- Email: [email protected]
The correlation of fixed negative charge with glycosaminoglycan content of human articular cartilage
492
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 2 6 1 0 9
T H E C O R R E L A T I O N OF F I X E D NEGATIVE C H A R G E W I T H GLYCOSAMINOGLYCAN CONTENT OF HUMAN A R T I C U L A R CARTILAGE ALICE MAROUDAS, H E L E N M U I R AND J O A N W I N G H A M
Biomechanics Unit, Mechanical Engineering Department, lmperial College of Science and Technology, London, SW. 7 (Great Britain) and Kennedy Institute of Rheumatology, London, W. 6 (Great Britain) (Received November I Ith, 1968)
SUMMARY
A physical method is described for measuring the concentration of fixed negatively charged groups in articular cartilage. Comparison is made with values of fixed charged density derived from chemical analysis of hexuronic acid and total hexosamine contents. The mean deviation between values derived from the two methods was IO %. This study was carried out on successive tangentially cut slices of human articular cartilage from the femoral condyle. The following observations were made. The fixed charge density increased with depth from the articular surface, as did the uronic acid and total hexosamine contents. In the 25-60 age group neither the hexuronic acid nor the total hexosamine contents showed any correlation with the age of the subject. However, there was considerable local variation and variation between individuals.
INTRODUCTION
A rapid physical method for measuring the concentration of fixed negatively charged groups ill cartilage has recently been developed b y one of the authors. The theory of the method and a preliminary description have been given 61sewhere 1. The primary purpose of the present work was to compare the results obtained by this method with those deduced from chemical analysis. In the course of this investigation a study was also made of the variation in the chemical composition of articular cartilage with depth from articular surface, and these results will be included and discussed.
Theory of the physical method The matrix of cartilage can be treated as a cation exchange membrane, since it contains fixed negative charges due to the sulphate and the carboxylate groups of chondroitin sulphate and keratan sulphate. When an electrolyte solution flows under a pressure gradient through an ion-exchange membrane, a potential is set up across the membrane ~. This potential is known as the streaming potential and is a function Biochim. Biophys. Acta, 177 (I969) 492-500
493
F I X E D C H A R G E D E N S I T Y OF C A R T I L A G E
of the fixed charge density, the electrical conductivity and the flow permeability of the membrane. The streaming potential is given by: A ~p
FcxK
Ap
K
(I)
where A~o = streaming potential; Ap = pressure differential; Cx = concentration of fixed negatively charged groups in cartilage; F = Faraday's constant; K = flow permeability; K = specific electrical conductivity. The present method makes use of Eqn. I to calculate Cx from the experimentally determined values of/l~p, K and K. A sample calculation is given as follows using typical values obtained for cartilage: A~o-----5.O.lO -4 V; A p = IO~ dynes/cmZ; K ~ - 3.5.IO -3 cm3.sec per g; K = 6.5.1o-3/2-1; F = 965o0. From Eqn. I : z]~p Cx =
•
A p " F-K =
5.0" lO -4 × 6. 5 "IO - s i o e × 9 . 6 5 ' 164 × 3.5"IO-1~
= o.o96 m e q u i v / c m 3
METHODS AND MATER~LS
Materials Healthy cartilage taken at postmortem from human femoral condyles was used throughout. The cartilage was obtained from 6 subjects varying in age between 26 and 6o years. Consecutive slices 400 p in thickness and about I cm in diameter were cut on a sledge microtome from different areas on the femoral condyle. The number of slices varied between 3 and 7 depending on the thickness of cartilage in each area. The specimens were stored at - - 2 0 ° and were kept at room temperature only for the duration of the experiments. No mucopolysaccharides were leached out under these conditions as detected by the presence of uronic acid in the fluid. Two consecutive series of slices were cut downwards from the surface of contiguous areas of cartilage; one series was subjected to chemical analysis, the other to physical measurements. In the series 26 A' and 41.1 and 41.2, b o t h physical and chemical determinations were performed on the same slices. In three series, 54.IC, 54.2B and 54.2C, chemical analyses alone were carried out. Lateral and medial regions of condyles from one limb only were used.
Physical method and apparatus The combined apparatus for the measurement of permeability and streaming potential is shown in Fig. I. The cartilage slice is clamped between two tubes one of which is connected to a compressed gas cylinder whilst the other ends in a precision bore capillary. The slice rests on a porous polytetrafluoroethylene disc which prevents the cartilage from bulging and thinning under pressure. In the calculation of the permeability coefficient a correction has to be applied for the reduction in effective surface area of the cartilage slice due to contact with the solid portion of the polytetrafluoroethylene disc. (This correction was omitted in previous permeability measurements 1 which are thus about 20% too low.) Biochim. Biophys. Acta, z77 (x969) 4 9 2 - 5 0 0
494
A. MAROUDAS et
al.
Ag-AgC1 electrodes are immersed in Ringer's solution on either side of the membrane and the potential at zero pressure differential is recorded. This potential should theoretically be equal to zero. In fact due to slight differences in the standard states of the electrodes a small potential difference, of the order of 2. lO.4 V, is usually observed. At PLATINUM CONTACTS B: PERSPEX STRIP C : CARTILAGE D:
RECESS 1crux 2ram x 0 5 m m
TO CONOUCTIVITY BRtDGE
SILVER- SILVER CHLORIDE ELECTRODE GLASS
r,,.~l~./I k : . / ~ ~"~/'~1 ~ - - T O ~ FLOW' PRESSURE ' I/ " / / " CYLINDER L '/. ~, ~ . ~ "
TUBE....l';;~l~.'"J~'~.
PRECISION GLASS J CAPILLARY
SiDE VIEW SHOWING CURVED ARMS TO PRESS DOWN GN CARTILAGE
=
•
a / V /
.
, /X/V T"/;"AV/>
GROUND PERSPEX' ' ~ l \ SINIERED PTFE FACES CARTILAGE SUPPORT SLICE
=5=J PLAN SHOWING RECESS TO HOLD STRIP OF CARTILAGE
Fig. z. Apparatus for the measurement of permeability and streaming potential (from Biophys. J., 8 (1968) 584, by permission of the Editors). PTFE, polytetrafluoroethylene. Fig. 2. Conductivity cell (from Biophys. J., 8 (z968) 582, by permission of the Editors).
The rate of flow of fluid under a given applied pressure is measured by the rate of rise of liquid in the capillary. A vernier microscope is used to measure liquid displacement. The potential developed between the two electrodes as a result of flow is the streaming potential and it is measured b y means of an electrometer capable of reading accurately down to lO-4 V. It is important to take as the true value of the streaming potential the rise in the potential difference between the two electrodes obtained immediately upon the application of pressure, as continued flow results in the production of extraneous potentials due to electrolyte filtration. The conductivity apparatus is shown in Fig. 2. The cartilage strip whose conductivity is to be measured is placed in the recess D. Curved perspex arms (B) with flat platinum contacts (A) cemented at the ends press down on the cartilage. In this way, good electrical contact is ensured. The distance between the electrodes remains always the same. Cartilage strips 0.2 cm in width, 4" IO-~ cm in thickness and about I cm long were cut from the middle portion of the cartilage slice under consideration. Before the strips were inserted into the cell, surface electrolyte was removed by gentle wiping with absorbent paper. To minimize the evaporation of water from the cartilage during the measurements, the cell was placed in a plastic box in which the air was kept saturated with water vapour. The platinum electrodes were connected to a direct-reading conductivity bridge (range 3 p~Q-Lo.3 ~Q-1 full scale deflection). Biochim. Biophys. Acta, 177 (I969) 492-500
FIXED
CHARGE DENSITY
495
OF CARTILAGE
Chemical analysis
The cartilage slices, suspended in Ringer's solution, were blotted with filter paper and immediately weighed. They were then dried to constant weight i n vacuo over PzO 5 for 24-48 h. Each slice was suspended in 3 ml of o.15 M borate buffer containing o.o2 M CaCI~ to which 6 mg of pronase P was added (Kaken Chemical Co., Tokyo), and the solution was covered with a layer of toluene, Digestion was complete after 12 h at 60 °. H e x u r o n i c acid
Using glucuronolactone as a standard, the automated method of BALAZS et al. a was used, with the following modifications. To reduce the consumption of the sulphuric acid reagent despite some loss in sensitivity, one Acidflex tube passing 1.44 ml/min, replaced two tubes passing 1.19 ml/min. The absorbance of a standard solution of glucuronolactone containing 20/zg/ml was o.I. The samples from each digest were analysed directly in triplicate at the rate of 12 per h, each sample being followed by three sample vol. of distilled water. Hexosamine
Duplicate aliquots of 0.5 ml from each cartilage digest were placed in tubes fitted with screw caps having Teflon seals (Kimax). An equal vol. (o.5 ml) of concentrated HC1 (sp. gr., 1.16) was added to each, and the tubes closed and heated at 95 ° for 4 h. Hydrolysis curves showed no further release of hexosamine beyond 4 h when pooled cartilage was hydrolysed under these conditions. The acid was removed i n vacuo over NaOH pellets, and PzOs, and the dry residue dissolved in I ml of distilled water. The hydrolysates were transferred quantitatively to centrifuge tubes, by washing the hydrolysis tubes twice more with I ml of water (final vol., 3 ml). Activated charcoal was added, the tubes shaken and allowed to stand for I h before centrifuging at 2500 rev./min for about 15 min. Each clear supernatant was analysed in triplicate for hexosamine b y the automated procedure of BALAZSet al. 4 with the following modiHEATING
BATH
(~ - -
DELIVERY
MIXING COIL j ~'dOOO" ~, N MIXING AND COOLING
[M,×,
CO,L
mt/mln 0-6 AIR 1.2 SAMPLE 1.2 BUFFER AC
.2EHRL,CN'S
'O'OO'--'O OOOO"
2.0 WASTE
HEATING BATH
RECORDER
Fig. 3. Flow diagram for the automated analysis of hexosamines. Biochim. Biophys. A~ta, 177 (I969) 492-500
A. MAROUDAS et
496
al.
£s
<
P
k=~.
61
I
¢1
I
",'. ~,,.... ,..
\¢ '
÷
m
÷;
,
~
to
,
~,, "<,
~__a
,
~o
,%_2~._1
,
~
_3°
, ,~,
~~ <
'o o
o©'i
~m
Oo
g [ I H-313~
im
13M I N 3 3
[IH913~ AaO ON a3a o f f ]
°
o
g
g
o
g
o
,.~
.
EI3d] IN31NO0 ~J]LW~ 3NIHVSO)f3H '~ OIDY 31NO~Irl
~8
~s ~6 o | I
I I
+
d ~ g'r~
o,o,
I
~'~
#
.a ~o a.
#
6o,
oi o
d
oI
t
ol
[.LHOI3M ,~3~ I N 3 3 ~ l ] d ] 1N31NOO ~31VA~ ~1H913~t~ A~lO O~l a 3 d 01~'~ 3NIINVSOX~IH 'll QIOY 3 l N O a n
Biochim. Biophys. dcta,
i77 (I969) 492-500
° 4-.8
FIXED CHARGE DENSITY OF CARTILAGE
497
fication of the flow system to increase the sensitivity as shown in the flow diagram (Fig. 3). The reagents were the same as those used by BALAZSet al. 4 but the consumption of Ehrlich reagent was halved. The sensitivity, however, was increased b y passing the solution after addition of Ehrlich reagent through the second coil of the heating bath at 95 ° in place of a time delay coil at 2o °, as used by BALAZSet al. 4. A standard solution of glucosamine hydrochloride containing 20 t~g/ml had an optical density of 0.2. RESULTS AND DISCUSSION
The hexosamine and hexuronic acid contents based on dry weight of cartilage are shown in Figs. 4a and 4b together with the proportion of water lost by each specimen on being dried to constant weight, expressed as a percentage of the wet wt. All specimens showed a similar trend in which both the hexosamine and hexuronic acid contents increased with depth, the relative differences being greatest between slices nearest the surface of the cartilage. On the other hand, the water content was higher in most of the superficial specimens, decreasing somewhat with depth. The chondroitin sulphate in the specimens would be estimated b y hexuronic acid, and both chondroitin sulphate and keratan sulphate by hexosamine, assuming that any hexosamine due to glycoproteins was negligible. Subtracting hexuronic acid from the values for total hexosamine would thus provide an approximate estimate of keratan sulphate. A value for the total negative charge per unit weight of cartilage contributed by each glycosaminoglycan was derived b y making the following approximations; that chondroitin sulphate provides two negative groups and keratan sulphate one per disaccharide repeating unit. The density of the cartilage studied in the present series was found to be 1.o8 (:t: o.oi), and this figure was used to convert the total concentration of negative groups from a weight to a volume basis. It was then possible to compare the values calculated from chemical analysis with those obtained b y the streaming potential method (Figs. 5 and 6). Fig. 5 shows the pattern of variation of fixed charge density with distance from articular surface for both sets of values. It can be seen (a) that the two sets lie close to one another and (b) that fixed charge density increases with depth from articular surface, in accordance with the previously reported observations by one of the authors 1. Fig. 6 shows a direct plot of the results obtained by the two methods versus one another. In this plot perfect agreement would result in the line, y = x, going through the origin. The best line drawn through the experimental points and the origin is given by the formula, y = I.IX. This agreement is close enough to justify the assumption that fixed negative charge is contributed by the glycosaminoglycans, and hence these can be estimated by the physical method. The reproducibility of the uronic acid determinations was within :t: 3 % ; that of the hexosamine determinations was less good because the errors of several manipulations were added to the usual analytical errors. In most instances reproducibility was within + 5 %, but occasionally it was within 4- 12 %. Nevertheless Figs. 4a and b show that differences between different cartilage slices were generally larger than these analytical errors. The relative proportion of keratan sulphate and the total Biochim. Biophys. Acta, I77 (1969) 49:2-500
498
A. MAROUDAS et al.
amount of keratan sulphate and of chondroitin sulphate generally varied between slices at different depths from the same area. There thus appears to be considerable local topographical variation in the glycosaminoglycan composition of this cartilage. 0.20 0'15 0 '16 0 q& 0'12 0,10 0'08 0'06 0"04
26A
351 B
522A
/
o/° & ..../ o"-o
/ ~'
.
~ C'20
26 A'
0.18 E 0.16 o 3 0,1t,
/o
.
.
.
.
.
.
.
.
.
.
.
.
.
351 C
oZ
0.04
I
.
J
I
522
J
I
C
/
/
.~ o.10
-_ 0.08 0"06
.
412
J~
/
z~O'02 I
E.20
.
.
352 8
26 B
.
.
I
.
521
to ~ q 8
0.16 TI- O"14 ,u 0-12 'Q 0"10 ~- 0"08
yx
0.0~
O.OL, 0"02 0
o
//
oJ
o14[ 0.16 I-
°~o
o/o
Y 0 0 64f 0.0 0.02
.
.
.
.
.
&
521B
,.c
.
54 1 B
/
. . . . . . .
I
A
f 60
¢
/
ZX
1 2 34 SLICE
567 NUMBER
1 2 34 567 FROM S U R F A C E
I 2 3 4567
Fig. 5. The variation of fixed charge density with depth from the articular surface. The numbers and letters above each graph as in Figs. 4 a and 4 b. O, from streaming potential; A , from chemical analysis.
In the present limited series, the total hexuronic acid content was quite variable and showed no correlation with the age of the subject, in agreement with previous findings on whole cartilage from the same sourceS, e. Furthermore, neither the amount nor relative proportion of keratan sulphate increased with age in agreement with findings in human costal cartilageS, 8 where a plateau was observed from the fourth decade onward. In most areas there was, however, an increase in the amount of keratan sulphate with depth from the articular surface, in agreement with results of STOCKWELL AND SCOTT9. Cartilage from different subjects showed considerable variation in the total c o n t e n t of k e r a t a n s u l p h a t e .
In some series of slices, such as all four series from the 35-year-old subject (Fig. 4a), the keratan sulphate content was markedly high and even rose above the Biochim. Biophys. Acla, z77 (1969) 492-500
499
FIXED CHARGE DENSITY OF CARTILAGE
chondroitin sulphate at certain depths. The analyses of specimens from the same condyle were more similar than those from different condyles, which might suggest that each subject's cartilage had a characteristic composition. 02 ~ .
o~
o
02C
~
o
o
X~ 0--°
o
~o
a°
o
o
8/
/
o °~0 /~"~--Theoreticai 0/.~/~ Line
© 0.1c
/
o
~=
o oy~°/o
°
o
o
~/o
0
!
0
010 FIXED
CHARGE ANALYSIS
!
0 20
DENSITY F R O M CHEM4CAL ( M d i i e q u i v o t e n t s per crn 3)
Fig. 6. Comparison between fixed charge density as determined b y physical and chemical m e t h o d s (pooled results).
The water contents of the specimens varied between 65 % and 85 % and were above those recorded by BOLLETAND NANCE% but similarly showed no correlation with age. Conclusion
In articular cartilage the fixed negative charge density as measured by the streaming potential method agrees closely with the value calculated from the concentration of acid glycosaminoglycans, which showed considerable local variation and variation between individuals. ACKNOWLEDGEMENTS
The work was supported by a grant from the British Medical Research Council. Thanks are due to Mrs. L. Rippon for competent assistance with part of the experimental work. REFERENCES I A. MAROUDAS, Biophys. J., 8 (i968) 5752 F. HELItlrERICH, Ion Exchange, McGraw-Hill, New York, I962, p. 393. 3 E. A. BALAZS, K. E. BERNSTEN, j. KAROSSA AND D. A. SWAI~IN,Anat. Biochem., 12 (1965) 547-
Biochim. Biophys. Acla, 177 (I969) 492-500
500
A. MAROUDAS et
al.
4 E. A. BALAZS, K. E. BE,RNSTEN, J. KAROSSA AND D. A. SWANN, Anal. Biochem., I2 (1965) 559 5 A. J. BOLLET, J. 1:{. HANDY AND B. C. STURGILL, 3". Clin. Invest., 42 (1963) 853. 6 A. J. BOLLET AND J. L. NANCE, J. Clin. Invest., 45 (1966) 117 o. 7 D. I{.APLAN AND K. MEYER, Nature, 183 (1959) 1267. 8 M. ]3. MATTHEWS AND S. GLAGOV, J. Clin. Invest., 45 (1966) 11o 3. 9 R. A. STOCKWELL AND J. E. SCOTT, Nature, 215 (1967) 1376.
Biochim. Biophys. Acta, i77 (1969) 492-500
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 2 6 1 0 9
T H E C O R R E L A T I O N OF F I X E D NEGATIVE C H A R G E W I T H GLYCOSAMINOGLYCAN CONTENT OF HUMAN A R T I C U L A R CARTILAGE ALICE MAROUDAS, H E L E N M U I R AND J O A N W I N G H A M
Biomechanics Unit, Mechanical Engineering Department, lmperial College of Science and Technology, London, SW. 7 (Great Britain) and Kennedy Institute of Rheumatology, London, W. 6 (Great Britain) (Received November I Ith, 1968)
SUMMARY
A physical method is described for measuring the concentration of fixed negatively charged groups in articular cartilage. Comparison is made with values of fixed charged density derived from chemical analysis of hexuronic acid and total hexosamine contents. The mean deviation between values derived from the two methods was IO %. This study was carried out on successive tangentially cut slices of human articular cartilage from the femoral condyle. The following observations were made. The fixed charge density increased with depth from the articular surface, as did the uronic acid and total hexosamine contents. In the 25-60 age group neither the hexuronic acid nor the total hexosamine contents showed any correlation with the age of the subject. However, there was considerable local variation and variation between individuals.
INTRODUCTION
A rapid physical method for measuring the concentration of fixed negatively charged groups ill cartilage has recently been developed b y one of the authors. The theory of the method and a preliminary description have been given 61sewhere 1. The primary purpose of the present work was to compare the results obtained by this method with those deduced from chemical analysis. In the course of this investigation a study was also made of the variation in the chemical composition of articular cartilage with depth from articular surface, and these results will be included and discussed.
Theory of the physical method The matrix of cartilage can be treated as a cation exchange membrane, since it contains fixed negative charges due to the sulphate and the carboxylate groups of chondroitin sulphate and keratan sulphate. When an electrolyte solution flows under a pressure gradient through an ion-exchange membrane, a potential is set up across the membrane ~. This potential is known as the streaming potential and is a function Biochim. Biophys. Acta, 177 (I969) 492-500
493
F I X E D C H A R G E D E N S I T Y OF C A R T I L A G E
of the fixed charge density, the electrical conductivity and the flow permeability of the membrane. The streaming potential is given by: A ~p
FcxK
Ap
K
(I)
where A~o = streaming potential; Ap = pressure differential; Cx = concentration of fixed negatively charged groups in cartilage; F = Faraday's constant; K = flow permeability; K = specific electrical conductivity. The present method makes use of Eqn. I to calculate Cx from the experimentally determined values of/l~p, K and K. A sample calculation is given as follows using typical values obtained for cartilage: A~o-----5.O.lO -4 V; A p = IO~ dynes/cmZ; K ~ - 3.5.IO -3 cm3.sec per g; K = 6.5.1o-3/2-1; F = 965o0. From Eqn. I : z]~p Cx =
•
A p " F-K =
5.0" lO -4 × 6. 5 "IO - s i o e × 9 . 6 5 ' 164 × 3.5"IO-1~
= o.o96 m e q u i v / c m 3
METHODS AND MATER~LS
Materials Healthy cartilage taken at postmortem from human femoral condyles was used throughout. The cartilage was obtained from 6 subjects varying in age between 26 and 6o years. Consecutive slices 400 p in thickness and about I cm in diameter were cut on a sledge microtome from different areas on the femoral condyle. The number of slices varied between 3 and 7 depending on the thickness of cartilage in each area. The specimens were stored at - - 2 0 ° and were kept at room temperature only for the duration of the experiments. No mucopolysaccharides were leached out under these conditions as detected by the presence of uronic acid in the fluid. Two consecutive series of slices were cut downwards from the surface of contiguous areas of cartilage; one series was subjected to chemical analysis, the other to physical measurements. In the series 26 A' and 41.1 and 41.2, b o t h physical and chemical determinations were performed on the same slices. In three series, 54.IC, 54.2B and 54.2C, chemical analyses alone were carried out. Lateral and medial regions of condyles from one limb only were used.
Physical method and apparatus The combined apparatus for the measurement of permeability and streaming potential is shown in Fig. I. The cartilage slice is clamped between two tubes one of which is connected to a compressed gas cylinder whilst the other ends in a precision bore capillary. The slice rests on a porous polytetrafluoroethylene disc which prevents the cartilage from bulging and thinning under pressure. In the calculation of the permeability coefficient a correction has to be applied for the reduction in effective surface area of the cartilage slice due to contact with the solid portion of the polytetrafluoroethylene disc. (This correction was omitted in previous permeability measurements 1 which are thus about 20% too low.) Biochim. Biophys. Acta, z77 (x969) 4 9 2 - 5 0 0
494
A. MAROUDAS et
al.
Ag-AgC1 electrodes are immersed in Ringer's solution on either side of the membrane and the potential at zero pressure differential is recorded. This potential should theoretically be equal to zero. In fact due to slight differences in the standard states of the electrodes a small potential difference, of the order of 2. lO.4 V, is usually observed. At PLATINUM CONTACTS B: PERSPEX STRIP C : CARTILAGE D:
RECESS 1crux 2ram x 0 5 m m
TO CONOUCTIVITY BRtDGE
SILVER- SILVER CHLORIDE ELECTRODE GLASS
r,,.~l~./I k : . / ~ ~"~/'~1 ~ - - T O ~ FLOW' PRESSURE ' I/ " / / " CYLINDER L '/. ~, ~ . ~ "
TUBE....l';;~l~.'"J~'~.
PRECISION GLASS J CAPILLARY
SiDE VIEW SHOWING CURVED ARMS TO PRESS DOWN GN CARTILAGE
=
•
a / V /
.
, /X/V T"/;"AV/>
GROUND PERSPEX' ' ~ l \ SINIERED PTFE FACES CARTILAGE SUPPORT SLICE
=5=J PLAN SHOWING RECESS TO HOLD STRIP OF CARTILAGE
Fig. z. Apparatus for the measurement of permeability and streaming potential (from Biophys. J., 8 (1968) 584, by permission of the Editors). PTFE, polytetrafluoroethylene. Fig. 2. Conductivity cell (from Biophys. J., 8 (z968) 582, by permission of the Editors).
The rate of flow of fluid under a given applied pressure is measured by the rate of rise of liquid in the capillary. A vernier microscope is used to measure liquid displacement. The potential developed between the two electrodes as a result of flow is the streaming potential and it is measured b y means of an electrometer capable of reading accurately down to lO-4 V. It is important to take as the true value of the streaming potential the rise in the potential difference between the two electrodes obtained immediately upon the application of pressure, as continued flow results in the production of extraneous potentials due to electrolyte filtration. The conductivity apparatus is shown in Fig. 2. The cartilage strip whose conductivity is to be measured is placed in the recess D. Curved perspex arms (B) with flat platinum contacts (A) cemented at the ends press down on the cartilage. In this way, good electrical contact is ensured. The distance between the electrodes remains always the same. Cartilage strips 0.2 cm in width, 4" IO-~ cm in thickness and about I cm long were cut from the middle portion of the cartilage slice under consideration. Before the strips were inserted into the cell, surface electrolyte was removed by gentle wiping with absorbent paper. To minimize the evaporation of water from the cartilage during the measurements, the cell was placed in a plastic box in which the air was kept saturated with water vapour. The platinum electrodes were connected to a direct-reading conductivity bridge (range 3 p~Q-Lo.3 ~Q-1 full scale deflection). Biochim. Biophys. Acta, 177 (I969) 492-500
FIXED
CHARGE DENSITY
495
OF CARTILAGE
Chemical analysis
The cartilage slices, suspended in Ringer's solution, were blotted with filter paper and immediately weighed. They were then dried to constant weight i n vacuo over PzO 5 for 24-48 h. Each slice was suspended in 3 ml of o.15 M borate buffer containing o.o2 M CaCI~ to which 6 mg of pronase P was added (Kaken Chemical Co., Tokyo), and the solution was covered with a layer of toluene, Digestion was complete after 12 h at 60 °. H e x u r o n i c acid
Using glucuronolactone as a standard, the automated method of BALAZS et al. a was used, with the following modifications. To reduce the consumption of the sulphuric acid reagent despite some loss in sensitivity, one Acidflex tube passing 1.44 ml/min, replaced two tubes passing 1.19 ml/min. The absorbance of a standard solution of glucuronolactone containing 20/zg/ml was o.I. The samples from each digest were analysed directly in triplicate at the rate of 12 per h, each sample being followed by three sample vol. of distilled water. Hexosamine
Duplicate aliquots of 0.5 ml from each cartilage digest were placed in tubes fitted with screw caps having Teflon seals (Kimax). An equal vol. (o.5 ml) of concentrated HC1 (sp. gr., 1.16) was added to each, and the tubes closed and heated at 95 ° for 4 h. Hydrolysis curves showed no further release of hexosamine beyond 4 h when pooled cartilage was hydrolysed under these conditions. The acid was removed i n vacuo over NaOH pellets, and PzOs, and the dry residue dissolved in I ml of distilled water. The hydrolysates were transferred quantitatively to centrifuge tubes, by washing the hydrolysis tubes twice more with I ml of water (final vol., 3 ml). Activated charcoal was added, the tubes shaken and allowed to stand for I h before centrifuging at 2500 rev./min for about 15 min. Each clear supernatant was analysed in triplicate for hexosamine b y the automated procedure of BALAZSet al. 4 with the following modiHEATING
BATH
(~ - -
DELIVERY
MIXING COIL j ~'dOOO" ~, N MIXING AND COOLING
[M,×,
CO,L
mt/mln 0-6 AIR 1.2 SAMPLE 1.2 BUFFER AC
.2EHRL,CN'S
'O'OO'--'O OOOO"
2.0 WASTE
HEATING BATH
RECORDER
Fig. 3. Flow diagram for the automated analysis of hexosamines. Biochim. Biophys. A~ta, 177 (I969) 492-500
A. MAROUDAS et
496
al.
£s
<
P
k=~.
61
I
¢1
I
",'. ~,,.... ,..
\¢ '
÷
m
÷;
,
~
to
,
~,, "<,
~__a
,
~o
,%_2~._1
,
~
_3°
, ,~,
~~ <
'o o
o©'i
~m
Oo
g [ I H-313~
im
13M I N 3 3
[IH913~ AaO ON a3a o f f ]
°
o
g
g
o
g
o
,.~
.
EI3d] IN31NO0 ~J]LW~ 3NIHVSO)f3H '~ OIDY 31NO~Irl
~8
~s ~6 o | I
I I
+
d ~ g'r~
o,o,
I
~'~
#
.a ~o a.
#
6o,
oi o
d
oI
t
ol
[.LHOI3M ,~3~ I N 3 3 ~ l ] d ] 1N31NOO ~31VA~ ~1H913~t~ A~lO O~l a 3 d 01~'~ 3NIINVSOX~IH 'll QIOY 3 l N O a n
Biochim. Biophys. dcta,
i77 (I969) 492-500
° 4-.8
FIXED CHARGE DENSITY OF CARTILAGE
497
fication of the flow system to increase the sensitivity as shown in the flow diagram (Fig. 3). The reagents were the same as those used by BALAZSet al. 4 but the consumption of Ehrlich reagent was halved. The sensitivity, however, was increased b y passing the solution after addition of Ehrlich reagent through the second coil of the heating bath at 95 ° in place of a time delay coil at 2o °, as used by BALAZSet al. 4. A standard solution of glucosamine hydrochloride containing 20 t~g/ml had an optical density of 0.2. RESULTS AND DISCUSSION
The hexosamine and hexuronic acid contents based on dry weight of cartilage are shown in Figs. 4a and 4b together with the proportion of water lost by each specimen on being dried to constant weight, expressed as a percentage of the wet wt. All specimens showed a similar trend in which both the hexosamine and hexuronic acid contents increased with depth, the relative differences being greatest between slices nearest the surface of the cartilage. On the other hand, the water content was higher in most of the superficial specimens, decreasing somewhat with depth. The chondroitin sulphate in the specimens would be estimated b y hexuronic acid, and both chondroitin sulphate and keratan sulphate by hexosamine, assuming that any hexosamine due to glycoproteins was negligible. Subtracting hexuronic acid from the values for total hexosamine would thus provide an approximate estimate of keratan sulphate. A value for the total negative charge per unit weight of cartilage contributed by each glycosaminoglycan was derived b y making the following approximations; that chondroitin sulphate provides two negative groups and keratan sulphate one per disaccharide repeating unit. The density of the cartilage studied in the present series was found to be 1.o8 (:t: o.oi), and this figure was used to convert the total concentration of negative groups from a weight to a volume basis. It was then possible to compare the values calculated from chemical analysis with those obtained b y the streaming potential method (Figs. 5 and 6). Fig. 5 shows the pattern of variation of fixed charge density with distance from articular surface for both sets of values. It can be seen (a) that the two sets lie close to one another and (b) that fixed charge density increases with depth from articular surface, in accordance with the previously reported observations by one of the authors 1. Fig. 6 shows a direct plot of the results obtained by the two methods versus one another. In this plot perfect agreement would result in the line, y = x, going through the origin. The best line drawn through the experimental points and the origin is given by the formula, y = I.IX. This agreement is close enough to justify the assumption that fixed negative charge is contributed by the glycosaminoglycans, and hence these can be estimated by the physical method. The reproducibility of the uronic acid determinations was within :t: 3 % ; that of the hexosamine determinations was less good because the errors of several manipulations were added to the usual analytical errors. In most instances reproducibility was within + 5 %, but occasionally it was within 4- 12 %. Nevertheless Figs. 4a and b show that differences between different cartilage slices were generally larger than these analytical errors. The relative proportion of keratan sulphate and the total Biochim. Biophys. Acta, I77 (1969) 49:2-500
498
A. MAROUDAS et al.
amount of keratan sulphate and of chondroitin sulphate generally varied between slices at different depths from the same area. There thus appears to be considerable local topographical variation in the glycosaminoglycan composition of this cartilage. 0.20 0'15 0 '16 0 q& 0'12 0,10 0'08 0'06 0"04
26A
351 B
522A
/
o/° & ..../ o"-o
/ ~'
.
~ C'20
26 A'
0.18 E 0.16 o 3 0,1t,
/o
.
.
.
.
.
.
.
.
.
.
.
.
.
351 C
oZ
0.04
I
.
J
I
522
J
I
C
/
/
.~ o.10
-_ 0.08 0"06
.
412
J~
/
z~O'02 I
E.20
.
.
352 8
26 B
.
.
I
.
521
to ~ q 8
0.16 TI- O"14 ,u 0-12 'Q 0"10 ~- 0"08
yx
0.0~
O.OL, 0"02 0
o
//
oJ
o14[ 0.16 I-
°~o
o/o
Y 0 0 64f 0.0 0.02
.
.
.
.
.
&
521B
,.c
.
54 1 B
/
. . . . . . .
I
A
f 60
¢
/
ZX
1 2 34 SLICE
567 NUMBER
1 2 34 567 FROM S U R F A C E
I 2 3 4567
Fig. 5. The variation of fixed charge density with depth from the articular surface. The numbers and letters above each graph as in Figs. 4 a and 4 b. O, from streaming potential; A , from chemical analysis.
In the present limited series, the total hexuronic acid content was quite variable and showed no correlation with the age of the subject, in agreement with previous findings on whole cartilage from the same sourceS, e. Furthermore, neither the amount nor relative proportion of keratan sulphate increased with age in agreement with findings in human costal cartilageS, 8 where a plateau was observed from the fourth decade onward. In most areas there was, however, an increase in the amount of keratan sulphate with depth from the articular surface, in agreement with results of STOCKWELL AND SCOTT9. Cartilage from different subjects showed considerable variation in the total c o n t e n t of k e r a t a n s u l p h a t e .
In some series of slices, such as all four series from the 35-year-old subject (Fig. 4a), the keratan sulphate content was markedly high and even rose above the Biochim. Biophys. Acla, z77 (1969) 492-500
499
FIXED CHARGE DENSITY OF CARTILAGE
chondroitin sulphate at certain depths. The analyses of specimens from the same condyle were more similar than those from different condyles, which might suggest that each subject's cartilage had a characteristic composition. 02 ~ .
o~
o
02C
~
o
o
X~ 0--°
o
~o
a°
o
o
8/
/
o °~0 /~"~--Theoreticai 0/.~/~ Line
© 0.1c
/
o
~=
o oy~°/o
°
o
o
~/o
0
!
0
010 FIXED
CHARGE ANALYSIS
!
0 20
DENSITY F R O M CHEM4CAL ( M d i i e q u i v o t e n t s per crn 3)
Fig. 6. Comparison between fixed charge density as determined b y physical and chemical m e t h o d s (pooled results).
The water contents of the specimens varied between 65 % and 85 % and were above those recorded by BOLLETAND NANCE% but similarly showed no correlation with age. Conclusion
In articular cartilage the fixed negative charge density as measured by the streaming potential method agrees closely with the value calculated from the concentration of acid glycosaminoglycans, which showed considerable local variation and variation between individuals. ACKNOWLEDGEMENTS
The work was supported by a grant from the British Medical Research Council. Thanks are due to Mrs. L. Rippon for competent assistance with part of the experimental work. REFERENCES I A. MAROUDAS, Biophys. J., 8 (i968) 5752 F. HELItlrERICH, Ion Exchange, McGraw-Hill, New York, I962, p. 393. 3 E. A. BALAZS, K. E. BERNSTEN, j. KAROSSA AND D. A. SWAI~IN,Anat. Biochem., 12 (1965) 547-
Biochim. Biophys. Acla, 177 (I969) 492-500
500
A. MAROUDAS et
al.
4 E. A. BALAZS, K. E. BE,RNSTEN, J. KAROSSA AND D. A. SWANN, Anal. Biochem., I2 (1965) 559 5 A. J. BOLLET, J. 1:{. HANDY AND B. C. STURGILL, 3". Clin. Invest., 42 (1963) 853. 6 A. J. BOLLET AND J. L. NANCE, J. Clin. Invest., 45 (1966) 117 o. 7 D. I{.APLAN AND K. MEYER, Nature, 183 (1959) 1267. 8 M. ]3. MATTHEWS AND S. GLAGOV, J. Clin. Invest., 45 (1966) 11o 3. 9 R. A. STOCKWELL AND J. E. SCOTT, Nature, 215 (1967) 1376.
Biochim. Biophys. Acta, i77 (1969) 492-500