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Comparison of the concentrations of pentosidine in the synovial fluid, serum and urine of patients with rheumatoid arthritis and osteoarthritis

Comparison of the concentrations of pentosidine in the synovial fluid, serum and urine of... Abstract Objective.Pentosidine, an advanced glycation end product (AGE), has recently been observed to be elevated in rheumatoid arthritis (RA). The aim was to elucidate which pentosidine levels, i.e. in serum, synovial fluid or urine, are more related to the disease status of RA. Methods.We measured levels of pentosidine in serum, synovial fluid or urine in RA compared with osteoarthritis (OA), and examined the relationship between pentosidine and RA disease activity. Subjects were 20 patients with RA and 22 patients with OA. Results.In total RA and OA patients combined, there was a significant correlation between pentosidine in serum, synovial fluid and urine. Pentosidine in serum and synovial fluid was significantly higher in RA than in OA. In RA, there were significant correlations between pentosidine in serum and synovial fluid and C-reactive protein, Lansbury index (LI) and erythrocyte sedimentation rate. Conclusions.These results demonstrate that pentosidine levels in body fluids correlated with each other, and pentosidine in serum and in synovial fluid is associated with the systemic inflammatory activity of RA. Higher or similar concentrations of pentosidine in serum compared with synovial fluids indicate that the elevated pentosidine levels in serum in RA are not derived from the synovial fluid, but from an increase in the formation of pentosidine in the whole body in RA. Among body fluids, serum pentosidine was the superior indicator for RA disease status. Rheumatoid arthritis, Osteoarthritis, Pentosidine, Oxygen radicals Pentosidine is one of a number of advanced glycation end products (AGEs) [1]. Its significant elevation was observed in tissues in diabetes mellitus [2], in uraemic patients with end-stage renal failure [2–4], and in accelerated aging [5–7]. Pentosidine is formed by sequential glycosylation and oxidation reactions. Therefore, it is hypothesized that pentosidine formation is accelerated in diseases related to oxidative stress. Rheumatoid arthritis (RA) is a chronic systemic disease, although its major clinical consequence is inflammation of the joints and contiguous structures. There is now considerable evidence for the involvement of oxygen-centred free radicals in acute and chronic inflammatory arthritis [8, 9]. The role of free radicals in the autoxidation of biological lipids is well established [10]. Patients with RA have depressed serum levels of the antioxidant, and a low antioxidant level is a risk factor for RA [11]. Pentosidine is formed by sequential glycosylation and oxidation reactions. Because oxidative stress and oxygen free radicals play a significant role in tissue damage and inflammation in RA, pentosidine is supposed to be implicated in this disease [12–15]. Several years ago, pentosidine was reported to be elevated in the articular cartilage of RA [12]. However, its significant relationship to RA has not been investigated until recently. Now, there have been several studies which provide evidence that pentosidine is elevated in patients with RA. Pentosidine in serum and urine is elevated, and reflects the disease activity of RA [13]. Pentosidine in synovial fluid is higher in RA than in osteoarthritis (OA), and its level also reflects the disease activity of RA [16]. Although all pentosidine levels in body fluids have been associated with RA disease activity, it has not been clear which pentosidine levels, i.e. in serum, synovial fluid or urine, are a superior indicator. In the present study, therefore, we investigated pentosidine levels in serum, synovial fluid and urine in OA and RA. Patients and methods Patients The study included 20 patients with RA and 22 patients with OA. RA was diagnosed according to the criteria of the American College of Rheumatology (ACR) [17]. The RA group consisted of 15 female and five male patients aged 25–69 yr (mean±s.d.=61.0±13.0). OA of the knee joints was diagnosed on the basis of clinical symptoms, examination and radiographic findings. These patients fulfilled the ACR criteria of OA. The OA group consisted of 13 female and nine male patients aged 46–79 yr (mean±s.d.=62.7±10.6). They were attending the out-patient clinic in the Department of Orthopaedic Surgery at Hamamatsu University School of Medicine. There was no significant difference in age between RA and OA patients. Patients who had diabetes mellitus, steroid users and those with abnormal levels of serum blood urea nitrogen (BUN) or creatinine were excluded from the study. Blood, synovial fluid and urine were collected on the same day from all RA and OA patients. After collection, blood samples were centrifuged at 3000 r.p.m. for 15 min. Synovial fluid was obtained by knee aspiration. Sera, synovial fluid and urine samples were kept at −30°C until analysis. Informed consent was obtained from all participants. The procedures followed were in accordance with the principles of the Declaration of Helsinki in 1975, as revised in 1983. Clinical features, blood biochemistry in RA Serum C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR) were measured by routine laboratory methods. A Lansbury index (LI) was determined based on the duration of morning stiffness, ESR (value at 1 h), grip strength (mmHg) and joint score [18, 19]. Urinary creatinine content was measured by a routine laboratory method. Measurements of pentosidine in serum, synovial fluid and urine The pre-treatment of samples before injection into the HPLC column was described previously [13, 16]. Pentosidine was measured by a direct HPLC method with column switching as described previously [13]. Briefly, the samples were injected into a gel-filtration pre-column (TSK pre-column PW, 4.6 mm×3.5 cm; TOSOH, Tokyo, Japan), the eluate fraction containing pentosidine selected, and this fraction introduced into a reversed-phased column (TSK-GEL ODS-80T, 4.6 mm×15 cm; TOSOH) by use of a switching valve. The urine sample was injected directly into the HPLC column without acid hydrolysis. The concentrations of pentosidine in serum and synovial fluid were expressed as nmol/l. The concentrations of pentosidine in urine were corrected by the concentrations of urine creatinine and expressed as μmol/mol creatinine. Standard pentosidine was synthesized [20] and the concentration was calibrated with pentosidine, which was a gift from Dr V. M. Monnier. Statistical analysis The statistical significance of difference was determined with non-parametric statistics using Mann–Whitney U-tests between two groups. Simple regression was performed for univariate correlation and the statistical significance of correlation was determined with the Spearman rank correlation test. The analysis was performed with StatView II software (Abacus Concepts, Inc., Berkeley, CA, USA) on a Macintosh computer (Apple Computer, Inc., Cupertino, CA, USA). Values of P<0.05 were considered significant. Results Pentosidine levels in serum and synovial fluid were significantly higher in RA than in OA (Table 1). In urine, pentosidine levels tended to be higher in RA than in OA, but not significantly so. In either RA or OA, pentosidine levels tended to be higher in serum than in synovial fluid, but there was no statistically significant difference. The correlations of pentosidine levels between serum, synovial fluid and urine were examined in total patients of RA and OA. Pentosidine levels in serum, synovial fluid and urine were significantly correlated to each other (Fig. 1). Pentosidine levels in serum and synovial fluid of RA were significantly correlated with CRP, ESR and LI. However, there was no significant correlation between urine pentosidine levels and CRP, ESR and LI (data not shown). When pentosidine levels were compared between subgroups of RA patients who were divided into high (CRP 20 mg/l and LI 40%) and low (CRP<20 mg/l and/or LI<40%) activity groups, pentosidine levels in serum and synovial fluid were significantly higher in the high-activity group than in the low-activity group (P<0.05). However, there was no significant difference in urine pentosidine levels between these two groups. When pentosidine levels were compared between subgroups of RA patients who were classified by stage (Stage I+II and Stage III+IV) or class (Class I+II and Class III+IV) according to Steinbrocker et al. [21], there were no statistically significant differences in serum, synovial fluid and urine pentosidine levels between the two subgroups of stage and the two subgroups of class (data not shown). Discussion Free radicals are implicated in tissue damage and inflammation in RA [8, 9], and the formation of pentosidine is associated with oxidation [22]. However, there have so far been a few reports studying pentosidine in RA [12–16]. To our knowledge, we were the first to report the increased levels of serum pentosidine in RA [13]. The present study demonstrates that pentosidine levels of several body fluids were elevated in RA. However, the mechanisms of these phenomena remain unclear. It is known that chronic hyperglycaemia leads to the accumulation of non-enzymatically derived glycosylation products of proteins, such as pentosidine in diabetes mellitus. In RA, the increased pentosidine concentrations are present in body fluids; however, glucose levels are normal. Interest in recent years has focused on the potential of glycated proteins as a source of free radicals [23]. Exposure of IgG and other proteins to oxygen radicals, generated by photolysis or by a mixture of iron or copper salts and hydrogen peroxide, induces fluorescence in the protein which is essentially indistinguishable from that attributed to Schiff base formation. Because pentosidine formation requires aerobic conditions, we propose that such free radicals could explain possible pathways for the elevation of pentosidine in RA. Thus, protein-linked fluorescence such as that of pentosidine may therefore not be the result of cumulative glycaemia over many years, but could be the result of successive exposure to free radical-induced oxidation, and this may be of importance in disease activity in RA. When we first observed the elevated levels of serum pentosidine in RA and its significant relationship to disease activity [13], we supposed that the elevated pentosidine in the blood circulation was derived from the inflammatory joints in RA. Actually, pentosidine in synovial fluid was observed to be increased greater in RA than OA in the successive study [16]. In that study, we proposed two possible pathways for the elevation of pentosidine in RA. One possibility is that because pentosidine is abundant in cartilage compared with the other tissues [24–26], inflammation of the joints may induce the breakdown of cartilage, releasing cartilaginous pentosidine into synovial fluid; the other is an increase in the formation of pentosidine in synovial fluid. It is known that total proteins increase in synovial fluid in severely inflamed joints, especially in those of RA. It is also known that free radical oxidation products of synovial fluid were higher in inflammatory than in degenerative joint disease, and in patients with RA than in normal controls [27]. A combination of elevated proteins (and amino acids) and the acceleration of free radicals in synovial fluid of RA stimulates Maillard reactions, then increases the formation of pentosidine in synovial fluid in RA. However, the present study and the previous one [15] showed that pentosidine levels in synovial fluid were relatively lower than those in serum because a certain retention of proteins in synovial membranes and a consumption of the protein in the joint make the concentrations of proteins in synovial fluid slightly lower than those in plasma [28, 29]. Therefore, the elevated pentosidine levels in serum of RA are not derived from the synovial fluid, but from an increase in the formation of pentosidine in the whole body in RA. The general increase in free radicals is likely to accelerate the formation of pentosidine in RA. Accordingly, pentosidine may not be a biochemical marker for joint degradation, but may be a marker for the disease activity of RA. However, further research is needed to verify this. The results in the present study showed that pentosidine levels in serum, synovial fluid and urine were significantly correlated with each other. However, serum and synovial fluid pentosidine were a more superior indicator for RA disease activity than urine pentosidine. Although the substances in synovial fluid can directly reflect the local joint status, such as the inflammation and degradation of the joint tissues, pentosidine may be carried into synovial fluid from the blood circulation. Therefore, among these body fluids, serum pentosidine is the best indicator for the disease status of RA. Table 1.  Comparison of pentosidine levels in serum, synovial fluid and urine in RA and OAa   RA  OA  P  aValues are the mean ± s.d . The values in parentheses are medians and interquantile ranges. P values are by Mann–Whitney U-test.  n  20  22    Age  61.0 ± 13.0  62.7 ± 10.6  0.98  Ser-pen (nmol/l)  152.0 ± 103.7 (123.6, 93.6)  66.7 ± 38.4 (55.0, 52.6)  0.0003  SP-pen (nmol/l)  98.9 ± 48.4 (86.3, 79.5)  56.6 ± 28.7 (52.8, 43.3)  0.023  Ur-pen (μmol/mol) creat  8.55 ± 5.56 (15.7, 15.6)  7.13 ± 3.03 (13.7, 8.8)  0.306    RA  OA  P  aValues are the mean ± s.d . The values in parentheses are medians and interquantile ranges. P values are by Mann–Whitney U-test.  n  20  22    Age  61.0 ± 13.0  62.7 ± 10.6  0.98  Ser-pen (nmol/l)  152.0 ± 103.7 (123.6, 93.6)  66.7 ± 38.4 (55.0, 52.6)  0.0003  SP-pen (nmol/l)  98.9 ± 48.4 (86.3, 79.5)  56.6 ± 28.7 (52.8, 43.3)  0.023  Ur-pen (μmol/mol) creat  8.55 ± 5.56 (15.7, 15.6)  7.13 ± 3.03 (13.7, 8.8)  0.306  View Large Fig. 1.  View largeDownload slide Correlation between serum, synovial fluid and urine pentosidine in all subjects with RA and OA. Equations: (A) Ser-pen (nmol/l)=1.67×(SF-pen nmol/l)−20.83, r=0.856, P=0.0001; (B) SF-pen (nmol/l)=4.88×(Ur-pen μmol/mol creat)+38.62, r=0.496, P=0.025; (C) Ser-pen (nmol/l)=9.68×(Ur-pen μmol/mol creat)+31.77, r=0.496, P=0.029. Fig. 1.  View largeDownload slide Correlation between serum, synovial fluid and urine pentosidine in all subjects with RA and OA. Equations: (A) Ser-pen (nmol/l)=1.67×(SF-pen nmol/l)−20.83, r=0.856, P=0.0001; (B) SF-pen (nmol/l)=4.88×(Ur-pen μmol/mol creat)+38.62, r=0.496, P=0.025; (C) Ser-pen (nmol/l)=9.68×(Ur-pen μmol/mol creat)+31.77, r=0.496, P=0.029. References 1  Sell DR, Monnier VM. Structure elucidation of a senescence cross-link from human extracellular matrix. J Biol Chem  1989; 264: 21597–602. Google Scholar 2  Sell DR, Monnier VM. End-stage renal disease and diabetes catalyze the formation of a pentose-derived crosslink from aging human collagen. J Clin Invest  1990; 85: 380–4. Google Scholar 3  Dyer DG, Dunn JA, Thorpe SR et al. Accumulation of Maillard reaction products in skin collagen in diabetes and aging. J Clin Invest  1993; 91: 2463–9. Google Scholar 4  Odetti P, Fogarty J, Sell DR, Monnier VM. Chromatographic quantitation of plasma and erythrocyte pentosidine in diabetic and uremic subjects. Diabetes  1992; 41: 153–9. Google Scholar 5  Takahashi M, Kushida K, Kawana K et al. Quantitation of the cross-link, pentosidine in serum from normal and uremic subjects. Clin Chem  1993; 39: 2162–5. Google Scholar 6  Takahashi M, Ohishi T, Aoshima H et al. The Maillard protein cross-link pentosidine in urine from diabetic patients. Diabetologia  1993; 36: 664–7. Google Scholar 7  Lyons TJ, Silvestri G, Dunn JA, Dyer DG, Baynes JW. Role of glycation in modification of lens crystallins in diabetic senile cataracts. Diabetes  1991; 40: 1010–5. Google Scholar 8  Blake DR, Lunec J. Copper, iron, free radicals and arthritis. Br J Rheumatol  1985; 24: 123–5. Google Scholar 9  Merry P, Winyard PG, Morris CJ, Grootveld M, Blake DR. Oxygen free radicals, inflammation, and synovitis: the current status. Ann Rheum Dis  1989; 48: 864–70. Google Scholar 10 Ansari NH, Awasthi YC, Srivastava SK. Role of glycosylation in protein disulphide formation and cataractogenesis. Exp Eye Res  1980; 31: 9–19. Google Scholar 11 Heliovaara M, Knekt P, Aho K, Aran RK, Alfthan G, Aromaa A. Serum antioxidants and risk of rheumatoid arthritis. Ann Rheum Dis  1994; 53: 51–3. Google Scholar 12 Takahashi M, Kushida K, Ohishi T et al. Quantitative analysis of crosslinks pyridinoline and pentosidine in articular cartilage of patients with bone joint disorders. Arthritis Rheum  1994; 37: 724–8. Google Scholar 13 Takahashi M, Suzuki M, Kushida K, Miyamoto S, Inoue T. Relationship between pentosidine levels in serum and urine and activity in rheumatoid arthritis. Br J Rheumatol  1997; 36: 637–42. Google Scholar 14 Rodriguez-Garcia J, Requena JR, Rodriguez-Segade S. Increased concentration of serum pentosidine in rheumatoid arthritis. Clin Chem  1998; 44: 250–5. Google Scholar 15 Miyata T, Ishiguro N, Yasuda Y et al. Increased pentosidine, an advanced glycation end product, in plasma and synovial fluid from patients with rheumatoid arthritis and its relation with inflammatory markers. Biochem Biophys Res Commun  1998; 244: 45–9. Google Scholar 16 Chen JR, Takahashi M, Suzuki M, Kushida K, Miyamoto S, Inoue T. Measurement of pentosidine in synovial fluid in patients with osteoarthritis and rheumatoid arthritis: Relationship with disease activity in rheumatoid arthritis. J Rheumatol  1998; 25: 2440–4. Google Scholar 17 Arnett FC, Edworthy SM, Bloch DA et al. The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum  1988; 31: 315–24. Google Scholar 18 Lansbury J. Methods for evaluating the status of rheumatoid arthritis. Ann Rheum Dis  1957; 16: 101–7. Google Scholar 19 Lansbury J. Methods for evaluating rheumatoid arthritis. In: Hollander JL, ed. Arthritis and allied conditions, 7th edn. Philadelphia: Lea and Febiger, 1966:269–91. Google Scholar 20 Grandhee SK, Monnier VM. Mechanism of formation of the maillard protein cross-link pentosidine. J Biol Chem  1991; 266: 11649–53. Google Scholar 21 Steinbrocker O, Traeger CH, Batterman RC. Therapeutic criteria in rheumatoid arthritis. J Am Med Assoc  1949; 140: 659–62. Google Scholar 22 Wolff SP, Jiang ZY, Hunt JV. Protein glycation and oxidative stress in diabetes mellitus and ageing. Free Radicals Biol Med  1991; 10: 339–52. Google Scholar 23 Kristal BS, Yu BP. An emerging hyothesis: synergistic induction of aging by free radicals and maillard reactions. J Gerontol  1992; 47: B107–14. Google Scholar 24 Monnier VM, Sell DR, Nagaraj RH et al. Maillard reaction-mediated molecular damage to extracellular matrix and other tissue proteins in diabetes, aging, and uremia. Diabetes  1992; 41(suppl. 2): 36–41. Google Scholar 25 Uchiyama A, Ohishi T, Takahashi M et al. Fluorophores from aging human articular cartilage. J Biochem  1991; 110: 714–8. Google Scholar 26 Takahashi M, Hoshino H, Kushida K, Inoue T. Direct measurement of crosslinks, pyridinoline, deoxypyridinoline, and pentosidine, in the hydrolysate of tissues using high-performance liquid chromatography. Anal Biochem  1995; 232: 158–62. Google Scholar 27 Lunec J, Halloran SP, White AG, Dormandy TL. Free-radical oxidation (peroxidation) products in serum and synovial fluid in rheumatoid arthritis. J Rheumatol  1981; 8: 233–45. Google Scholar 28 Nettelbladt E, Sundblad L. Protein patterns in synovial fluid and serum in rheumatoid arthritis and osteoarthritis. Arthritis Rheum  1959; 2: 144–9. Google Scholar 29 Willumsen L, Friis J. A comparative study of the protein pattern in serum and synovial fluid. Scand J Rheumatol  1975; 4: 234–40. Google Scholar © 1999 British Society for Rheumatology http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Rheumatology Oxford University Press

Comparison of the concentrations of pentosidine in the synovial fluid, serum and urine of patients with rheumatoid arthritis and osteoarthritis

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Publisher
Oxford University Press
Copyright
© 1999 British Society for Rheumatology
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1462-0324
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1462-0332
DOI
10.1093/rheumatology/38.12.1275
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Abstract

Abstract Objective.Pentosidine, an advanced glycation end product (AGE), has recently been observed to be elevated in rheumatoid arthritis (RA). The aim was to elucidate which pentosidine levels, i.e. in serum, synovial fluid or urine, are more related to the disease status of RA. Methods.We measured levels of pentosidine in serum, synovial fluid or urine in RA compared with osteoarthritis (OA), and examined the relationship between pentosidine and RA disease activity. Subjects were 20 patients with RA and 22 patients with OA. Results.In total RA and OA patients combined, there was a significant correlation between pentosidine in serum, synovial fluid and urine. Pentosidine in serum and synovial fluid was significantly higher in RA than in OA. In RA, there were significant correlations between pentosidine in serum and synovial fluid and C-reactive protein, Lansbury index (LI) and erythrocyte sedimentation rate. Conclusions.These results demonstrate that pentosidine levels in body fluids correlated with each other, and pentosidine in serum and in synovial fluid is associated with the systemic inflammatory activity of RA. Higher or similar concentrations of pentosidine in serum compared with synovial fluids indicate that the elevated pentosidine levels in serum in RA are not derived from the synovial fluid, but from an increase in the formation of pentosidine in the whole body in RA. Among body fluids, serum pentosidine was the superior indicator for RA disease status. Rheumatoid arthritis, Osteoarthritis, Pentosidine, Oxygen radicals Pentosidine is one of a number of advanced glycation end products (AGEs) [1]. Its significant elevation was observed in tissues in diabetes mellitus [2], in uraemic patients with end-stage renal failure [2–4], and in accelerated aging [5–7]. Pentosidine is formed by sequential glycosylation and oxidation reactions. Therefore, it is hypothesized that pentosidine formation is accelerated in diseases related to oxidative stress. Rheumatoid arthritis (RA) is a chronic systemic disease, although its major clinical consequence is inflammation of the joints and contiguous structures. There is now considerable evidence for the involvement of oxygen-centred free radicals in acute and chronic inflammatory arthritis [8, 9]. The role of free radicals in the autoxidation of biological lipids is well established [10]. Patients with RA have depressed serum levels of the antioxidant, and a low antioxidant level is a risk factor for RA [11]. Pentosidine is formed by sequential glycosylation and oxidation reactions. Because oxidative stress and oxygen free radicals play a significant role in tissue damage and inflammation in RA, pentosidine is supposed to be implicated in this disease [12–15]. Several years ago, pentosidine was reported to be elevated in the articular cartilage of RA [12]. However, its significant relationship to RA has not been investigated until recently. Now, there have been several studies which provide evidence that pentosidine is elevated in patients with RA. Pentosidine in serum and urine is elevated, and reflects the disease activity of RA [13]. Pentosidine in synovial fluid is higher in RA than in osteoarthritis (OA), and its level also reflects the disease activity of RA [16]. Although all pentosidine levels in body fluids have been associated with RA disease activity, it has not been clear which pentosidine levels, i.e. in serum, synovial fluid or urine, are a superior indicator. In the present study, therefore, we investigated pentosidine levels in serum, synovial fluid and urine in OA and RA. Patients and methods Patients The study included 20 patients with RA and 22 patients with OA. RA was diagnosed according to the criteria of the American College of Rheumatology (ACR) [17]. The RA group consisted of 15 female and five male patients aged 25–69 yr (mean±s.d.=61.0±13.0). OA of the knee joints was diagnosed on the basis of clinical symptoms, examination and radiographic findings. These patients fulfilled the ACR criteria of OA. The OA group consisted of 13 female and nine male patients aged 46–79 yr (mean±s.d.=62.7±10.6). They were attending the out-patient clinic in the Department of Orthopaedic Surgery at Hamamatsu University School of Medicine. There was no significant difference in age between RA and OA patients. Patients who had diabetes mellitus, steroid users and those with abnormal levels of serum blood urea nitrogen (BUN) or creatinine were excluded from the study. Blood, synovial fluid and urine were collected on the same day from all RA and OA patients. After collection, blood samples were centrifuged at 3000 r.p.m. for 15 min. Synovial fluid was obtained by knee aspiration. Sera, synovial fluid and urine samples were kept at −30°C until analysis. Informed consent was obtained from all participants. The procedures followed were in accordance with the principles of the Declaration of Helsinki in 1975, as revised in 1983. Clinical features, blood biochemistry in RA Serum C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR) were measured by routine laboratory methods. A Lansbury index (LI) was determined based on the duration of morning stiffness, ESR (value at 1 h), grip strength (mmHg) and joint score [18, 19]. Urinary creatinine content was measured by a routine laboratory method. Measurements of pentosidine in serum, synovial fluid and urine The pre-treatment of samples before injection into the HPLC column was described previously [13, 16]. Pentosidine was measured by a direct HPLC method with column switching as described previously [13]. Briefly, the samples were injected into a gel-filtration pre-column (TSK pre-column PW, 4.6 mm×3.5 cm; TOSOH, Tokyo, Japan), the eluate fraction containing pentosidine selected, and this fraction introduced into a reversed-phased column (TSK-GEL ODS-80T, 4.6 mm×15 cm; TOSOH) by use of a switching valve. The urine sample was injected directly into the HPLC column without acid hydrolysis. The concentrations of pentosidine in serum and synovial fluid were expressed as nmol/l. The concentrations of pentosidine in urine were corrected by the concentrations of urine creatinine and expressed as μmol/mol creatinine. Standard pentosidine was synthesized [20] and the concentration was calibrated with pentosidine, which was a gift from Dr V. M. Monnier. Statistical analysis The statistical significance of difference was determined with non-parametric statistics using Mann–Whitney U-tests between two groups. Simple regression was performed for univariate correlation and the statistical significance of correlation was determined with the Spearman rank correlation test. The analysis was performed with StatView II software (Abacus Concepts, Inc., Berkeley, CA, USA) on a Macintosh computer (Apple Computer, Inc., Cupertino, CA, USA). Values of P<0.05 were considered significant. Results Pentosidine levels in serum and synovial fluid were significantly higher in RA than in OA (Table 1). In urine, pentosidine levels tended to be higher in RA than in OA, but not significantly so. In either RA or OA, pentosidine levels tended to be higher in serum than in synovial fluid, but there was no statistically significant difference. The correlations of pentosidine levels between serum, synovial fluid and urine were examined in total patients of RA and OA. Pentosidine levels in serum, synovial fluid and urine were significantly correlated to each other (Fig. 1). Pentosidine levels in serum and synovial fluid of RA were significantly correlated with CRP, ESR and LI. However, there was no significant correlation between urine pentosidine levels and CRP, ESR and LI (data not shown). When pentosidine levels were compared between subgroups of RA patients who were divided into high (CRP 20 mg/l and LI 40%) and low (CRP<20 mg/l and/or LI<40%) activity groups, pentosidine levels in serum and synovial fluid were significantly higher in the high-activity group than in the low-activity group (P<0.05). However, there was no significant difference in urine pentosidine levels between these two groups. When pentosidine levels were compared between subgroups of RA patients who were classified by stage (Stage I+II and Stage III+IV) or class (Class I+II and Class III+IV) according to Steinbrocker et al. [21], there were no statistically significant differences in serum, synovial fluid and urine pentosidine levels between the two subgroups of stage and the two subgroups of class (data not shown). Discussion Free radicals are implicated in tissue damage and inflammation in RA [8, 9], and the formation of pentosidine is associated with oxidation [22]. However, there have so far been a few reports studying pentosidine in RA [12–16]. To our knowledge, we were the first to report the increased levels of serum pentosidine in RA [13]. The present study demonstrates that pentosidine levels of several body fluids were elevated in RA. However, the mechanisms of these phenomena remain unclear. It is known that chronic hyperglycaemia leads to the accumulation of non-enzymatically derived glycosylation products of proteins, such as pentosidine in diabetes mellitus. In RA, the increased pentosidine concentrations are present in body fluids; however, glucose levels are normal. Interest in recent years has focused on the potential of glycated proteins as a source of free radicals [23]. Exposure of IgG and other proteins to oxygen radicals, generated by photolysis or by a mixture of iron or copper salts and hydrogen peroxide, induces fluorescence in the protein which is essentially indistinguishable from that attributed to Schiff base formation. Because pentosidine formation requires aerobic conditions, we propose that such free radicals could explain possible pathways for the elevation of pentosidine in RA. Thus, protein-linked fluorescence such as that of pentosidine may therefore not be the result of cumulative glycaemia over many years, but could be the result of successive exposure to free radical-induced oxidation, and this may be of importance in disease activity in RA. When we first observed the elevated levels of serum pentosidine in RA and its significant relationship to disease activity [13], we supposed that the elevated pentosidine in the blood circulation was derived from the inflammatory joints in RA. Actually, pentosidine in synovial fluid was observed to be increased greater in RA than OA in the successive study [16]. In that study, we proposed two possible pathways for the elevation of pentosidine in RA. One possibility is that because pentosidine is abundant in cartilage compared with the other tissues [24–26], inflammation of the joints may induce the breakdown of cartilage, releasing cartilaginous pentosidine into synovial fluid; the other is an increase in the formation of pentosidine in synovial fluid. It is known that total proteins increase in synovial fluid in severely inflamed joints, especially in those of RA. It is also known that free radical oxidation products of synovial fluid were higher in inflammatory than in degenerative joint disease, and in patients with RA than in normal controls [27]. A combination of elevated proteins (and amino acids) and the acceleration of free radicals in synovial fluid of RA stimulates Maillard reactions, then increases the formation of pentosidine in synovial fluid in RA. However, the present study and the previous one [15] showed that pentosidine levels in synovial fluid were relatively lower than those in serum because a certain retention of proteins in synovial membranes and a consumption of the protein in the joint make the concentrations of proteins in synovial fluid slightly lower than those in plasma [28, 29]. Therefore, the elevated pentosidine levels in serum of RA are not derived from the synovial fluid, but from an increase in the formation of pentosidine in the whole body in RA. The general increase in free radicals is likely to accelerate the formation of pentosidine in RA. Accordingly, pentosidine may not be a biochemical marker for joint degradation, but may be a marker for the disease activity of RA. However, further research is needed to verify this. The results in the present study showed that pentosidine levels in serum, synovial fluid and urine were significantly correlated with each other. However, serum and synovial fluid pentosidine were a more superior indicator for RA disease activity than urine pentosidine. Although the substances in synovial fluid can directly reflect the local joint status, such as the inflammation and degradation of the joint tissues, pentosidine may be carried into synovial fluid from the blood circulation. Therefore, among these body fluids, serum pentosidine is the best indicator for the disease status of RA. Table 1.  Comparison of pentosidine levels in serum, synovial fluid and urine in RA and OAa   RA  OA  P  aValues are the mean ± s.d . The values in parentheses are medians and interquantile ranges. P values are by Mann–Whitney U-test.  n  20  22    Age  61.0 ± 13.0  62.7 ± 10.6  0.98  Ser-pen (nmol/l)  152.0 ± 103.7 (123.6, 93.6)  66.7 ± 38.4 (55.0, 52.6)  0.0003  SP-pen (nmol/l)  98.9 ± 48.4 (86.3, 79.5)  56.6 ± 28.7 (52.8, 43.3)  0.023  Ur-pen (μmol/mol) creat  8.55 ± 5.56 (15.7, 15.6)  7.13 ± 3.03 (13.7, 8.8)  0.306    RA  OA  P  aValues are the mean ± s.d . The values in parentheses are medians and interquantile ranges. P values are by Mann–Whitney U-test.  n  20  22    Age  61.0 ± 13.0  62.7 ± 10.6  0.98  Ser-pen (nmol/l)  152.0 ± 103.7 (123.6, 93.6)  66.7 ± 38.4 (55.0, 52.6)  0.0003  SP-pen (nmol/l)  98.9 ± 48.4 (86.3, 79.5)  56.6 ± 28.7 (52.8, 43.3)  0.023  Ur-pen (μmol/mol) creat  8.55 ± 5.56 (15.7, 15.6)  7.13 ± 3.03 (13.7, 8.8)  0.306  View Large Fig. 1.  View largeDownload slide Correlation between serum, synovial fluid and urine pentosidine in all subjects with RA and OA. Equations: (A) Ser-pen (nmol/l)=1.67×(SF-pen nmol/l)−20.83, r=0.856, P=0.0001; (B) SF-pen (nmol/l)=4.88×(Ur-pen μmol/mol creat)+38.62, r=0.496, P=0.025; (C) Ser-pen (nmol/l)=9.68×(Ur-pen μmol/mol creat)+31.77, r=0.496, P=0.029. Fig. 1.  View largeDownload slide Correlation between serum, synovial fluid and urine pentosidine in all subjects with RA and OA. 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Rheumatology – Oxford University Press

Published: Dec 1, 1999

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