concluded in the present research.

Regarding the MMP12 level, the results did not show any significant differences between the studied groups. Matrix metalloproteinases (MMPs) are considered degrading enzymes. These have an essential function in extracellular matrix remodeling³⁰. Most of the MMP enzymes are expressed by cartilage and bone cells under physiological or pathological conditions such as osteoporosis, rheumatoid arthritis, and osteoarthritis. In the present study, MMP-12 did not appear to contribute to bone cell degradation, while in other studies, it contributed to other tissues such as in the heart and lungs. The MMP-12 level in serum varied from case to case¹⁸. Meanwhile, other studies showed a high level of MMP-12 in hepatocellular carcinoma patients³¹ and patients with lung injuries³².

CONCLUSION:

LOX, ALP, and Mg were significantly associated with osteoporosis patients; whereas MMP12 and Vit D were found to affect the density of the bone. Therefore, the studied parameters may be used in a bone mineral density decline index. Further, we suggest using serum LOX for the early diagnostic testing of patients with osteoporosis, considering 10.3 ng/ml as the best cutoff value to distinguish between the patients and controls.

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Received on 11.04.2021 Modified on 07.09.2021

Research J. Pharm. and Tech. 2022; 15(6):2655-2660.

DOI: 10.52711/0974-360X.2022.00444

Fractures cause significant morbidity; they can cause mortality in men in particular. Moreover, it is a common, age-dependent, silent illness that affects a large number of women, causing serious problems6,7.

Fractures cause significant morbidity; they can cause mortality in men in particular. Moreover, it is a common, age-dependent, silent illness that affects a large number of women, causing serious problems^6,7.