GROUND SUBSTANCE OF THE CONNECTIVE

POSTGRAD. MED. J. (1965), 41, 382

RECENT TRENDS IN THE BIOCHEMISTRY OF THEGROUND SUBSTANCE OF THE CONNECTIVE

TISSUESA. G. LLOYD, B.Sc., Ph.D., A.R.I.C.

Senior Lecturer in Biochemistry and Member of the M.R.C. Research Group for Studies on ConnectiveTissue and Lung, University College, Cardiff.

THE UBIQUITY of members of the anatomicalgroup loosely defined as connective tissues isa matter of long-standing record. The samecomment applies to the convention that, despitethe differing biological roles which they arerequired to play and their characteristicallydifferent mechanical character at macroscopiclevel, the individual members of the connectivetissue group exhibit certain basic similaritieswhich first become apparent at microscopiclevel. A common biochemical approach is todevelop these features of similarity to a pointwhere an idealised composite of all connectivetissues can be devised. Use of such a modelallows initial broad generalisations to be drawn.On the other hand, the same model must sub-sequently be employed to establish not onlymorphological but fundamental biochemicaldifferences which exist between the tissues.A model connective tissue is usually con-

ceived as containing relatively few cells perunit volume compared to a highly cellularorganization such as liver and additionallydifferentiated from the latter by the presenceof a significant quantity of extracellularmaterial. This material is characterised by thepresence of organised fibrillar elements, com-posed variously of the biochemically distinctproteins collagen, elastin and reticulin, thesebeing embedded in an amorphous mediumtermed the ground substance.

In this context, it is fitting that laboratoriesin the Cardiff area should be connected instudies on all aspects of pulmonary dust dis-ease and in particular of coal miner's pneumo-coniosis, progressive massive fibrosis andsilicosis. The need for such work is presentedwith considerable impact in the observationsof Gough 1(1947). 'Much 'has already beenestablished about the statistical incidence andpathological characteristics of such dust dis-eases, but their true aetiology is still a matterfor conjecture (Policard, 1962). However, fromthe simplified histological standpoint the humanlung lesion in such conditions shows character-istically an increased 'fibrosis' when compared

to the surrounding tissue, the fibres usuallybeing described as collagenous. As yet thereis no clear indication whether such fibrosesarise according to the partially establishedpattern of inflammatory response in which theearliest recognisable biochemical changes in-clude alterations in the ground substance ofconnective tissues followed only subsequentlyby de novo formation of additional fibrousmaterial. It is for this reason that the researchgroup housed in the Department of Biochemis-try is designed to assist in the clarificationof fundamental biochemical changes associatedwith general lung dust diseases from the aspectof disturbed connective tissue metabolism. Suchan approach must obviously take into accountthe many recent developments concerning thenature and properties of the structural elementsof the connective tissues and in particular theintrinsic complexity of the ground substance.

The Ground Substance

There is now no doubt that histologicalexamination of connective tissues using con-ventional techniques provides a wholly incom-plete and frequently artificial picture of thecomplexity of the ground substance. It has evenbeen established that long-used methods oftissue fixation may even facilitate the extrac-tion of ground substance constituents (seeSzirmai, 1963). Thus, even for relatively grosshistological observations such methods mustbe replaced by the use of modified tissue fixa-tives (Quintarelli, Scott and Dellovo, 1964a)or alternatively frozen section techniques inwhich 'fixation' is virtually carried out in thedye bath (Szirmai, 1963). Methods have be-come available for differentiating some of theground substance components by selectivephysico-chemical blocking and unblocking inthe presence of quaternary ammonium com-pounds (Kelly, Bloom and Scott, 1963), byselective staining in the presence of solutionsof varying pH or electrolyte concentration(Szirmai, 1963; Saunders, 1964; Quintarelli,

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LLOYD: Biochemistry of Ground Substance of Connective Tissues

Scott and Dellovo, 1964b) and by selectiveenzymic digestion ,(Zugibe, 1962 a and b).However, such techniques continue to be theprovince of the specialist in the field, ratherthan methods of routine applicability. As such,much reliance must still be placed onapproaches based on the extraction of connect-ive tissue components followed by theircharacterisation using physical, chemical andbiochemical methods. Even in this regardstudies initiated during the middle of the lastcentury and subsequently prosecuted withincreasing vigour are still the subject of con-tinuous contemporary reappraisal.Polysaccharide-Protein Complexes of theGround SubstanceThe Polysaccharides

Early studies on these materials invariablyinvolved treatment of connective tissues underrelatively vigorous conditions such as pro-longed digestion with proteolytic enzymesfollowed by extraction with strong salt solutionsor even dilute alkali. The resultant extractsyielded, by several methods of purification,varying amounts of one or more of a group ofcarbohydrate high polymers. These have beenvariously and synonymously termed acid muco-polysaccharides, acid aminopolysaccharides ormore recently acid glycosaminoglycans (Jeanloz,1960). In defining the purity of such materialsearly workers attached much significance tothe absence of amino acid and polypeptideresidues from the polysaccharide preparationsand when demonstrated such 'contamination'was frequently attributed to inefficiency ofpurification procedures. Structural studies (re-viewed by Jeanloz, 1963) on these compoundsreveal a pattern of similar molecular organiza-tion. Thus, in the main they are relativelyhigh molecular weight polysaccharides havinglinear chains containing hexosamine residues.The latter may be either D-glucosamine orD-galactosamine each occurring in a state inwhich the amine grouping is blocked. Thehexosamines alternate regularly in the polymerchains with other monosaccharide residueswhich may be hexuronic acids, such as D-glucuronic acid and L-iduronic acid or ahexose such as D-galactose.

All of the polysaccharides are also poly-anions, the negative charge arising either fromthe carboxyl group of the above uronic acidsor from the presence of ester bound sulphate,or both. Accepted structures show the poten-tially charged groups arranged regularly along

the polymer chains. As both carboxyl andsulphate groups are fully ionized at physiologi-cal pH the resultant highly negative characteris likely to have considerable bearing on thefunctions attributed to the polymers in thenative state.The acidic glycosaminoglycans recognised to

date fall into four classes, differentiated byfeatures of chemical fine structure:

A. Polysaccharides having anionic characterconferred solely by hexuronic acid constituentse.g., hyaluronic acid and chondroitin.

B. Polysaccharides having anionic characterresulting from the dual presence of hexuronicacid and O-sulphate groups e.g., chondroitin4-sulphate (syn. chondroitin sulphate-A),chondroitin 6-sulphate (syn. chondroitin sul-phate-C) and dermatan sulphate (syn. chond-roitin sulphate-B, P-heparin).

C. Polysaccharides having O-sulphate groupsas sole functional anionic entities e.g., keratansulphate (syn. keratosulphate).

D. Polysaccharides characterised by the pre-sence of hexuronic acid, O-sulphate and N-sulphate residues e.g., heparan sulphates (syn.heparitin sulphates). Heparin and o-heparinmay also be included in this class for the sakeof completeness.The compositions of each of these materials isshown in Table 1 and the major repeatingperiods of the polysaccharides from Classes A,B and C are summarised in Figs. 1, 2 and 3respectively. These should be considered to bestructures derived on the principle of bestanalytical evidence. While there is little doubtthat they account for the format of the majorportion of the polysaccharide chains there areindications that some regions of the polymersare not constructed in this way. Such evidencewill be dealt with later. It should also benoted that variability of sulphate analysis isnot an uncommon occurrence and that acidglycosaminoglycan preparations with sulphurto nitrogen ratios both greater and less thanunity have been isolated. Conclusive dataallowing the assignment of wholly acceptablerepeating period structures for heparansulphates (and for heparin and o-heparin)has yet to be obtained (see Muir, 1964).

Aside from the significant contributionswhich they have made in structural work onthe acid glycosaminoglycans, elegant studiesmade by Professor Karl Meyer and his col-leagues lay a foundation for the concept thatconnective tissues fulfilling distinctive biologicalfunctions are also provided with differing

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384 POSTGRADUATE MEDICAL JOURNAL July, 1965

TABLE 1MAJOR CONSTITUENTS OF THE ACIDIC GLYCOSAMINOGLYCANS

Polysaccharides Hexosamine Other N-Acetyl O-Sulphate N-SulphateMonosaccharide Group Group (s) GroupCLASS A

Hyaluronic Acid D-Glucosamine D-Glucuronic Acid +--Chondroitin D-Galactosamine D-Glucuronic Acid +

CLASS BChondroitin 4-Sulphate D-Galactosamine D-Glucuronic Acid + +Chondroitin 6-Sulphate D-Galactosamine D-Glucuronic Acid + +Dermatan Sulphate D-Galactosamine L-Iduronic Acid + +

CLASS CKeratan Sulphate D-Glucosamine D-Galactose + +

CLASS DHeparan Sulphates D-Glucosamine D-Glucuronic Acid + + +Heparip D-Glucosamine D-Glucuronic Acid - + +

CH2OHH20H

OH H

(a) ;-0O oH H NH.COCH3

FIG. 1.-Structures of the repeating peiods in

Tio

H20HCOOH HO

H 0 H 0

hyaluronic acid (a) and chondroitin (b).

complements of the acidic polysaccharides(Meyer, Davidson, Linker and Hoffman, 1956).Table 2 is a summary of part of the originalwork plus some additional information, givinganalyses for tissues of average chronologicalage. On the other hand, it should also bestressed that the content of acid glycosamino-glycans must not be considered to be staticthroughout life. Thus, apart from variationsdue to metabolic turnover, changes both inoverall content and in the concentration ratiosof individual polysaccharides as a function ofage have also been observed. For example,embryonic pig skin polysaccharides are com-prised of 9.5% dermatan sulphate, 71%hyaluronic acid and about 20% of a mixtureof chondroitin 4-sulphate and chondroitin6-sulphate. Analyses of adult pigskin revealsa sharp change in ratio corresponding to 64%

CH0 H

COOH HOSO (33

-H HH H NHH.COCH3

CH2OSO3H

OHH0 0 (b)OH NH.COCH3

H HO

FIG. 2.-Structures of the repeating periods in chon-droitin 4-sulphate (a), chondroitin 6-sulphate (b)and dermatan sulphate (c).

CH20H CHHOSo3HHO H0H H

-O H

H OH H NH.COCH

----'l~

FIG. 3.-Structure of the repeating period in keratansulphate.



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July, 1965 LLOYD: Biochemistry of Ground Substance of Connective Tissues 385

TABLE 2

DISTRIBUTION OF ACIDIC GLYCOSAMINOGLYCANS IN MAMMALIAN CONNECTIVE TISSUEPOLYSACCHARIDES

Tissues Hyaluronic Chondroitin Chondroitin Chondroitin Dermatan Keratan Heparan HeparinAcid 4-Sulphate 6-Sulphate Sulphate Sulphate Sulphate

Synovial Fluid + - -Cartilage + + +Bone + + - +Tendon + + +Ligamentum Nuchae + -+ -+ ++Skin + + + + +Aorta + -+ - + +Vitreous Humor +Cornea - + + +Sclera + +Umbilical Cord +

dermatan sulphate, 30%/ hyaluronic acid andsome 0.1% of the chondroitin 4-sulphate-chondroitin 6-sulphate mixture (Loewi andMeyer, 1958). Similar effects have been ob-served in rat and in man (Loewi, 1961; Prodi,1964). In addition Kaplan and Meyer (1959)recorded that in human costal cartilage thereis a decrease in the overall content of acidicglycosaminoglycans with age. The same authorshave shown that in phase with this decreasethe constituent polysaccharides change fromexclusively chondroitin 4-sulphate in foetalcostal cartilage to a mixture of chondroitin 6-sulphate and keratan sulphate in cartilagefrom the same location in adults. These selectedexamples can be considered to be representativeof phenomena which are likely to become therule for all connective tissues rather than ex-ceptional observations.The distributions summarized in Table 2 are

based on analyses of 'bulk' samples of materialwhere the morphological integrity of the tissueis destroyed at an early stage of the extractionprocedure and therefore correspond to averageresults for fairly large volumes of tissue. Assuch they give no indication whether the poly-saccharides are arranged homogeneouslythroughout the whole ground substance orwhether in fact they are the subject of concent-ration and localisation effects in relation to thefine structure of the tissue. Such informationis clearly of prime importance to assessmentsof the functional significance of individualacid glycosaminoglycans. Antonopoulos, Gar-dell, Szirmai and de Tyssonsk (1964) haverecognised the need for means to carry outprecise surveys. These authors are responsiblefor the development of methods which allowalternate tissue sections (10-40 A thick) to beexamined qualitatively by histological meansand quantitatively by micro-scale modificationsof established chemical methods. Data collected

in this way allows the construction of concentra-tion (and tentative identity) profiles througha given tissue. The methods have been appliedto studies on bovine nasal septum and rabbitintervertebral disc (Antonopoulos and others,1964) and to epiphyseal plates from normal andrachitic dogs (Hjertquist, 1964a, b). The result-ing observations establish that the acid gly-cosaminoglycans are subject not only to grossvariation from tissue to tissue and to agefluctuations in the same tissue but also tothree dimensional variations in a given tissueof defined age.

The ComplexesThe 'pure' polysaccharides required for

structural studies do not exist as such at tissuelevel and are, in a sense, chemical artifactsproduced by the relatively vigorous meansused for extraction. Application of mildermethods, such as physical disintegration of thetissue by high speed homogenisation, yieldsthe acid glycosaminoglycans linked firmly toprotein moieties by covalent bonds (see forexample Shatton and Schubert, 1954; Gerber,Franklin and Schubert, 1960). Erroneous im-plications should not be drawn from thepersistent use of the generic nomenclatureof polysaccharide-protein complexes for thesematerials when obviously the term compoundwould be far more appropriate.

In recent years the most extensively studiedcomplex is that derived from various kinds ofcartilage. Preliminary work conceived thecartilage chondromucoprotein complex as con-sisting of numerous polysaccharide chains(largely chondroitin 4-sulphate or 6-sulphateplus some keratan sulphate) firmly attachedto a median, non-collagenous protein core(Partridge, Davis and Adair, 1961). It is acharacteristic of the cartilage complexes that



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the polysaccharide moieties are cleaved ir-reversibly from the protein chain by treatmentwith dilute alkali (Muir, 1958; Partridge andDavis, 1958; Malawista and Schubert, 1958;Partridge and Elsden, 1961). However, thework of Muir (1958) established that whenthe isolated complex from cartilage was ex-haustively degraded with proteolytic enzymesin vitro the polysaccharide preparations sub-sequently isolated were still firmly associatedwith serine-rich polypeptide stubs. Such obser-vations focussed considerable attention on thepossibility that serine residues in the originalcore material were intimately concerned in thelinkage of polysaccharide and protein. The valueof this initial work is illustrated by the recentisolation from a chondroitin sulphate-proteincomplex of carbohydrate residues linked directlyto serine (Roden, Gregory and Laurent, 1964).Evidence has been presented (Dr. L. Rod6n,personal communication) that a novel carbo-hydrate linkage sequence, comprising xyloseand galactose moieties, is involved as a bridgebetween the serine hydroxyl and the character-istic repeating periods of the glycosaminoglycanchain. These observations are by no meansunique to the chondroitin sulphate-proteincomplexes as similar linkage arrangements arealso found in heparin-polypeptide complexes(Lindahl and Roden, 1964). Heparan sulphate-protein complexes are likely to be bonded in ananalogous fashion (Jacobs and Muir, 1964).

Treatment of glycosaminoglycan-proteincomplexes with alkali and analysis of theproducts of reaction provides a relatively rapidmeans for assessing the participation of serineand threonine in glycoprotein linkage (Ander-son, Seno, Sampson, Riley, Hoffman andMeyer, 1964; Anderson, Hoffman and Meyer,1965). Studies using these alkaline degradationmethods reveal the involvement of hydroxylatedalkyl amino acid residues in linkages betweenprotein and chondroitin 4-sulphate, chondroitin6-sulphate and skeletal keratan sulphate. How-ever, bonds in the corresponding complexes ofchondroitin, dermatan sulphate and cornealkeratan sulphate are suggested to be of theasparaginyl-glycosyl type as found in ao-glyco-protein (Professor K. Meyer, personal com-munication).There is no clear indication whether a single

type of acid glycosaminoglycan may beattached to protein cores of different aminoacid constitution. However, it has been shownthat bovine nasal septum cartilage yields twovarieties of chondroitin sulphate-protein com-plex, distinguished by their differential sedimen-

tation in the ultracentrifuge (Gerber, Franklinand Schubert, 1960). The large particle frac-tion (designated PP-H) has a substantiallyhigher protein content than the lighter (so-called PP-L) fraction. Both PP-H and PP-Lalso contain small amounts of materials re-cognisable as keratan sulphate and sialic acid.Differences have been noted in the PP-H: PP-Lratio in cartilages from varying anatomical loca-tions. Thus, human costal cartilage, in contrastto bovine nasal septum, contains a large pro-portion of PP-H and a correspondingly smalleramount of PP-L, both fractions having ahigher content of keratan sulphate and pro-portionately less chondroitin sulphate. Further-more, human costal cartilage PP-L shows twodistinct components on electrophoresis(Schubert, 1964). No information is yet avail-able regarding a similar pattern of behaviourfor the protein complexes of other acidglycosaminoglycans.

Lastly, it is necessary to record that hyalu-ronic acid exists naturally in firm combinationwith protein (Rogers, 1961). Even in thisinstance complicating features have arisensince it has been suggested that up to 10%of the hyaluronic acid molecule may not beconstructed according to the accepted repeatingperiod plan (Montgomery and Nag, 1963).The examples given are designed to show

the new approaches which are being made instudies on the chemical constitution of theground substance. However, they also serveto illustrate the complexity of the situationwhich exists in a highly vascular organ suchas lung where biochemical events take placesimultaneously at the interfaces of severaldistinct types of connective tissue.The Biological Significance of the Complexes

It is now known that the biosynthesis of thecomplexes takes place inside the appropriateconnective tissue cells. For example, studieswith inorganic [35S]-sulphate and [14C]-labelledamino acids reveal the concentration of theradioisotopes predominantly in the chondro-cytes of bovine costal cartilage slices in vitro(Campo and Dziewiatkowski, 1962). The samestudies point to the simultaneous formationof the polysaccharide and the protein moietiesof the chondroitin sulphate-protein complex.However, it is still not clear at what stage inthe synthesis union between polysaccharide andprotein is achieved. Godman and Lane (1964)have shown the localisation of [35S]-sulphate,visualised autoradiographically, in vesicles ofthe juxtanuclear Golgi apparatus of chondro-

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cytes, suggesting that the sulphation of thecarbohydrate moieties at least occurs in thisregion of the cell. Subsequently, it is believedthat the molecules of the intact complex areconveyed through the cytoplasm to the peri-phery of the cell in vesicle-like structures andthen discharged into the extracellular matrixvia stomata in the cell membrane (Godmanand Porter, 1960; Sheldon and Robinson, 1960;Godman and Lane, 1964).Once in the extracellular environment

there is no doubt, for the chondromucoproteincomplex of cartilage at least, that the biologicalstability and consequent functional significanceof the polysaccharide chains is directly depend-ent on the integrity of the protein core. Theremarkable finding that intravenous injectionin young animals of the proteolytic enzymepapain produces the now renowned 'flop-ear'rabbits (see Thomas, 1956) may be explainedit terms of in situ degradation of the proteincore of the ear cartilage complex. In additionto the effect on ear cartilage the simultaneousand more widespread action of the enzymethroughout the 'body is illustrated by the dis-appearance of the matrix in all forms ofcartilage as evidence by basophilic and meta-chromatic staining reactions. Similar experi-ments show the disappearance of extractablechondroitin sulphate from these tissues (Tsaltas,1958) and its appearance in blood and urine(Bryant, Leder and Stetten, 1958). Papainintroduced in this way shows greatest activityin young animals and towards cartilage, olderanimals becoming progressively more resistantpresumably in phase with changes in the cons-titution of the ground substance. It is nowknown that such findings are not restricted tothe injection of papain but also accompanythe administration of ficin and bromelin(McCluskey and Thomas, 1958), whilechondroitin sulphate is also liberated fromcartilage by the action of plasmin (Lack andRogers, 1958; Anderson, 1962). An endogen-ous protease, liberated in vivo or in vitro frommammalian lysozomes by the action ofvitamin A etc. and its action on chondroitinsulphate-protein complexes, has been the sub-ject of extensive examination (see Dingle, 1964,for the most recent review). To date attentionhas been devoted almost exclusively to studieson the degradation of cartilage protein-poly-saccharide complexes and in consequencethere is no appropriate information regardingthe biological stabilities of the other materials.

In terms of percentage dry weight the proteincomplexes of the acidic polysaccharides appear

to represent in many instances only an insignifi-cant part of the ground substance of theconnective tissues. However, considered ascentres of intense anionic character there canbe little doubt that in the tissues they arelikely to exist as a focus for the concentrationof inorganic cations. They must also participatein electrostatic interactions with proteins ex-hibiting cationic properties at physiological pH.Obviously, highly ionic arrangements of thissort can only exist at tissue level in a statesolvated with water molecules. The conceptof 'dry weight' thus has little bearing on thephysical existence of the complexes in the bio-logical environment. It is conceived (seeSchubert, 1964) that the complexes occurnaturally as diffuse, extended macromolecularstructures, each occupying an aqueous 'domain'that is large in comparison to the absoluteweight of the complex. The interaction of eachdomain is envisaged as producing a threedimensional network, the pore size of whichmust obviously be governed by the degree ofsolvation, the intensity of charge and thechemical and physical fine structure of eachcomplex. Experimental evidence suggests thatapart from a differential ability to bind cationsaccording to the nature of the constituentglycosaminoglycans (Dunstone, 1960, 1962;Mathews, 1964) such three-dimensional molecu-lar organizations should also be able to entangleparticles (Laurent, 1964) and exclude largesolutes (Gerber and Schubert, 1964). Viewed inthis way it is tempting to suggest that theapparently precise biological allocation ofstructurally different anionic polysaccharide-protein complexes is a direct function of theirvariable properties in controlling ion-binding,water content and even the porosity of thetissue as a whole.

Opinions vary as to the extent to which theacidic glycosaminoglycans and their proteincomplexes participate in collagen fibrogenesis,both in the normal tissue and during the courseof an inflammatory response. In some instancesit has 'been suggested that because of theirhighly regular repeating character the poly-saccharides may function intimately astemplates or organizers in determining thesize and geometrical arrangement of collagenfibres in 'the connective tissues (Meyer, 1960).Studies designed to explore the effect ofchemically-pure acidic polysaccharides on theformation of collagen fibres in vitro tend tocontradict this view. Gross (1959) found thata number of acidic glycosaminoglycans were

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POSTGRADUATE MEDICAL JOURNAL

without measurable effect on the rate of collagenfibril formation. The observations of Wood(1960) were partially at variance with theearlier findings in so far as chondroitin 4-sulphate, chondroitin 6-sulphate and a pre-paration rich in keratan sulphate acceleratedfibril formation markedly. Dermatan sulphateand hyaluronic acid had relatively little effect,while heparin actually inhibited precipitation.It is now obviously essential to repeat suchin vitro studies on collagen fibre formation asa function of the presence of the intact poly-saccharide-protein complexes and not merelythe polysaccharides themselves. In the mean-while it must be considered tentatively thatthe complexes are concerned with fibre bindingand the limitation of collagen mobility in thethree dimensional net of the ground substance.Acid Glycosaminoglycans in the InflammatoryResponse.The last two decades have seen an expanding

interest in the biochemical events involvedin the processes of inflammation and woundrepair. Perhaps the most astonishing featureof the ensuing work is the diversity of tech-niques and materials used in the induction ofthe inflammatory focus. Broadly speaking thesefall under four major headings.

a. Wounding. Included in this section areobservations on various cutaneous incisionsand burns induced in mouse, rat, guinea pig,rabbit and man, together with studies on re-generating tendon in rat and guinea pig andbone fractures in various experimental animals.

b. Injection or implantation of 'resorbable'substances. The most widely examined res-ponses here are granulomata induced sub-cutaneously, intraperitoneally and even in thecornea of several species of experimentalanimal by the introduction of agar, carrageenin,alginic acid, croton oil, oil of turpentine etc.

c. Injection or implantation of 'non-resorb-able substances'. Materials used in suchexperiments have included sections of polyvinylsponge, cotton pellets, perforated stainless steelcylinders, mineral particles including talc, silica,coal and asbestos these being located in sub-cutaneous, intraperitoneal and pleural regionsof various animals.

d. Lesions induced topically by irritation orother means. Examples here include skin irrita-tion produced by various hydrocarbon oils andexposure to high frequency current or ionizingradiations.

It is surprising to note that the biochemicalchanges associated with connective tissue com-

ponents apparent in each of these instancesfollows an almost identical broad sequenceirrespective of the species of animal employed,differences in anatomical location and variationsin the causative agent. Observed deviationsfrom the sequence are almost invariably ofdegree rather kind.Where measured it has been found that the

earliest detectable biochemical event involves asharp, time-based increase in the nucleic acid(both DNA and RNA) content of the tissuein the vicinity of the inflammatory focusevidencing considerably elevated cellularactivity. Such observations 'have been attributedboth to the invasion of the area by cells andto increased cell division.

Within a very short period after injury histo-chemical observations also show pronouncedchanges associated with the acid glycosamino-glycans of the ground substance. Bothmetachromatic and colloidal iron staining re-actions are considerably intensified, a maximumusually being reached in 72-96 hours. There-after, there is a gradual decline in the intensityof stain in healing wounds and in areas ofresorbing granulomata but a persistence in focisurrounding non-resorbable material.The results of microscopic examination are

substantiated by biochemical studies. In-variably the latter have involved analyses madefor total hexosamine as a measure of overallglycosaminoglycan content, or for the individualhexosamines D-glucosamine and D-galactos-amine as an index of the presence of thecorresponding parent polysaccharides. Alter-natively, the magnitude of the uptake ofinorganic [5S] sulphate has been used as anindication of altered synthesis of sulphatedglycosaminoglycans. The amounts of totalhexosamine and [35S] sulphate in inflamed areashave been shown to increase abruptly, in someinstances in parallel with the content of DNAand usually within 24 hours after injury.Maxima are reached in both series of deter-minations in a matter of days, and may persistat these levels even after the nucleic acidcontent has declined to 'normal' values. Therate of decline of hexosamine and [35S] sulphatevalues, like the staining reactions, is a functionof the material or technique used to producethe lesion and a persistence at levels above'normal' is noted in areas surrounding non-resorbable material. Usually, in healing woundsand resorbable granulomata a return ofhexosamine level to average values for thetissue is recorded by the time active formation

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July, 1965 LLOYD: Biochemistry of Ground Substance of Connective Tissues

of collagen fibres is already under way. Insome instances the actual nature of the acidicglycosaminoglycans responsible for theseanalytical changes has been examined but thepolymers produced and their ratios one toanother are not unexpectedly varied accordingto the site of injury. However, as a generalrule non-sulphated polymers such as hyaluronicacid tend to appear before the sulphated poly-saccharides. No observations have yet beenmade concerning the protein moieties associatedwith the polysaccharides. In this regard it hasbeen shown that the acid glycosaminoglycansof inflamed areas tend to become progressivelymore resistant with time to extraction withneutral salt solutions.The significance of such changes in the

ground substance in relation to fibrogenesisremains unclear, but may be considered ten-tatively as preparing the extracellular environ-ment for the appearance of newly formedcollagen. (Because of the large number ofpublications in this field individual referenceshave been excluded to ensure continuity).Studies on Normal and Diseased LungThere are few recorded attempts to apply

such methods to the study of normal lung andto pulmonary dust diseases in experimentalanimals and man. The interpretation of theresults of such studies is also likely to be acomplex matter. The connective tissues of theaverage normal lung are considered to containdermatan sulphate, heparan sulphates andheparin (see Muir, 1964), each presumablystabilised as its protein complex. The presenceof the chondroitin sulphates and keratansulphate has also been detected in the vicinityof major airways, the content of the polymersincreasing in progressive massive fibrosis inman (A. G. Lloyd, unpublished results).Constitutional studies will also have to takeinto account the various mucous secretions(Jakowska, 1963).

Studies on age-related changes in the con-nective tissues of lung have been directedlargely to observations on the fibrous elements.Thus, it is now accepted generally that theproportion of lung 'collagen', measured interms of hydroxyproline analyses per unitweight, increases in age in mice, rats, guineapigs and humans (Elster and Lowry, 1950;Slutskii and Sheleketina, 1959; Briscoe, LoringChvapil, 1957, 1960; Chvapil and Roth, 1964;and McClement, 1959; Boucek, Noble andMarks, 1961). However, there is also tentativeevidence for the doubling of the elastin content

of human lungs between the ages of 25 and 75with no alteration in collagen content (Pierce,1962).

Variation in the polysaccharide constitutionof lung in experimental animals and man hasbeen studied only through the medium ofhexosamine analysis. Thus, Saltzman, Schaubleand Siecker (1961) record little change withage of overall hexosamine content and in theratio of galactosamine and glucosamine in lungparenchyma in rabbit and man. The sameauthors indicate statistically significant altera-tion of these values in lung parenchyma incertain cases of chronic disease. In contrast tosuch regions Saltzman, Siecker and Green (1963)indicate that there are significant age relateddecreases in bronchial cartilage hexosaminecontent and the galactosamine: glucosamineratio in the absence of lung disease. It isinteresting to compare these results withanalogous age-related effects in rib cartilageand nucleus pulposus (Shetlar and Masters,1955; Stidworthy, Masters and Shetlar, 1958;Kaplan and Meyer, 1959; Davidson andWoodhall, 1959). On the other hand, bronchialcartilage from emphysematous lung maintainsa persistently higher ratio of galactosamine toglucosamine with advancing age in contrast tothe progressive decrease observed with sene-scence in specimens derived from normal lung.In relation to pulmonary dust diseaseHarington, Marasas, Melamed, Sutton andDreosti (1960) were unable to show appreciableincrease in total hexosamine content of guinea-pig lung after dusting for periods up to 70days. However, these observations should beconsidered simultaneously with the report ofBaily, Kilroe-Smith and Harington (1964) whichshows a substantial increase in lung weight industed guinea pigs, the increase being duelargely to materials low in hydroxyprolinecontent. Lastly note should also be taken ofobservations of excessive production ofhyaluronic acid in pleural mesotheliomas(Wagner, Munday and Harington, 1962).Conclusions

It must be clear from the foregoing thatmuch remains to be elucidated regarding thenature and properties of connective tissues,both in the normal lung and during pulmonarydust diseases. Furthermore, such work mustcontinue to proceed hand in hand with advancesin general biochemical knowledge in relatingto the connective tissue field as a whole, dueregard always being given to studies on themucous secretions of the respiratory tract.

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390 POSTGRADUATE MEDICAL JOURNAL July, 1965

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