L. Tsyhanyk, O. Abrahamovych, U. Abrahamovych, O. Romanyuk, S. Guta
Danylo Halytsky Lviv National Medical University
Introduction. Systemic Lupus Erythematosus (SLE) is a systemic autoimmune condition induced by endogenous and exogenous factors and is characterized by multifunctional affection, including bones.
The majority of patients with SLE are the women having gender-peculiar risk of bone mass loss. Apart from this, different factors make their impact on the mineral density of bone tissue (MDBT) and they are associated with the condition and the treatment . It is expected that numerous investigations have proved osteoporosis (OP) frequency increase and systematic fractures for the patients with SLE in comparison to the controlled group (CG) of healthy people of relevant age and gender [4, 5].
It is generally considered that one of the major pathogenic factor of OP development of the patients with SLE is accelerated osteoclast genesis induced by inflammatory cytokines which are redundantly produced in the phase of disease aggravation and the violation of osteoblast genesis as a result of the suppression of VMR-2-induced differentiation of osteoblasts which eventually violate bone metabolism processes . Bone remodeling intensity (bone tissue development and its destruction) may be evaluated if to determine the contents of biochemical enzymes which are produced as a result of osteoclasts or osteoblasts activity. Osteocalcin is one of biochemical markers of bone development. It is a bone glutamine albumen which is revealed by osteoblasts during the process of bone tissue development. P1NP – is amino thermal procolagen propeptide of I type, which is specific for I type collagen development and is also a biochemical marker of bone development. The process of bone resorption is characterized by β-crossLaps, this is an isomeric C-terminal telopeptide specific to I type collagen degradation in the bone. The role of bone interrelation indexes as prognostic markers of osteopenia has been studied in numerous researches. In particular, it has been discovered that the decrease of P1NP contents in blood serum is predictor of bone mass loss during the next 12 months for the women with SLE in the premenopausal stage . As a result of five years of research, T. Y. Zhu and co-authors  discovered the relation between the indexes of MDBT indexes for the patients with SLE, disease activity and organ damage index.
In our recent research  of separate indexes influence of SLE course and its treatment on MDBT we have managed to prove that the state of bone tissue does not depend on the activity index of SLE (AISLE). Obviously, OP development is a sustained process and even hard aggravation does not make any «immediate» impact which would immediately influence bone state and might be evaluated by densitometry. That is why there is a necessity of the search of more informative factors which would able to immediately become a specific marker of SLE activity and OP complication.
A comprehensive research of the patients’ bone state dependence with SLE evaluated according to the results of bone remodeling markers on a separately determined parameter and total AISLE will enable us to find new opportunities to discover pathogenic peculiarities of OP development along with treatment and prevention measures.
The Aim of the Research. To analyze the interrelations between bone remodeling markers and activity index of systemic lupus erythematous.
Data and Methods of Research. The research under consideration has been fulfilled on the basis of rheumatic department of KZ LOR of «Lviv Regional Hospital». Having received a written permit for the research conduction according to the principles of Helsinki Human Rights Declaration and the Convention of European Council on the matter of human rights and biomedicine and the laws of Ukraine, we have examined 123 patients with the condition of SLE according to the criteria of American Rheumatology Collegium (1997) considering the previous stratification of the thesis and the pre-menopause status. The research was conducted with the application of randomized way of examination. The patients have had all their organs and systems tested systemically and with the application of instruments according to the order of the Ministry of Health Protection of Ukraine № 676 since 12.10.2006 and «The Declaration of Minutes of First Aid on the specialist «Rheumatology», European League against Rheumatism (2010) and American Rheumatologists Collegium (2010, 2012). The age of patients of a research group (RG) was from 21 to 51 (average age for the moment of investigation – 41.13 ± 12.04); average condition duration was 10.08 ± 0.72 years; 100.0 % of patients took methylprednisolone in the dose (in recount into prednisolone) from 5.0 to 30.0 mg/24 hrs. (average dose 8.99 ± 0.65 mg/24 hrs., сaverage course dose 224.69 ± 97.60 gr) and medicines of calcium (the dose for 24 hours was 1000.0 mg) in combination with vitamin D (24 hour dose 400.0 MO); average treatment duration by glucocorticostiroids and combined medicines of calcium was relevant to average disease duration.
Controlled group (CG) consisted of 25 mostly healthy women in premenopausal stage of a certain age.
To evaluate SLE activity we applied AISLE according to C. Bombardier et al. (1992).
Systemic Lupus Erythematous Activity Index according to C. Bombardier et al. (1992)
To evaluate the speed of bone tissue remodeling in both groups we have been researching the markers of bone tissue development: osteocalcin and P1NP, and also biochemical marker of bone resorption – β-crossLaps. Bone tissue remodeling markers and antidsDNA in blood serum were determined by the method of immune chemical analysis with the help of automatic chemical immune analyzer СOBAS E 411 made by Roche (Switzerland) with the use of commercial test-kit of the same company according to the instructions provided.
The norms were based on the referent values provided by the producer of test-systems in the instruction: osteocalcin (women to 50 years old – 11.00–43.00 ng/ml; after 50 years – 15.00–46.00 ng/ml), P1NP (women from 14 years old – 15–50 ng/ml, from 24 to 30 years old – 22.50–120.00 ng/ml, from 30 years old – 10.20–95.00 ng/ml), β-crossLaps (women <0.57 ng/ml).
To achieve this goal we made three steps. The first step was dedicated to the research of the state of osteoblast function according to the results of the evaluation of the contents of osteocalcin, P1NP and osteoclast in blood serum according to the results of their contents β-crossLaps in blood serum in both RG and CG and their comparison. The second step was dedicated to the research of the dependence of the markers of bone metabolism on disease activity according to AISLE, the third one was to determine the indexes of AISLE and their constellations which made the most considerable influence on bone remodeling markers.
The data processing of the research results has been fulfilled with the help of MS Excel and EViews. In order to analyze quantitative indexes we counted average values, standard deviations and also applied percentile analysis to evaluate the distribution of the result values of laboratory and instrumental methods of research in both RG and CG. We have conducted a T-test to determine the reliability of the difference between average values calculated for both groups. To analyze category indexes we have calculated absolute and relative values (general number and a part), and also we have evaluated the connection between category and quantity indexes with the help of the method of point-bead correlation. To analyze the interrelations between quantity indexes we have applied the method of regressive analysis. We have also been analyzing the sensitivity and specificity of the indexes of AISLE to the markers of bone remodeling on the basis of the schemes of conjugation. The indexes of chances correlation have been calculated opposite the risk and prognostic value: the risk of receiving an altered research index when the symptoms are either present or absent; relative risk – the degree of risk of receiving an altered index when the symptoms either present or absent; absolute and relative risk decrease – the correlation of risk of having an altered index when the symptoms are either present or absent; prognostic value of the result – the prediction of probability to receive an altered index when the symptom are either present or absent; prognostic value of negative result – the prediction of probability of receiving unchanged index when there symptoms are absent. We have also researched the coefficient of associations (the relation with the coefficient of association >0.5 was considered a proved one). In the course of T-test for the sample collection, under the bead-point correlation, regressive analysis and the comparison of parts we considered the connection, influence or difference reliable and as р < 0.05.
The Results of Research and its Discussion. The First step was dedicated to the research of the markers of osteoblast and osteoclast functions of bone tissue for the patients with SLE (RG) and healthy people (CG) (tabl. 1).
Notes: *– р < 0.001 according to the t-criterion by Student in comparison to the indexes of mostly healthy people of relevant age and gender; ***– р <0.05 according to t-criterion by Student in comparison to the indexes of mostly healthy people of relevant age and gender.
Evaluating the markers of osteoblast function we have discovered the difference between the indexes in RG and CG which meant that average value of osteocalcin for the patients was reliably lower than for healthy people and was relatively 13.42 ± 5.43 ng/ml (р <0.001) and 18.3 ± 0.37 ng/ml. A reliable difference between average values of P1NP for women from RG and CG was not found – 39.86 ± 22.85 ng/ml (р > 0.05) and 39.67 ± 11.70 ng/ml accordingly.
Concerning the evaluation of osteoclast function indexes, according to average value of β-crossLaps we observed a reliable difference between two groups under investigation with higher indexes for the patients with SLE – 0.40 ± 0.25 ng/ml in comparison to healthy ones – 0.26 ± 0.08 ng/ml (р < 0.05).
To determine the range and variability of values distribution of the markers of osteoblast and osteoclast functions in RG and CG we have conducted their percentile analysis (tabl. 2).
The Indexes of Bone Tissue Remodeling in the Blood Serum of the Patients with Systemic Lupus Erythematous and Healthy People of Relevant Age and Gender according to Percentile Analysis
The patients’ of RG osteocalcin contents was 7.51–22.73 ng/ml, mediana – 12.39 ng/ml, when healthy women’s – від 15.22 до 27.76 ng/ml, mediana – 17.70 ng/ml (fig. 1).
Fig. 1. Percentile analysis of osteocalcin.
According to the results of percentile analysis the for the patients with SLE P1NP was 10.34–84.20 ng/ml, mediana – 32.82 ng/ml, for healthy women – 19.53–58.34 ng/ml, mediana – 41.20 ng/ml (fig. 2).
Fig. 2. Percentile analysis of P1NP.
For the patients of RG β-crossLaps made 0.11–1.01 ng/ml (P5–P95), mediana – 0,34 ng/ml, CG– 0.15–0.42 ng/ml (P5–P95), mediana – 0.23 ng/ml (fig. 3).
Fig. 3. Percentile analysis of β-crossLaps.
To evaluate the frequency of the distribution of the received results of bone metabolism markers for the patients of RG and the people of CG on the basis of percentile scale we have outlined three nominal groups: with low (0–25), medium (25–75), and high (75–100) indexes.
The indexes of osreocalcin which are considered low in a CG (15.22–15.50 ng/ml) considerably exceeded the indexes of analogical group and coincided to the average indexes in RG (9.24–16.71 ng/ml), which testifies to the fact f osteoclast function suppression for the patients with SLE.
According to the results of percentile analysis, average and high indexes of P1NP for the patients in RG (56.98–67.20 ng/ml) coincided to high and very high for the patients of the CG (50.72–58.34 ng/ml).
Comparing β-crossLaps according to analogical groups if indexes in RG and CG, higher indexes were discovered in the groups with medium (0.250–0.480 ng/ml і 0.210–0.310 ng/ml accordingly) and high (0.480–0.710 ng/ml і 0.310–0.380 ng/ml accordingly) indexes for the patients in RG with SLE and were almost equally low when the indexes of CG patients were not very higher and it allows us claiming that osteoclast function strengthening for the patients with SLE.
To make the second step and study the interrelation between the markers of bone metabolism and AISLE we have applied the method of linear correlation.
According to the results of the research (tabl. 3), a total activity index according to AISLE intercorrelates with the contents of osteocalcin in blood serum (r = ((-0.21), p < 0.05), when the increase of activity index according to AISLE makes a negative impact on the marker of bone tissue development. The contents of osteocalcin decreases up to 0.19 ng/ml when the AISLE rises per one point.
We have fixed a reliable direct connection between AISLE activity and β-crossLaps (0.30; p < 0.01). The increase of SLE activity according to AISLE per 1 point leads to the increase of the contents of β-crossLaps in blood serum 0.010 ng/ml, that is why one can claim that in case of disease aggravation bone tissue is under intensive destruction.
The Interrelation between Total Score of Activity Index of Systemic Lupus Erythematous according to C. Bombardier et al. (1992) and the Markers of Bone Remodeling
Notes: **– р < 0.01; ***– р < 0.05.
The third step is dedicated to the determination of the indexes according to AISLE and their constellations which had the most considerable influence on the markers of bone remodeling (tabl. 4).
Notes: * – р < 0.001; ** – р < 0.01; *** – р < 0.05.
According to the results of the research, the patients who with SLE who suffer from such complications as psychosis have a reliably lower contents of osteocalcin in blood serum. It means that we have discovered a reversed dependence between the psychosis as SLE manifestation and the marker of osteoblast function of bone tissue – osteocalcin (r = (-0.20), p < 0.05).
The patients with SLE who suffer from nerve system violation which is a brain organic symptoms, have also a reliably lower contents of osteocalcin and P1NP, as there is a reversed connection between the presence of this feature and the markers of bone tissue development (r = (-0.27), p < 0.01 and (-0.29), p < 0.01 accordingly).
The patients with vacuities diagnosed for 2.0 % of women with RG, we expect a higher contents of osteocalcin in blood serum (r = 0.19, p < 0.05).
Myositis, which is diagnosed for 14.3 % of patients with SLE, was associated with two biochemical markers of bone development under research: osteocalcin (r = 0.31, p < 0.001) and P1NP (r = 0.26, p < 0.01). So, the patients with myositis the indexes of both markers of osteoblast function were reliably higher.
The diagnosis of proteinuria which was diagnosed for 8.6 % of patients with SLE directly depends on the bone development markers, namely, P1NP (r = 0.29, p < 0.01), and bone tissue resorption – β-crossLaps (r = 0.45, p < 0.001).
For the patients with SLE, alopecia was one of its manifestations, and we have diagnosed the deviation of osteoblast function. Alopecia was also observed in 40.0 % of the patients with SLE and it correlated with P1NP (r = (-0.32), p < 0.001).
The decrease of thrombocytes amount in the blood of 16.2 % patients with SLE is associated with the indexes of osteoblast function of bone tissue (r = (-0.22), p < 0.05) і P1NP (r = (-0.22), p < 0.05).
There is a direct dependence between leukopenia which was diagnosed for 12.9 % of women out of RG during the research, and the marker of bone tissue destruction (r = 0.36, p < 0.01).
The patients with a high amount of antidsDNA there is intensive bone tissue destruction as a result of the increase of the amount of β-crossLaps – the resorption bone tissue marker in blood serum.
So, the indexes of bone tissue remodeling for the patients with SLE differed from the analogical ones for the healthy people, namely: the contents of the marker of bone tissue development – osteocalcin – is lower, when resorption – β-crossLaps – is higher which coincides with the results of the research by J. Bogaczewicz et al. .
We have found out that the indexes of AISLE which have the highest specificity, sensitivity and the coefficient of association with the marker of bone remodeling (tabl. 5).
Diagnostic Value of Activity Index of Systemic Lupus Erythematous according to C. Bombardier et al. (1992) for Osteocalcin Characteristics
The indexes for osteocalcin are brain organic syndromes (sensitivity 19.0 %, specificity 97.0 %, preciseness 0.63, association coefficient 0.77) and thrombocytopenia (sensitivity 26.0 %, specificity 96.0 %, preciseness 0.65, association coefficient 0.83). A small amount of osteocalcin in blood serum was 7 times more often diagnosed for the patients with organic brain syndrome and 10 times more often with thrombocytopenia. The evidence of brain organic syndromes and thrombocytopenia for 84.0 and 86.0 % of cases testifies to the fact that there is a decreased amount of osteocalcin in the blood serum, while their lack for 61.0 % – about unchanged bone development marker.
Taking into consideration all the highest indexes of sensitivity and specificity we have been determining the constellation of symptoms which were the most associated with the decrease of the content of osteocalcin in the blood serum. Namely 65.0 % of the patients with the combination of such symptoms of SLE as psychosis, brain organic syndromes, headache, thrombocytopenia, leukopenia have a decreased amount of osteocalcin in the blood serum. 77.0 % of the patients with unchanged marker of bone tissue development do not possess the above mentioned symptoms (sensitivity 65.0 %, specificity 78.0 %, preciseness 0.72, association coefficient 0.74). The evidence of the combination of the above mentioned symptoms in 69.0 % of cases testifies to a small amount of osteocalcin while the lack of it for 74.0 % speaks about normal indexes of bone tissue development. The decrease of the amount of bone development marker in blood serum is 6 times more often found out for the patients with psychosis, brain organic syndromes, headaches, thrombocytopenia and leukopenia at once.
β-crossLaps (tabl. 6) possesses the highest coefficient of association with the evidence of ulcers of mucous membranes and proteinuria. This way, 23.0 % of the patient who have been diagnosed SLE with ulcers of mucous membranes had a high level of β-crossLaps. The patients with SLE, who did not suffer from mucous membrane ulcers had their β-crossLaps contents thrice higher in the blood serum (sensitivity 23.0 %, specificity 92.0 %, preciseness 80.0 %, association coefficient 0.59). The evidence of mucous membrane ulcers in 41.0 % of cases testifies to the increased amount of β-crossLaps in blood serum, while its lack for 84.0 % – is a normal index of bone resorption. 47.0 % of patients with a higher index of the marker bone tissue destruction suffered from proteinuria, while 95.0 % of patients with normal β-crossLaps – did not have any. This way, a higher index of bone destruction marker arises 20 times more often for the patients with proteinuria than without it. The risk of having a higher amount of β-crossLaps in the blood serum is 6 times higher for the patients with proteinuria. The evidence of proteinuria for 71.0 % of cases testifies to the increase of the contents of bone resorption marker in the blood serum and the absence of albumen in urine 89.0 % – which means β-crossLaps in the limits of referential values (sensitivity – 47.0 %, specificity 95.0 %, preciseness 0.87, association coefficient 0,91).
Diagnostic Value of Activity Index of Systemic Lupus Erythematous Indexes according to C. Bombardier et al. (1992) for β-crossLaps Analysis
We have also determined a constellation of indexes which are the most associated with the increase of bone destruction marker in blood serum. Headache, proteinuria and ulcers were observed in 76.0 % of patients with the increased content of β-crossLapsin blood serum, while 82.0 % of the patients with normal indexes of bone resorption did not possess such indexes combination (sensitivity 76.0 %, specificity 82.0 %, preciseness 81.0 %, association coefficient 0.88). The combination of mucous membrane ulcers, headaches and proteinuria for the patients with SLE enables us to reliably predict the changes of β-crossLaps in 81.0 %. The increased content of bone destruction marker in blood serum was diagnosed 15 times more often for the patients with the above mentioned symptoms (correlation of chances – 15.6). Prognostic value of the result which means the evidence of decreased amount of bone destruction marker in blood serum for the patients with mucous membrane ulcers, headaches, proteinuria is 50.0 %, while the absence of these three symptoms in 93.0 % testifies to the fact that the content of β-crossLaps in blood serum is within the limits of referential indexes.
The influence of complex immune inflammation on the bone remodeling indexes has been discovered according to the results of our research and has also been mentioned in the papers by other researchers [2, 5, 6]. Namely Q. Guo  demonstrated a negative correlation between the indexes of disease activity and the content of osteocalcin in blood serum as well as a direct interrelation between antidsDNA and β-crossLaps, which indicates that along with the increase of SLE activity, the process of bone remodeling is violated: osteoblast and osteoclast functions are weakened which leads to the loss of MDBT while aggravation number and duration increase lead to OP . We may consider a chronic autoimmune inflammation process a pathogenic basis for OP development which is induced by T-lymphocytes deviation and hyper development of inflammation factors (insulin-like growth factor, granulocytic – macrophage risk factor, tumor necrosis factor-α/β, interleukin (IL 1, IL 6, IL 17)) and other cytokines which influence the maturity, proliferation, osteoblast and osteoclast differentiation and their function as well.
Conclusions. Having researched the interrelation between bone remodeling markers with the index of the activity of systemic lupus erythematosus according to C. Bombardier et al. (1992), we have discovered that average content value of osteocalcin in blood serum for the patients was reliably lower than for healthy ones (13.42 ± 5.43; р < 0.05 and 18,3 ± 0.37; р < 0.05 accordingly), when β-crossLaps – reliably higher (0.40 ± 0.25 р < 0.05 and accordingly 0.26 ± 0.08), which testifies to the fact of the prevalence of bone resorption process over the process of bone development for the patients with SLE.
A total index by the scale of systemic lupus erythematosus interrcorelates with the content of osteocalcin in blood serum (r = (-0.21), p < 0.05), the increase of the index up to 1 point leads to the decrease of osteocalcin content in blood serum to 0.19 ng/ml, and also has a direct reliable correlation with β-crossLaps content in blood serum (0.30, p < 0.01), the increase of the index up to 1 point leads to the increase of β-crossLaps in blood serum up to 0.01 ng/ml, which testifies to the fact that in case of disease aggravation, osteoblast genesis is weakened and osteoclast genesis is intensified.
There exists a correlation between the markers of bone remodeling both with separate indexes of systemic lupus erythematosus activity (for the patients with such manifestations as psychosis osteoblast function is deviated and manifested by a reliable decrease of the content of osteocalcin in blood serum; alopecia – P1NP, when brain organic syndromes, myositis, thrombocytopenia, proteinuria – of osteocalcin and P1NP); for the patients with leukopenia, proteinuria, the increase of titers antidsDNA and the violation of osteoclast functions there is an increase of β-crossLaps content in blood serum), and with indexes constellation which have the highest coefficient of association with the markers of bone metabolism (the combination of psychosis, organic brain syndrome, headaches, thrombocytopenia and leukopenia with osteocalcin; headaches, proteinuria and mucous membranes ulcers – із β-crossLaps). It enables us to outline the risk groups concerning the evidence of the deviations of bone remodeling and choose a correct strategy of additional examination and the treatment of the patients with systemic lupus erythematosus accordingly.
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