Laboratory diagnostics of osteoarthritis

In most cases, patients with osteoarthritis have no changes in blood and urine tests, except for cases of synovitis with significant effusion, when there may be an increase in ESR, hypergammaglobulinemia, an increase in the level of acute phase indices - CRP, fibrinogen, etc. In the study of synovial fluid, significant differences from normal indicators do not reveal.

In recent years, an intensive search for possible biological markers (BM) for the degeneration and repair of joint tissues (mainly cartilaginous and bone) is under way. BM should reflect these dynamic changes, serve as predictors of osteoarthritis prognosis and markers of the effectiveness of pathogenetic treatment. The discovery of new and more in-depth study of known biological markers will allow us to better understand the mechanisms of the pathogenesis of osteoarthritis. However, the main task of using biological markers of cartilaginous metabolism is to assess the chondroprotective properties of drugs and to monitor treatment with drugs belonging to the DMO AD group - "disease modifying".

In osteoarthritis, pathological changes occur mainly in the articular cartilage, as well as in the subchondral bone, the synovial membrane, and other soft tissues of the joint. Since our ability to directly investigate these structures is limited, the most important sources for the collection of biological markers are blood, urine and synovial fluid.

Urine examination is most preferred, since it is not associated with any invasive procedures. In our opinion, the ideal material for the study is daily urine. Analysis of the morning portion of urine would be more appropriate, but the possibility of using it is based only on the fact that it is this analysis that is used to determine biological markers of bone metabolism in osteoporosis: it is known that biological markers are subject to circadian rhythms, and the peak concentration of biological markers of bone metabolism is night hours. To date, there is no information in the literature on circadian rhythms of biological markers of soft tissues, cartilage, so the final decision on the selection of an adequate urine test will be taken after appropriate studies.

The blood test refers to routine clinical analyzes. Some biological markers in the blood determine today, for example, indicators of the acute phase, others, perhaps in the near future will be included in the standard list of biochemical tests. For each biological marker, it is necessary to specify in which component of the blood it should be determined - in plasma or serum. The results of the studies indicate that the concentration of biological markers in blood plasma is significantly different from that in serum. Usually, biological markers are determined in serum. According to V. Rayan and co-authors (1998), the concentration of biological markers in the blood taken from a vein near the affected joint and from a more distant vein is different. These data indicate the need to standardize blood sampling for the study of biological markers.

According to LJ Attencia et al. (1989), the cartilage of synovial joints in an adult is only 10% of the total mass of the hyaline cartilage of the body, including intervertebral discs. Thus, the determination of biological markers in the blood and urine reflects a systemic metabolism rather than local changes in the joint affected by osteoarthritis. Synovial fluid is closest to the pathological focus in osteoarthritis and probably most accurately reflects the processes occurring in the affected joint. The concentration of biological markers in the synovial fluid can be significantly higher than in the blood, and therefore it is easier to determine. Examples are the epigope 846 aggrecan - in the synovial fluid it is 40 times larger than in the blood serum, cartilage oligomeric matrix proteins (HOMB) - 10 times greater than in serum. The degradation products in the synovial fluid more accurately reflect catabolic processes in the articular cartilage. Drainage of molecules from the synovial fluid through the local lymphatic system can lead to a decrease in their size and even to their destruction.

Despite the invasiveness of the technique of samovial fluid intake, coupled with a number of possible complications, the value of determining biological markers in it is obvious. To avoid problems with the so-called dry joint, just before the removal of fluid in the joint, you can enter 20 ml of isotonic NaCl solution. Immediately after the injection of the isotonic solution, the patient must perform 10 fold flexion-extension of the limb in the joint, followed by rapid aspiration of the diluted synovial fluid. According to EM-JA Thonar (2000), such a dilution of synovia affects metabolism in articular cartilage. However, the results of the study by FC Robion and co-authors (2001) indicate that repeated lavage of the horse's knee joints does not cause significant changes in cartilage metabolism. These data, of course, require confirmation. Therefore, for each biological marker at the preclinical research stage in animals it is necessary to determine the effect of lavage of the joint on the change in its concentration.

The next important point is the determination for each biological marker of half-life in the synovial fluid and in the blood. Without such data, interpretation of the test results will be difficult. Typically, the half-life of biologically active substances in the blood is less than in other liquid media, due to effective clearance of the liver and kidneys. Thus, for each biological marker it is also necessary to determine the path of elimination. Thus, the N-propeptide of type III collagen is secreted by the liver via receptor-mediated endocytosis, and the non-glycosylated fragments of collagen are excreted mainly by wetting, as well as by cacosteocalcin. The endothelial cells of the sinuses of the hepatic lobules have receptors for glycosaminoglycans, so hyaluronic acid and proteoglycans are eliminated by the liver. The half-life of hyaluronic acid in the blood is 2-5 minutes. The presence of synovitis may accelerate clearance of biological markers from the joints, although the study in rabbits showed no significant differences in the clearance of proteoglycans in the presence and absence of synovitis. Thus, it is necessary to investigate the effect of inflammation on changes in the concentration of biological markers in body fluids.

Kidneys selectively filter biological markers. Thus, glycosaminoglycans bearing a large negative charge may not penetrate the renal basement membrane, whereas glycosaminoglycans such as chondroitin-6-sulfate and chondroitin-4-sulfate are detected in the urine.

In addition to pathology (in particular, osteoarthrosis), the concentration of biological markers in body fluids can be affected by a number of factors:

1. Circadian rhythms have been studied only for a small number of biological markers. For markers of bone metabolism, they are studied. Thus, the peak concentration of osteocalcin falls on the night hours, and the collagen cross-links for the morning - for 8 hours. In rheumatoid arthritis, the peak of activity of IL-6 also occurs at night hours (about 2 hours), and earlier than osteocalcin. These data are of some interest in the involvement of IL-6 in inflammation and in the physiology of bone tissue. TNF, on the contrary, does not have circadian rhythms. However, the receptors of this cytokine can be subordinated to them.
2. Peristalsis. Hyaluronic acid is synthesized by synovial cells (as well as many other cells) and is a potential marker of synovitis in osteoarthritis and rheumatoid arthritis. However, the highest concentration of hyaluronate is found in the lymphatic system of the intestine. It is not surprising that the concentration of circulating hyaluronic acid can increase after eating. Thus, blood sampling for determining biological markers should be performed on an empty stomach or 3 hours after eating. And the influence of peristalsis on the level of biological markers in the blood requires study.
3. Physical activity in the morning after sleep leads to an increase in the concentration of hyaluronic acid in the blood, MMP-3 and the epitope of keratan-sulfate in healthy individuals. Physical load can change the concentration of some markers in both synovial fluid and serum. This increase is more pronounced in patients with rheumatoid arthritis, moreover, the concentration of biological markers correlates with the clinical state of these patients.
4. Diseases of the liver and kidneys. Cirrhosis causes a significant increase in the level of hyaluronic acid in the blood serum and, probably, affects the elimination of proteoglycans. It is known that kidney diseases affect the concentration of osteocalcin. This problem also requires a deeper study.
5. Age and sex. During the growth of the body, the activity of growth plate cells increases, which is accompanied by an increase in the concentration of skeletal biological markers in the blood serum. An example is the increase in the concentration of fragments of aggrecan and collagen type II in peripheral blood and urine in growing animals. Thus, the interpretation of analyzes of biological markers in children and adolescents with diseases of the musculoskeletal system is difficult. For many biological markers, an increase in the concentration with aging has been found. In men, the concentration of biological markers is significantly higher than that of women in the cartilaginous and bone tissues. In addition, in women in the menopausal and postmenopausal periods, changes in the concentration of biological markers of the metabolism of cartilaginous tissue can be expected in a manner similar to that observed in bone tissue.
6. Surgical operations can also affect the level of biological markers, moreover, this effect can last several weeks.

The basis of the concept of biological markers of osteoarthritis is the assumption that they reflect certain aspects of metabolic processes in the tissues of the joints. However, the relationship between the concentrations of biological markers in the liquid media of the body and the metabolism of cartilaginous, synovial and other tissues proved to be very complicated.

Thus, for example, the concentration of markers for the degradation of ECM of articular cartilage in synovial fluid can depend not only on the degree of degradation of the matrix itself, but also on other factors such as the degree of elimination of fragments of molecules from synovia, as already mentioned above, cartilage tissue left in the joint.

Despite the above facts, the concentration of biological markers in the synovial fluid generally correlates with the metabolism of VKM molecules of the articular cartilage. For example, changes in the concentration of fragments of aggrecan, 846 epitope, HOMB and C-propeptide of collagen II in synovial fluid after joint trauma and in the development of osteoarthritis are consistent with changes in the intensity of metabolism of aggrecan, HOMB and collagen II in experimental models of osteoarthrosis in animals / and vivo and in the articular cartilage of patients with osteoarthritis / and vitro.

Identification of specific sources of molecular fragments is a complex process. An increase in the degree of release of fragments of molecules can occur both as a result of a general increase in the degradation processes that are not compensated by synthetic processes or due to enhanced degradation, while simultaneously increasing the synthesis of the same ECM molecules; in the latter case the concentration of VKM molecules does not change. Thus, it is necessary to search for markers specific for degradation and for synthesis. Examples of the former may be fragments of aggrecan, and the second - C-propeptide of collagen 11.

Even if the biological marker is associated with a certain aspect of metabolism, it is necessary to take into account the specific features of this process. For example, identifiable fragments can be formed as a result of degradation of a de novo synthesized molecule that has not yet had time to integrate into the functional ECM, a molecule that has just been built into the ECM and finally a constant ECM molecule that is an important functional part of the mature matrix. The problem is also the determination of a specific matrix zone (pericellular, territorial and interterritorial matrix), which served as a source of biological markers found in synovial fluid, blood or urine. Studies in vitro indicate that the metabolic rate in the individual zones of articular cartilage ECM may be different. The study of some epitopes associated with the sulfation of chondroitin sulfates can help identify the population of de novo-synthesized aggrecan molecules.

It can be assumed that the appearance in the joint fluid of fragments of molecules, normally present in cartilage VKM, is associated with the metabolism of the cartilaginous matrix. However, this is not always so, because it depends on a number of factors, in particular, on how much the concentration of this molecule in the articular cartilage exceeds that in other tissues of the joint and how much its cartilage metabolism exceeds that in the other tissues of the joint. Thus, the total mass of aggrecan in the articular cartilage is much greater than that, for example, in the meniscus of the knee joint, while the total mass of HOMB in the meniscus practically does not differ from that in the articular cartilage. Both chondrocytes and synovitises produce stromelysin-1, but the total number of cells in the synovial membrane exceeds that in the cartilage, so a significant part of the stromelysin-1 found in the synovial fluid is probably of synovial origin. Thus, the identification of a specific source of biological markers is extremely complex and often impossible.

In the study of biological markers in serum and urine, the problem of determining a possible extra-articular source appears. In addition, in case of monoarticular damage, biological markers allocated by the affected joint can be mixed with markers allocated by intact joints, including contralateral ones. The composition of articular cartilage is less than 10% of the total mass of the hyaline cartilage of the body. Thus, the determination of biological markers in the blood and urine can be justified in polyarticular, or systemic, diseases (for osteoarthrosis, in generalized osteoarthritis).

Requirements for biological markers depend on the purpose for which they are used - as a diagnostic, prognostic or evaluation test. For example, a diagnostic test identifies the differences between healthy individuals and patients with osteoarthritis, which is expressed by the concept of sensitivity and specificity of the test. A prognostic test reveals in the cohort of persons who are most likely to progress rapidly. Finally, the evaluation test is based on the ability of the marker to monitor changes in time in an individual patient. In addition, biological markers can be used to determine the sensitivity of patients to a particular drug.

Initially, it was assumed that biological markers can serve as diagnostic tests that will help to distinguish the joint affected by osteoarthritis from intact, as well as conduct differential diagnosis with other joint diseases. So, the determination of serum keratan sulfate concentration was considered as a diagnostic test for generalized osteoarthritis. However, subsequent studies have shown that this biological marker can only reflect the degradation of cartilage proteoglycans in some situations. It turned out that the concentration of biological markers in the serum depends on the age and sex of the subject.

Alleged biological markers of joint tissue metabolism in synovial fluid and serum of patients with osteoarthritis

Biological marker	Process	In synovial fluid (links)	Serum (links)
1. The cartilage
Aggrecan
Fragments of the core protein	Aggrecan Degradation	Lohmander LS. Et al., 1989; 1993	Thonar EJMA et al., 1985; Campion GV et al., 1989; MehrabanF. Et al., 1991; Spector TD et al., 1992; Lohmander LS., Thonar EJ-MA, 1994; PooleAR et al., 1994) t (Poole AR et al., 1994)
Epitopes of the core protein (specific neoepitopes of the cleavage zone)	Aggrecan Degradation	Sandy JD et al., 1992; LohmanderLS. Etal., 1993; LarkM.W. Etal., 1997
Epitopes of keratan sulfates	Aggrecan Degradation	Campion GV et al., 1989; Belcher C et al., 1997
Epitopes of chondroitin sulfate (846, ZVZ, 7D4 and DR.)	Synthesis / degradation of aggrecan	Poole AR et al., 1994; HazellP.K. Et al., 1995; Slater RR Jr. Etal., 1995; Plaas AHK et al., 1997; 1998; Lohmander LS. Etal., 1998
The ratio of chondroitin-6-and chondroitin-4-sulfates	Synthesis / degradation of aggrecan	Shinme iM. Et al .. 1993
Small proteoglycans	Degradation of small proteoglycans	Witsch-PrehmP. Et al., 1992
Cartilage matrix proteins
HOMP	Degradation of HOMP	Saxne T., Heinegerd D., 1992 "LohmanderLS, et al., 1994; Petersson IF et al., 1997	Sharif M. Et al., 1995
Collagen cartilage
C-propeptide of type II collagen	Synthesis of collagen II	ShinmeiM. Etal., 1993; YoshiharaY. Et al., 1995; LohmanderLS. Etal., 1996
Fragments of the a chain of type II collagen	The degradation of collagen II	Hollander AP et al., 1994; Billinghurst RC etal., 1997; AtleyLM. Etal., 1998
MMP and their inhibitors	Synthesis and secretion	From synovia or articular cartilage?
II. Menisci
HOMP	Degradation of HOMP	From articular cartilage, meniscus or synovia?
Small proteoglycans	Degradation of small proteoglycans
III. The synovium
Hyaluronic acid	Synthesis of hyaluronic acid		Goldberg RL et al., 1991; HedinP.-J. Et al., 1991; Sharif M. Etal., 1995
MMP and their inhibitors
Stromelysin (MMP-3)	Synthesis and secretion of MMP-3	LohmanerLS. Et al., 1993	ZuckerS. Et al., 1994; YoshiharaY. Etal., 1995
Interstitial collagenase (MMP-1)	Synthesis and secretion of MMP-1	Clark IM et al., 1993; LohmanderLS. Etal., 1993	Manicourt DH et al., 1994
TIMP	Synthesis and secretion of TIMP	Lohmander LS. Et al., 1993; Manicourt DH et al., 1994	Yoshihara Y. Et al., 1995
N-propeptide of type III collagen	Synthesis / degradation of collagen III	Sharif M. Et al., 1996	Sharif M. Et al., 1996

A number of studies have demonstrated differences in the concentrations of fragments of Aggrecan, HOMP and MMP and their inhibitors in articular fluid of knee joints in healthy volunteers, patients with rheumatoid arthritis, reactive arthritis or osteoarthrosis. Although the authors show significant differences in mean concentrations of biological markers, interpretation of data is difficult because The comparative analysis carried out was profile and retrospective. The prognostic properties of these tests need to be confirmed in prospective studies.

Biological markers can be used to assess the severity of a disease or the staging of a pathological process. With regard to osteoarthrosis, the severity of the disease and its stages are judged by the results of x-ray studies, arthroscopy, as well as by the severity of the pain syndrome, restriction of the function of affected joints and the functional capacity of the patient. L. Dahlberg and co-authors (1992) and T. Saxne and D. Heinegard (1992) proposed the use of certain molecular markers of articular cartilage metabolism for additional characterization of the stages of osteoarthritis. However, for the introduction of such biological markers into medical practice, further research is needed in this direction.

There are reports of the possible use of biological markers as prognostic tests. For example, it was shown that the concentration of hyaluronic acid (but not keratan sulfate) in the blood serum in patients with knee osteoarthritis at the beginning of the study indicates a progression of gonarthrosis during 5 years of follow-up. In the same population of patients, it was demonstrated that elevated serum levels of HMB in patients with gonarthrosis were associated with radiological progression within 5 years of follow-up within the first year after the onset of the study. Studies of biological markers in patients with rheumatoid arthritis showed that the concentration in the serum of HOMB, epitope 846, chondroitin sulfate is associated with faster disease progression. These results, obtained in small groups of patients, often do not demonstrate the strength of the relationship between the level of biological markers and the progression of the disease, ie, further studies are needed, prospective and in large cohorts of patients.

TD Spector and co-authors (1997) found a small increase in serum CRP levels in patients with early osteoarthrosis and reported that CRP can serve as a predictor of progression of osteoarthritis. In this case, the increase in the level of CRP reflects the processes of damage to joint tissues and may be associated with an increase in the level of hyaluronic acid, also indicative of the progression of the disease. It is possible that the synovial membrane is responsible for most of the hyaluronic acid detected in the blood serum, which indicates the presence of a weak synovitis. An increase in the concentration of MMP stromelysin in the synovial fluid and serum of patients with osteoarthritis and after joint trauma may also be associated with a weak synovitis.

Finally, biological markers can be used as efficacy criteria in clinical trials of drugs, as well as for monitoring pathogenetic treatment. However, there are two interrelated problems: the lack of drugs with the proven properties of "modify the structure" or "modify the disease" is largely due to the lack of reliable biological markers and vice versa, the lack of specific markers of joint tissue metabolism is largely due to the lack of controlled studies of the drugs of these groups.

[1], [2], [3], [4]