Postgrad Med J: first published as 10.1136/pgmj.41.477.382 on 1 July 1965. Downloaded from POSTGRAD. MED. J. (1965), 41, 382 RECENT TRENDS IN THE BIOCHEMISTRY OF THE GROUND SUBSTANCE OF THE CONNECTIVE TISSUES A. 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 and Lung, University College, Cardiff. THE UBIQUITY of members of the anatomical to the surrounding tissue, the fibres usually group loosely defined as connective tissues is being described as collagenous. As yet there a matter of long-standing record. The same is no clear indication whether such fibroses comment applies to the convention that, despite arise according to the partially established the differing biological roles which they are pattern of inflammatory response in which the required to play and their characteristically earliest recognisable biochemical changes in- different mechanical character at macroscopic clude alterations in the ground substance of level, the individual members of the connective connective tissues followed only subsequently tissue group exhibit certain basic similarities by de novo formation of additional fibrous which first become apparent at microscopic material. It is for this reason that the research level. A common biochemical approach is to group housed in the Department of Biochemis- develop these features of similarity to a point try is designed to assist in the clarification where an idealised composite of all connective of fundamental biochemical changes associated tissues can be devised. Use of such a model with general lung dust diseases from the aspect allows initial broad generalisations to be drawn. of disturbed connective tissue metabolism. Suchby copyright. On the other hand, the same model must sub- an approach must obviously take into account sequently be employed to establish not only the many recent developments concerning the morphological but fundamental biochemical nature and properties of the structural elements differences which exist between the tissues. of the connective tissues and in particular the A model connective tissue is usually con- intrinsic complexity of the ground substance. ceived as containing relatively few cells per unit volume compared to a highly cellular The Ground Substance organization such as liver and additionally differentiated from the latter by the presence There is now no doubt that histologicalhttp://pmj.bmj.com/ of a significant quantity of extracellular examination of connective tissues using con- material. This material is characterised by the ventional techniques provides a wholly incom- presence of organised fibrillar elements, com- plete and frequently artificial picture of the posed variously of the biochemically distinct complexity of the ground substance. It has even proteins , and reticulin, these been established that long-used methods of being embedded in an amorphous medium tissue fixation may even facilitate the extrac- termed the substance. tion of substance constituents ground ground (see on September 23, 2021 by guest. Protected In this context, it is fitting that laboratories Szirmai, 1963). Thus, even for relatively gross in the Cardiff area should be connected in histological observations such methods must studies on all aspects of pulmonary dust dis- be replaced by the use of modified tissue fixa- ease and in particular of coal miner's pneumo- tives (Quintarelli, Scott and Dellovo, 1964a) coniosis, progressive massive and or alternatively frozen section techniques in silicosis. The need for such work is presented which 'fixation' is virtually carried out in the with considerable impact in the observations dye bath (Szirmai, 1963). Methods have be- of Gough 1(1947). 'Much 'has already been come available for differentiating some of the established about the statistical incidence and ground substance components by selective pathological characteristics of such dust dis- physico-chemical blocking and unblocking in eases, but their true aetiology is still a matter the presence of quaternary ammonium com- for conjecture (Policard, 1962). However, from pounds (Kelly, Bloom and Scott, 1963), by the simplified histological standpoint the human selective staining in the presence of solutions lung lesion in such conditions shows character- of varying pH or electrolyte concentration istically an increased 'fibrosis' when compared (Szirmai, 1963; Saunders, 1964; Quintarelli, Postgrad Med J: first published as 10.1136/pgmj.41.477.382 on 1 July 1965. Downloaded from July, 1965 LLOYD: Biochemistry of Ground Substance of Connective Tissues 383 Scott and Dellovo, 1964b) and by selective the polymer chains. As both carboxyl and enzymic digestion ,(Zugibe, 1962 a and b). sulphate groups are fully ionized at physiologi- However, such techniques continue to be the cal pH the resultant highly negative character province of the specialist in the field, rather is likely to have considerable bearing on the than methods of routine applicability. As such, functions attributed to the polymers in the much reliance must still be placed on native state. approaches based on the extraction of connect- The acidic recognised to ive tissue components followed by their date fall into four classes, differentiated by characterisation using physical, chemical and features of chemical fine structure: biochemical methods. Even in this regard A. Polysaccharides having anionic character studies initiated during the middle of the last conferred solely by hexuronic acid constituents century and subsequently prosecuted with e.g., and chondroitin. increasing vigour are still the subject of con- B. Polysaccharides having anionic character tinuous contemporary reappraisal. resulting from the dual presence of hexuronic acid and O-sulphate groups e.g., chondroitin Polysaccharide-Protein Complexes of the 4-sulphate (syn. chondroitin sulphate-A), Ground Substance chondroitin 6-sulphate (syn. chondroitin sul- The Polysaccharides phate-C) and dermatan sulphate (syn. chond- Early studies on these materials invariably roitin sulphate-B, P-heparin). involved treatment of connective tissues under C. Polysaccharides having O-sulphate groups relatively vigorous conditions such as pro- as sole functional anionic entities e.g., keratan longed digestion with proteolytic enzymes sulphate (syn. keratosulphate). followed by extraction with strong salt solutions D. Polysaccharides characterised by the pre- or even dilute alkali. The resultant extracts sence of hexuronic acid, O-sulphate and N- yielded, by several methods of purification, sulphate residues e.g., heparan sulphates (syn.by copyright. varying amounts of one or more of a group of heparitin sulphates). Heparin and o-heparin carbohydrate high polymers. These have been may also be included in this class for the sake variously and synonymously termed acid muco- of completeness. polysaccharides, acid aminopolysaccharides or The compositions of each of these materials is more recently acid glycosaminoglycans (Jeanloz, shown in Table 1 and the major repeating 1960). In defining the purity of such materials periods of the polysaccharides from Classes A, early workers attached much significance to B and C are summarised in Figs. 1, 2 and 3 the absence of amino acid and polypeptide respectively. These should be considered to be residues from the polysaccharide preparations structures derived on the principle of best http://pmj.bmj.com/ and when demonstrated such 'contamination' analytical evidence. While there is little doubt was frequently attributed to inefficiency of that they account for the format of the major purification procedures. Structural studies (re- portion of the polysaccharide chains there are viewed by Jeanloz, 1963) on these compounds indications that some regions of the polymers reveal a pattern of similar molecular organiza- are not constructed in this way. Such evidence tion. Thus, in the main are will be dealt with later. It should also be molecular they relatively high weight polysaccharides having noted that variability of sulphate analysis is on September 23, 2021 by guest. Protected linear chains containing hexosamine residues. not an uncommon occurrence and that acid The latter may be either D-glucosamine or preparations with sulphur D-galactosamine each occurring in a state in to nitrogen ratios both greater and less than which the amine grouping is blocked. The unity have been isolated. Conclusive data hexosamines alternate regularly in the polymer allowing the assignment of wholly acceptable chains with other monosaccharide residues repeating period structures for heparan which may be hexuronic acids, such as D- sulphates (and for heparin and o-heparin) glucuronic acid and L-iduronic acid or a has yet to be obtained (see Muir, 1964). hexose such as D-galactose. Aside from the significant contributions All of the polysaccharides are also poly- which they have made in structural work on anions, the negative charge arising either from the acid glycosaminoglycans, elegant studies the carboxyl group of the above uronic acids made by Professor Karl Meyer and his col- or from the presence of ester bound sulphate, leagues lay a foundation for the concept that or both. Accepted structures show the poten- connective tissues fulfilling distinctive biological tially charged groups arranged regularly along functions are also provided with differing Postgrad Med J: first published as 10.1136/pgmj.41.477.382 on 1 July 1965. Downloaded from 384 POSTGRADUATE MEDICAL JOURNAL July, 1965 TABLE 1 MAJOR CONSTITUENTS OF THE ACIDIC GLYCOSAMINOGLYCANS Polysaccharides Hexosamine Other N-Acetyl O-Sulphate N-Sulphate Monosaccharide CLASS A Group Group (s) Group Hyaluronic Acid D-Glucosamine D-Glucuronic Acid +-- Chondroitin D-Galactosamine D-Glucuronic Acid + CLASS B Chondroitin 4-Sulphate D-Galactosamine D-Glucuronic Acid + + Chondroitin 6-Sulphate D-Galactosamine D-Glucuronic Acid + + Dermatan Sulphate D-Galactosamine L-Iduronic Acid + + CLASS C Keratan Sulphate D-Glucosamine D-Galactose + + CLASS D Heparan Sulphates D-Glucosamine D-Glucuronic Acid + + + Heparip D-Glucosamine D-Glucuronic Acid - + +

CH0 H CH2OHH20H COOH HO33SO (

OH H -H H (a) ;-0O o H H NHH.COCH3 H H NH.COCH3

Tio by copyright. CH2OSO3H H20H HO OH COOH 0 (b) H 0 H H0 0 OH NH.COCH3 http://pmj.bmj.com/ FIG. 1.-Structures of the in repeating peiods O hyaluronic acid (a) and chondroitin (b). H H complements of the acidic polysaccharides (Meyer, Davidson, Linker and Hoffman, 1956). Table 2 is a summary of part of the original work plus some additional information, giving on September 23, 2021 by guest. Protected analyses for tissues of average chronological FIG. 2.-Structures of the repeating periods in chon- age. On the other hand, it should also be droitin 4-sulphate (a), chondroitin 6-sulphate (b) stressed that the content of acid glycosamino- and dermatan sulphate (c). glycans must not be considered to be static throughout life. Thus, apart from variations due to metabolic turnover, changes both in ----'l~ overall content and in the concentration ratios CH20H CHHOSo3H of individual polysaccharides as a function of HO H0H H age have also been observed. For example, -O H embryonic pig skin polysaccharides are com- prised of 9.5% dermatan sulphate, 71% H OH H NH.COCH hyaluronic acid and about 20% of a mixture of chondroitin 4-sulphate and chondroitin 6-sulphate. Analyses of adult pigskin reveals FIG. 3.-Structure of the repeating period in keratan a sharp change in ratio corresponding to 64% sulphate. Postgrad Med J: first published as 10.1136/pgmj.41.477.382 on 1 July 1965. Downloaded from July, 1965 LLOYD: Biochemistry of Ground Substance of Connective Tissues 385 TABLE 2 DISTRIBUTION OF ACIDIC GLYCOSAMINOGLYCANS IN MAMMALIAN CONNECTIVE TISSUE POLYSACCHARIDES Tissues Hyaluronic Chondroitin Chondroitin Chondroitin Dermatan Keratan Heparan Heparin Acid 4-Sulphate 6-Sulphate Sulphate Sulphate Sulphate Synovial Fluid + - - + + + + + - + + + + Ligamentum Nuchae + -+ -+ + Skin + + + + + Aorta + -+ - + + Vitreous Humor + Cornea - + + + Sclera + + Umbilical Cord + dermatan sulphate, 30%/ hyaluronic acid and in this way allows the construction of concentra- some 0.1% of the chondroitin 4-sulphate- tion (and tentative identity) profiles through chondroitin 6-sulphate mixture (Loewi and a given tissue. The methods have been applied Meyer, 1958). Similar effects have been ob- to studies on bovine nasal septum and rabbit served in rat and in man (Loewi, 1961; Prodi, intervertebral disc (Antonopoulos and others, 1964). In addition Kaplan and Meyer (1959) 1964) and to epiphyseal plates from normal and recorded that in human costal cartilage there rachitic dogs (Hjertquist, 1964a, b). The result- is a decrease in the overall content of acidic ing observations establish that the acid gly- glycosaminoglycans with age. The same authors cosaminoglycans are subject not only to gross have shown that in phase with this decrease variation from tissue to tissue and to ageby copyright. the constituent polysaccharides change from fluctuations in the same tissue but also to exclusively chondroitin 4-sulphate in foetal three dimensional variations in a given tissue costal cartilage to a mixture of chondroitin 6- of defined age. sulphate and keratan sulphate in cartilage from the same location in adults. These selected examples can be considered to be representative The Complexes of phenomena which are likely to become the The 'pure' polysaccharides required for rule for all connective tissues rather than ex- structural studies do not exist as such at tissue ceptional observations. level and are, in a sense, chemical artifacts http://pmj.bmj.com/ The distributions summarized in Table 2 are produced by the relatively vigorous means based on analyses of 'bulk' samples of material used for extraction. Application of milder where the morphological integrity of the tissue methods, such as physical disintegration of the is destroyed at an early stage of the extraction tissue by high speed homogenisation, yields procedure and therefore correspond to average the acid glycosaminoglycans linked firmly to results for fairly large volumes of tissue. As protein moieties by covalent bonds (see for such they give no indication whether the Shatton and Schubert, poly- example 1954; Gerber, on September 23, 2021 by guest. Protected saccharides are arranged homogeneously Franklin and Schubert, 1960). Erroneous im- throughout the whole ground substance or plications should not be drawn from the whether in fact they are the subject of concent- persistent use of the generic nomenclature ration and localisation effects in relation to the of polysaccharide-protein complexes for these fine structure of the tissue. Such information materials when obviously the term compound is clearly of prime importance to assessments would be far more appropriate. of the functional significance of individual In recent years the most extensively studied acid glycosaminoglycans. Antonopoulos, Gar- complex is that derived from various kinds of dell, Szirmai and de Tyssonsk (1964) have cartilage. Preliminary work conceived the recognised the need for means to carry out cartilage chondromucoprotein complex as con- precise surveys. These authors are responsible sisting of numerous polysaccharide chains for the development of methods which allow (largely chondroitin 4-sulphate or 6-sulphate alternate tissue sections (10-40 A thick) to be plus some keratan sulphate) firmly attached examined qualitatively by histological means to a median, non-collagenous protein core and quantitatively by micro-scale modifications (Partridge, Davis and Adair, 1961). It is a of established chemical methods. Data collected characteristic of the cartilage complexes that Postgrad Med J: first published as 10.1136/pgmj.41.477.382 on 1 July 1965. Downloaded from 386 LLOYD: Biochemistry of Ground Substance of Connective Tissues July, 1965 the polysaccharide moieties are cleaved ir- tation in the ultracentrifuge (Gerber, Franklin reversibly from the protein chain by treatment and Schubert, 1960). The large particle frac- with dilute alkali (Muir, 1958; Partridge and tion (designated PP-H) has a substantially Davis, 1958; Malawista and Schubert, 1958; higher protein content than the lighter (so- Partridge and Elsden, 1961). However, the called PP-L) fraction. Both PP-H and PP-L work of Muir (1958) established that when also contain small amounts of materials re- the isolated complex from cartilage was ex- cognisable as keratan sulphate and sialic acid. haustively degraded with proteolytic enzymes Differences have been noted in the PP-H: PP-L in vitro the polysaccharide preparations sub- ratio in from varying anatomical loca- sequently isolated were still firmly associated tions. Thus, human costal cartilage, in contrast with serine-rich polypeptide stubs. Such obser- to bovine nasal septum, contains a large pro- vations focussed considerable attention on the portion of PP-H and a correspondingly smaller possibility that serine residues in the original amount of PP-L, both fractions having a core material were intimately concerned in the higher content of keratan sulphate and pro- linkage of polysaccharide and protein. The value portionately less chondroitin sulphate. Further- of this initial work is illustrated by the recent more, human costal cartilage PP-L shows two isolation from a chondroitin sulphate-protein distinct components on electrophoresis complex of carbohydrate residues linked directly (Schubert, 1964). No information is yet avail- to serine (Roden, Gregory and Laurent, 1964). able regarding a similar pattern of behaviour Evidence has been presented (Dr. L. Rod6n, for the protein complexes of other acid personal communication) that a novel carbo- glycosaminoglycans. hydrate linkage sequence, comprising xylose Lastly, it is necessary to record that hyalu- and galactose moieties, is involved as a bridge ronic acid exists naturally in firm combination between the serine hydroxyl and the character- with protein (Rogers, 1961). Even in this istic repeating periods of the glycosaminoglycan instance complicating features have arisenby copyright. chain. These observations are by no means since it has been suggested that up to 10% unique to the chondroitin sulphate-protein of the hyaluronic acid molecule may not be complexes as similar linkage arrangements are constructed according to the accepted repeating also found in heparin-polypeptide complexes period plan (Montgomery and Nag, 1963). (Lindahl and Roden, 1964). Heparan sulphate- The examples given are designed to show protein complexes are likely to be bonded in an the new approaches which are being made in analogous fashion (Jacobs and Muir, 1964). studies on the chemical constitution of the Treatment of glycosaminoglycan-protein ground substance. However, they also serve complexes with alkali and analysis of the to illustrate the complexity of the situationhttp://pmj.bmj.com/ products of reaction provides a relatively rapid which exists in a highly vascular organ such means for assessing the participation of serine as lung where biochemical events take place and threonine in linkage (Ander- simultaneously at the interfaces of several son, Seno, Sampson, Riley, Hoffman and distinct types of connective tissue. Meyer, 1964; Anderson, Hoffman and Meyer, 1965). Studies using these alkaline degradation The Biological Significance of the Complexes methods reveal the involvement of It is now known that the hydroxylated biosynthesis of the on September 23, 2021 by guest. Protected alkyl amino acid residues in linkages between complexes takes place inside the appropriate protein and chondroitin 4-sulphate, chondroitin connective tissue cells. For example, studies 6-sulphate and skeletal keratan sulphate. How- with inorganic [35S]-sulphate and [14C]-labelled ever, bonds in the corresponding complexes of amino acids reveal the concentration of the chondroitin, dermatan sulphate and corneal radioisotopes predominantly in the chondro- keratan sulphate are suggested to be of the cytes of bovine costal cartilage slices in vitro asparaginyl-glycosyl type as found in ao-glyco- (Campo and Dziewiatkowski, 1962). The same protein (Professor K. Meyer, personal com- studies point to the simultaneous formation munication). of the polysaccharide and the protein moieties There is no clear indication whether a single of the chondroitin sulphate-protein complex. type of acid glycosaminoglycan may be However, it is still not clear at what stage in attached to protein cores of different amino the synthesis union between polysaccharide and acid constitution. However, it has been shown protein is achieved. Godman and Lane (1964) that bovine nasal septum cartilage yields two have shown the localisation of [35S]-sulphate, varieties of chondroitin sulphate-protein com- visualised autoradiographically, in vesicles of plex, distinguished by their differential sedimen- the juxtanuclear Golgi apparatus of chondro- Postgrad Med J: first published as 10.1136/pgmj.41.477.382 on 1 July 1965. Downloaded from July, 1965 LLOYD: Biochemistry of Ground Substance of Connective Tissues 387 cytes, suggesting that the sulphation of the to represent in many instances only an insignifi- carbohydrate moieties at least occurs in this cant part of the ground substance of the region of the cell. Subsequently, it is believed connective tissues. However, considered as that the molecules of the intact complex are centres of intense anionic character there can conveyed through the cytoplasm to the peri- be little doubt that in the tissues they are phery of the cell in vesicle-like structures and likely to exist as a focus for the concentration then discharged into the of inorganic cations. They must also participate via stomata in the cell membrane (Godman in electrostatic interactions with ex- and Porter, 1960; Sheldon and Robinson, 1960; proteins Godman and Lane, 1964). hibiting cationic properties at physiological pH. Once in the extracellular environment Obviously, highly ionic arrangements of this there is no doubt, for the chondromucoprotein sort can only exist at tissue level in a state complex of cartilage at least, that the biological solvated with water molecules. The concept stability and consequent functional significance of 'dry weight' thus has little bearing on the of the polysaccharide chains is directly depend- physical existence of the complexes in the bio- ent on the integrity of the protein core. The logical environment. It is conceived (see remarkable finding that intravenous injection Schubert, 1964) that the complexes occur in young animals of the proteolytic enzyme naturally as diffuse, extended macromolecular papain produces the now renowned 'flop-ear' structures, each occupying an aqueous 'domain' rabbits (see Thomas, 1956) may be explained that is large in comparison to the absolute it terms of in situ degradation of the protein weight of the complex. The interaction of each core of the ear cartilage complex. In addition domain is envisaged as producing a three to the effect on ear cartilage the simultaneous dimensional network, the pore size of which and more widespread action of the enzyme must obviously be governed by the degree of throughout the 'body is illustrated by the dis- solvation, the intensity of charge and the by copyright. appearance of the matrix in all forms of chemical and physical fine structure of each cartilage as evidence by basophilic and meta- complex. Experimental evidence suggests that chromatic staining reactions. Similar experi- apart from a differential ability to bind cations ments show the disappearance of extractable according to the nature of the constituent chondroitin sulphate from these tissues (Tsaltas, glycosaminoglycans (Dunstone, 1960, 1962; 1958) and its appearance in blood and urine Mathews, 1964) such three-dimensional molecu- (Bryant, Leder and Stetten, 1958). Papain lar organizations should also be able to entangle introduced in this way shows greatest activity particles (Laurent, 1964) and exclude large in young animals and towards cartilage, older solutes (Gerber and Schubert, 1964). Viewed in http://pmj.bmj.com/ animals becoming progressively more resistant this way it is tempting to suggest that the presumably in phase with changes in the cons- apparently precise biological allocation of titution of the ground substance. It is now structurally different anionic polysaccharide- known that such findings are not restricted to protein complexes is a direct function of their the injection of papain but also accompany variable properties in controlling ion-binding, the administration of ficin and bromelin water content and even the porosity of the tissue as a whole. (McCluskey and Thomas, 1958), while on September 23, 2021 by guest. Protected chondroitin sulphate is also liberated from Opinions vary as to the extent to which the cartilage by the action of plasmin (Lack and acidic glycosaminoglycans and their protein Rogers, 1958; Anderson, 1962). An endogen- complexes participate in collagen fibrogenesis, ous protease, liberated in vivo or in vitro from both in the normal tissue and during the course mammalian lysozomes by the action of of an inflammatory response. In some instances vitamin A etc. and its action on chondroitin it has 'been suggested that because of their sulphate-protein complexes, has been the sub- highly regular repeating character the poly- ject of extensive examination (see Dingle, 1964, saccharides may function intimately as for the most recent review). To date attention templates or organizers in determining the has been devoted almost exclusively to studies size and geometrical arrangement of collagen on the degradation of cartilage protein-poly- fibres in 'the connective tissues (Meyer, 1960). saccharide complexes and in consequence Studies designed to explore the effect of there is no appropriate information regarding chemically-pure acidic polysaccharides on the the biological stabilities of the other materials. formation of collagen fibres in vitro tend to In terms of percentage dry weight the protein contradict this view. Gross (1959) found that complexes of the acidic polysaccharides appear a number of acidic glycosaminoglycans were Postgrad Med J: first published as 10.1136/pgmj.41.477.382 on 1 July 1965. Downloaded from

388 POSTGRADUATE MEDICAL JOURNAL July, 1965 without measurable effect on the rate of collagen ponents apparent in each of these instances fibril formation. The observations of Wood follows an almost identical broad sequence (1960) were partially at variance with the irrespective of the species of animal employed, earlier findings in so far as chondroitin 4- differences in anatomical location and variations sulphate, chondroitin 6-sulphate and a pre- in the causative agent. Observed deviations paration rich in keratan sulphate accelerated from the sequence are almost invariably of fibril formation markedly. Dermatan sulphate degree rather kind. and hyaluronic acid had relatively little effect, Where measured it has been found that the while heparin actually inhibited precipitation. earliest detectable biochemical event involves a It is now obviously essential to repeat such sharp, time-based increase in the nucleic acid in vitro studies on collagen fibre formation as (both DNA and RNA) content of the tissue a function of the presence of the intact poly- in the vicinity of the inflammatory focus saccharide-protein complexes and not merely evidencing considerably elevated cellular the polysaccharides themselves. In the mean- activity. Such observations 'have been attributed while it must be considered tentatively that both to the invasion of the area by cells and the complexes are concerned with fibre binding to increased cell division. and the limitation of collagen mobility in the Within a very short period after injury histo- three dimensional net of the ground substance. chemical observations also show pronounced Acid Glycosaminoglycans in the Inflammatory changes associated with the acid glycosamino- Response. glycans of the ground substance. Both The last two decades have seen an expanding metachromatic and colloidal iron staining re- interest in the biochemical events involved actions are considerably intensified, a maximum in the processes of inflammation and wound usually being reached in 72-96 hours. There- the most feature after, there is a gradual decline in the intensity repair. Perhaps astonishing by copyright. of the ensuing work is the diversity of tech- of stain in healing wounds and in areas of niques and materials used in the induction of resorbing granulomata but a persistence in foci the inflammatory focus. Broadly speaking these surrounding non-resorbable material. fall under four major headings. The results of microscopic examination are a. Wounding. Included in this section are substantiated by biochemical studies. In- observations on various cutaneous incisions variably the latter have involved analyses made and burns induced in mouse, rat, guinea pig, for total hexosamine as a measure of overall rabbit and man, together with studies on re- glycosaminoglycan content, or for the individual tendon in rat and and generating guinea pig hexosamines D-glucosamine and D-galactos-http://pmj.bmj.com/ bone fractures in various experimental animals. amine as an index of the presence of the b. Injection or implantation of 'resorbable' corresponding parent polysaccharides. Alter- substances. The most widely examined res- natively, the magnitude of the uptake of ponses here are granulomata induced sub- inorganic [5S] sulphate has been used as an cutaneously, intraperitoneally and even in the indication of altered synthesis of sulphated cornea of several species of experimental glycosaminoglycans. The amounts of total animal by the introduction of agar, carrageenin, hexosamine and [35S] sulphate in inflamed areas alginic acid, croton oil, oil of turpentine etc. have been shown to increase abruptly, in some on September 23, 2021 by guest. Protected c. Injection or implantation of 'non-resorb- instances in parallel with the content of DNA able substances'. Materials used in such and usually within 24 hours after injury. experiments have included sections of polyvinyl Maxima are reached in both series of deter- sponge, cotton pellets, perforated stainless steel minations in a matter of days, and may persist cylinders, mineral particles including talc, silica, at these levels even after the nucleic acid coal and asbestos these being located in sub- content has declined to 'normal' values. The cutaneous, intraperitoneal and pleural regions rate of decline of hexosamine and [35S] sulphate of various animals. values, like the staining reactions, is a function d. Lesions induced topically by irritation or of the material or technique used to produce other means. Examples here include skin irrita- the lesion and a persistence at levels above tion produced by various hydrocarbon oils and 'normal' is noted in areas surrounding non- exposure to high frequency current or ionizing resorbable material. Usually, in healing wounds radiations. and resorbable granulomata a return of It is surprising to note that the biochemical hexosamine level to average values for the changes associated with connective tissue com- tissue is recorded by the time active formation Postgrad Med J: first published as 10.1136/pgmj.41.477.382 on 1 July 1965. Downloaded from July, 1965 LLOYD: Biochemistry of Ground Substance of Connective Tissues 389 of collagen fibres is already under way. In of human lungs between the ages of 25 and 75 some instances the actual nature of the acidic with no alteration in collagen content (Pierce, glycosaminoglycans responsible for these 1962). analytical changes has been examined but the Variation in the polysaccharide constitution polymers produced and their ratios one to of lung in experimental animals and man has another are not unexpectedly varied according been studied only through the medium of to the site of injury. However, as a general hexosamine analysis. Thus, Saltzman, Schauble rule non-sulphated polymers such as hyaluronic and Siecker (1961) record little change with acid tend to appear before the sulphated poly- age of overall hexosamine content and in the saccharides. No observations have yet been ratio of galactosamine and glucosamine in lung made concerning the protein moieties associated parenchyma in rabbit and man. The same with the polysaccharides. In this regard it has authors indicate statistically significant altera- been shown that the acid glycosaminoglycans tion of these values in lung parenchyma in of inflamed areas tend to become progressively certain cases of chronic disease. In contrast to more resistant with time to extraction with such regions Saltzman, Siecker and Green (1963) neutral salt solutions. indicate that there are significant age related The significance of such changes in the decreases in bronchial cartilage hexosamine ground substance in relation to fibrogenesis content and the galactosamine: glucosamine remains unclear, but may be considered ten- ratio in the absence of lung disease. It is tatively as preparing the extracellular environ- interesting to compare these results with ment for the appearance of newly formed analogous age-related effects in rib cartilage collagen. (Because of the large number of and nucleus pulposus (Shetlar and Masters, publications in this field individual references 1955; Stidworthy, Masters and Shetlar, 1958; have been excluded to ensure continuity). Kaplan and Meyer, 1959; Davidson and Woodhall, 1959). On the other hand, bronchial Studies on Normal and Diseased Lung cartilage from emphysematous lung maintainsby copyright. There are few recorded attempts to apply a persistently higher ratio of galactosamine to such methods to the study of normal lung and glucosamine with advancing age in contrast to to pulmonary dust diseases in experimental the progressive decrease observed with sene- animals and man. The interpretation of the scence in specimens derived from normal lung. results of such studies is also likely to be a In relation to pulmonary dust disease complex matter. The connective tissues of the Harington, Marasas, Melamed, Sutton and average normal lung are considered to contain Dreosti (1960) were unable to show appreciable dermatan and increase in total hexosamine content of guinea- sulphate, heparan sulphates http://pmj.bmj.com/ heparin (see Muir, 1964), each presumably pig lung after dusting for periods up to 70 stabilised as its protein complex. The presence days. However, these observations should be of the chondroitin sulphates and keratan considered simultaneously with the report of sulphate has also been detected in the vicinity Baily, Kilroe-Smith and Harington (1964) which of major airways, the content of the polymers shows a substantial increase in lung weight in increasing in progressive massive fibrosis in dusted guinea pigs, the increase being due man G. largely to materials low in (A. Lloyd, unpublished results). content. note hydroxyproline Constitutional studies will also have to take Lastly should also be taken of on September 23, 2021 by guest. Protected into account the various mucous secretions observations of excessive production of (Jakowska, 1963). hyaluronic acid in pleural mesotheliomas Studies on age-related changes in the con- (Wagner, Munday and Harington, 1962). nective tissues of lung have been directed largely to observations on the fibrous elements. Conclusions Thus, it is now accepted generally that the It must be clear from the foregoing that proportion of lung 'collagen', measured in much remains to be elucidated regarding the terms of hydroxyproline analyses per unit nature and properties of connective tissues, weight, increases in age in mice, rats, guinea both in the normal lung and during pulmonary pigs and humans (Elster and Lowry, 1950; dust diseases. Furthermore, such work must Slutskii and Sheleketina, 1959; Briscoe, Loring continue to proceed hand in hand with advances Chvapil, 1957, 1960; Chvapil and Roth, 1964; in general biochemical knowledge in relating and McClement, 1959; Boucek, Noble and to the connective tissue field as a whole, due Marks, 1961). However, there is also tentative regard always being given to studies on the evidence for the doubling of the elastin content mucous secretions of the respiratory tract. Postgrad Med J: first published as 10.1136/pgmj.41.477.382 on 1 July 1965. 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