Serum nitrate and nitrite levels in patients with rheumatoid arthritis, ankylosing spondylitis, and osteoarthritis

OBJECTIVE: To assess and compare serum nitrate and nitrite levels in patients with ankylosing spondylitis (AS), rheumatoid arthritis (RA), and osteoarthritis (OA). METHODS: Thirty five patients with RA, 32 patients with AS, and 36 patients with OA were entered into this study. In addition, 30 healthy volunteers acted as a control group. Concentrations of nitrate and nitrite in serum were determined by direct and indirect Griess reactions. C reactive protein and erythrocyte sedimentation rate levels were determined as markers of systemic activity of disease (SAD) in RA and AS groups. RESULTS: Serum nitrate and nitrite levels were found to be higher in patients with AS and RA than in the OA group (p<0.01). In addition, serum nitrate and nitrite levels were higher in all three groups than in the control group (p<0.01). Moreover, serum nitrate and nitrite levels were higher in patients who had SAD than in those who had not in the RA and AS groups (p<0.01 and p<0.05, respectively), and there was a correlation between serum nitrate and nitrite concentrations and SAD variables in patients with RA (Spearman's r(s)=0.414, p<0.05 and r(s)=0.408, p<0.05, respectively) and AS (r(s)=0.421, p<0.05 and r(s)=0.412, p<0.05, respectively). CONCLUSION: The findings suggest that nitrate and nitrite production is enhanced in patients with inflammatory arthritis compared with OA. In addition, serum nitrate and nitrite levels are enhanced in patients with RA, AS, and OA compared with healthy subjects. Furthermore, there is a correlation between the SAD variables and serum nitrate and nitrite levels in patients with RA and AS.     

SERUM BIOCHEMISTRY IN RHEUMATOID ARTHRITIS, SERONEGATIVE ARTHROPATHIES, OSTEOARTHRITIS, SLE AND NORMAL SUBJECTS

Most arthritic conditions are characterized by chronic inflammation,resulting in secondary changes in serum biochemistry. In anattempt to profile different mechanisms of inflammation whichmight account for the clinical diversity of rheumatic diseases,we have measured C-reactive protein (CRP), plasma viscosity,serum histidine and total serum sulphydryl in 259 patients withrheumatoid arthritis (RA), 84 with ankylosing spondylitis (AS),76 with osteoarthritis, 69 with psoriatic arthritis, 34 withsystemic lupus erythematosus (SLE), 36 with Reiter's syndromeand 121 normal controls. The most extreme abnormalities were seen in rheumatoid arthritisand the least in osteoarthritis. The seronegative spondarthritidesand SLE occupied a midway position, emphasizing a correlationbetween biochemical abnormality and severity of inflammation. A low serum histidine characterized both RA and SLE. The formerwas more likely to be associated with a raised CRP. Plasma viscositywas characteristically raised in psoriatic arthritis and CRPin AS. KEY WORDS: Serum biochemistry, Rheumatoid arthritis, Seronegative arthropathies     

Serum and synovial fluid levels of angiotensin converting enzyme in polyarthritis.

Serum levels of angiotensin converting enzyme (ACE) activity in patients with rheumatoid arthritis (RA) (n = 48), osteoarthritis (OA) (n = 11), ankylosing spondylitis (n = 24), psoriatic arthritis (n = 12), and Behçet''s syndrome (n = 20) were not significantly different from those of normal controls (n = 26). Synovial fluid ACE activity was lower in OA than in RA but was similar when corrected for protein levels. An increase in serum ACE concentration in patients with RA receiving captopril therapy is in agreement with previous results. There was some correlation of ACE with erythrocyte sedimentation rate (ESR) and C reactive protein (CRP) but not with clinical indices in captopril treated patients. It is suggested that the beneficial actions of captopril in the treatment of RA are not due to its activity as an ACE inhibitor, but more probably a result of captopril being an aliphatic thiol.     

Humoral immunity to link protein in patients with inflammatory joint disease, osteoarthritis, and in non-arthritic controls.

Cartilage link protein of high purity was prepared and used in an enzyme linked immunosorbent assay (ELISA). Antibodies to link protein were sought in the sera of 98 patients with rheumatic disorders; 38 with rheumatoid arthritis (RA), 29 with osteoarthritis (OA), 13 with psoriatic arthritis (PA), nine with ankylosing spondylitis (AS), nine with systemic lupus erythematosus (SLE), and in 83 healthy controls. Antibodies were detected in all groups with the following prevalences: 21/83 normals, 9/38 RA, 7/29 OA, 7/13 PA, 3/9 AS, and 4/9 SLE. No statistically significant differences existed between the groups with regard to either prevalence or mean titre of anti-link antibodies. Serum antibodies to proteoglycan link protein appear to be no more common in patients with rheumatic disorders than in healthy controls.     

THE RELEVANCE OF CHONDROITIN AND KERATAN SULPHATE MARKERS IN NORMAL AND ARTHRITIC SYNOVIAL FLUID

This study investigated the synovial fluid concentrations ofglycosaminoglycan (GAG), keratan sulphate (KS) epitope 5D4 andchondroitin sulphate (CS) sulphation patterns in healthy volunteersand patients with osteoarthritis (OA) and rheumatoid arthritis(RA). Synovial fluids were collected from knee joints of healthyvolunteers (n = 24), and patients with OA (n = 28) and RA (n= 29). Concentrations of GAG and the keratan sulphate epitopeSEW were measured in 15 of the healthy volunteers, and all ofthe OA and RA synovial fluids. Total GAG was measured usinga dye-binding method and 5D4 by an ELISA. The unsaturated CSdisaccharides     

Increased matrix metalloproteinase-3 serum levels in rheumatic diseases: relationship with synovitis and steroid treatment

OBJECTIVE: To determine matrix metalloproteinase-3 (MMP-3) serum levels in patients with rheumatic diseases and to study the relation between MMP-3 and C reactive protein (CRP) levels. METHODS: MMP-3 serum levels were determined by enzyme linked immunosorbent assay (ELISA) in (a) patients with active inflammatory rheumatic diseases: rheumatoid arthritis (RA), psoriatic arthritis, polymyalgia rheumatica, acute crystal arthritis, and ankylosing spondylitis; (b) patients with active inflammatory systemic diseases: cutaneo-articular or renal systemic lupus erythematosus (SLE), systemic sclerosis, and vasculitides; (c) patients with non-inflammatory rheumatic diseases: osteoarthritis and fibromyalgia; (d) critically ill patients without rheumatic diseases, representing an acute inflammatory control group; (e) healthy controls. RESULTS: MMP-3 serum levels were significantly increased in patients with active RA, psoriatic arthritis, and polymyalgia rheumatica, whether treated or not by corticosteroids, and in female patients with acute crystal arthritis. MMP-3 serum levels were normal in steroid-free patients with active cutaneo-articular or renal SLE, systemic sclerosis, and vasculitides but were significantly increased in steroid treated patients. MMP-3 levels were normal in fibromyalgia, osteoarthritis, ankylosing spondylitis, and acute inflammatory controls. MMP-3 was significantly correlated with CRP in RA (r=0.5, p=0.0004) but not in any of the other disease groups. CONCLUSIONS: MMP-3 serum levels are increased in inflammatory rheumatic diseases characterised by joint synovitis, such as RA, polymyalgia rheumatica, psoriatic arthritis, and acute crystal arthritis-that is, whether the diseases are acute or chronic, erosive or not. They are normal in SLE, systemic sclerosis, and vasculitides as well as in non-rheumatic inflammatory controls, but are significantly increased by steroids. These data strongly suggest that serum MMP-3 reflects synovial inflammation.     

C reactive protein and immunoglobulin G in synovial fluid and serum in joint disease.

C reactive protein (CRP) and immunoglobulin G (IgG) were measured in synovial fluid and serum of 72 patients (29 with rheumatoid arthritis (RA), 17 with osteoarthritis, 11 with crystal synovitis, seven with undifferentiated arthritis, and eight with seronegative arthritis). The synovial fluid:serum (SF:S) ratios were compared with those calculated from the SF:S ratios of transferrin, caeruloplasmin, and alpha 2 macroglobulin, using the binomial test within groups and the Mann-Whitney test between groups. In RA synovial fluid CRP concentrations were lower than expected and IgG concentrations higher than expected. In osteoarthritis CRP concentrations were higher than expected. In seronegative arthritis IgG concentrations were raised. The ratio of CRP:IgG was depressed in RA. These findings are consistent with a role for CRP in the inflammatory process of RA, while the CRP:IgG ratio may be of value in the differential diagnosis of joint disease.     

Comparative studies of serum and synovial fluid C reactive protein concentrations.

The relation between serum and synovial fluid (SF) C reactive protein (CRP) concentrations was investigated in a variety of arthritides, including rheumatoid arthritis (RA), psoriatic arthritis, reactive arthritis, and osteoarthritis. SF CRP levels were significantly reduced compared with serum levels in the inflammatory arthritides, but there was good correlation between serum and SF values. SF CRP values were all at the lower limit of the detectable range in osteoarthritis. In patients with RA or psoriatic arthritis followed up serially through an exacerbation of arthritis, changes in SF CRP reflected closely changes in serum CRP. In patients with RA SF/serum ratios of proteins of different molecular weight were used to derive a regression equation between SF/serum ratio and molecular mass. SF/serum values for CRP were significantly less than predicted from its molecular weight, suggesting that CRP is either being selectively bound in synovium or specifically consumed in SF and may be playing an important part in the inflammatory process in RA.     

Serum hyaluronic acid and aminoterminal procollagen III peptide in inflammatory and degenerative joint diseases

Serum levels of hyaluronic acid (HA) and the amino-terminal type III procollagen peptide (NP-III-P) were determined simultaneously by specific immunoassays in patients with rheumatoid arthritis (n = 41), osteoarthritis (n = 43), ankylosing spondylitis (n = 7), psoriatic arthritis (n = 6), and reactive arthritis (n = 6). Increased serum levels of both HA and NP-III-P, were found in rheumatoid arthritis and - although less pronounced - in osteoarthritis, differing significantly from age- and sex-matched controls (n = 77). Furthermore, patients suffering from active rheumatoid arthritis showed higher serum levels of both antigens than patients with inactive disease, and significant correlations were found in rheumatoid arthritis between acute phase plasma proteins, HA and NP-III-P, respectively. In contrast, determination of low molecular weight fractions of NP-III-P by Fab- assay proved not to be useful in regard to clinical application. No significant effects of anti-inflammatory treatment were evident in any of the parameters. In rheumatoid patients, the serum concentrations of HA were found to correlate positively with the serum reactivity of NP-III-P related antigens (r = 0.692) and with the excretion of urinary pyridinoline (r = 0.455). Thus, both parameters seem to reflect similar mechanisms of connective tissue activation and may be related to inflammatory activity in joint diseases.     

Serum IgA, acute phase proteins, and glycosylation of alpha 1-acid glycoprotein in ankylosing spondylitis.

Several investigators have suggested that gastrointestinal inflammation has a role in the pathogenesis of ankylosing spondylitis. To test this hypothesis markers of gastrointestinal immunostimulation, as manifested by serum IgA concentrations, were compared with serum markers of inflammation, as manifested by acute phase proteins. Serum samples from 45 unrelated Caucasian patients with ankylosing spondylitis (AS) were tested for correlation of serum IgA and six acute phase proteins: C reactive protein (CRP), alpha 1-antitrypsin, alpha 1-antichymotrypsin, caeruloplasmin, alpha 1-acid glycoprotein (AGP), and haptoglobin. Serum IgA was shown to be significantly positively correlated with four of these six acute phase proteins: CRP (r = 0.58, p less than 0.001), alpha 1-antitrypsin (r = 0.29, p less than 0.05), AGP (r = 0.61, p less than 0.01), and haptoglobin (r = 0.58, p less than 0.001), suggesting that gastrointestinal immunostimulation does have a role in the pathogenesis of inflammation in AS. In addition, the microheterogeneity of the pattern of glycosylation of AGP, expressed as reactivity coefficients, was examined. The AGP reactivity coefficient has been shown to increase in infection, remain the same in systemic lupus erythematosus, and decrease in rheumatoid arthritis. It was found that the AGP reactivity coefficient was significantly decreased in patients with AS as compared with healthy controls (p less than 0.006). As recent studies have indicated that patterns of glycosylation reflect intrahepatocellular biosynthetic processes induced by cytokines our data suggest that cytokine-hepatocellular mechanisms in AS may be similar to those occurring in rheumatoid arthritis, but different from those in systemic lupus erythematosus or infection.     

Further studies on serum lipoproteins in connective tissue diseases.

Men with ankylosing spondylitis (AS) and females with systemic lupus erythematosus (SLE) were found to have low total serum lipid concentrations similar to results previously obtained in patients with rheumatoid arthritis (RA). In AS men total serum TG was about 50% of control values and in AS men and SLE women total serum cholesterol was 78% of control values but close to corresponding RA concentrations. This was explained mainly by low LDL concentrations. There was a marked difference between RA patients and AS and SLE patients in that the two latter groups had normal HDL cholesterol concentrations whereas in RA patients the HDL cholesterol concentration was only 70% of control values. Thus in spite of similar and low total serum lipid concentrations, differences in lipoprotein composition were found in the three different rheumatic diseases, underlining the importance of lipoprotein analyses in the study of dyslipoproteinaemia.     

Incidence of serum antibodies to native type I and type II collagens in patients with inflammatory arthritis.

Using a new solid-phase double-antibody radioimmunoassay we have determined the incidence of serum IgG antibodies to native bovine type I and type II collagens in patients with rheumatoid arthritis (RA), ankylosing spondylitis (AS), and psoriatic arthritis. Raised serum IgG antibody levels to native type I collagen were present in 49% of patients with RA, 30% with AS, and none of the patients with psoriatic arthritis. Raised serum IgG antibody levels to native type II collagen were present in 42% of patients with RA, 30% with AS, and none of the patients with psoriatic arthritis. In rheumatoid arthritis there was a lack of correlation between IgG antibody levels to collagen and the erythrocyte sedimentation rate, IgG rheumatoid factor, and circulating immune complexes measured by the Clq-binding activity. In ankylosing spondylitis IgG antibody levels to native type II collagen were raised only in patients with peripheral joint involvement. The significance of IgG anticollagen antibodies is not certain, but parallels with rheumatoid factor are discussed.     

CARTILAGE OLIGOMERIC MATRIX PROTEIN: A NOVEL MARKER OF CARTILAGE TURNOVER DETECTABLE IN SYNOVIAL FLUID AND BLOOD

Cartilage oligomeric matrix protein (COMP) is a tissue specificnon-collagenous matrix protein. We have developed an enzyme-linkedimmunosorbent assay for the detection of this protein in synovialfluid and serum. The protein has been quantified in these fluidsin patients with rheumatoid arthritis (RA), reactive arthritis,juvenile chronic arthritis, osteoarthritis and in sera of controlsubjects. The protein was detectable in all fluids and the synovialfluid levels were always higher than in serum in paired samples.The highest knee joint synovial fluid levels were found in reactivearthritis patients and the lowest in RA patients with advanceddestruction of the knee joint. However, the relative synovialfluid content of COMP was higher in these RA patients than inpatients with advanced osteoarthritis. In patients with longstandingreactive synovitis the concentrations decreased. This decrease,however, was less marked than for proteoglycan concentrations. The serum concentrations were low in patients with juvenilechronic arthritis and in patients with RA with advanced cartilagedestruction of the studied knee joint. In the other groups serumlevels did not differ between groups or from controls. KEY WORDS: Cartilage matrix, Noncollagenous protein, Rheumatoid arthritis, Proteoglycans     

Correlation of immunoglobulin and C reactive protein levels in ankylosing spondylitis and rheumatoid arthritis.

Serum C reactive protein (CRP), IgG, and IgA levels were measured in 22 patients with ankylosing spondylitis (AS) and in 20 patients with rheumatoid arthritis (RA) to study the regulation of these proteins in inflammatory disease states. In both RA and AS the mean CRP, IgG, and IgA levels were raised above normal values. Although IgA and CRP levels showed a significant positive correlation in RA (r = 0.53, p = 0.02), there was no correlation between these values in AS (r = 0.24, p = 0.29). The difference in correlation coefficients between the AS and RA groups was significant at a p = 0.05 level. In RA the raised IgA levels may be another manifestation of the acute phase response, as shown by the good correlation between IgA and CRP in that disease. In AS, however, the IgA levels, although raised, do not correlate with CRP levels, suggesting that the mechanism of increase of IgA in the two diseases is different. Gut mediated immune stimulation has been proposed as a cause of raised IgA levels in AS.     

Reactivity of serum antibodies to the keratin layer of rat esophagus in patients with rheumatoid arthritis

Serum antibodies reactive with the keratin layer of rat esophagus (AKA) were found in 46 of 80 (57.5%) rheumatoid arthritis (RA) patients. In contrast, AKA were present in only 7 of 82 (9.5%) patients with other types of rheumatic disorders and in 2 of 47 (4.2%) healthy subjects. AKA were not specific for RA, however, because in the former group, AKA were present in 4 of 20 (20%) systemic sclerosis patients and in 3 of 12 (25%) ankylosing spondylitis patients. AKA belong predominantly to the IgG class and are complement fixing. Although found in some RA joint fluids, AKA were not selectively concentrated in the joint fluid. Absorption of RA serum with type I human collagen or with human epidermal keratin did not remove AKA activity. The frequency of AKA in RA patients both negative and positive for DR4 was equal. There was no relationship between the frequency of AKA and the occurrence of other serum autoantibodies such as antibodies to intermediate filaments, smooth muscle, and nuclear antigens. Serum antibody reactive with human stratum corneum found in patients with psoriatic arthritis was shown to be different from AKA. Rabbit antiserum to human keratin did not inhibit the reaction of AKA against the keratin layer of rat esophagus. Autoimmunity to structural proteins including collagen, vimentin intermediate filaments, smooth muscle antigens, and keratin is a characteristic feature of RA.     

Impairment of neutrophil Fc gamma receptor mediated transmembrane signalling in active rheumatoid arthritis.

Neutrophil Fc gamma receptor (Fc gamma R) signalling responses were compared in healthy subjects, patients with definite rheumatoid arthritis (RA), ankylosing spondylitis, and osteoarthritis. The patients with A were subdivided into those with active synovitis and those with quiescent disease. Basal intracellular calcium ion concentrations in patients with inactive RA were significantly higher than in control subjects, which in turn were greater than in patients with active RA. Transient cytosolic calcium ion fluxes were observed after binding Fc gamma RII or Fc gamma RIII with specific monoclonal antibodies and cross linking with the F(ab')2 fragment of antimouse IgG. Response times were significantly faster for Fc gamma RII than for Fc gamma RIII. Peak concentrations of intracellular calcium ions after neutrophil stimulation were comparable for Fc gamma RII and RIII in healthy subjects. Neutrophils in patients with ankylosing spondylitis and osteoarthritis responded to Fc gamma R triggering, but in the group with active RA fluxes of calcium ions were severely depressed. Neutrophils isolated from patients with RA with quiescent disease showed exaggerated responses when compared with controls. Expression of all three Fc gamma R types on neutrophils from patients with active RA, as measured by monoclonal antibody binding, was comparable with control cells. Impairment of neutrophil Fc gamma R cytosolic signalling in active RA could reflect a receptor signalling defect with potential effects on Fc mediated functions, or a fundamental defect in calcium ion homeostasis within these cells.     

Immunoglobulin isotype composition of circulating and intra-articular immune complexes in patients with inflammatory joint disease

Summary The immunoglobulin (Ig) heavy chain isotype composition of intra-articular and circulating immune complexes (ICs) were determined by a Raji cell flow cytometric assay in paired serum and synovial fluid samples from 15 patients with rheumatoid arthritis (RA) and 15 patients with other articular diseases (osteoarthritis, ankylosing spondylitis, gout, psoriatic arthritis, Reiter's discase). ICs were most prevalent in synovial fluid samples of patients with RA but were infrequently detected in serum and synovial fluid samples from the non-RA patients. ICs in patients with RA were heterogeneous both in the prevalence of Ig subclasses identified and in the distribution of the respective Ig isotypes within the complexes. Furthermore, differences were observed in the Ig isotype composition of ICs in paired serum and synovial fluid samples in dicating that circulating ICs may not always arise simply by spill-over from articular sites. The possible mechanisms for IC formation in RA are discussed with reference to four patients who displayed features of extra-articular disease.     

Comparison between serum alpha 2-pregnancy-associated globulin and activity of rheumatoid arthritis and ankylosing spondylitis during pregnancy

A possible association between alpha 2-pregnancy-associated globulin (alpha 2-PAG) and activity of rheumatoid arthritis (RA) during pregnancy was investigated. In a prospective study, the correlation between disease activity and serum alpha 2-PAG levels during and after pregnancy was evaluated in 11 women with RA. For comparison, 12 women with ankylosing spondylitis (AS) and 14 healthy women were also studied. No correlation between improvement of disease activity and serum alpha 2-PAG levels could be detected in pregnant RA or AS patients. In contrast, a positive association between total disease activity score and serum alpha 2-PAG levels was found in pregnant RA and AS patients.     

Autoantibodies to type II collagen: occurrence in rheumatoid arthritis, other arthritides, autoimmune connective tissue diseases, and chronic inflammatory syndromes.

Serum IgG antibodies to native and denatured human type II collagen (Col II) were measured using an enzyme linked immunosorbent assay (ELISA). One hundred and thirty one patients with various forms of arthritis such as rheumatoid arthritis (RA), ankylosing spondylitis (AS), psoriatic arthritis (PSA). Reiter's Syndrome (RS), osteoarthritis (OA), and gout, 60 with autoimmune connective tissue disease, and 37 with the chronic inflammatory conditions--graft versus host disease and leprosy--were studied. With the exception of RS, PSA, OA, and gout, significant levels of Col II antibodies were detected in each disease group. Blocking studies with types I and II collagen on selected serum samples confirmed the specificity to native Col II, though some cross reactivity was apparent with denatured collagen. The patients with RA who were Col II antibody positive tended to fall into stage III of disease progression. There was, however, no correlation with rheumatoid factor, erythrocyte sedimentation rate, or disease duration and this, together with the finding that Col II antibodies are present in a wide array of diseases, makes their role in the pathogenesis of RA questionable. They may arise as a secondary disease perpetuating mechanism in some patients, or in turn may be an epiphenomenon secondary to generalised disturbed immunoregulation or B cell hyperreactivity, or both, that characterises these clinical conditions.     

Raised serum vascular endothelial growth factor levels are associated with destructive change in inflammatory arthritis

OBJECTIVE: To determine whether elevated levels of the angiogenic cytokine vascular endothelial growth factor (VEGF), detected on presentation to an early arthritis clinic, are associated with the development of chronic and erosive arthritis. METHODS: Concentrations of VEGF and its soluble receptor, soluble fms-like tyrosine kinase 1 (sFlt-1), were measured by enzyme-linked immunosorbent assay in serum samples from patients with early (<2 years from onset) arthritic symptoms in the peripheral joints, namely early rheumatoid arthritis (RA), self-limiting arthritis (viral, reactive, and idiopathic inflammatory arthritis), or psoriatic arthritis. In addition, measurements were made in random samples from patients with longstanding (>3 years from symptom onset) RA treated with disease-modifying antirheumatic drugs, from patients with osteoarthritis (OA), and from patients with polyarthralgia without arthritis, as well as from nonarthritic controls. RESULTS: Serum VEGF levels at presentation were elevated in patients with inflammatory arthritis (RA, psoriatic, and self-limiting arthritis) as well as in patients with OA, in comparison with nonarthritic controls. Moreover, serum VEGF concentrations were significantly higher in patients with early RA than in patients with self-limiting arthritis. Serum VEGF levels at presentation in patients with early RA correlated significantly with the development of radiographic damage after 1 year. Improvement in the clinical symptoms of RA was associated with a reduction in serum VEGF levels. Serum sFlt-1 levels were raised in patients with early and longstanding RA and in those with self-limiting arthritis, and correlated positively with the serum VEGF concentrations in patients with inflammatory arthritis. CONCLUSION: These findings implicate the proangiogenic cytokine VEGF in the persistence of inflammatory arthritis, and support the hypothesis that expansion of the synovial vasculature is important for the development of joint destruction in RA.