|Year : 2016 | Volume
| Issue : 4 | Page : 1130-1135
Serum undercarboxylated osteocalcin and bone mineral density in premenopausal patients with rheumatoid arthritis
Abd El Samad I Al Hewala1, Samar G Soliman2, Heba A Esaily2, Dalia H Abo Ela3, Eman A Galbat MBBCh 2
1 Department of Rheumatology, Physical Medicine and Rehabilitation, Faculty of Medicine, Zagazig University, Zagazig, Egypt
2 Department of Physical Medicine and Rehabilitation, Faculty of Medicine, Menoufia University, Menoufia, Egypt
3 Department of Clinical Pathology, Faculty of Medicine, Menoufia University, Menoufia, Egypt
|Date of Submission||24-Apr-2015|
|Date of Acceptance||08-Jun-2015|
|Date of Web Publication||21-Mar-2017|
Eman A Galbat
Department of Physical Medicine and Rehabilitation, Menoufia University, Al Shohdaa, Menoufia, 32958
Source of Support: None, Conflict of Interest: None
The aim of this study was to measure serum undercarboxylated osteocalcin (ucOC) and bone mineral density (BMD) in premenopausal patients with rheumatoid arthritis (RA) and their correlation with each other and with disease activity.
Osteocalcin is a molecule synthesized as a prepro-osteocalcin. After proteolytic cleavage and vitamin K-dependent carboxylation processes, it is released and circulates in the blood as carboxylated osteocalcin or ucOC.
Participants and methods
In total, 60 premenopausal patients with RA and 30 healthy premenopausal controls with matched age were included in this study. All patients and controls were subjected to demographic data, clinical examination, and laboratory investigations including serum level of ucOC, disease activity assessment by DAS-28 score, and BMD measurement in the lumbar spine L2–L4, hip, and distal radius by dual-energy X-ray absorptiometry equipment.
Results; The level of ucOC was significantly higher in patients of RA than controls (P < 0.001). BMD in patients was found to be significantly lower than in controls in spine, femoral neck, and distal radius areas. The most frequent osteoporotic site according to Z-score was the spine (16.7%) followed by the femoral neck (8.3%) and then the distal radius (6.7%). Moreover, the commonest osteopenic site according to Z-score of -1 or less was the spine (31.7%) followed by the femoral neck (21.7%) and then (16.7%) the distal radius. This work showed that ucOC level was found to be high in premenopausal patients with RA with higher DAS values than those with lower DAS value (P < 0.001). In this work, BMD measured by dual-energy X-ray absorptiometry scan was found to be lower with higher DAS values, and vice versa.
Serum level of ucOC (which is a mirror of vitamin K deficiency) was found to be higher in premenopausal patients with RA than controls and correlated positively with disease activity and inversely with BMD measurement.
Keywords: bone mineral density, disease activity, rheumatoid arthritis, undercarboxylated osteocalcin
|How to cite this article:|
Al Hewala AI, Soliman SG, Esaily HA, Abo Ela DH, Galbat EA. Serum undercarboxylated osteocalcin and bone mineral density in premenopausal patients with rheumatoid arthritis. Menoufia Med J 2016;29:1130-5
|How to cite this URL:|
Al Hewala AI, Soliman SG, Esaily HA, Abo Ela DH, Galbat EA. Serum undercarboxylated osteocalcin and bone mineral density in premenopausal patients with rheumatoid arthritis. Menoufia Med J [serial online] 2016 [cited 2020 Jun 1];29:1130-5. Available from: http://www.mmj.eg.net/text.asp?2016/29/4/1130/202498
| Introduction|| |
Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease that causes chronic inflammation of the synovium with subsequent destruction and deformity of the joints .
Osteoporosis is a multifactorial skeletal disease characterized by a reduction in bone mass and deterioration of the microarchitectural structure of bone tissue, with resulting increase in bone fragility and fracture risk .
Osteoporosis is more common in patients with RA than in the general population. The prevalence of concurrent osteoporosis is 50%. Osteoporosis can cause pain and loss of height, and it increases the risk of fractures after falling .
The chronic synovial inflammation in RA can promote osteoclastogenesis, leading directly to both focal and generalized bone loss and increased risk of fractures .
Vitamin K is a cofactor of γ-carboxylase, which converts three glutamic acid (Glu) residues in osteocalcin (OC) to γ-carboxyglutamic acid (Gla) and is thus essential for γ-carboxylation of OC. Without this modification, OC becomes undercarboxylated osteocalcin (ucOC), which lacks structural integrity and the ability to bind to the mineral hydroxyapatite. Vitamin K deficiency impairs γ-carboxylation of OC, resulting in high serum concentrations of ucOC .
| Participants and Methods|| |
The study was done by Clinical Pathology Department in collaboration with Physical Medicine and Rehabilitation Department.
Participants were classified into two groups: group I included
60 premenopausal patients diagnosed as having RA according ACR/EULAR 2010 criteria , with age greater than 16 years and disease duration of more than 2 years, and group II included
30 age-matched healthy premenopausal female patients as control who were free of earlier fractures and chronic diseases and were not taking medications that influence bone metabolism (e.g., anticonvulsants and thyroxine).
Postmenopausal female patients with RA, male patients with RA, those experiencing serious cardiovascular and/or pulmonary disease, those having an abnormal thyroid function or a serious infection, those with hyperparathyroidism, those with hepatic or renal dysfunction, those taking any drugs or hormones that affect bone metabolism (e.g., sex steroids) were excluded.
All patients and controls were subjected to the following:
- Demographic data recording
- Full history taking and thorough clinical examination
- Disease activity assessment.
Disease Activity Score (DAS-28) with three variables [erythrocyte sedimentation rate (ESR), the number of swelled joints, and number of tender joints] was used .
The level of disease activity was interpreted as follows:
- DAS-28 ≤ 3.2 = low;
- 3.2 <DAS-28 ≤ 5.1 = moderate;
- DAS-28 > 5.1 = high
- DAS < 2.6 = disease being in remission according to the American Rheumatism Association (ARA) criteria .
- Laboratory investigations
ESR  was obtained by Westergren method, rheumatoid factor and C-reactive protein levels by latex agglutination ,, and serum levels of ucOC and anti-CCP level were measured by using Cobas Elecsys 2010 (Roche Diagnostics GmbH, Mannheim, Germany).
Serum levels of undercarboxylated osteocalcin
Serum levels of ucOC were obtained using the sandwich principle test (enzyme-linked immunosorbent assay). The total duration of the assay was 18 min.
A 20 µl sample, a biotinylated monoclonal N-MID OC-specific antibody, and a monoclonal N-MID OC-specific antibody labeled with a ruthenium complex [Tris (2,2’-bipyridyl) ruthenium (II)-complex (Ru (bpy)] react to form a sandwich complex.
After addition of streptavidin-coated microparticles, the complex becomes bound to the solid phase through interaction of biotin and streptavidin. The reaction mixture is aspirated into the measuring cell where the microparticles are magnetically captured onto the surface of the electrode. Unbound substances are then removed with ProCell/ProCell M. Application of a voltage to the electrode then induces chemiluminescent emission which is measured by a photomultiplier.
Results are determined using a calibration curve, which is an instrument specifically generated by two-point calibration and a master curve provided through the reagent barcode. Normal level is less than 4.5 ng/ml.
Under complete aseptic conditions, 5 ml of venous blood was collected. Then, 4 ml of whole blood was added to a sterile plain tube and allowed to clot at 37°C. Serum was separated by centrifugation and used for measuring levels of ucOC, anti-CCP, rheumatoid factor, and C-reactive protein. Moreover, 0.8 ml of whole blood was added to 0.2-ml sodium citrate tube to measure ESR.
- Radiographic assessment
Bone mineral density (BMD) was measured in the lumbar spine L2–L4, hip, and distal radius by dual-energy X-ray absorptiometry (DEXA) equipment .
BMD was expressed in SD from the mean of healthy age-matched and sex-matched people (the Z-score) and as the number of SD from the mean of healthy, young sex-matched people (the T-score). By the WHO classification and the 2005 International Society for Clinical Densitometry (ISCD) official positions, a T- score of -2.5 or lower in postmenopausal women was classified as osteoporosis, a T-score between >-2.5 and <-1 as osteopenia, and a T-score ≥-1 as normal, whereas osteoporosis according to Z-score of -2.0 or lower in females before menopause was defined as 'below the expected range for age' .
The data collected were tabulated and analyzed by SPSS (Statistical Package for the Social Science software) statistical package version 20 on IBM compatible Computer (IBM Corp. Released 2013. IBM SPSS Statistics for Windows, Version 22.0. Armonk, NY: IBM Corp., USA). A total of two types of statistics were done: descriptive statistics included percentage (%), range, mean (X), and SD, and analytical statistics included Student's t- test, Mann–Whitney test, and Fisher's exact test. P value greater than 0.05 was considered a nonsignificant difference, P value less than 0.05 as a significant difference, and P value less than 0.001 as a highly significant difference.
| Results|| |
In this work, the level of ucOC was significantly higher in patients with RA than in controls (P < 0.001, [Table 1]) and higher in patients with osteoporosis than those with osteopenia and those with normal BMD ([Table 2]). BMD in patients was found to be significantly lower than controls in all sites (spine, femoral neck, and distal radius areas). The most frequent osteoporotic site according to Z-score was the spine (16.7%) followed by the femoral neck (8.3%) and then the distal radius (6.7%). According to T-score, the commonest osteoporotic site was the spine (13.3%), whereas the commonest osteopenic site according to Z-score of -1 or less was the spine (31.7%) followed by the femoral neck (21.7%) and then the distal radius (16.7%) ([Table 3]). Level of ucOC was found to be positively correlated with DAS score (P < 0.001, [Table 4]). BMD measured by DEXA scan was found to be lower with higher DAS values and vice versa, as shown in [Table 5].
|Table 1 A comparison between the studied patient group and controls regarding undercarboxylated osteocalcin (ng/ml)|
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|Table 2 A comparison between female patients with osteoporosis, female patients with osteopenia, and normal bone mineral density patients regarding undercarboxylated osteocalcin (ng/ml)|
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|Table 3 A comparison between the studied patient group and control regarding dual-energy X-ray absorptiometry parameters(n (%))|
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|Table 4 Relationship between disease activity and undercarboxylated osteocalcin level (ng/ml) among studied group of patients (n=60)|
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|Table 5 Relationship between disease activity and dual-energy X-ray absorptiometry parameters among studied group of patients (n=60)|
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| Discussion|| |
RA is a chronic inflammatory autoimmune disease that causes chronic inflammation of the synovium with subsequent destruction and deformity of the joints. Osteoporosis is more common in patients with RA than in the general population. Vitamin K is a cofactor of γ-carboxylase, which converts three glutamic acid (Glu) residues in OC to γ-carboxyglutamic acid (Gla) and is thus essential for γ-carboxylation of OC. Without this modification, OC becomes ucOC, which lacks structural integrity and the ability to bind to the mineral hydroxyapatite, and secreted OC can no longer be carboxylated. Vitamin K deficiency impairs γ-carboxylation of OC, resulting in high serum concentrations of ucOC.
This study revealed that the level of ucOC was significantly higher in patients with RA than in controls. This is in accordance with Sakai et al.  who showed that serum levels of ucOC were increased in higher state of bone turnover as indicated by biochemical markers, and vice versa.
No other searchers discussed ucOC in adult patients with RA, although van Summeren et al.  found that children with JIA (Juvenile Idiopathic Arthritis) have a high ratio of ucOC/carboxylated osteocalcin, indicating low vitamin K status, and associated with low bone ultrasound parameters, whereas children with a high vitamin K status had markedly higher bone properties.
Moreover, Iwamoto et al.  found that serum ucOC is an index of vitamin K nutritional status in treatment-naïve postmenopausal women with osteoporosis and concluded that high level of ucOC is a risk factor for osteoporotic fracture, which is decreased by vitamin K intake.
In addition, in this work, ucOC level was found to be positively correlated with disease activity in premenopausal patients with RA.
To our knowledge, no other studies discuss the relationship between ucOC in premenopausal patients with RA and DAS score.
In this work, BMD in patients was found to be significantly lower than in controls in the spine, the femoral neck, and the distal radius areas. The most frequent osteoporotic site according to Z-score was the spine (16.7%) followed by the femoral neck (8.3%) and then the distal radius (6.7%), and the commonest osteopenic site according to Z-score of -1 or less was spine (31.7%) followed by the femoral neck (21.7%) and then the distal radius (16.7%).
This is in accordance with Lee et al. , who found that the prevalence of osteoporosis in patients with RA was 1.9 times higher than in healthy subjects.
Moreover, Lodder and colleagues found that the frequency of osteoporosis (T-score≤−2.5 SD) is 12.6 and 6.5% in the spine and the femoral neck, respectively. Reduced bone mass or osteopenia occurred in the spine and the femoral neck at 20.7 and 18.9%, respectively .
Laan and colleagues examined BMD in 97 patients with recent-onset RA and a mean disease duration of 30 months. Low bone mass (Z-score≤-1.0) was found in 32.0% of the patients in the lumbar spine and in 24.2% in the hip. They confirm that in comparison with healthy age-matched and sex-matched controls, low bone mass occurs more frequently in patients with RA .
Another research by Haugeberg and colleagues studied a representative sample of 394 female patients with RA with a mean disease duration of 13 years, using both T- scores and Z- scores. The prevalence of osteoporosis (T-score≤-2.5) was 16.8% in the spine, 14.7% in the femoral neck, and 14.7% in the total hip. The prevalence of reduced bone mass (Z-score≤-1.0) was greater than expected (15.9%) in the femoral neck (27.6%), the total hip (31.6%), and the spine (19.6%) .
However, Hamalainen et al.  concluded that according to bone mineral concentration, premenopausal women with RA both with and without prednisolone treatment and controls lost bone statistically similarly.
Hui et al.  had followed up 130 healthy premenopausal white women for 1–9 years and found a significant decrease in femoral neck BMD over time.
In our work, BMD measured by DEXA scan was found to be lower with higher DAS values, and vice versa.
This is in accordance with studies by Lodder et al. , Shenstone et al. , and Gough et al. ; their studies concluded that patients with active disease lost more bone at the spine and hip than patients with inactive disease.
However, Hamalainen et al.  conducted their study on premenopausal women and found that women in the RA with PRED group had lower BMD values than those in the RA without PRED group at the start of follow-up, in both lumbar spine and in femoral neck; however, finally in the premenopausal women, bone status is stable or bone loss minimal in the lumbar spine, whereas some bone loss takes place in the femoral neck.
| Conclusion|| |
From the present study, we concluded that patients with RA are more susceptible to develop generalized osteoporosis than other population even if they are in premenopausal phase. Serum level of ucOC is an indicator of BMD in premenopausal patients with RA. Patients with high serum level of ucOC are more susceptible to osteoporosis than other patients are. Patients with active RA disease are associated with high level of ucOC and lower BMD values. The commonest sites of osteoporosis in premenopausal patients with RA were in lumbar spine (16.7%) followed by hip (8.3%) and the distal radius (6.7%) according to Z-score.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]