COMPOSITION OF TRACHEOBRONCHIAL SECRETIONS IN CYSTIC FIBROSIS · POSTGRAD. MED. J. (I961), 37, 596 Symposium on Chest Diseases in Children (continued) THE COMPOSITION OF TRACHEOBRONCHIAL

POSTGRAD. MED. J. (I961), 37, 596

Symposium on Chest Diseases in Children (continued)

THE COMPOSITION OF TRACHEOBRONCHIALSECRETIONS IN CYSTIC FIBROSIS

WARREN S. CHERNICK, D.Sc.The Children's Hospital of Philadolp,'ia, Pa., U.S.A.

SINCE this disease was named in I939, manyother terms have been given to this syndromenotably ' mucoviscidosis '. For want of a betterdescriptive term, we most generally refer to it asfibrocystic disease or cystic fibrosis of the pancreas.It is obvious that, with the generalized nature of thedisease, as we now view it, most of these terms aretoo restrictive. Cystic fibrosis is a generalizedexocrine gland dysfunction with the intensity ofthis dysfunction varying in the different glandularsites, as well as among different patients.

In all exocrine glands that have been thoroughlystudied, some type of derangement has been found.

In the pancreas, the classical pathological lesionand the resultant deficiency of pancreatic enzymesin the duodenal fluid characterize the involvementof this area. In addition, the duodenal fluid is notonly deficient in pancreatic enzymes but manifestsan abnormal viscosity.One of the pronounced deviations from the

normal occurs in the sweat glands where patientswith cystic fibrosis manifest a much higher sodiumand chloride concentration than the normal child.Such abnormalities in the sweat are utilized asdiagnostic criteria and there is no question thatsuch tests have resulted in the emergence of thisdisease from the category of a rare malabsorptionsyndrome to a major disease classification inpaediatrics.

Studies conducted on parotid gland secretoryactivity (Barbero and Chernick, I958) haveindicated that children with cystic fibrosis show asignificantly higher flow rate than control children.The exact significance of this effect and its inter-relationship to other exocrine gland abnormalitiesis not known at the present time but it does, oncemore, indicate the generalized nature of involve-ment by this disease.Another glandular site which we have recently

studied is the submaxillary gland (Chernick andBarbero, in press). Analyses have shown thepresence of a turbid, insoluble material occurringin the submaxillary saliva of children with cysticfibrosis as compared to a clear, transparent saliva

observed in control children. The secretoryresponse of this gland shows a definite deviation,in many respects, from the normal glandularsecretory pattern.

All of these exocrine gland dysfunctions men-tioned form an integral part of the overall diseaseprocess and are of vital research significance in theeventual elucidation of the pathophysiologicalmechanism associated with this disease; however,from a therapeutic viewpoint, the effects can becontrolled or are of little clinical significance.

Unfortunately, the abnormality associated withthe tracheobronchial secretions is difficult, if notimpossible at the present time, to control and thecontinual accumulation of these secretions con-stitutes the major problem in arresting theprogression of this disease.The extent of accumulation associated with

these secretions, I think, can best be seen in thefollowing illustrations:

Fig. I is a top view of the larynx and tracheataken of a patient with cystic fibrosis at autopsy.The thick, muco-purulent secretions occupy andobstruct the tracheobronchial area.

Fig. 2 illustrates the presence of such secretionsthroughout the entire tracheobronchial tract asindicated by the emergence of this muco-purulentsecretion from the terminal bronchioles on thiscut section.

Interestingly, we were able in a cut section ofa bronchiole to obtain Figure 3-indicating thecentral position of bacteria completely surroundedby mucus.The extent of pulmonary damage associated

with this progression is varied and Fig. 4 indicatesthe characteristic pathology seen in advanced casesat autopsy. The emphysematous lungs showpatches of atelectasis.The serious obstruction produced by the

secretions of the tracheobronchial tract in cysticfibrosis indicated the necessity for an investigationof some of the physical and chemical characteristicsof this material.

Utilizing tracheobronchial secretions obtained

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October i961 CHERNICK: The Composition of Tracheobronchial Secretions in Cystic Fibrosis

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TABLE IORGANIC ANALYSIS OF TRACHEOBRONCHIAL

SECRETIONS0/o /0 %

% % N CH Hexo- Li- %samine pids DNA

Dry Dry Dry Dry Dry DryW t. Wt. Wt. Wt. Wt.

Cystic fibrosis 13.16 12.o 3. 2.09 I6.4 10.2

No. of samples 39 I5 I 8 5 14Bronchiectasis 6.oI 11.4 3.6 2. I8 I9.9 7.4

No. of samples 38 12 10 6 5 10

by bronchoscopy from patients with cysticfibrosis and bronchiectasis, an investigation wasmade of some of the major inorganic and organicconstituents of these secretions (Chernick andBarbero, I959).

Values for some of the organic components aregiven in Table i.As can be seen, the mean per cent. dry weight in

cystic fibrosis secretion is twice that observed in the

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TABLE 2INORGANIC ANALYSIS OF TRACHEOBRONCHIAL

SECRETIONS

% % Ash % NA % K

Dry Wet Dry Wet Dry Wet DryWt . Wt. Wt. Wt. Wt. Wt.

Cystic fibrosis 13.16 o.88 6.58 0.25 1.7 o.o8 o.5i

No. of samples 39 34 34 23 23 24 24

Bronchiectasis 6.oi o.96 13.42 0.34 4.7 o.oI 1.31No. of samples 38 33 33 26 26 26 26

bronchiectatic secretions. It is evident that thefluid component in the bronchiectatic specimens ismuch greater than that found in the cystic fibrosissecretions. The means and ranges for content ofnitrogen, total carbohydrates, hexosamine andlipids, when based on per cent. dry weight are verysimilar. DNA was the only component found tobe significantly different in the two groups, witha higher content in the cystic fibrosis secretions.

Looking at the inorganic constituents (Table 2)

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598 POSTGRADUATE MEDICAL JOURNAL October i961

TABLE 3INORGANIC ANALYSIS OF TRACHEOBRONCHIAL

SECRETIONS

I-% CA % P

Wet Dry Wet DryWeight Weight Weight Weight

Cystic fibrosis .. o.o24 o.I85 o. I7 0.89No. of samples .. I 5 15 5 5

Bronchiectasis .. .oo.o8o 0.07 0.74No. of samples .. 17 I7 5 5

these data show that the amount of sodium found,based on the total secretion and the amountavailable to the solid components, is much less inthe cystic fibrosis secretion than in the bron-chiectatic material. No significant difference wasnoted in the potassium concentration between thetwo groups.The calcium content in the total cystic fibrosis

secretion was found to be twice that found in thebronchiectatic material (Table 3).The striking difference in the content of sodium,

computed on a dry weight basis, found in these twotypes of secretion raises an interesting question asto the relationship of sodium to the peculiarphysical characteristics of cystic fibrosis secretion.Kwart and Shashoua (I957) have proposed astructure (Fig. 5) for snail mucus in which amucopolysaccharide is cross-linked to a muco-protein by the divalent calcium ion. They foundthat snail mucus could be extracted by solutions ofsalts containing any type of monovalent cation,whereas no extraction was possible by solutions ofsalts of divalent cations. They postulated thatmonovalent cations can exchange with the calciumand hence diminish the branched or cross-linkednature of the mucus to increase solubility. Theyalso observed that the intrinsic viscosity of snailmucus was at a maximum in a solution of calciumchloride and offered the explanation that a lack ofhydrolysis or ion-exchange of calcium was mani-fested in the presence of excess calcium.

Experiments performed in our laboratory indi-cated that the viscosity of homogenated tracheo-bronchial secretions obtained from patients withcystic fibrosis was substantially increased upon theaddition of calcium chloride. As previouslyindicated, the total cystic fibrosis secretion containsat least twice the amount of organic constituentsand calcium ions as the total bronchiectaticsecretion. Such a proportionality between anincrease in dry weight and increase in calciumcontent lends substantial credence to the postula-

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04-- SO,- Na+

Ca++

c=o too,C=O IOOHCH2 R CH2

--NH-CH-CO-NH-CH-Co-NH----CO- NH-CH-CO---

FIG. 5.

tion that the calcium ion functions as a crosslinkingagent in human mucoid material as well as in snailmucus. Consequently, it is not inconsistent toenvisage an important role for calcium in additionto sodium with respect to the physical propertiesof the cystic fibrosis secretion. The difference inthe ratio of ionic to organic constituents noted inthe more viscous cystic fibrosis secretions ascompared to the less viscous bronchiectaticmaterial suggests the possibility that the physicaldifferences exhibited in the bronchial tract couldresult from an alteration in the physico-chemicalbonding characteristics of the secretion.

High-molecular-weight polymers are greatlyinfluenced by the surrounding ionic concentration.In cystic fibrosis secretions, the quantity of sodiumions which are influencing the polymeric structuresis less than the number affecting structures in thebronchiectatic material and, as a result of thisalteration, the bonding characteristics of thecalcium ions may also be affected.The results of this investigation reveal that

patients with cystic fibrosis manifest some type ofdefect which results in a decrease in the number ofsodium ions available to affect the solid con-stituents of the tracheobronchial secretions whencompared with the less viscous secretions inbronchiectasis. These ionic alterations couldresult primarily from the presence of an abnormalconfiguration of mucoprotein or mucopolysac-charide, which might affect the hydration pro-perties of the material and in this manner explainthe ionic alterations observed. Another possi-bility is that there might be a defect mediatedthrough some portion of the ion-transport systemof the secretory glands in the presence of secretionof mucoid material possessing normal polymericstructure. This latter hypothesis is interesting in

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October I96i CHERNICK: The Composition of Tracheobronchial Secretions in Cystic Fibrosis

view of the electrolyte disturbance exhibited byother exocrine glands in cystic fibrosis. Timedoes not permit a discussion of the pros and cons of

these two alterations but present research indicatesthat one of the postulates may be eliminated inthe near future.

REFERENCESBARBERO, G. J., and CHERNICK, W. S. (I958): Function of the Salivary Gland in Cystic Fibrosis of the Pancreas,

Pediatrics, 22, 945.CHERNICK, W. S., BARBERO, G. J., and PARKINS, F. (96 I): Studies on Submaxillary Saliva in Cystic Fibrosis,J. Pedicat. (in

press)., and BARBERO, G. J. (1959): Composition of Tracheobronchial Secretions in Cystic Fibrosis of the Pancreas andBronchiectasis, Ibid., 24, 739.

KWART, H., and SHASHOUA, V. E. (1957): The Structure and Constitution of Mucus, Trans. N. Y. Acad. Sci., 19, 595.

DR. LYNNE REID, M.B.(Melb.), M.R.A.C.P., M.R.C.P.(Senior Lecturer, Institute of Diseases of the Chest, Brompton Hospital)

First I would like to thank Dr. Chernick for find-ing time in a very brief stay in London to come andtell us his fascinating biochemical story, becausethis is a disease in which discovery is being madequite fast and it is quite difficult for us to incorporateit into our clinical thinking. I shall largely be con-cerned just to mention how the mucus does in factgive rise to an increase of bronchial disease. Themucus is a good culture medium for a number ofbacteria, and a large amount of experimental workthat was done with introducing infection into thelung in fact depended on the introduction ofbacteria suspended in mucin. It has been shownthat the effect of mucin was not in any way to lowerresistance or to enhance virulence, whatever thosetwo terms mean, but to be related to the size of theinoculum. Injecting the bacteria suspended inmucus in fact gave them time to multiply to a large-sized colony before they invaded. I have beenprivileged to examine quite a lot of Dr. Anderson'smaterial from the Babies' Hospital in New York,and though clearly the basic thing here is a bio-,chemical disturbance, once the mucus is in thebronchi it brings its own results. In both chronicbronchitis and bronchiectasis we have beeninterested to find that you can show a differencebetween the size of the bronchial glands in thenormals and in patients who do produce a large:amount of secretion, and one of the questions thatpresented in relation to these fibrocystic children-was-Do they develop a gland hypertrophy at anystage, and if so at what stage ?-and when I saygland hypertrophy perhaps I should divide it upinto the mucus glands in the wall of the bronchialtree and the goblet cells on the surface. To sum-marize briefly the findings in the material we haveexamined to date, it would seem that at birth youdo not get an abnormal number of goblet cells in.the bronchial tree. The normal lung at birth has

very few goblet cells even in bronchi and certainlyhardly any in the bronchiolar region of the lung.The gland ratio we have actually measured inbronchitis and in normals: the mucus gland/wallratio, and I shall refer to this ratio as an index ofthe degree of hypertrophy of the glands. In thenew-born fibrocystic, and up to some months, wehaven't been able to show any increase in glandsize, but once these children have become infectedand this has continued for six years or more it ispossible to find that a degree of hypertrophy hasoccurred, so that this does not only involve theglands in the wall but also the goblet cells inthe peripheral bronchioli. It would seem that theamount of secretion produced is somehow relatedto what has happened after birth rather than to theintrinsic biochemical disturbance, but perhaps Ican ask Dr. Chernick a question there-whetherthey do in fact find in the parotid and sub-maxillary, two glands in which infection is notclinically a problem, associated with the bio-chemical disturbance there is any increase in size ofthe gland ? Some experimental work we havedone using non-specific irritants to try andseparate the effects of irritation from those of in-fection-it is possible to induce in the bronchialtree of the experimental animal a hypersecretion ofmucus together with hypertrophy of mucus-secreting cells while the lungs are in fact sterile, sothat clearly the effect in bronchitis and bronchiec-tasis is largely there a question of recurrent in-fection and environmental conditions causinggland hypertrophy. But in this condition it maybe possible to sort out whether or not the enzymechange of itself is responsible, and I would like toend by saying that in this condition as the sweattest and as the disturbance in the sweat glandsbecame clear one hoped that from the geneticpoint of view we were getting close to the basic

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600 POSTGRADUATE MEDICAL JOURNAL October i961

enzyme involved and hence to the chromosomeand gene concerned with this condition, but clearlythe sweat test hasn't fulfilled all the early hopes ofdiagnosis-some patients with fibrocystic diseaseare at the upper range of normal limits, and theabnormality in sweat hasn't helped to separate outthe heterozygotes, so I wonder if I could ask Dr.Chernick a second question-whether he feelsfrom what he has done whether we are getting anycloser to which is the basic overall enzyme dis-turbance in this condition-is it perhaps con-cerned with calcium rather than with sodium ?

DR. CHERNICK: It should be emphasized thatwe have concentrated most of our attention onsubmaxillary gland secretion recently becausetracheobronchial secretions contain a variety ofcontaminants, notably bacteria and polymorpho-nucleocytes, which hinder a detailed biochemicalanalysis. Therefore, the utilization of submaxillarysecretion provides an uncontaminated source ofa sero-mucoid secretion which may be moreuseful in the eventual elucidation of this ab-normality

In the submaxillary gland, there is a definiteenlargement associated with the obstruction' of

the ductules by mucoid material. In this gland,there is no evidence of infection, merely anaccumulation of material resulting presumablyfrom some type of abnormality and not associatedwith infection.

Dr. Reid's second question relating to the basicenzyme defect is difficult to answer at the presenttime. I think, in the very near future, we may beable to ascertain whether the alteration in exocrinegland secretion in this disease is related to anabnormal mucoprotein or mucopolysaccaride orwhether it is primarily a defect in ionic transport.Unfortunately, the basic enzyme systems whichare responsible for the active transport of theseions are not known and, until such informationbecomes available, a definitive answer cannot begiven.My own personal opinion, at the present time,

is that the alteration in ionic transport is primarilyresponsible for all symptoms observed in thisdisease including the increased viscosity of themucoid secretions. As to whether the defectinvolves primarily sodium or calcium ions, thedata thus far is too incomplete and much morework is needed to clarify this point.

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