The aim of this study is to re-evaluate urinary collagen cross-links, previously proposed as markers of osteoarthritis (OA). The urinary excretion of collagen cross-links, pyridinoline (PYD) and deoxypyridinoline (DPD), was measured using high-performance liquid chromatography (HPLC) in 114 patients with OA, 19 patients with rheumatoid arthritis (RA) and 40 healthy subjects. An increase in PYD and DPD, expressed per millimole of creatinine, was confirmed in RA. However, PYD and DPD in patients with hip OA, knee OA and polyOA were similar, and did not differ from controls. In patients with radiographic end-stage OA, PYD and DPD were significantly higher than in patients with an early OA, but not significantly higher than in controls. The PYD/DPD ratio did not vary with the OA stage. Thus, urinary collagen cross-links are not elevated in OA, but could reflect bone sclerosis and/or erosion in late O

DALLARD, M. C.

HELLIO LE GRAVERAND, M. P.

ICHOU, M.

RICHARD, M.

TRON, A. M.

UEBELHART, D.

VIGNON, E.

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British Journal of Rheumatology 1996;35:1091-1095ASSESSMENT OF URINARY HYDROXYPYRIDINIUM CROSS-LINKSMEASUREMENT IN OSTEOARTHRITISM. P. HELLIO LE GRAVERAND, A. M. TRON, M. ICHOU,* M. C. DALLARD, M. RICHARD,*D. UEBELHARTf and E. VIGNONPavilion F and * Biochemistry Laboratory, Claude Bernard University, Edouard Herriot Hospital, place d'Arsonval,69437 Lyon Cedex 03, France and f University Hospital of Geneva, Switzerland and Rush Presbyterian, St Luke'sMedical Center, Chicago, IL, USASUMMARYThe aim of this study is to re-evaluate urinary collagen cross-links, previously proposed as markers of osteoarthritis (OA). Theurinary excretion of collagen cross-links, pyridinoline (PYD) and deoxypyridinoline (DPD), was measured usinghigh-performance liquid chromatography (HPLC) in 114 patients with OA, 19 patients with rheumatoid arthritis (RA) and 40healthy subjects. An increase in PYD and DPD, expressed per millimole of creatinine, was confirmed in RA. However, PYDand DPD in patients with hip OA, knee OA and polyOA were similar, and did not differ from controls. In patients withradiographic end-stage OA, PYD and DPD were significantly higher than in patients with an early OA, but not significantlyhigher than in controls. The PYD/DPD ratio did not vary with the OA stage. Thus, urinary collagen cross-links are not elevatedin OA, but could reflect bone sclerosis and/or erosion in late OA.KEY WORDS: Pyridinium cross-links, Osteoarthritis, Rheumatoid arthritis.THE covalent bridges or cross-links between theadjacent molecules of collagen stabilize the extracellu-lar matrix of connective tissues. Owing to their naturalfluorescent properties, the pyridinium cross-links,pyridinoline (PYD) and deoxypyridinoline (DPD),have been identified in many connective tissues,essentially in bone and cartilage [1, 2]. DPD is mostlyfound in bone, while PYD is found both in skeletaland vascular connective tissues [1, 3]. Unlike hydroxy-proline, pyridinium cross-links are released followingmature collagen degradation in peptide-free formsand are excreted unchanged in urine. Pyridiniumcross-links are measured by high-performance liquidchromatography (HPLC), paper chromatographyand enzyme-linked immunoassay (ELISA) [4]. Bothpaper chromatography and ELISA do not allow PYDand DPD to be distinguished. Quantitative analysisusing ion-paired reversed-phase liquid chromato-graphy followed by fluorescent detection has beenimproved [5].Pyridinium cross-links have already been proposedas an index of bone resorption in Paget's disease [6],primary hyperparathyroidism [7], bone metastasis [8]and osteoporosis [9]. PYD and DPD were alsoinvestigated in osteoarthritis (OA) and rheumatoidarthritis (RA). They were suggested as markers of jointdegradation [10-15]. These studies led us to correlateurinary pyridinium cross-links with the measurementof joint space narrowing in patients with hip OA [16].However, initial results obtained in such patientsclearly conflicted with reported studies. The presentSubmitted 21 December 1995; revised version accepted 26 April1996.Correspondence to: E. Vignon, Claude Bernard University,Edouard Herriot Hospital, Pavilion F, place d'Arsonval, 69437 LyonCedex 03, France.work is a re-evaluation of urinary concentrations ofpyridinium cross-links in OA patients.PATIENTS AND METHODSPatientsThe OA group was made up of 114 consecutivepatients, referred to the rheumatology clinic forsymptomatic OA. There were 47 hip OA (coxOA), 31knee OA (gonOA) and 36 patients with generalized OA(polyOA). All these patients fulfilled the ACR criteriafor OA [17]. A normal control group was composed of40 healthy volunteers. A joint disease control groupwas composed of 19 patients with classic RA,according to the ARA criteria. Age, sex, menopausalstatus, weight and body mass index are given inTable I.Anteroposterior roentgenograms of lumbar rachis,pelvis and knees taken in the weight-bearing position,a sunrise view of the patellofemoral joints and dorsalX-ray of the hands were performed in OA patients andnormal controls. Films were read by a singleexperienced observer who was blinded to the patient'sstatus. X-ray OA was scored as follows: stage 1,minimal joint space loss and/or osteophytosis; stage 2,substantial but incomplete joint space loss; stage 3,total and focal joint space narrowing; stage 4, nearlycomplete joint space narrowing and subchondral boneerosion (intraobserver reproducibility: k = 0.76) [18].In patients with bilateral OA, the most severely affectedside was analysed.Westergren erythrocyte sedimentation rate (ESR)(mm/h), routine blood haematology and biochemistrytests (red and white cell counts, alkaline phosphatase,alanine aminotransferase, calcium, phosphorus, gly-caemia, albumin and creatinine) were performed in OAand RA patients. None of the patients suffered fromrenal impairment, diabetes or a metabolic bone disease.© 1996 British Society for Rheumatology10911092ControlsOARACharacteristicsJV4011419BRITISH JOURNAL OF RHEUMATOLOGY VOL- 35TABLE Iof patients with osteoarthritis (OA), rheumatoid arthritis (RA) andAge42.9 (12)62.44(11.5)58.3 (18)Weight70.56 (13.4)73.72 (12.5)66.67 (15)BMI24.37 (4.2)27.74 (4.2)24.75 (4)NO. 11controls (medianSex ratioM/F21/1949/658/11andiD.)Post-menopausal/non-post-menopausal9/1050/157/4Measurement of urinary pyridinoline and deoxypyridino-lineMorning urine samples were collected from allpatients and stored at — 20°C prior to analysis. Urinesamples were centrifuged at 1000 g during 10 min andan aliquot of urine was hydrolysed by heating with anequal volume of 12 M HC1 at 116°C during 16 h, toconvert all cross-links to the free form. The hydro-lysates were fractionated by partition chromatographyon CFl cellulose and the pyridinium compounds werequantified by reverse-phase, isocratic, ion-pairedHPLC as described by James et al. [19]. The ion-pairingagent used was the octane sulphonic acid. Concen-trations of urinary pyridinium cross-links wereexpressed relative to values of urinary creatinine(nmol/mmol Cr). The coefficient of variation of thePYD and DPD assay was 14 and 15%, respectively.Statistical analysisA comparison between two groups was performedusing Student's f-test. Analysis of variance was usedwhen there were more than two groups. Correlationswere studied using simple linear regression.RESULTSFigure 1 shows the individual values of PYD andDPD for each group. Means and standard deviationsaccording to age and sex are given in Table II. PYDand DPD excretion, as well as the PYD/DPD ratio,were unrelated to sex, age, menopausal status, weightor body mass index (BMI) in each of the control,OA and RA groups. In healthy control individuals,mean values and standard deviations of PYD andDPD/mmol of creatinine were 34.5 ± 4.9 and6.9 ± 1.1 nmol/mmol, respectively. The PYD/DPDratio was 8.1 ± 1.9 nmol/mmol. The strong correlationbetween PYD and DPD (r = 0.76; /> < 0.001) isillustrated in Fig. 2.Urinary concentrations of PYD and DPD in OApatients were slightly but not significantly lower thanthose in the control group (30.2 ±1.7 and6.2 ± 0.4 nmol/mmol, respectively). The PYD/DPDratio was unmodified in OA (5.5 ± 0.2 nmol/mmol).PYD was sometimes increased in polyOA, but nosignificant difference was found between patients withhip OA, knee OA and polyOA (Fig. 3). Urinaryconcentrations of hydroxypyridinium cross-links ac-cording to radiographic scores were studied in patientswith hip or knee OA. They clearly increased withradiographic scores, but the difference between the fourgroups was not significant. When pooling patients withearly OA (X-ray score 1 and 2) and patients with lateOA (X-ray score 3 and 4), PYD and DPD were,respectively, 1.47- and 1.4-fold higher in late OA thanin early OA (P = 0.003 and 0.04, respectively) (Fig. 4).The PYD/DPD ratios were 4.4 ± 1.3 and 6.1 ±2.7,respectively. The urinary excretion of PYD and DPDwas unrelated to NSAID uptake.PYD and DPD were increased by 42.5 and 29.7%,respectively, in RA patients, when compared withnormal controls. The difference was significantS 150£u"5EoEcaa.c3100 -50 -OA RA Controlsoceo"5EoC4030 -20 -10 -OA RA ControlsFIG. I.—Urinary concentrations of pyridinoline (PYD) (a) anddeoxypyridinoline (DPD) (b) in patients with osteoarthritis (OA) andrheumatoid arthritis (RA) and controls. All concentrations areexpressed relative to urinary creatinine.HELLIO LE GRAVERAND ET AL.: PYRIDINIUM CROSS-LINKS AND OA 1093TABLE IIPyridinium cross-link values (median and S.D.) in patients with osteoarthritis (OA), rheumatoid arthritis (RA) and controls relative to sex andmenopausal status. Values are expressed relative to urinary creatinineControls OA RAPYD DPD(nmol/mmol) (nmol/mmol)PYD DPDN (nmol/mmol) (nmol/mmol)PYD DPDN (nmol/mmol) (nmol/mmol)MalesNon-post-menopausalfemalesPost-menopausal femalesTotal211094033.77 ±3.9 6.71 ± 1.9 49 27.45 ± 2.2 4.9 ± 0.4 8 47.61 ± 3.9 8.58 ± 1.632.86 ± 6.637.78 ± 8.634.5 ± 4.97.89 ± 2.46.34 ± 1.96.92 ± 1.1155011422.9 ±2.133.17 ±2.830.17 ± 1.74.9 ± 0.77.42 ± 0.76.21 ± 0.4471952.3 ± 10.953.2 ± 10.550.66 ± 5.97.72 ± 1.4910.4 ± 2.28.98 ± 1.04{P < 0.02) for PYD only. The difference with the OAgroup was significant for both parameters (P = 0.018and P = 0.02, respectively). The PYD/DPD ratio was6 ± 0.5. PYD and DPD were significantly (P < 0.05)higher, by 104.1 and 69.7%, respectively, in RApatients treated with steroids than in RA patientswithout steroids (Table III). The latter were notsignificantly different from the normal controls. ThePYD/DPD ratio did not vary with the steroid intake.There was no correlation between cross-links and ESR.DISCUSSIONThe present work does not confirm the previouslyreported increased urinary concentrations of PYD andDPD m OA [13, 15].The difference between our results and those ofprevious studies does not seem to be in relation tothe controls. Our number of normal controls israther higher than that of previous studies, in some ofwhich the same control group was used repeatedly[11,12,15]. Means and standard deviations of thepresent normal control group were also close to thoseof previous studies in which the same HPLC methodwas used [10, 19]. There were more women and oldpatients in our OA group than in the control group.The urinary excretion of pyridinium cross-links issimilar in men and pre-menopausal women. It was alsofound to be unrelated to age. An increased excretion ofPYD and DPD in peri- and post-menopausal womenwas demonstrated [20, 21]. However, increasing thenumber of menopaused women in our control groupwould have increased the normal values accordingly.The selection of OA patients could be an explanationFIG. 2.—Correlation between urinary pyridinoline (PYD) andurinary deoxypyridinoline (DPD) values in controls. Spearman test:P< 0.001 and r = 0.76.for our results. MacDonald et al. [15] found a higherurinary excretion of PYD in gonOA than in coxOAand polyOA. Astbury et al. [14] demonstrated that theurinary excretion of pyridinium cross-links could berelated to joint damage. The present work does notsupport the findings of MacDonald et al. and ratherIooEEonc3150100 "50 "knee hip potyOA£ 40re£o•5o302010Knee hip potyOAFIG. 3.—Urinary pyridinoline (PYD) (a) and deoxypyridinoline(DPD) (b) values in osteoarthritis (OA) subgroups. All concen-trations are expressed relative to urinary creatinine.1094 BRITISH JOURNAL OF RHEUMATOLOGY VOL. 35 NO. 11soPYD• early OA• late OACO£uIIcDPDFIG. 4.—Urinary pyridinoline (PYD) and deoxypyridinoline (DPD)values relatively to radiographic scores. Patients were divided intoearly OA (X-ray scores 1 or 2) and late OA (X-ray scores 3 or 4).PYD and DPD increased significantly in late OA: P •= 0.003 andP •» 0.04, respectively.suggests an increase in some cases of polyOA. Itdemonstrates that PYD and DPD excretion increasedsignificantly with the radiological stage of OA.Although there was no significant difference betweenthe late OA subgroup and the normal group, theselection of a larger number of patients with late OAmight have made the difference between OA and thecontrol significant.The tissular topography of hydroxypyridiniumPyridinium cross-linkRA + steroidsRA — steroidsTotalN91019TABLE IIIvalues (median and s.D.)intakePYD78.7 ± 7.238.5 ± 2.350.6 ± 5.9relative to steroidDPD12.9 ±7.6 ±8.8 ±1.80.41.04DPD/PYD7.1 ±0.95.3 ± 0.36.1 ±0.4cross-links has been studied in detail [1,2]. DPD couldbe considered as a sensitive marker of bonedegradation since it is mostly found in bone anddentine, and the turnover of the latter tissue isnegligible [1]. PYD is a major collagen cross-link inbone, cartilage and other connective tissues such asmuscle and intervertebral discs [1, 22]. Although boneresorption is probably the major source of urinehydroxypyridinium cross-links [13, 14], the PYD/DPDratio may help to define collagen degradation incartilage relative to other tissues. In agreement withprevious studies in OA patients [10, 13], PYD andDPD were highly correlated (r = 0.75, P < 0.001), andthe PYD/DPD ratio was unchanged. In patients withadvanced OA, both PYD and DPD were significantlyincreased and the PYD/DPD ratio was also un-changed. Thus, most probably, the observed increasedexcretion of pyridinium cross-links in patients withlate OA reflects the bone erosion and/or the increasedsclerotic bone remodelling of joint epiphysis thatoccurs in the advanced stages of OA. Unfortunately,bone sclerosis was not graded in our score of OA [18].The unchanged excretion of PYD and DPD in earlierstage OA suggests that the markers certainly donot reflect collagen degradation of the articularcartilage.An increased urinary excretion of pyridiniumcross-links has been reported in RA [12, 13, 14, 23, 24].A small control group of RA patients was included inthis study to test the validity of our measurements. Wewere able to confirm a dramatic increase in the urinaryexcretion of PYD and DPD in RA patients.Interestingly, this increase was only found in patientstreated with steroids. Since joint destruction in RApatients was not investigated, the results suggest thatan increase in urinary PYD and DPD was related tosevere forms of RA justifying steroids, and/or with theincreased bone resorption induced by steroids.ACKNOWLEDGEMENTPart of this work was supported by grant no.32-35582.92 from the Swiss National Foundation forScientific Research to DU.REFERENCES1. Eyre DR, Koob JJ, Van Ness KB. Quantitation ofhydroxypyridinium crosslinks by HPLC. Anal Biochem1984;137:380-8.2. Eyre DR, Paz MA, Galop PM. Crosslinking in collagenand elasu'n. Annu Rev Biochem 1984;53:717-48.3. Robins SP, Duncan A. Pyridinium crosslinks of bonecollagen and their location in pcptides isolated from ratfemur. Biochim Biophys Ada 1987^14:233-9.4. Robins SP. An enzyme-linked immunoassay for thecollagen crosslink pyridinoline. Biochem J 1982;207:617-20.5. Black D, Duncan A, Robins SP. Quantitative analysis ofthe pyridinium crosslinks of collagen in urine usingion-paired reversed-phase high-performance liquid chro-matography. Anal Biochem 1988;169:197-203.6. Uebelhart D, Gineyts EC, Chapuy MC, Delmas PD.Urinary excretion of pyridinium crosslinks: A newHELLIO LE GRAVERAND ET AL.\ PYRIDIN1UM CROSS-LINKS AND OA 1095marker of bone rcsorption in metabolic bone disease.Bone Miner 1990;8:87-96.7. Seibel MJ, Gartenberg F, Silverberg SJ, Ratcliffe A,Robins SP, Bilizikian JP. 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ASSESSMENT OF URINARY HYDROXYPYRIDINIUM CROSS-LINKS MEASUREMENT IN OSTEOARTHRITIS

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