ARTHRITIS & RHEUMATISMVol. 46, No. 8, August 2002, pp 2105–2108DOI 10.1002/art.10424© 2002, American College of Rheumatology

The Effects of Age and Sex on Chondroitin Sulfates in NormalSynovial Fluid

Yoshihito Nakayama,1 Tetsuya Narita,1 Atsushi Mori,1 Shinji Uesaka,1 Kyosuke Miyazaki,2

and Hiromoto Ito1

Objective. To examine how age and sex influencechondroitin sulfates (CS) in normal synovial fluid, wemeasured the concentrations of chondroitin 6-sulfate(C6S), chondroitin 4-sulfate (C4S), and hyaluronic acid(HA) in healthy subjects of different ages.

Methods. Synovial fluid samples were obtainedfrom 82 healthy volunteers, ages 20–79 years.

Results. The concentrations of CS and HA andthe C6S:C4S ratio varied with age. Their values werehighest between 20 and 30 years of age, and thereafterthey showed a tendency to decrease. Statistically, theC6S concentration and the C6S:C4S ratio at ages 60–70years were significantly lower than those at 20–30 yearsof age. There was also a clear between-sex difference, inwhich the CS concentrations and the C6S:C4S ratio inwomen were significantly lower than those in men (P �0.0003 for C6S, P � 0.02 for C4S, P � 0.002 for C6S:C4Sratio). In sharp contrast, little between-sex differencewas found in the HA concentration. In multiple regres-sion analysis, age correlated strongly with the C6Sconcentration and the C6S:C4S ratio (r � �0.521 andr � �0.617, respectively), weakly with the C4S concen-tration (r � �0.202), and moderately with the HAconcentration (r � �0.483). Sex showed a weak corre-lation with the concentrations of C6S and C4S and theC6S:C4S ratio (r � 0.307, r � 0.225, and r � 0.237,respectively), and little correlation was seen between sexand the HA concentration.

Conclusion. The CS concentrations and the sul-fation patterns in normal synovial fluid vary with age

and sex, and these physiologic variations need to betaken into account when using synovial fluid CS asmarkers for arthritic conditions.

Glycosaminoglycans (GAGs) in synovial fluid areimportant for joint lubrication and for cartilaginousnutrition (1). Their concentrations in articular cartilagehave been published recently (2), to reflect joint-tissuemetabolism under normal and pathologic conditions.Chondroitin sulfates (CS) in synovial fluid have beeninvestigated at the center of arthritic diseases, and theyhave attracted considerable interest as possible markersfor joint disease (3).

Changes in the concentration of chondroitin6-sulfate (C6S) and chondroitin 4-sulfate (C4S) and theC6S:C4S ratio have been reported to represent thepathologic process in the cartilage of patients withrheumatoid arthritis (RA) and osteoarthritis (OA) (4–8). However, these arthritic diseases occur more com-monly in women, and increase with age; it is importantthat the changes associated with joint pathology aredistinguished from normal age-related and sex-relatedevents. To our knowledge, there have been few studiesfocusing on the effects of these two demographic vari-ables in normal synovial fluid.

The aim of this study was to examine CS in thesynovial fluid of healthy subjects of different ages, and toassess how age and sex influence the CS concentrationsand the sulfation patterns.

SUBJECTS AND METHODS

Subjects. Synovial fluid was obtained from 82 healthyvolunteers with no history of joint diseases or injuries. Theirconsent was obtained in accordance with the guidelines of theethics committee of Nippon Medical School. These 82 volun-teers consisted of 46 men and 36 women, with a mean age of45.3 years (range 20–79 years).

Synovial fluid preparation. Synovial fluids were aspi-rated utilizing a lateral infrapatellar approach, using a 21-

1Yoshihito Nakayama, MD, Tetsuya Narita, MD, AtsushiMori, MD, Shinji Uesaka, MD, Hiromoto Ito, MD: Nippon MedicalSchool, Tokyo, Japan; 2Kyosuke Miyazaki: Tokyo Research Institute,Seikagaku Corporation, Tokyo, Japan.

Address correspondence and reprint requests to YoshihitoNakayama, MD, Department of Orthopaedic Surgery, Nippon Medi-cal School, Daini Hospital, 1-396 Kosugi-cho, Nakahara-ku, Kawasaki,Kanagawa 211-8533, Japan. E-mail: nakayama_y/orthop@nms.ac.jp.

Submitted for publication August 15, 2001; accepted inrevised form April 4, 2002.

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gauge needle. In our experience, this approach is superior tothe conventional, lateral suprapatellar approach in terms ofthe yield of synovial fluid. Samples were collected in steriletubes and centrifuged at 10,000g for 15 minutes at 4°C toremove cells and joint debris. The supernatants were stored at�80°C until subjected to the biochemical assay for CS isomersand hyaluronic acid (HA).

Enzymatic digestion of synovial fluid. The volume ofsynovial fluid required for the assay was 0.2 ml. The unsatur-ated disaccharides of CS were measured by high-performanceliquid chromatography (HPLC) (9) and those of HA by theMorgan-Elson method (10). Chondroitinase ABC (50 �l, 5units/ml), 10 mM sodium acetate buffer (80 �l, pH 8.0), anddistilled water (70 �l) were added to 10-fold–diluted synovialfluid (200 �l). The mixture was incubated at 37°C for 2 hours,and then ultrafiltered by the Ultrafree C3GC system(molecular-size cutoff value of 10,000; Japan Millipore, Tokyo,Japan). To completely reduce HA to the disaccharide, Strep-tococcus dysgalactiae hyaluronidase (30 �l, 0.5 units/ml; Seika-gaku Corporation, Tokyo, Japan) and 100 mM sodium acetatebuffer (30 �l, pH 6.0) were added to the ultrafiltrate of 300 �l,and incubated at 37°C for 2 hours. After ultrafiltrating, themixture was analyzed by HPLC.

HPLC analysis. The unsaturated disaccharides in eachsample were eluted with a gradient of 0–100 mM sodiumsulfate for 60 minutes at a flow rate of 0.5 ml/minute. Theeluant from the column was mixed with 100 mM sodiumtetraborate buffer containing 10 mg/ml 2-cyanoacetamide at aflow rate of 0.5 ml/minute, and the mixture was passed througha polyetheretherketone reaction coil (0.8 mm � 10m) in adry-reaction bath set at 137°C. The effluent was monitored byspectrofluorometer, set at an excitation wavelength of 331 nmand an emission wavelength of 383 nm. The coefficient ofvariance of the assay was 4.4% for C6S and 7.1% for C4S in 10repetitions of measurement of the unsaturated disaccharidestandards. The unsulfated disaccharide chondroitin (C0S) insynovial fluid could not be quantified by the HPLC methodused in this study because of overlap of HA disaccharide withC0S.

Statistical analysis. The data were analyzed by Stat-View 4.0 (Brain Power, Calabasas, CA), and a P value less than0.05 was accepted as the minimum level of significance.Multiple comparison was performed using Scheffe’s methodwith analysis of variance. Multiple regression analysis wascarried out, and the effects of variables were expressed aspartial correlation coefficients.

RESULTS

The overall results of the concentrations of CSand HA and the C6S:C4S ratio in 82 healthy subjects aresummarized in Table 1. The values of all these markerswere highest between 20 and 30 years of age, andthereafter they showed a tendency to decrease. Statisti-cally, the C6S concentration and the C6S:C4S ratio at60–70 years were significantly lower than those at 20–30years of age. With respect to the concentrations of C4Sand HA, a significant difference was observed only in theHA concentration, between ages 20 years and 70 years.

As for a sex-related difference, the concentra-tions of C6S and C4S and the C6S:C4S ratio in womenwere significantly lower than those in men (P � 0.0003,P � 0.02, and P � 0.002, respectively) (Table 2). Incontrast, there was little between-sex difference withrespect to the HA concentration (P � 0.586). When thesubjects studied were divided into 3 age groups, the C6Sconcentration in women tended to be lower than that inmen, irrespective of the age group.

When the effects of age and sex on the markerswere expressed as partial correlation coefficients (Table3), age strongly correlated with the C6S concentrationand the C6S:C4S ratio (r � �0.521 and r � �0.617,respectively), whereas there was only a weak correlation

Table 1. Synovial fluid concentrations of chondroitin sulfates (CS) and hyaluronic acid (HA) of healthy subjects of various ages*

Age, years

Number of subjects

Volume, ml C6S, nmoles/ml C4S, nmoles/ml C6S:C4S HA, mg/mlTotal Men Women

20–29 22 13 9 0.6 � 0.3 125.8 � 46.1 18.9 � 4.4 6.5 � 1.1 3.5 � 0.630–39 15 10 5 0.6 � 0.1 121.9 � 34.7 20.3 � 6.9 6.1 � 0.9 3.5 � 0.840–49 11 6 5 0.6 � 0.3 101.6 � 22.5 18.4 � 3.7 5.5 � 0.7 3.2 � 0.550–59 11 7 4 0.6 � 0.4 99.4 � 28.3 18.4 � 5.9 5.6 � 1.4 3.0 � 0.760–69 13 6 7 0.4 � 0.3 76.8 � 18.5† 17.5 � 4.4 4.4 � 0.6‡ 2.8 � 0.670–79 10 4 6 0.7 � 0.5 64.9 � 18.7† 15.3 � 2.7 4.2 � 1.0‡ 2.6 � 0.6§Total 82 46 36 0.6 � 0.4 103.1 � 38.8 18.4 � 5.0 5.6 � 1.3 3.2 � 0.7

* Except where otherwise indicated, values are the mean � SD.† P � 0.005 for 20 years versus 60 years, P � 0.0008 for 20 years versus 70 years, P � 0.03 for 30 years versus 60 years, P � 0.005 for 30 years versus70 years.‡ P � 0.0001 for 20 years versus 60 years, P � 0.0001 for 20 years versus 70 years, P � 0.003 for 30 years versus 60 years, P � 0.001 for 30 yearsversus 70 years.§ P � 0.04 versus 20 years.

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with the C4S concentration (r � �0.202) and a moder-ate correlation with the HA concentration (r � �0.483).Sex showed a weak correlation with the concentrationsof C6S and C4S and the C6S:C4S ratio (r � 0.307, r �0.225, and r � 0.237, respectively). Little correlation wasobserved between sex and the HA concentration (r �0.135).

DISCUSSION

Some of the GAGs in synovial fluid are nowbeing reported to be putative molecular markers of jointdiseases. To understand the true nature of markers, it isimportant that the changes associated with joint pathol-ogy are distinguished from physiologic variations. In thepresent study, we focused on investigating the effects ofage and sex on CS in the synovial fluid of healthysubjects. The data reported herein are of great impor-tance for the study of diseased synovial fluid as well asfor the assessment of the effects of pharmaceuticalintervention in a variety of joint diseases.

HA has been described to play an important rolein joint lubrication as well as to have beneficial effects on

the joint tissues, including an antiinflammatory effect,an inhibition of cartilage degeneration, and a positiverole in cartilage repair. Balazs (11) reported that the HAconcentration in healthy subjects is highest (3.4–3.8mg/ml) between the ages of 18 and 27 years, after whichit decreases. Balazs found that the synovial fluid retainsan HA level of 2.5 mg/ml (results in those ages 36–51years were not shown). In the present study, our resultsin the 20–30-years age group were consistent with theresults of Balazs. However, during subsequent aging(40–70 years), the HA concentration gradually de-creased from 3.2 mg/ml to 2.6 mg/ml, showing a signifi-cant difference between 20 years and 70 years.

In measurements of age-related changes of CS insynovial fluid, Yamada et al (5) reported that in patientswith hip OA, there was an inverse correlation of age withthe C6S concentration and the C6S:C4S ratio. Sharif etal (6) demonstrated that in a series of 80 subjects,including patients with RA and OA and healthy subjects,the C6S:C4S ratio appeared to decrease with age. How-ever, when 24 healthy subjects were considered sepa-rately, they found no relationship between age and theC6S:C4S ratio. In the present study, we noted a stronginverse correlation of age with the C6S concentrationand the C6S:C4S ratio. These markers especially showeda decreasing tendency between ages 60 and 70 years, andat 70 years, the concentrations of C6S and the C6S:C4Sratio decreased 48.4% and 35.4%, respectively, com-pared with those at 20 years of age.

With respect to aging changes in the adult carti-lage, animal studies have demonstrated a decrease in theCS content and in the C6S:C4S ratio (12) with age. Incontrast, Bayliss et al (13) reported that in humancartilage, the C6S:C4S ratio remained relatively constant

Table 2. Difference in the synovial fluid concentrations of CS and HA by sex*

C6S, nmoles/ml C4S, nmoles/ml C6S:C4S HA, mg/ml

Men (n � 46) 116.3 � 41.4 19.5 � 5.4 6.0 � 1.2 3.1 � 0.8Women (n � 36) 86.2 � 27.7 16.9 � 4.0 5.1 � 1.2 3.2 � 0.6P 0.0003 0.020 0.002 0.586Age 20–39 years

Men (n � 23) 135.9 � 44.5 20.9 � 5.8 6.4 � 0.3 3.4 � 0.8Women (n � 14) 104.9 � 27.4 17.1 � 3.9 6.1 � 0.8 3.6 � 0.4P 0.025 0.047 0.343 0.493

Age 40–59 yearsMen (n � 13) 111.6 � 25.5 19.3 � 5.5 5.9 � 1.0 2.9 � 0.7Women (n � 9) 84.5 � 13.0 16.9 � 3.4 5.1 � 0.9 3.4 � 0.4P 0.009 0.259 0.082 0.076

Age 60–79 yearsMen (n � 10) 77.2 � 11.9 16.3 � 2.5 4.8 � 0.9 2.7 � 0.7Women (n � 13) 67.2 � 22.7 16.7 � 4.7 4.0 � 0.5 2.7 � 0.6P 0.221 0.801 0.011 0.888

* Except where otherwise indicated, values are the mean � SD. See Table 1 for definitions.

Table 3. Relationship between the synovial fluid concentrations ofCS and HA and demographic variables*

C6S C4S C6S:C4S HA

AgePartial correlation �0.521 �0.202 �0.617 �0.483P 0.0001 0.064 0.0001 0.0001

SexPartial correlation 0.307 0.225 0.237 0.135P 0.0007 0.040 0.005 0.180

* See Table 1 for definitions.

CHONDROITIN SULFATES IN NORMAL SYNOVIAL FLUID 2107

from age 20 years to age 85 years. The reason why ourage-related changes in the synovial fluid are not propor-tional to those in the cartilage might be explained, inpart, by topographic and zonal variations of the sulfationpatterns in the cartilage. In addition, joint tissues otherthan articular cartilage may contribute to the CS insynovial fluid. Bayliss et al (14) reported that the diffi-culties in interpreting synovial fluid analyses of CS arenot only that the molecular and tissue sources areunknown, but also that the clinical stage of joint diseaseundoubtedly has a differential effect on each tissue.Further investigation is needed on the origin of synovialfluid CS.

Sex factors have been considered to have sys-temic, metabolic, or local effects on the development ofarthritic diseases. There have been some observationsconcerning sex differences in normal articular cartilage.Independent of body and bone size, women have asmaller cartilage volume than do men (15). Moreover,the cartilage from female animals has a lower concen-tration of proteoglycan than that from male animals(16). In the present study concerning normal synovialfluid, irrespective of age, the C6S concentration inwomen tended to be lower than that in men. Our resultsand those reported previously suggest that a sex differ-ence in the metabolic capacity of the cartilage matrix islinked, in some way, to the development of arthriticdiseases.

In summary, this study showed, for the first time,the presence of age- and sex-related variations in thesynovial fluid CS of normal subjects. These physiologicvariations need to be taken into account when usingsynovial fluid CS as markers for arthritic conditions.

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