The role of Human xanthine oxidoreductase (HXOR), anti-HXOR
antibodies and microorganisms in synovial fluid of patients with joint
inflammation
Najah Al- Muhtaseb
1*
, Elham Al-Kaissi
2*
, Abdul Jalil Thawaini 3, Zuhair Muhi
eldeen 1 , Sabah Al- Muhtaseb 4, Badiee Al-Saleh5
1
Department of Medicinal chemistry and Pharmacognosy, Faculty of Pharmacy, Petra
University, Amman, Jordan
2
Department of Pharmaceutics and biotechnology, Faculty of Pharmacy, Petra University,
Amman, Jordan
3
Department of Basic sciences, Faculty of Pharmacy, Philadelphia University, Amman,
Jordan
4
Department of Allied Medical Sciences, Faculty of Al-Balqa , University of Zarqa,
Zarqa, Jordan
5
Outpatient Clinic, Rheumatologist, Amman, Jordan
P.O. Box 961343 Amman, Jordan, Fax 00962-6-5715551, Tel. 00962-6-05715546
E mails:
1
naj_nim@yahoo.com
2
Kaielham@hotmail.com
3
ajthaini@hotmail.com
1b
4
5
Eldeenz@hotmail.com
sabahesa@yahoo.com
badie_saleh@yahoo.com
* Contributed equally
1
Abstract
Objectives: This work is to investigate the levels of human xanthine oxidoreductase
(HXOR), its antibodies and microorganisms in synovial fluid of patients with untreated
rheumatoid joint diseases and to assess a possible relationship between severity of the
inflammatory condition and the level of anti-XOR titers.
Methods: Synovial fluids were collected from sixty four patients with rheumatoid joint
diseases who had not been treated with corticosteroid or disease modifying antirheumatic
drugs prior to study. Sixty four age matched individuals were included as control. XOR
proteins level and anti XOR antibodies were determined in the blood and synovial fluid,
using human XOR as antigen, by enzyme linked immunosorbent assay (ELISA
technique). C-reactive protein, ESR and rheumatoid factors were measured in patient's
blood, and were culture for bacteria and fungi.
Results: The titers of XOR protein in the synovial fluid of patients with rheumatoid
arthritis were 90.43 ± 23.37 µg/ml (mean ± SD, n=29) and up to 62.42 ± 8.74 µg/ml
(mean ± SD, n=35) in other joint inflammation. Anti-HXOR antibodies titers in patients
were (167.72 ± 23.64 µg/ ml, n= 64) which was significantly higher in rheumatoid
arthritis patients compared to patients with other joint inflammations. No microorganisms
were detected.
Conclusions: A good correlation was found between the severity of the disease and the
levels of HXOR protein. In contrast, it seems that anti-HXOR antibodies in synovial
fluids have a protective role as high concentrations against XOR were detected in
inflammatory arthritis. These antibodies play role in eliminating XOR from synovial
fluids (positive role). However, immune complex formation could activate complement
2
and participate in propagating the inflammatory cycle. Synovial aspirate Ordinary
microbial cultures were negative for any bacteria or fungi but that does not exclude
organisms of special culture requirements.
Keywords: synovial fluid, human xanthine oxidoreductase (HOXR), ELISA, rheumatoid
joint diseases. free radical.
.
3
Introduction
In recent years, free radicals reactions and other reactive intermediates produced in
normal metabolic processes have been implicated in the pathogenic mechanism of a wide
range of diseases, including inflammatory diseases [1] as acute coronary syndrome [2]
and others cardiovascular diseases [3]. Similarly pathogenic organisms such as
parvovirus B19, rubella, hepatitis B and C, herpes [4-10], Epstein-Barr virus [11] and
retroviruses could also serve as infectious causes of Rheumatoid arthritis like disease.
Synovial human T lymphotropic virus type 1 (HTLV-1) infection is associated with
chronic arthritis [4].
Rheumatic joint disease is a chronic syndrome of ambiguous aetiology and is
characterized by non- specific inflammation of the peripheral joints with joint swelling,
morning stiffness, destruction of articular tissues and joint deformities. One particular
type of tissue injury from free radicals is reoxygenation injury following reperfusion of
ischemic tissue [12-17]. Rheumatoid synovitis is usually accompanied by increased
synovial effusions. Damage to bone and cartilage of synovial tissue is mediated by
metabolic free radical sources such as xanthine oxidoreductase (XOR) and others.
Xanthine oxidoreductase (XOR) is a complex metalloflavoprotein which plays a key role
in catalysing the oxidation of a wide range of substrates (purine, pterins, dinucleotides,
and aldehydes) [18]. The enzyme is a homodimer of approximately 300KDa [19]. XOR
exists into 2 different but interconvertible forms; xanthine dehydrogenase (XDH, EC
1.1.1.204) and xanthine oxidase (XO, EC 1.1.3.22) [19-20]; the former is the dominant
form. XOR produces reactive oxygen species (ROS), superoxide (O -2) or hydrogen
peroxide (H2O2) respectively [21]. The capacity to generate such ROS has led to a great
4
deal of interest in XOR as a pathogenic factor in many instances of ischaemia –
reperfusion injury in various organs [19, 22-25] including inflammatory joint and
rheumatic diseases[15-17]. Xanthine oxidase is the first registered biological source of
oxygen free radicals and plays the leading role in the pathogenesis of tissue damage by
production of superoxide anion, hydrogen peroxide, and hydroxyl radical [26]. There is
substantial evidence that XOR participates in the pathophysiology of many diseases [27].
Rheumatoid arthritis (RA) is the most common chronic inflammatory arthritis that affects
about 1% of adults [28]. There are no specific laboratory tests specific for RA; diagnosis
depends on a constellation of signs and symptoms that can be supported by serology and
radiographs [29-31]. Some RA patients show evidence of autoimmunity long before the
appearance of clinical arthritis [32].
.
The presence of human anti XOR antibodies was initially reported by Oster et al [33].
Bruder et al [34] confirmed the existence of such antibodies, and showed them to be IgG.
The amount of anti-XOR antibody in normal healthy humans represents 1-8% of total
IgG. Levels of these antibodies were shown to be elevated in patients suffering from
myocardial infarction [35]. Ng et al [36] found by ELISA technique that the IgM antiXOR antibodies in healthy humans varied from 0.32-1.8% of total IgM, whereas IgG
anti-XOR level represented only 0.02-0.4% of total IgG. Ng and Lewis [37] reported the
presence of circulating XOR-anti-XOR immune complexes (XORICs) in healthy
humans. The complex containing antibodies of IgG and IgM classes, representing < 20%
of total anti-XOR antibodies. The levels of these complexes correlate with the anti-XOR
antibody titers [38]. Rheumatoid arthritis (RA) is characterized by the appearance of autoantibodies of types IgM, IgG and IgA known as rheumatoid factors (RF) that react
5
essentially against autologous IgG [39]. Immune complexes activate complement.
Complement activation induces inflammatory reaction [40], which contributes to the
constitution of a vascular formation leading to cartilage destruction and bone erosion [4143]. XOR present in the synovium could be liberated by synovium destruction and could
play a role in post- ischemic reperfusion of rheumaroid synovium [17] contributing to the
characteristics signs and symptoms of radical attack present in synovial fluid. XOR
concentration is significantly raised in patients suffering from RA, up to 60 times in
synovium [16] compared to healthy humans or patients with other non- rheumatic
diseases.
The detection of xanthine oxidoreductase in human synovium is therefore of importance
as a first step in the investigation of possible mechanisms of radical generation peculiar to
a reperfusion phase of synovial injury. The aim of this work is to investigate the presence
of human xanthine oxidoreductase, its antibodies in synovial fluid and the possible
microbial aetiology of the inflammation in untreated patients suffering from rheumatoid
arthritis (rheumatoid factor + ve) and other joint inflammatory diseases (rheumatoid
factor – ve).
.
6
Materials and methods
Subjects
Sixty four patient with rheumatoid joint disease and 64 age and sex matched controls
were included in the study. All patients fulfilled the revised American College of
Rheumatology (ACR) criteria for rheumatoid arthritis [23]. Consent for all procedures
was obtained from each individual. The study was approved by the hospital ethics
committee. Preliminary evaluation consisting of a brief medical history, smoking, alcohol
habits and physical examinations was performed. Patients with any history of liver
diseases, diabetes mellitus, respiratory disorders and cardiovascular diseases were
excluded. None had been treated with corticosteroids or disease modifying anti-rheumatic
drugs prior the study.
A pooled high titer anti-XOR and immune complexes (XORIC) human sera from
volunteer donors were served to build standard curves.
Rheumatoid factor detection
Rheumatoid factor is detected by latex agglutination test using appropriate plates from
Behring (Germany) according to the manufacturer recommendations. Fifty µL of latex
coated with human IgG was added to different dilutions from each serum sample.
Negative and positive sera were used as controls. After two minutes a clear agglutination
is observed in the positive indicating the presence of RF. Sera with titer less than 20
UI/ml were considered negative according to the manufacturer recommendation.
Milk and reagents:
Human breast milk was kindly donated by mothers who had that in excess at the
special care unit of the maternity hospitals; human milk was freshly collected and stored
7
at -20oC until use.
Unless otherwise stated, all other reagents were purchased from sigma (Poole, UK).
CRP latex agglutination was purchased from Genzyme diagnostics (Kent, UK).
Purification of human xanthine oxidoreductase (XOR)
Purification of human XOR was carried out according to the previously described
protocol for human milk [22]. The purified enzyme was dialysed overnight against 3 L of
50 mM sodium / Bicine buffer pH8.3 and processed as reported by Bejamin et al [44].
Enzyme activity tests were then carried out. Purified XOR enzyme was found to have
more than 75% in the oxidase form.
Total protein estimation
Protein estimation for XOR enzyme was carried out according to the method of
Bradford [45]. The enzyme purity was assessed on protein / flavin ratio (PFR=A
280nm
/
A450nm). An enzyme sample with a PFR value 5-5.2 is widely accepted to be pure.
Xanthine oxidase activity assay
Total xanthine oxidase activity of the synovial fluid was determined by measuring
the rate of oxidation of xanthine to uric acid spectrophotometrically at 295 nm in a Cary
100 spectrophotometer, using a molar absorption coefficient (ε ) of 9.6 mM-1 [46].
Assays were performed at 25.0 ± 0.2oC in air-saturated 50 mM Na / Bicine buffer, pH
8.3, containing 100µM xanthine. Total (oxidase plus dehydrogenase) activity was
determined as above but in the in presence of 500 µm NAD+. Dehydrogenase content of
an enzyme sample was determined from the ratio of oxidase and total activities.
C - reactive protein (CRP) concentration determination
CRP concentration was determined as recommended by the manufacturer. 2.4 µl of patient
8
serum is added to 120 µl buffer solutions (pH 8.5) and mixed with 120 µl suspension of mouse
anti-human CRP monoclonal antibody that is bound to latex (2mg/ml) and incubated for 5
minutes. CRP binds to the latex-bound antibody and agglutinates. The resulting agglutination was
measured spectrophotometrically at 580 nm, negative and positive control samples were included.
Values higher than 9.4 mg/l for females and 8.55 mg/l for males were consider as positive.
Single radial immunodiffusion assay (SRID) to determine levels of total IgG & IgM
Commercially available plates of agarose containing anti-human IgG or anti- human IgM
(Behring, Germany) were used as described by Benboubetra et al [38]. Serial dilutions of
purified human IgM or IgG were used to generate standard curves. Human synovial
fluids or sera were dialyzed against the commercial buffer and run against anti-human
IgG or anti-human IgM on the same plates as the standard curves. Plates were placed in a
humidified box and stored for 48-72 hours at room temperature until the sizes of the
precipitate rings were stable. Standard curve was plotted and used to determine total IgG
and IgM anti- human XOR contents in synovial fluids.
Enzyme linked immunosorbent assay (ELISA)
a- For anti-HXOR antibodies
Specific human anti-XOR antibodies were determined as previously described by
Harrison R, et al [35] and Benboubetra et al [38] with slight difference in enzyme
substrate.
Orthophenylene
diamine(OPD)
[47]
was
used
instead
of
3,3,5,5
tetramethylbenzidine (TMB). In addition to synovial fluids, each plate (Coster, Spain)
included serial dilutions (200 - 6400 fold in PBS-Tween) of a standard high-titer pooled
serum, in duplicate wells (100 µL / well). The absorbance was measured at 492nm, in
each well using a 96 well plate reader (Diagnostics Pasteur LP200).
9
.
b- For XOR immune complexes (XORIC)
.
To determine (XORIC) polystyrene 96 well microtiter plates (Coster, Spain) were
coated (100 µL / well) with diluted (1 in 40) rabbit anti-human XOR serum in sodium
hydrogencarbonate (pH 9.6), then incubated overnight at 4oC and processed as described
by Stevens et al [17]. XORIC concentrations were calculated from plotted standard
curves of absorbance against log concentration of the standard serum for each plate, and
the linear part of the curve was used to calculate the titres as a percentage of the standard.
Data for ELISA was expressed as mean ± SD.
Data from patient groups were compared to each other using students t test. SigmaStat
Software were used for statistical analyses, Probability values of 0.05 were considered
significant
Microbiologic testing
Synovial fluids were cultured on Blood agar (BA), MacConkey's agar (MA),
Chocolate Agar (CA) and Sabouraud's dextrose agar (SA) (Oxoid). BA and MA plates
were incubated aerobically at 37oC, CA plates were incubated under microaerophilic
condition (3% CO2), SA plates were incubated at room temperature (25o C) for 72 hours.
Synovial fluids were centrifuged at 500 rpm for 5 minutes and smears from the deposit
were Gram and Acid fast stained. The slides were examined by light microscopy with oil
immersion lens.
10
Results
Latex agglutination used in determining IgM-RF factor showed that, of the 64
patients 29 were RA+ (Seropositive) and 35 were RA- (Seronegative). The Clinical and
biochemical characters of the participated subjects are shown in table 1. The mean age
and BMI for both rheumatoid arthritis and other joint inflammation and control subjects
were comparable. ESR (after 1 hour) and CRP values were significantly elevated in
patients with rheumatoid arthritis (RA+) compared with the values in patient with other
joint inflammation (RA-) and to those of the control values.
The total IgG and IgM titres in the synovial fluid of patient with rheumatoid arthritis
(RA+) and patients with other joint inflammation (RA-) as determined by single radial
immunodiffusion assay (SRIDA) are shown in table 2.
Total IgG and IgM (mg/ml) levels were significantly higher in patient with rheumatoid
arthritis in comparison to the levels in patient with other joint inflammation respectively.
Total IgG and IgM titers in blood circulation of patient with rheumatoid arthritis (RA+),
other joint inflammation (RA-) and in healthy volunteers, using SRIDA are shown in
table 3.
Both IgG and IgM were significantly higher in rheumatoid arthritis patients than the
healthy controls. Similarly, the levels were higher in serum of patient with other joint
inflammation than controls. IgG levels were more elevated in serum of patient with
rheumatoid arthritis compared to other joint inflammation (P< 0.001). On the other hand
total IgM was more elevated in serum of patients with other joint inflammation compared
to rheumatoid arthritis.
Human XOR protein, its activity and anti-human XOR (IgG and IgM) in patient's
11
synovial fluid were measured; the results are shown in table 4.
Human xanthine oxidase protein (HXO) concentrations were significantly elevated in
patient with rheumatoid arthritis compared with the concentrations in patient with other
joint inflammation (P< 0.01).
Human xanthine oxidase (HXO) activity was also significantly elevated in patient with
rheumatoid arthritis (100.00 ± 47.24 nmol.min-1.mg-1) compared with the activity in
patients with other joint inflammation (81.42 ± 33.22 nmol.min-1.mg-1) (P< 0.01).
Both IgG and IgM anti-HXOR levels are remarkably high, representing, in the case of
anti- HXOR IgM antibodies, 6.6% of the total IgM immunoglobulin in patient with
rheumatoid arthritis and 4% in patient with other joint inflammation. However antiHXOR IgG was higher in patients with other joint inflammation compared with patients
with rheumatoid arthritis.
Levels of free and immune complexes IgG and IgM anti-HXOR antibodies determined
by ELISA are shown in (Table 5).
Both the free and immune complexes of anti-HXOR (IgG and IgM) were lower in
patients with rheumatoid arthritis compared with the levels in patients with other joint
inflammation.
Direct examination as well as aerobic and microaerophilic cultures for bacteria and fungi
did not reveal the presence of microorganisms.
12
.
Discussion
The elevated levels of ESR (erythrocyte sedimentation rate) and CRP (C-reactive
protein) reported in this study indicate the presence of inflammation in both patient
groups. Patients positive for rheumatoid factor were labeled as rheumatoid arthritis
(RA+) while seronegative patients were labeled as other joint inflammation (RA-).
The level of human xanthine oxidase protein and its activity is significantly high in the
synovial fluid of patient with rheumatoid arthritis (90.43ug/ml, 100.00 nmol.min-1.mg-1)
(Table 4) .The normal level in healthy individual as reported by others (48, 49) is 0.160.38 mU/g. This may be due to upregulation of the enzyme by characteristically high
levels of cytokines and hypoxic nature of rheumatoid synovium. Xanthine oxidase is the
first registered biological source of oxygen free radicals and plays the leading role in the
pathogenesis of tissue damage by production of superoxide anion, hydrogen peroxide and
hydroxyl radical. The roles of XOR in cytokine induced bone erosion promoting
vasculitis have been well documented by others [15-16].
The slightly higher levels of total IgG and IgM titres in patients sera determined by
SRIDA in RA+ patients compared to the levels in other joint inflammation and to the
levels in the serum of control healthy are in agreement with [35, 37, 50].This is expected
because of the auto-immune nature of the disease. On the other hand the results showed
that synovial fluids of patients suffering from rheumatoid arthritis and other joint
inflammation contained lower levels of total IgG and IgM compared to the levels of these
immunoglobulines in patients serum. This disagrees with the finding of others [35, 37,
50]. This may indicate that some of those immunoglobulins were bound to the tissue or
are bound in immune complexes which need to be detected by other techniques. In fact
13
the pathology of rheumatoid arthritis is attributed to an Arthus like reaction in which
immune complexes mediate the crucial role.
The high anti-HXOR titres in RA patients may be due to elevated serum and synovial
fluid XOR level. In most cases, more than 50% of synovial XOR is present as oxidase
form [51]. The detection of relatively high concentration of XOR enzyme in its oxidative
form in rheumatoid arthritis patient synovial fluids compared to the concentration of the
enzyme in other joint inflammation indicates that the enzyme XOR is liberated with
lysosomal enzymes from the synovium and could be involved in cartilage destruction and
bone erosion suggesting a positive correlation between the level of the enzyme and the
severity of the disease.
The anti-HXOR (IgM) titres, in the synovial fluid samples, were found to be significantly
higher in other joint inflammation than in RA+ patients. This finding which is also
reported by others [50] could be explained by the fact that IgM- Anti XOR are more
efficient in immune complex formation and consequently damage in RA+ patients due to
activation of the complement system.
The levels of IgM-containing XORICs were considerably higher than those of IgG,
reflecting the relative levels of the free specific antibodies. In the case of both IgM and
IgG, a high proportion of specific antibodies were in the complex form (90.34, 77.06
respectively). It is worth noting that, despite the relatively high levels of circulating
(complexes) XOR, corresponding enzymic activity is seldom detectable, which most
probably reflects the low specific activity of the human enzyme. This was also reported
by Abadeh et al [52] and Yamomoto et al [53].
Synovial aspirate were never positive for any bacteria or fungi. This was also reported by
14
other [54].A blood culture may be more revealing if an association between RA disorder
and infection with a microorganism exists. Some have reported improvement after
treatment of RA patient with antibiotics [55] suggesting a microbial role. It may be useful
to look for unconventional organisms such as Chlamydia and Mycoplasma as suggested
by other [4, 56].
Rheumatoid arthritis may have complex pathology. In addition to the obvious effect of
the rheumatoid factor, Proteus mirabilis was also incriminated. Newkirk et al [57] have
found that IgM and IgA anti-Proteus mirabilis antibodies were significantly higher in
patients
with
rheumatoid
arthritis
RA+
Compared
with
RA-
arthritis,
spondyloarthropathy and undifferentiated arthritis. Similarly, Tani et al [58] have found
that patients with rheumatoid arthritis showed elevated levels of antibodies to Proteus
mirabilis compared to ankylosing spondylitis and controls. They suggested an amino acid
homology between an outer membrane haemolysin protein of Proteus mirabilis and
susceptibility sequence in HLA-DR1, and DR4 subtypes. Also, Senior et. al [59] found
that 33% of patients with rheumatoid arthritis had asymptomatic insignificant bacteriuria
compared to 4% of age and gender matched controls. Similarly patients had significantly
higher levels of IgA, IgM and IgG antibodies to Proteus mirabilis in blood and urine than
controls. It is suggested that this may be the trigger for the rheumatoid arthritis condition.
15
Conclusions
These findings suggest that XOR may play an important role as a source of ROS in
RA and in other joint inflammation which participate in self-maintenance of the diseases.
Antibodies against XOR may play a major role in RA by inhibiting both xanthine and
NADH oxidase activities of XOR. They may also play a key role in eliminating XOR
from serum and synovial fluid. However, immune complex formation could activate the
complement system and participate in self maintenance of the diseases.
16
Abbreviations
XOR, xanthine oxidoreductase; HXOR, human xanthine oxidoreductase; anti-XOR,
antibodies (IgM or IgG) against xanthine oxidoreductase; anti-HXOR, antibodies (IgM or
IgG) against human xanthine oxidoreductase; ESR, erythrocytes sedimentation rate;
CRP, C- reactive protein; SD, standard deviation; ELISA, enzyme linked immunosorbent
assay; HTLV-1, human T lymphotropic virus type 1; ROS, reactive oxygen species;
H2O2, hydrogen peroxide; O-2, superoxide; XDH, xanthine dehydrogenase; RA,
rheumatoid arthritis; XORICs, xanthine oxidoreductase -anti- xanthine oxidoreductase
immune complexes; HXORICs, human xanthine oxidoreductase -anti- xanthine
oxidoreductase immune complexes; RF, rheumatoid factors; ACR, american college of
rheumatology; PFR, protein / flavin ratio; NAD+, nicotinamide adenine dinucleotide;
NADH, nicotinamide adenine dinucleotide hydrogen; SRID, single radial
innunodiffusion; OPD, orthophenylene diamine; TMB, tetramethyl benzidine; BA, blood
agar; SA, sabouraud's dextrose agar; BA, blood agar; CA, chocolate; IgM-RF, IgM –
rheumatoid factor; RA+, positive rheumatoid factor;RA-, negative rheumatoid factor;
BMI, body mass index; HLA-DR1, human leukocyte antigen subtypeDr1; DR4, human
leukocyte antigen subtype DR4; PBS, phosphate buffer solution.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
None
Acknowledgements
None
17
References
1. Chung HY, Baek BS, Song SH, Kim MS, Huh JI, Shim KH, Kim KW, and Lee KH:
Xanthine dehydrogenase / Xanthine oxidase and oxidative stress. Age 1997, 20:
127- 140.
2. Cheng-xing S, Hao-zhu C, and Jun-bo G: The role of inflammatory stress in acute
coronary syndrome. Chinese medical J 2004, 117: 133-139.
3. Dawson J, and Walters M: Uric acid and xanthine oxidase: future therapeutic
targets in the prevention of cardiovascular disease Br J Clin Pharmacol.2006 ,62: 633644.
4. Carty SM, Snowden N, Silman AJ: Should infection still be considered as the most
likely triggering factor for rheumatoid arthritis. Ann Rheum Dis 2004, 63: ii46ii49
5. Griffiths DJ: Rheumatoid arthritis: a viral aetiology ?. Hosp Med. 2000, 61: 378379.
6. Masuko-Hongo K, Kato T, Nishioka K: Virus-associated arthritis. Best Pract Res
Clin Rheumatol. 2003, 17: 309-318.
7. Phillips PE: Viral arthritis. Curr Opin Rheumatol 1997, 9: 337-344.
8. Zhang L, Nikkari S, Skurnik M, Ziegler T, Luukainen R, Mottonen T, Toivanen P:
Detection of herpesviruses by polymerase chain reaction in lymphocytes frm
patients with rheumatoid arthritis. Arthritis Rheum.1993, 36: 1080-1086.
9. Naides SJ: Rheumatic manifestations of parvovirus B19 infection. Rheum Dis Clin
North Am. 1998, 24: 375-401.
10. Katsumi E: Involvement of viral infection in rheumatoid arthritis. Pharma Med
18
1999, 17: 19-26.
11. Hasunuma T, Sumida T, Nishioka K: Human T cell leukemia virus type-1 and
rheumatoid arthritis. Int Rev Immunol 1998, 17: 291-307.
12. Zweier JL: Measurement and characterization of post-ischemic free radical
generation in isolated perfused heart. J Biol Chem 1988, 263: 1353-1357.
13. Kennedy TP, Rao NV, Hopkins C, Pennington L, Tolley E, and Hoidal JR: Role of
reactive oxygen species in reperfusion injury of the rabbit lung. J Clin Invest
1989, 83: 1326-35.
14. Sussman MS, and Bulkley GB: Oxygen-derived free radicls in reperfusion injury.
Methods in Enzymology 1990, 186: 711-723.
15. Miesel R, and Zuber M: Elevated levels of xanthine oxidase in serum of patients
with inflammatory and auto-immune rheumatic diseases. Inflammation 1993, 17:
551-560.
16. Blake DR, Stevens CR, Sahinoglu T, Ellis G, Gaffney K, Edmonds S, Benboubetra
M, Harrison R, Jawed S, Kanczler J, Millar TM, Winyard PG, and Zhang Z: Xanthine
oxidase; four roles for the enzyme in rheumatoid pathology. Biochem Soc Trans
1997, 25: 812-816.
17. Stevens CR, Benboubetra M, Harrison R, Sahinoglu T, Smith EC, and Blake DR:
Localization of xanthine oxidase to synovial endothelium. Ann Rheum Dis 1991,
50: 760-762.
18. Bray RC: Molybdenum iron-sulfur flavin hydroxylases and related enzymes. In:
Boyer PD, Editor. The enzymes. New York, Academic Press 1975: 299-419.
19. Harrison R: Structure and function of xanthine oxidoreductase: where are we
19
now? Free Radic Biol Med 2002, 33: 774-797.
20. Martin HM, Hancock JT, Salisbury V, and Harrison R: Role of xanthine
oxidoreductase as an antimicrobial agent. Infect Immun 2004, 72: 4933-4939.
21. Khan AU, Wilson T: Reactive oxygen species as cellular messengers. Chem Biol
1995, 2: 437-445.
22. Sanders SA, Eisenthal R, Harrison R: NADH oxidase activity of human xanthine
oxidoreductase – generation of superoxide anion. Eur J Biochem 1997, 245:541548.
23. Baghiani A, Harrison R, Benboubetra M: Purification and partial characterization
of camel milk xanthine oxidoreductase. Arch Physiol Biochem 2003, 111: 407-414.
24. Jarasch ED Grund C, Bruder G, Heid HW, Keenan TW, Franke WW: Localization
of xanthine oxidase in mammary-gland epithelium and capillary endothelium.
Cell 1981, 25: 67-82.
25. Meneshian A, and Bulkley GB: The physiology of endothelial xanthine oxidase:
from urate catabolism to reperfusion injury to inflammatory signal transduction.
Microcirculation 2002, 9: 162-175.
26. Kundalic S, Kocic G, Cosic V, Jevtovic-stoimenova t, Dordevic VB: The role of
xanthine dehydrogenase L xanthine oxidase in atherogenesis in patients with
hyperlipidaemia . Jugoslov Med biochem 2003, 22: 151-158
27.Berry CE and Hare JM: Xanthine oxidoreductase and cardiovascular diseases:
molecular mechanisms and pathophysiology . J physiol 2004, 555: 589-606
28. Mitchell KL, Pisetsky DS: Early rheumatoid arthritis. Curr Opin Rheumatol CME
2007, 19: 278-283.
20
29. Machold KP, Stamm TA, Eberl GJM, Nell VKP, Dunky A, Uffmann M, et al: Very
recent onset arthritis: clinical, laboratory, and radiological findings during the
first year disease. J Rheumatol 2002, 29: 2278-2287.
30. Hichon CA, Peschken CA, Shaikh S, El-Gabalawy HS: Early undifferentiated
arthritis. Rheum Dis Clin N Am 2005, 31: 605-626.
31. Van Aken J, Van Dongen H, Le Cessie S, Allaart CF, Breedveld FC, Huizinga TWJ,
et al: Comparison of long term outcome of patients with rheumatoid arthritis
presenting with undifferentiated arthritis or with rheumatoid arthritis: an
observational cohort study. Ann Rheum Dis 2006, 65: 20-25.
32. Kary S, Fritz J, Scherer HU and Burmester GR: Do we still miss the chance of
effectively treating early rheumatoid arthritis? New answers from a new study.
Rheumatol 2004, 43: 819-820.
33. Oster KA, Oster JB, Ross DJ: Immune response to bovine xanthine oxidzse in
atherosclerotic patients. Am Lab 1975, 6: 41-47.
34. Bruder g, Jarasch ED, Heid HW: High concentration of antibodies to xanthine
oxidase in human and animal sera. J Clin Invest 1984, 74: 783-794.
35. Harrison R, Benboubetra M, Bryson S, Thomas RD, Elwood PC: Antibodies to
xanthine oxidase: elevated levels in patients with acute myocardial infarction.
Cardioscience 1990, 1: 183-189.
36. Ng YLE, Lewis WHP, Chui SH: Enzyme linked immunosorbent assay for
determination of antibodies to xanthine oxidase. Med Lab Invest 1990, 47: 30-35.
21
37. Ng YLE, Lewis WHP: Circulating immune complexes of xanthine oxidase in
normal subjects. Br j biomed Sci 1994, 51: 124-127.
38. Benboubetra M, Gleeson A, Harris CPD, Khan J, Arrar L, Brennand D et al:
Circulating anti-(xanthine oxidoreductase) antibodies in healthy human adults.
Eur J clin invest 1997, 27: 611-619
39. Venn AJ, Dresser DW: Limitation of haemolytic plaque assay for IgG anti-IgG
rheumatoid factor-producing cells. J Immunol Meth 1987, 102: 195-204.
40. Simon L, Claustre j, Picard JJ, Marty m, blotman F: Intra-articular osteoid osteoma
of the elbow. Rheumatologie 1972, 24: 377-382.
41. Zvaifler NJ: The immunopathology of joint inflammation in rheumatoid
arthritis. Adv Immunol 1973, 16: 265-336.
42. Palmer DG, Hogg N, Revell PA: Lymphocytes, polymorphonuclear leukocytes,
macrophages and platelets in synovium involvef by rheumatoid arthritis. a study
with monoclonal antibodies. Pathology 1986, 18: 431-437.
43. Zubler RH, Nydegger U, Perrin LH, Fehr K, McCormick J, Lambert PH, et al.
Circulating and intra-articular immune complexes in patients with rheumatoid
arthritis. Correlation of 125I-CIq binding activity with clinical and biological
features of the disease. J Clin Invest 1976, 57: 1308-1319.
44. Godber BLJ, Schwarz G, Mendel RR, Lowe DJ, Bray RC, Eisenthal R, Harrison R:
Molecular characterization of human xanthine oxidoreductase: the enzyme is
grossly deficient in molybdenum and substantially deficient in iron-sulphur
centres. Biochem J 2005, 388: 501-508.
22
45. Bradford MM: A rapid and sensitive method for the quantitation of microgram
quantitative of proteins. Anal Biochem 1976, 72: 248-254.
46. Avis PG, Bergel F, Bray RC: Cellular constituents. The chemistry of xanthine
oxidase. Part III. Estimations of the cofactors and the catalytic functions of
enzyme fractions of cows' milk. J Chem Soc 1956, (Perkins I): 1219-1226.
47. Rousseaux-Prevost R, De Almeida M, Arrar L, Hublau P, Rousseaux J: Antibodies
to sperm basic nuclear protein detected in infertile patients by dotimmunobinding and ELISA. Am J Reproductive Immunol 1989, 20: 17-20.
48. Allen RE, Outhwaite JM, Morris CJ, and Blake DR: Xanthine oxidoreductase is
present in human synovium. Annals of the Rheumatic Diseases 1987, 46: 843-845.
49. Parks DA, Granger DN: Xanthine oxidase: biochemistry distribution and
physiology. Acta Physiol Scand Suppl 1986, 548: 87-99.
50. Arrar L, Hanachi N, Rouba K, Charef N, Khennouf S, Baghiani A: Anti-xanthine
oxidase antibodies in sera and synovial fluid of patients with rheumatoid arthritis
and other joint inflammations. Saudi Med J 2008, 29: 803-807.
51. Hanachi N, Charef N, Baghiani A, Khennouf S, Derradji Y, Boumerfeg s , et al:
Comparison of xanthine oxidase levels in synovial fluid from patients with
rheumatoid arthritis and other joint inflammations. Saudi Med j 2009, 30: 14221425.
52. Abadeh S, Killacky J, Benboubetra M, Harrison R: Purification and partial
characterization of xanthine oxidase from human milk. Biochem Biophys Acta
1992. 1117: 25-32.
53. Yamomoto T, Moriwaki Y, Takahashi S et al: Determination of human plasma
23
xanthine oxidase activity by high- performance liquid chromatography. J
Chromatog B 1996, 681: 395-400
54. Hall GS, Gordon S, Schroeder S, Smith K, Anthony K, and Procop W: Case of
Synovitis potenitially causedby Dolosigranulum pigrum. J Clin Microbiol 2001,
39: 1202-1203.
55. Ogrendik M: Rheumatoid arthritis is linked to oral bacteria: etiology association.
Modern Rheumatollogy 2009, 19: 453-456.
56. Schaeverbeke T, Renaudin H, Clerc M, Lequen L, Vernhes JP, De Barbeyrac B,
Bannwarth B, Bebear CH, and Dehais J: Systematic detection of mycoplasmas by
culture and polymerase chain reaction (PCR) procedures in 209 synovial fluid
samples. Br J Rheum 1997, 36: 310-314.
57. Newkirk, MM, Goldbach-Mausky, R, Senior, BW, Klippel, J, Schumucher, HR, AlGebalawy, HS: Elevated levels of IgM and IgA antibodies to Proteus mirabilis
and IgM antibodies to E. coli are associated with early RF- Positive rheumatoid
arthritis. Rheumatology 2005, 44 (11): 1433-1441.
58. Tani, Y; Tiwana, H; Hukudu,s; Nishioka,J; Fielder,M; Wilson, C; Bausal,S;
Ebriager,A: Antibodies to Klebsiella, Proteus and HLA-B27 peptides in Japanese
patients withAnkylosing spondylitis and rheumatoid arthritis. J. Rheumatology.
1997, 24(1): 109-114
59. Senior BW, Anderson GA, Mortey KD, and Kerv MA: Evidence the patients with
rheumatoid arthritis have a symptomatic non-significant Proteus mirabilis
bacteriuria more frequently than healthy controls J infect 1999, 38(2): 99-106.
24
Table 1: Clinical and biochemical characters of the participated subjects
Type of
No.
Age
Height
Weight
disease
of
(years)
(cm)
(Kg)
BMI
.
ESR
CRP
(mm)
Subjects
RA+
29
39.1± 8.9
159.8±5.8
69.6±2.3
27.3±9.2
66.6±45.2
+
RA -
35
41.04± 9.1
161.6±5.6
69.1±6.0
26.9±9.7
40.3±30.4
+
Controls
64
42.04± 7.7
162.3±4.7
68.7±5.8
25.8±2.3
12.8±4.01
25
_
Table 2: Total IgG and IgM titres in synovial fluid of patients with rheumatoid arthritis
(RA+) and other joint inflammation (RA-), using SRIDA**
Patients
Number of
Total IgG (mg/ml)
Total IgM (mg/ml)
patients
RA+*
29
13.71 ± 4.41a
0.97± 0.34 b
Other joint
35
9.77 ± 2.71
0.55 ± 0.20
inflammation
* RA = Rheumatoid arthritis latex agglutination positive
** SRIDA = Single radial immunodiffusion assay
a P < 0.001 significantly higher with RA+ group
b
P < 0.05 significantly higher with RA+ group
26
Table 3: Total IgG and IgM titres in sera of patients with rheumatoid arthritis (RA+),
other joint inflammation (RA-), and healthy volunteers, using SRIDA**
Patients
Number of
Total IgG (mg/ml)
Total IgM (mg/ml)
patients
RA+*
29
24.52 ± 9.53a
2.23± 1.25 a
Other joint
35
19.37 ± 5.6 b
3.42 ± 0.06 b
inflammation
Pools from healthy
60
13.5 ± 4.41
individuals
* RA = Rheumatoid arthritis latex agglutination positive
** SRIDA = Single radial immunodiffusion assay
a P < 0.0001 significantly differ from the control subjects
b P < 0.01 significantly differ from the other joint inflammation
Results are means ± S.D in milligrams per deciliter
27
1.5 ± 0.52
Table 4: Human xanthine oxidase protein, (HXOR) activity and anti-HXOR (IgG and
IgM) in patient synovial fluids
Type of disease
No. of
HXOR
HXOR activity
Anti-HXOR
Anti-
patients
protein
nmol.min-1.mg-1
(IgG)
HXOR
μg/ml
(IgM)
(μg/ml)
μg/ml
RA+
29
90.43±
100.00 ± 47.24
36.37 ± 22.35
23.37
Other joint
inflammation
P
35
62.42±
131.35 ±
30.39
81.42 ± 33.22
46.43±23.62
105.6 ±
31.36
< 0.05
< 0.001
8.74
< 0.01
< 0.01
28
Table 5: IgG and IgM anti-HXOR antibodies, free (HXORAb) and immune
complexes (HXORIC) determined by ELISA ( µg ml
-1
) in patient synovial fluid.
.
HXORAb
RA+
Others joint
29
35
IgG
IgM
IgG
IgM
36.77±22.35
131.35
77.06±
90.34± 74.96
±30.39
38.74
141.09±
86.13±
31.36
14.30
< 0.01
< 0.05
46.43±23.62
inflammation
p
HXORIC
< 0.001
29
134.94±43.61
< 0.001