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A simplified technique for the analysis of tissue acid mucopolysaccharides
CLINICA
CKIMICA
A S~~~PLII~IED
ACTA
TECHNIQUE
FOR
THE
ANALYSIS
OF TISSIX
ACID
aIUC,OPC)LYSACCIIARIDES
A method
is described
for the isolation,
fractionation,
and assay of acid muco-
polysaccharides. It is usable for quantities of tissue containing as little as 5 ,~g uranic acid as mucopolysaccharide, and offers a considerable improvement in simplicity and speed over existing techniques. ~Itllou~l~ developed in this laboratory pears probable that this method changes in other tissues.
for the analysis
of skin biopsies,
will prove of value in the study
it ap-
of pathological
Our knowledge of the structure and chemical hehaviour of the acid mucopolysaccharides has increased rapidly during the past decade. At present, at least eight individual members of this group of compounds have been isolated from tissues and characterised. These include the non-sulphated polysaccharicles hyaluronic acid and chondroitin; those carrying one sulphate group per repeating unit, namely the condroitin sulphates and heparin monosulphate; and the more heavily sulphated polysaccharide, heparin. ~~erat~~sulpllate is also generally included as an acid mucopoI~saccharicle, although it is atypical in that it does not contain a uranic. acid. There are indications that gross changes in the metabolism of these con~pouncls accompany a number of diseases involving connective tissue; this field has been Progress in this direction, however, has been reviewed by Rrimacombc and Stacy’. restricted by the absence of simple and reliable analytical methods. Many techniques for the extraction, fractionatit~n, and assay of indi~?idual acid muccll~~~l~saccl~arides have been described in the literature. In general, however, these procedures have been designed for preparative rather than analytical work. V%%en used for analytical purposes they have proved far too time-consuming for routine use: often they require large quantities of tissue, and cannot be satisfactorily scaled down. In addition, tfte recovery of added material is sometimes low and variable. A critical r-c-evaluation of several procedures has therefore been carried out in this laboratory, with the object of establishing a method which is sufficiently sensitive to handle small tissue biopsies and at tlic same time is simple enough to use for the
106
3111:1<,\VOOI)
routine investigation of large numhrs of specimens. The teclmicluc rcportt~tl lrcma, althougll developed primaril,- for skin, appwi-s to 1w cclually satisfactor!. for nu1ii~w)us other tissu:x-.
Papain (2 < recrystallized) was obtained front the Sigma Clicmiic-al C‘onilmn\-. Xl1 otlrcr reagents wel’e obtained from Hopkins M \I~illiams 12ttl. ; tllc carhazolc &d cet\-1 cllloridc were of General I’urpose, _ pm-idiniuln . Ahxdytical Reagent grade. The dialysis n~tnibrnne Sojas tubing, obtained from Hudcs Merchandising distilled
\\ater for I0 minutes
I. (‘ysteine
and tlw remaining nmtcrials of was 8 ,‘32 in. tliametvr \yisliing Corporation; tl1i.i was Imikd in
lwfore use.
hydroclh-ide
was dissolved in 0.2 JI sodium a&ate
(L nlginll) and
the pH adjusted to 7.0 b!r the addition of sodiunl l~ydroside. Tllis solution \Y;IY prepared freshly on the da>- of USE. 2. Trichloracetic acid, poo TV:\.. chloride (w/v) in 0.3 ;II sodiunl cllloritk. This solution 3. I()“,, cetyl pvridinium ” was filtered repeatedly until clear. _+, \Vashed celitc. The crude material was freed front fines b!r repeated tlccxrltation, 20;;
and tile remaining
material
suspended
in water at a concmtration
of ahut
Iv,;\-. 5.
6. 7. 8. Each of
0.03 41 sodium chloridr. o-p ,I1 sodium chloridc~. 1.2 -21 sodium chloride. 2.1 III sodium chloride. reagents g-8 contained c-et!.1 pyridiniunl
chloride at a c,oncentration
of o. I 'I,,
W/T'. 0.
I';,,
10. o.I”,
sodium tctraborate (w/x,) in concentrated carbazolr in absolute etliancol.
sulpliuric
acid.
The intact specimen (containing not less tllan 20 /‘g uranic acid as mu~~q~ol\~saccharide) was rinsed briefly with awtonc, and defntted in j: 1 ctller aictoiw mixture for 1 11 at 37O. After clrying, it vxs transferred to a ccntl-ifup tulxl. One 1111of cysteine sodium acetate solution and 2 mg of papain mm-e added, and tile tube phcctl in an incubator at 65’. Total disintegration of tile tissue occurred within 0 II ; tIri\ could be speeded by occasional shaking, or when more conxw~ient, could 1~ left ovt’rnight without loss of polysaccliaridc. When disintegration was complete, 0.2 ml of trichloracetic acid solution was added, and the mixture centrifugctl. The rtAduc VYIY waslied with 0.5 ml water, and the combined supernatant and wahliing was dinl~wtl against running tap water overnight. Tile dialysatc was transferred to a 3._j-l~ll conical centrifuge tulxx, and 0.2 1111 each of cetl-1 pyridinium chloride solution and of the wlitr suspension wow xldc~l. After thorough mixing, the tube was kept at 3T” for r Ii to ensure coagulation of tllc,
TISSI-I:
ACID
precipitate,
hip-COPOLTSACCHARII)~S
re-mixed,
and
centrifuged;
107 it \vas iinportant
that
the
supernatant
was
absolutely clear at tliis point. The residue was washed with I nil of 0.03 ‘11 sodium chloride, the supernatant being again discarded. The residue was finally extracted successix~ely \\-it11 o.+inl, 0.4-1111 and o.z-in1 portions of each of reagents 6-8, the extracts cwrresponding to each individual reagent being pooled. T11c three pooled extracts (containing the non-sulphated, nionosulpl~nted and “lliglll>.” sulphated mucol)ol!.sacclIaridrs, respectively) and a water blank were placed in 0 x z” test tubes, and 5 ml of I,oratc,~sulplluric acid aclcled with shaking. Tlrcy nere llented for IO min in a boiling water-bath, cooled, and 0.2 ml of carbazole solution added. After mixing, the tubes were returned to tlic water-bath for a furtller 15 min. The tubes were finall\~ renioved, cooled, and the absorbance of tile solution measured on a “Spckker” colarimeter using a green filter. The uranic acid content of each fraction \Y~S calculated from a standard wrve prepared using wglucuronic acid. Iii certain experiments it wits necessary to assay samples containing less tllan 20 ,c.g nronit acid as mucop”l~saccliaritle. The procedure found to he most satisof about 0.5 ml. T11c factory was to c\xporate tile dial!-sate ifr XKIIO to a \wlume suhequent precipitation, \vaslling:, extraction and assav were then scaled down to one-half or one-quarter of the volumes in microcells of I cm light path.
described
above,
and the alxorbances
measured
RISSULTS
X series of analyses were carried out using skin and other tissues from guinea pigs with the object of testing the validity, reproducibility and general applicability of the proposed techniques. I *alidity qf t/z _fmctiountio~2 fwoccdztrrs. Samples of the two major fractions obtained from guinea pig skin (0.4 dl and 1.2 31 sodium chloride extracts) were examined by a number of methods. Electrophesis was carried out on cellulose acetate membrane at pH 8.6. The two extracts each gave a single, compact hand, with no evidence of cross-contamination. The o.4 J1 extract co-ran with an authentic specimen of hyaluronic acid (human umbilical co-running ical Co.),
cord) ; the 1.2 11I extract sl~~wed a much with a marker of chondroitin sulphate
greater (“mixed
electrophoretic mobility, isomers”, Sigma Chenl-
The sensitivity to liyaluroniclase (bovine testes), carbazole: orcinol ratio, amino sugar content, and approximate molecular weights of the fractions were also examined in order to obtain positive identification of these two extracts. The results are
108
.\IIliI?,
summarised
\VOOl)
in Table I. So attempt was made to ol)tain positive, identification of tllcx chloride extract from skin, because of the relatiwly small cluantit~~ of
2.1 31 sodium
this fraction. Rt;hrodztcibility. Seven specimens of skin were removed from a singk guinw l+, The four larger samples were assayed as described, and the tlrrw smaller s;~n~I~lcsh!, the scaled down procedure (half scale). The uranic, a( id contents of tile 0.4 M ant1 I .L A11extracts, togetller with means and standard deviatinns, are sl10wn in Table I I licroiu~y~ cv,hui77w72ts. I\ series of anal>ws \vere carried out in wliicl~ 2j ; g quantities of liyaluronic acid, c~hondroitin sulphatc, and Iieparin \VertLa&M to slwcinens of skin. The mean figures for tlic rccovcrv of tliwe subxtanws in tile approlxiatc cxtrnct were 62”,,, 67°,2, and Oq”,, reslxctivcl7~ for tlic overall z+a\~ prowdur-0. A,bl)lic~ahilit~~to tisslfcs othr thfr77 ski7l. Slwcimcks in geiicral of Loo-j(x) nig Crwll wici ass;~~~tl$15dewrihc~tl. were remo~ecl from a number of diffeiwit guinea-pig tissws, c The results are sllown in Table I II ; WCII figure rq~r~wnts tlltl 111can of samplt~s frollr two animals.
years,
The quantitative extraction of mucopolvsaccharides from tissues has, in recent invariably been carried out with the aid of proteolytic enzymes, because tile
TISSUE
ACID
109
MUCOPOLYSrlCCHARIDES
earlier methods
using alkaline
hydrolysis
were either incomplete
or caused consider-
able damage to the polysaccharide. Even using proteolytic enzymes, however, it has I>roved difficult to free the polysaccharides of amino acids; most published techniques have tlrcrefore advocated prolonged hydrolysis, often using two enzymes in succession. However, our present aim was merely to determine the minimal conditions for complete extraction of the polysaccharide; providing that any protein which remained bound to the polysaccharide did not interfere with either the fractionation I>roccdure or the subsequent calorimetry, further hydrolysis appeared pointless. 11-e 11avc shown, in fact, that this is achieved in 6 11 with papain under the conditions described. Longer periods of hy~droly$s, or tli e subsequent use of trypsin, did not affect the analytic results for skin; this point should, of course, be verified if the metlrod is to be used routinely for other tissues. The fractionation procedures offer a much wider clioice. Elcctrophoresis, such as that on cellulose acetate, is ideal for the separation of micro-quantities? but is difficult to quantitate reproducibly. Ion exchange techniques seem to be unsurpassed in their powers of resolution” but are obviously unsuited to routine batch analysis. The technique selected was therefore based on complex formation with cetylpyridinium chloride’, and followed the method described by Schiller5 with slight modification. The modification of Bitter and Muir6 of the carbazole reaction for uranic acid was employed for the final assay. This offers several advantages over the more generally used technique of Dische’, notablv 1 greater sensitivity, more uniform colour yields from individual mucopolysaccharides, and a more stable colour. The overall method which is described in this communication offers a considerable advantage in speed, simplicity and sensitivity. over existing techniques. The reproducibility is excellent, and the overall recovery figures of f50-70”;, are acceptable. \Ihen applied to skin, the major fractions have been identified as hyaluranic acid (0.4 M sodium chloride extract) and chondroitin sulphate H (1.2 AT sodiuni chloride extract). T11e method has been used successfully iii this laboratory for the study of human skin biopsies and for certain irz ~i;jo studies of guinea-pig skin; these results will be published elsewhere. It appears that it would be equally suitable for the study of pathological changes of other tissues. The main disadvantages are two-fold. Firstly, no separation is effected between heparin monosulphate and the three isomeric chondroitin sulphates. Fortunately, in skin, as in certain other tissues, only one member of this group is present in substantial quantity. In tissues where more than one member of this group occurs, it should be feasible to use enzymes of appropriate specificity for their individual quantitation. Secondly, keratosulphate is not assayed by the proposed technique. This, however, is a feature common to most general analytic schemes for niucopolysaccharides; it is probable that in tissues where keratosulphate is predominant it is simplest to isolate and assay this compound individually.
The authors wish to acknowledge the generous support of the Medical Research Council for the \vorlc described in this report,
110
CKIMICA
A S~~~PLII~IED
ACTA
TECHNIQUE
FOR
THE
ANALYSIS
OF TISSIX
ACID
aIUC,OPC)LYSACCIIARIDES
A method
is described
for the isolation,
fractionation,
and assay of acid muco-
polysaccharides. It is usable for quantities of tissue containing as little as 5 ,~g uranic acid as mucopolysaccharide, and offers a considerable improvement in simplicity and speed over existing techniques. ~Itllou~l~ developed in this laboratory pears probable that this method changes in other tissues.
for the analysis
of skin biopsies,
will prove of value in the study
it ap-
of pathological
Our knowledge of the structure and chemical hehaviour of the acid mucopolysaccharides has increased rapidly during the past decade. At present, at least eight individual members of this group of compounds have been isolated from tissues and characterised. These include the non-sulphated polysaccharicles hyaluronic acid and chondroitin; those carrying one sulphate group per repeating unit, namely the condroitin sulphates and heparin monosulphate; and the more heavily sulphated polysaccharide, heparin. ~~erat~~sulpllate is also generally included as an acid mucopoI~saccharicle, although it is atypical in that it does not contain a uranic. acid. There are indications that gross changes in the metabolism of these con~pouncls accompany a number of diseases involving connective tissue; this field has been Progress in this direction, however, has been reviewed by Rrimacombc and Stacy’. restricted by the absence of simple and reliable analytical methods. Many techniques for the extraction, fractionatit~n, and assay of indi~?idual acid muccll~~~l~saccl~arides have been described in the literature. In general, however, these procedures have been designed for preparative rather than analytical work. V%%en used for analytical purposes they have proved far too time-consuming for routine use: often they require large quantities of tissue, and cannot be satisfactorily scaled down. In addition, tfte recovery of added material is sometimes low and variable. A critical r-c-evaluation of several procedures has therefore been carried out in this laboratory, with the object of establishing a method which is sufficiently sensitive to handle small tissue biopsies and at tlic same time is simple enough to use for the
106
3111:1<,\VOOI)
routine investigation of large numhrs of specimens. The teclmicluc rcportt~tl lrcma, althougll developed primaril,- for skin, appwi-s to 1w cclually satisfactor!. for nu1ii~w)us other tissu:x-.
Papain (2 < recrystallized) was obtained front the Sigma Clicmiic-al C‘onilmn\-. Xl1 otlrcr reagents wel’e obtained from Hopkins M \I~illiams 12ttl. ; tllc carhazolc &d cet\-1 cllloridc were of General I’urpose, _ pm-idiniuln . Ahxdytical Reagent grade. The dialysis n~tnibrnne Sojas tubing, obtained from Hudcs Merchandising distilled
\\ater for I0 minutes
I. (‘ysteine
and tlw remaining nmtcrials of was 8 ,‘32 in. tliametvr \yisliing Corporation; tl1i.i was Imikd in
lwfore use.
hydroclh-ide
was dissolved in 0.2 JI sodium a&ate
(L nlginll) and
the pH adjusted to 7.0 b!r the addition of sodiunl l~ydroside. Tllis solution \Y;IY prepared freshly on the da>- of USE. 2. Trichloracetic acid, poo TV:\.. chloride (w/v) in 0.3 ;II sodiunl cllloritk. This solution 3. I()“,, cetyl pvridinium ” was filtered repeatedly until clear. _+, \Vashed celitc. The crude material was freed front fines b!r repeated tlccxrltation, 20;;
and tile remaining
material
suspended
in water at a concmtration
of ahut
Iv,;\-. 5.
6. 7. 8. Each of
0.03 41 sodium chloridr. o-p ,I1 sodium chloridc~. 1.2 -21 sodium chloride. 2.1 III sodium chloride. reagents g-8 contained c-et!.1 pyridiniunl
chloride at a c,oncentration
of o. I 'I,,
W/T'. 0.
I';,,
10. o.I”,
sodium tctraborate (w/x,) in concentrated carbazolr in absolute etliancol.
sulpliuric
acid.
The intact specimen (containing not less tllan 20 /‘g uranic acid as mu~~q~ol\~saccharide) was rinsed briefly with awtonc, and defntted in j: 1 ctller aictoiw mixture for 1 11 at 37O. After clrying, it vxs transferred to a ccntl-ifup tulxl. One 1111of cysteine sodium acetate solution and 2 mg of papain mm-e added, and tile tube phcctl in an incubator at 65’. Total disintegration of tile tissue occurred within 0 II ; tIri\ could be speeded by occasional shaking, or when more conxw~ient, could 1~ left ovt’rnight without loss of polysaccliaridc. When disintegration was complete, 0.2 ml of trichloracetic acid solution was added, and the mixture centrifugctl. The rtAduc VYIY waslied with 0.5 ml water, and the combined supernatant and wahliing was dinl~wtl against running tap water overnight. Tile dialysatc was transferred to a 3._j-l~ll conical centrifuge tulxx, and 0.2 1111 each of cetl-1 pyridinium chloride solution and of the wlitr suspension wow xldc~l. After thorough mixing, the tube was kept at 3T” for r Ii to ensure coagulation of tllc,
TISSI-I:
ACID
precipitate,
hip-COPOLTSACCHARII)~S
re-mixed,
and
centrifuged;
107 it \vas iinportant
that
the
supernatant
was
absolutely clear at tliis point. The residue was washed with I nil of 0.03 ‘11 sodium chloride, the supernatant being again discarded. The residue was finally extracted successix~ely \\-it11 o.+inl, 0.4-1111 and o.z-in1 portions of each of reagents 6-8, the extracts cwrresponding to each individual reagent being pooled. T11c three pooled extracts (containing the non-sulphated, nionosulpl~nted and “lliglll>.” sulphated mucol)ol!.sacclIaridrs, respectively) and a water blank were placed in 0 x z” test tubes, and 5 ml of I,oratc,~sulplluric acid aclcled with shaking. Tlrcy nere llented for IO min in a boiling water-bath, cooled, and 0.2 ml of carbazole solution added. After mixing, the tubes were returned to tlic water-bath for a furtller 15 min. The tubes were finall\~ renioved, cooled, and the absorbance of tile solution measured on a “Spckker” colarimeter using a green filter. The uranic acid content of each fraction \Y~S calculated from a standard wrve prepared using wglucuronic acid. Iii certain experiments it wits necessary to assay samples containing less tllan 20 ,c.g nronit acid as mucop”l~saccliaritle. The procedure found to he most satisof about 0.5 ml. T11c factory was to c\xporate tile dial!-sate ifr XKIIO to a \wlume suhequent precipitation, \vaslling:, extraction and assav were then scaled down to one-half or one-quarter of the volumes in microcells of I cm light path.
described
above,
and the alxorbances
measured
RISSULTS
X series of analyses were carried out using skin and other tissues from guinea pigs with the object of testing the validity, reproducibility and general applicability of the proposed techniques. I *alidity qf t/z _fmctiountio~2 fwoccdztrrs. Samples of the two major fractions obtained from guinea pig skin (0.4 dl and 1.2 31 sodium chloride extracts) were examined by a number of methods. Electrophesis was carried out on cellulose acetate membrane at pH 8.6. The two extracts each gave a single, compact hand, with no evidence of cross-contamination. The o.4 J1 extract co-ran with an authentic specimen of hyaluronic acid (human umbilical co-running ical Co.),
cord) ; the 1.2 11I extract sl~~wed a much with a marker of chondroitin sulphate
greater (“mixed
electrophoretic mobility, isomers”, Sigma Chenl-
The sensitivity to liyaluroniclase (bovine testes), carbazole: orcinol ratio, amino sugar content, and approximate molecular weights of the fractions were also examined in order to obtain positive identification of these two extracts. The results are
108
.\IIliI?,
summarised
\VOOl)
in Table I. So attempt was made to ol)tain positive, identification of tllcx chloride extract from skin, because of the relatiwly small cluantit~~ of
2.1 31 sodium
this fraction. Rt;hrodztcibility. Seven specimens of skin were removed from a singk guinw l+, The four larger samples were assayed as described, and the tlrrw smaller s;~n~I~lcsh!, the scaled down procedure (half scale). The uranic, a( id contents of tile 0.4 M ant1 I .L A11extracts, togetller with means and standard deviatinns, are sl10wn in Table I I licroiu~y~ cv,hui77w72ts. I\ series of anal>ws \vere carried out in wliicl~ 2j ; g quantities of liyaluronic acid, c~hondroitin sulphatc, and Iieparin \VertLa&M to slwcinens of skin. The mean figures for tlic rccovcrv of tliwe subxtanws in tile approlxiatc cxtrnct were 62”,,, 67°,2, and Oq”,, reslxctivcl7~ for tlic overall z+a\~ prowdur-0. A,bl)lic~ahilit~~to tisslfcs othr thfr77 ski7l. Slwcimcks in geiicral of Loo-j(x) nig Crwll wici ass;~~~tl$15dewrihc~tl. were remo~ecl from a number of diffeiwit guinea-pig tissws, c The results are sllown in Table I II ; WCII figure rq~r~wnts tlltl 111can of samplt~s frollr two animals.
years,
The quantitative extraction of mucopolvsaccharides from tissues has, in recent invariably been carried out with the aid of proteolytic enzymes, because tile
TISSUE
ACID
109
MUCOPOLYSrlCCHARIDES
earlier methods
using alkaline
hydrolysis
were either incomplete
or caused consider-
able damage to the polysaccharide. Even using proteolytic enzymes, however, it has I>roved difficult to free the polysaccharides of amino acids; most published techniques have tlrcrefore advocated prolonged hydrolysis, often using two enzymes in succession. However, our present aim was merely to determine the minimal conditions for complete extraction of the polysaccharide; providing that any protein which remained bound to the polysaccharide did not interfere with either the fractionation I>roccdure or the subsequent calorimetry, further hydrolysis appeared pointless. 11-e 11avc shown, in fact, that this is achieved in 6 11 with papain under the conditions described. Longer periods of hy~droly$s, or tli e subsequent use of trypsin, did not affect the analytic results for skin; this point should, of course, be verified if the metlrod is to be used routinely for other tissues. The fractionation procedures offer a much wider clioice. Elcctrophoresis, such as that on cellulose acetate, is ideal for the separation of micro-quantities? but is difficult to quantitate reproducibly. Ion exchange techniques seem to be unsurpassed in their powers of resolution” but are obviously unsuited to routine batch analysis. The technique selected was therefore based on complex formation with cetylpyridinium chloride’, and followed the method described by Schiller5 with slight modification. The modification of Bitter and Muir6 of the carbazole reaction for uranic acid was employed for the final assay. This offers several advantages over the more generally used technique of Dische’, notablv 1 greater sensitivity, more uniform colour yields from individual mucopolysaccharides, and a more stable colour. The overall method which is described in this communication offers a considerable advantage in speed, simplicity and sensitivity. over existing techniques. The reproducibility is excellent, and the overall recovery figures of f50-70”;, are acceptable. \Ihen applied to skin, the major fractions have been identified as hyaluranic acid (0.4 M sodium chloride extract) and chondroitin sulphate H (1.2 AT sodiuni chloride extract). T11e method has been used successfully iii this laboratory for the study of human skin biopsies and for certain irz ~i;jo studies of guinea-pig skin; these results will be published elsewhere. It appears that it would be equally suitable for the study of pathological changes of other tissues. The main disadvantages are two-fold. Firstly, no separation is effected between heparin monosulphate and the three isomeric chondroitin sulphates. Fortunately, in skin, as in certain other tissues, only one member of this group is present in substantial quantity. In tissues where more than one member of this group occurs, it should be feasible to use enzymes of appropriate specificity for their individual quantitation. Secondly, keratosulphate is not assayed by the proposed technique. This, however, is a feature common to most general analytic schemes for niucopolysaccharides; it is probable that in tissues where keratosulphate is predominant it is simplest to isolate and assay this compound individually.
The authors wish to acknowledge the generous support of the Medical Research Council for the \vorlc described in this report,
110