J. E. Scott: Research in Britain on Connective Tissues -3-

and Tooth Society was in vigorous life， dating back to 1950. This probably makes it

the oldest connective tissue c1ub in the wOrld， and it too was responsible for organis-

ing collaboration between national Bone and Tooth Societies on an international scale.

It seems inevitable that all three British groups will coalesce， and then we can expect

a very lively and rather large connective tissue society， with more than 500 members.

Reference

1) Scott， J. E. : Connective tissue research and organization. Calc. Tiss. Res.， 3: 198-210， 1969.

II. Critical Electrolyte Concentration Phenomena

in Connective Tissue Research

In 1953， as a post graduat巴 inthe Department of Chemical Pathology， Manchester

University， 1 was given the problem of analysing arterial tissue for “mucopolysac-

charides" as a part of an investigation of atherosc1erosis. At that time， th巴 structures

of the mucopolysaccharides were uncertain， and no systematic methods were available

for their analysis. 1 was to try out various techniques. Paper chromatography was

very popular， although very little used on polymeric materials. Kerby had achieved

separations of impure chondroitin sulphates， and this was followed up. The commoner

solvent systems did not move the polysaccharides from the origin， and on the principle

that the more soluble the compounds were in the solvent， the faster they would move，

amines were incorporated into the solvent systems. The best amines should thus have

a large organic portion， and on the shelf was a preparation of I.C.A. cetrimide (impure

cetyltrimethyl ammonium). With this in the solvent the results were puzzling. It was

impossible to stain the polysaccharide with toluidine blue， although no difficulty had

been encountered with other solvent systems. Other results suggested that the cetri-

mide was not moving with the solvent front， but some distance behind it， and there

would be a time during which the polymer was extracted with solvent without amine.

1 therefore added cetrimide to a solution of chondroitin sulphate before applying it to

the paper and was rewarded by the sight of a fiocculant white precipitate. This com-

plex was very insoluble， and could be obtained from solutions of chondroitin at con-

centrations of less than 1 ppm. This offered a basis for recovering polysaccharides

for analysis， and one important difficulty would be overcomeY

It was soon found that the precipitates w巴resoluble in salt solutions. The poly-

anions could then be recovered as inorganic salts by precipitation with ethanol， or by

absorbing or extracting the cetrimide in organic solvents such as chloroform， and then

dialyzing away the salt. We then had a practical method for rapidly isolating very

small amounts of chondroitin sulphate， etc.2)

- 4 Connective Tissue

The solubility of the precipitates in salt solutions was investigated to optimise

conditions for the practical procedures. It was found that the salt concentration neces-

sary to dissolve the precipitate of a given polyanion was clearly defined， with a cor-

relation between the structure of the polyanion and the concentration of salt needed

to dissolve the complex. Polyanions with sulphat巴 estergroups required much higher

concentrations of salt to dissolve them than those polyanions with carboxylate or

phosphate ester groups.s) The solubility phenomenon was very sharp， the solubility

of the complex being nil at one salt concentration， and then almost complete at a

slightly higher salt concentration， the critical electrolyte concentration (CEC). This

became the basis of a standard method of fractionating the polysaccharides of connec-

tive tissues，4) since the characteristic polysaccharide anions contain carboxylate and

sulphate ester groups in varying proportions. This procedure proved of great value

in commerce. Most of the world's heparin was prepared on this principle， and probably

still is. Since neutral polysaccharides are not precipitated， it is possible to purify many

neutral polysaccharides from contaminating polyanions， and Sepharose was among the

polymers so obtained.

The variabl巴sin the reaction， i.e. chain length of the paraffin chain， the nature of

the ammonium or onium group at the cationic head， the molecular w巴ightof the poly-

anion， and an increasing number of polymers were investigated. It was obvious that

this precipitation reaction was completely general for all polyanions.

Various practical versions of the salt fractionation scheme were devised. From a

solution containing high concentrations of salt， by adding water， one obtains sequential

precipitates with diminishing ratios of sulphate to carboxylate or phosphate ester， as

an alternative to dissolving up the precipitates in increasing concentrations of salt

solutions. The latter procedure was converted into a column process in which the

cetylpyridinium complex of the polyanion was adsorbed to a cellulose support and then

eluted in a salt gradient.5) This procedure has now gone through at least two genera-

tions of modifications. It was devised partly to avoid the difficulties of centrifuging

colloidal suspensions of cetylpyridinium complexes of polyanions in magnesium chlorid

The critical electrolyte concentration principle in histochemistry

The considerable success of the CEC principle， using quaternary ammonium deter-

gents in th巴 fractionationof polyanions， led to a search for an understanding of the

principle in physical chemical terms. Clearly an ion exchange is involved， and the

affinity of the different cations (e必 quaternaryammonium， magnesium， etc.)， for the

anionic sites of the polyanion， determines to a large extent the outcome of the com-

P巴tition.3) The extreme sharpness of the phenomena was recognized to b巴 dueto the

polymeric nature of the "substrateぺ6) The law of mass action applied to a simple

]. E. Scott: Critical Electrolyte Concentration Phenomena - 5ー

model showed that all the basic features of the CEC phenomenon could be readi1y

understood.7l As a consequence of this treatment， it was apparent that any organic

precipitant could be substituted for quaternary ammonium ions， and that they could

as well be coloured as colour1ess. Thus it seemed likely that fractionation of poly-

anions could be performed with coloured reagents， and this led naturally to the pos-

sibility of developing entirely new methods in histochemistry and histology based upon

the CEC phenomenon. In 1965， the first pap巴rwas published in which the principle

was applied to connective tissue polyanions， making use of the phthalocyanin dye，

alcian blue，8l as an exact analogue to， e.g. cetylpyridinium. The results were very

satisfactory， and the method has since become very widely established in histochemical

work. Fig. 1 shows how one can distinguish between polyanions in connective tissues.

The theoretical treatment and the new application， opened up an enormous range

of molecules which might be used in CEC techniques， and a considerable programme

was launched to investigate properties of both well-known and entirely new reagents.

It was found that dyestuffs used by histologists and histochemists could be c1assified

according to their behaviour in the ion exchange reaction system， to give great insight

into observations made by histologists over the last 100 years， but not otherwise ex-

plicable so far.9l It was possible to define two categories of cationic dye， those in

which the charge was due to an -onium type group， and those in which a chelated

metallic cation was involved (e.g. alcian blue and haematoxylin respectively). Thus，

it is possible to choose a reagent which binds to sulphated polyanions at high salt

concentrations (alcian blue， etc.) or alternatively one which is bound to carboxylated

polyanions at high salt concentrations (haematoxylin， etcよ

The ion exchange reaction is a very general phenomenon but other than electro-

static forces may be operative. Perhaps the most important example to the histologist

is that between planar， aromatic dyes and the Watson-Crick base pairs present in

nuc1eic acids. This combination involves at least electrostatic and Van der Waals

attractions and 'hydrophobic bonds'， and it is therefore more stable than a complex

formed only via electrostatic bonds. It resists dissociation by cations such as sodium

or magnesium. Thu

- 6 Connective Tissue

a b

c

Fig. 1

]. E. Scott: Critical Electrolyte Concentration Phenomena -7-

a b

Fig. 2. The CEC effect in the staining of nucleic acids in mouse liver. The dye cation is copper N， N/， N"， N'II tetramethyl tetrapyridinotetraazaporphin lO J• (a) dye at 0.05%

w jv in sodium acetate buffer pH 5. 7 (0.025 M) containing 0.05 M MgCl2 and 4%

glutaraldehyde. Un且xedfrozen liver， cut at 15，11， stained for 2 hrs.， processecl into Araldite， and sectioned for E.M. (x 3， 700). Note the dense staining of nucleic acids in nuclear bodies and in the cytoplasm. (b) as in (a) but with 0.5 M MgCl2 (instead of 0.05 M MgCl2). (x 8， 900). Note th巴 veryc1ense staining of small (ribosomal precursor?) bodies in the nucleus， and the absence of DNA staining of the type seen in (a).

biosynthesized polysaccharide material， which otherwise would be obscured by the

large amount of ambient nucleic acid. The dyes we use are tetraazaporphins， which

include in their structure a chelated metal atom. This increases electron density， and

some interesting results have already been obtained with th巴m.l!) Fig. 2 gives ex帽

amples.

By making use of electrolyte competition phenomena with the large variety of

organic cations available， there are few appIications in histochemistry which would

not yield more interpretable results than would previously available histological tech-

niques. It should also be possible to devise much improved biochemical reagents based

upon som巴 ofthe interaction effects already discovered.

Fig. 1. The CEC effect in the demonstration of glycosaminoglycuronans in connective tissue (human new.bo1'11 lung， formol fixecl). Alcian blue (0.05% w jv) in soclium acetate buffer pH 5. 7 (0.025 M)， containing (a) 0.0 M MgC12; (b) 0.2 M MgC12; (c) 0.5 M MgC12; (d) 0.7 M MgC12・ (¥160).

Note the increasingly specific patt巴1'11 of c1ye.bincling as the salt concentration in・

creases. At 0.5 M only chonclroitin sulphate in the cartilage is well stainecl. The changes in patte1'11 are sharp.

- 8ー Connective Tissue

References

1) Scott， J. E. : Ph. D. Thesis， University of Manchester， 1956.

2) Scott， J. E. : The reaction of long-chain quaternary ammonium compounds with acidic poly-

saccharides. Chem. and Ind. (London)， 168， 1955. 3) Scott， J. E. : The solubility of cetylpyridinium complexes of biological polyanions in solution

of salts. Biochim. Biophys. Acta， 18: 428-429， 1955.

4) Scott， J. E. : Aliphatic ammonium salts in the assay of acidic polysaccharides from tissues.

In， Methods in Biochemical Analysis， Vo1. 8 (Glick， D. Ed.)， Interscience Publishers， Inc.

New York， 1960， 146-198.

5) Antonopoulos， C. A.， Borelius， E.， Gardel1， S.， Hamnstr凸m，B. and Scott， J. E. : The precipita-

tion of polyanions by long-chain aliphatic ammonium compounds. IV. Elution in salt solu-

tions of mucopolysaccharide咽 quaternary ammonium complexes adsorbed on a support.

Biochim. Biophys. Acta， 54: 213-226， 1961.

6) Scott， J. E. : The fractionation of polyanions by long司chainaliphatic ammonium salts. Bio-

chem. J.， 78: 24-32， 1961.

7) Scott， J. E. : Affinity， competition and specific interactions in the biochemistry and histo-

chemistry of polyelectrolytes. Biochem. Soc. Trans.， 1: 787-806， 1973.

8) Scott， J. E. and Dorling， J. : Differential staining of acid glycosaminoglycans (mucopolysac-

charides) by a1cian blue in salt solutions. Histochemie， 5: 221-233， 1965.

9) Scott， J. E. and Wi11ett， I. H. : Binding of cationic dyes to nuc1eic acids and other biological

polyanions. Nature， 209: 985-987， 1966.

10) Scott， J. E. : Histochemistry of alcian blue. III. The molecular biological basis of staining

by a1cian blue 8GX and analogous phthalocyanins. Histochemie， 32: 191-212， 1972.

11) Scott， J. E. : Histochimie ultrastructurale des protるoglycanes. Ann. Med. Reims， 13: 63-67，

1976.