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Glycosaminoglycans in the bronchial mucus of patients with chronic bronchitis and mucoid impaction of the bronchus
Life Sciences, Voi.36, pp. 2287-2293 Printed in the U.S.A.
Pergamon Press
GLYCOSAMINOGLYCANS IN THE BRONCHIAL MUCUS OF PATIENTS WITH CHRONIC BRONCHITIS AND MUCOID IMPACTION OF THE BRONCHUS Dimitrios A. Theocharis Laboratory of Biological Chemistry,School of Health University of Patras,Patras, Greece.
Sciences,
(Received in final form April 3, 1985) Summary Glycosaminoglycans were isolated from mucus of patients with chronic bronchitis and mucoid impaction of the bronchus,whose contents were approximately 56 ~moles and 80 ~moles of hexosamine per g of dry weight of mucus respectively. Electrophoretic and chemical characterization and enzymatic susceptibility demonstrated that the glycosaminoglycans in mucus from bothgroups of the patients contained hyaluronic acid as the main constituent, with undersulphated chondroitin as a minor component. In addition,in mucus from the patient with mucoid impaction of the bronchus chondroitin sulphate a n d h e p a r a n sulphate or heparan sulphate-like substance were identified. It is known that all major glycosaminoglycan types are found in lung tissue or lung fibroblasts from various species (I-4). Little is known about the glycosaminoglycans present in pleural fluids from patients with various pulmonary diseases other than mesothelioma (5,6). Only a few biochemical investigations on the respiratory mucus glycosaminoglycans have been published. Hyaluronic acid is present in the pulmenary secretions of patients with asthma (7). The present study reports the isolation and characterization of various types of glycosaminoglycans from bronchial mucus of patients with chronic bronchitis and one case of a patient with mucoid impaction of the bronchus. Materials and methods Seven patients of either sex (aged 30-65 yr) with chronic bronchitis which were on medication for bronchitis, and one woman (age 42 yr ) with mucoid impaction of the bronchus were used for this study under the direction of Dr. E.Nikolaou. The patients were studied during a stable phase of the disease and were hospitalized at the time of the study to avoid as far as possible variations in environmental factors. Particularly in the case of the patient with mucoid impaction of the bronchus, the following information is given: G.P a 42 year-old,housewife had been admitted because of a sudden onset of a severe cough, which subsided only after excretion of great quantities of mucus casts, which were plugs themselves. The length of the excreted mucus material was between 3 and 15 cm. There was no history of previous drug or irritant substances exposure. Complete and 0024-3205/85 $3.00 + .00 Copyright (c) 1985 Pergamon Press Ltd.
2288
Glycosaminoglycans in Human Bronchial Mucus
Vol. 36, No. 24, 1985
detailed radiological,microbiological (sputum cultures for Aspergillus, Mucobacterium tuberculosis and other aerobic or anaerobic microorganisms),cytological and parasitological studies were negative, as were serological studies for the detection of antifungal antibodies and other examinations. The episode was not repeated and she is in excelent health, with no medication whatever for an observation period of 18 months. We believe that our mucoid impaction case cannot be correlated with any of the diseases reported by Irwin and Thomas (8), namely asthma, chronic bronchitis, cystic fibrosis and allergic bronchopulmonary aspergillosis. Materials and chemicals. Sephadex G-100 was purchased from Pharmacia Fin-e Chemicals ( Uppsala, Sweden). ECTEOLA-cellulose (ET-11) and cellulose powder (CF-11) were Whatman (W and R.Balston Ltd.,U.K.). Papain twice recrystallized 16-40 U/ mg of protein, testicular hyaluronidase and chondroitinase ABC were obtained from Sigma Chemicals Co.,St.Louis,MO,USA. All other reagents were of analytical grade,except for glucosaminehydrochloride, galactosamine hydrochloride, glucuronolactone and carbazole which were of reagent grade. Analytical methods. Uronic acid was determined by an automated modification (9) of the carbazole reaction (10) with glucuronolactone as standard. Glucosamine and galactosamine were determined by using a Beckman amino acid analyser (11). Hexoses were determined by the anthrone method (12),with galactose as standard and sulphate was determined by a modification of the benzidine method (13). Isolation of glycosaminoglycans as ethanolic precipitate. Mucus from each patient separately was collected as described by LopezVidriero and Reid (14) and then immediately mixed with 10 vol. of acetone and after standing overnight at 4°C,the mixture was centrifuged washed once with acetone and dried in air. Known amounts of dry mucus were exhaustively digested with papain and then the glycosaminoglycans were precipitated by addition of 4 volumes of ethanol as described previously (15). After standing for at least 8 hr in the cold,the precipitate formed was collected by centrifugation and dried in air. Each sample was dissolved in a known volume of water. Aliquots of individual samples were used for chemical analysis and the remainders were pooled in two groups (group A: the patients with chronic bronchitis and B: the patient with mucoid impaction of the bronchus). Groups A and B were used for further purification and characterization of the glycosaminoglycans. Fractionation of glycosaminoglycans. Samples from each group were fractionated by the cetylpyridinium chloride ( CPC )-- cellulose chromatographic procedure (16). The columns were eluted with two volumes of I% CPC-0.005M-Na2SO~ , 0.3M-NaCI, 0.5M-NaCI and 1.5M-NaCI; all salt solutions contained 0.05% CPC. The I% CPC fractions were first freed from CPC by extraction with isoamyl alcohol and then chromatographed on Epichlorohydrin Triethanolamine (ECTEOLA)-cellulose columns (I~). The columns were eluted with two bed vol. of 0.02M-HCI and 2M-NaCI. Each fraction from both chromatographies were concentrated by dialysis through Amicon UM-2 membranes and then analysed. The 2M-NaCI fractions contained no hexosamine. The recovery of hexosamine from combined chromatography was 94-95%. Enzymatic treatments. Samples of glycosaminoglycans were subjected to hyaluronidase digestion as described elsewhere (18), or to
Vol. 36, No. 24, 1 9 8 5
Glycosaminoglycansin Human Bronchial Mucus
2289
chondroitinase ABC digestion as described by the method of Saito et ai.,(19). The digests were chromatographed on a Sephadex G-100 column and the eluted fractions were analysed for uronic acid. Gel chromatography. Samples of glycosaminoglycans were c h r o m a t ~ graphed on columns (150 X 0.65 cm) packed with Sephadex G-100, equilibrated and eluted with 0.2M-sodium acetate pH 6.5. Fractions of 1.5 ml were collected and analysed for uronic acid. Electrophoresis. Samples of glycosaminoglycans from variousSractions were subjected to electrophoresis on cellulose acetate (20) and the strips were stained with 0.5% Toluidine blue in 15% of ethanol. Results and Discussion Analyses for determination of the chemical composition of the isolated macromolecular hexosamine-containing material from mucus of patients with chronic bronchitis and one patient with mucoid impaction of the bronchus,were carried out on known amounts of dry material, which was previously exhaustively digested with papain. The results from these analyses are shown in Table I. TABLE I Chemical analysis of the isolated glycosaminoglycans from mucus of patients with chronic bronchitis and mucoid impaction of the bronchus. The results are expressed as mg of each compound per g dry weight of mucus. Patients Compounds
Chronic bronchitis mean ± SD
with Mucoid impaction of the bronchus
Total hexosamine(T.HexN)
10.8 _+ 2.35
14.2
Uronic acid
10.1 -+ 1.77
15.1
0.4 + 0.09
0.7
Hexoses GlcN/GalN*(molar ratio)
13.1 -+ 0.4
8.1
S04/T.HexN
0.02
0.16
(molar ratio)
_+ 0 . 0 0 3
* GlcN:Glucosamine and GalN:Galactosamine. The mucus from the patient witb mucoid impaction of the bronchus contained greater amounts of uronic acid and hexoses than that from the patients with chronic bronchitis and different molar ratios of glucosamine to galactosamine and sulphate to total hexosamine. Gel chromatography of the isolated macromolecular material from mucus of patients with chronic bronchitis revealed two peaks,I and II (Fig. IA), which when subjected to hyaluronidase digestion and rechromatographed,disappeared completely. In contrast,gel chromatography of the respective isolated macromolecular material from the patient with mucoid impaction of the bronchus revealed three peaks I,II and III. Peaks I and II disappeared completely when rechromatographed after digestion with hyaluronidaseand chondroitinase
2290
Glycosaminoglycans in Human Bronchial Mucus
ABC, w h i l e p e a k ABC d i g e s t i o n .
Vol. 36, No. 24, 1985
III was r e s i s t a n t to h y a l u r o n i d a s e and This p e a k (III) r e p r e s e n t s 9% of total
chondroitase hexosamine.
I
25
A
2O --.
15
¢J
f
¢~ 25
~ 0
f
I
20
B
5 0 F RA C
ON
NO
~/~
FIGURE I. Sephadex G-100 gel chromatography of glycosaminoglycans isolated from human bronchial mucus of patients withchronic bronchitis (A) and patient with mucoid impaction of the bronchus (B); before (~ o) and after hyaluronidase or chondroitinase ABC digestion (= ~). Vo, V t ~dicate void and total volume of the column respectively. The aminosugar in this peak was identified as glucosamine. The fractions of this peak were pooled,concentrated and then electrophoresed on a cellulose acetate strip (Fig.2,1ane RF). One spot was observed with an electrophoretic mobility similar to heparan sulphate standard. In order to identify and determine the proportion of each type of hexosamine-containing material, fractionation on CPC-cellulose and ECTEOLA-cellulose columns was performed on known amounts from
Vol. 36, No. 24, 1 9 8 5
Glycosaminoglycansin Human Bronchial Mucus
2291
each group of patients. The results of this fractionation are shown in Table II. The fractionation of the hexosamine-containing material from patients with chronic bronchitis indicated that, 93% of the total hexosamine was eluted in the 0.3M-NaCI fraction. The analytical TABLE II Chemical composition of glycosaminoglycans purified on combined chromatography (details see in text). The results are expressed as ~moles/g of dry weight of mucus; figures in brackets refer to percentage of total hexosamine. Group of patients with
Fraction
Chronic bronchitis(A)
Uronic acid
GaIN
0.3M-NaCI
51.8
0
0.SM-NaCI
4.2
1.5M-NaCL
0
4.0(7) 0
Mucoid impaction 0.3M-NaCI
67.6
of the
0.5M-NaCI
13.5
5.9(7)
1.5M-NaCI
3.3
3.5(4)
bronchus
(B)
0
GIcN
S04/T.HexN (molar ratio)
52.1(93)
0
traces
0.3
0
0
68.0(80)
0
7.5(9)
0.8
0
0.9
data of this fraction were typical of hyaluronic acid i.e.the hexosamine and uronic acid occur in equimolar amounts, the hexosamine was identified as glucosamine, no sulphate was found in the fraction. The electrophoretic mobility was the same as the standard of the hyaluronic acid (Fig.2,1ane IA). The remainder hexosamine (7%) was eluted in 0.5M-NaCI. The aminosugar was identified as galactosamine. The fraction had a low sulphate content (molar ratio S04/ /hexosamine= 0.3). The electrophoretic mobility was slower than the chondroitin sulphate standard (Fig.2,1ane 2A). The respective fractionation of the hexosamine-containing material from the patient with mucoid impaction of the bronchus, revealed that there are quantitative and qualitative differences between the patients of the two groups. The fractionation of the glycosaminoglycans from mucus of the patient with mucoid impaction of the bronchus, indicate that, 80% of total hexosamine was eluted in 0.3M-NaCI fraction. The analytical data of this fraction were typical of hyaluronic acid as in the previous case. In contrast, 16% of total hexosamine was eluted in 0.5M-NaCI fraction where the 7% was identified as galactosamine and the remainder 9% identified as glucosamine. The fraction had a sulphate content higher than the respective fraction in the previous case (molar ratio SO4/hexosamine=0.8). Upon electrophoresis of this fraction two spots were observed (Fig.2,1ane 2B). One of these spots had electrophoretic mobility similar to heparan sulphate and the other spot was slower than the chondroitin sulphate as in the case of the 0.5M-NaCI fraction from patients with chronic bronchitis. A little proportion of hexosamine (about 4%) was eluted in 1.5M-NaCI fraction. It was identified as galactosamine with molar ratio S04/hexosamine=0.9. The electrophoretic mobility was the same as
2292
Glycosaminoglycans in Human Bronchial Mucus
chondroitin
sulphate
standard
(Fig.2,1ane
Vol. 36, No. 24, 1985
3B).
On the basis of these analytical and electrophoretic data it may be concluded with safety that, the degraded substances by hyaluronidase in bronchial mucus of the chronic bronchitic patients (Fig. IA) are hyaluronic acid as the main constituent and undersulphated chondroitin. In bronchial mucus from the patient with
Chondroitin
Heparin--nD sulphate-b
mmm qUlP
Heparan s u l p h a t e - ~ Hyaluronic acid--~
origin--~
.........................
G St IA 2A FIGURE
I B 2B 3B RF 2.
Electrophoretic pattern of glycosaminoglycans in various fractions from the combined chromatography. IA, 2A, IB, 2B, 3B indicate the 0.3M-NaCI, 0.5M-NaCI, 0.3M-NaCI, 0.5M-NaCI, and 1.5M-NaCI fractions as it appears in Table 2. RF: Resistant fractions (Fig.l, peak III) to hyaluronidase and chondroitinase ABC digestion. St: Standards of various types of glycosaminoglycans mucoid impaction of the bronchus,the analytical and electrophoretic data and the behaviour of the glycosaminoglycan types to hyaluronidase and chondroitinase ABC digestion (Fig. IB) suggest that, there are, in addition,chondroitin sulphate and heparan sulphateor a heparan sulphate-like substance. The presence of various types of glycosaminoglycans in lung ~tissue or lung fibroblasts have been reported by Other investigators, concerning different species (I-4). Results similar to those found in patients with chronic bronchitis have also been obtained by SAHU and LYNN (7) whose found that, hyaluronic acid was the only glycosaminoglycan present in the pulmonary secretion of patients with asthma. The case of a patient with mucoid impaction of the bronchus shows particular interest,because the presence of heparan sulphate and chondroitin sulphate may be specific for this particular disease and prove to be of clinical significance. It is obviously, difficult to propose the glycosaminoglycan pattern found in a single patient, as a criterion for differential diagnosis. Nevertheless, we feel that this is a significant observation and the study of more cases could be valuable. The mechanism of accumulation and the role of
Vol. 36, No. 24, 1 9 8 5
Glycosaminoglycansin Human Bronchial Mucus
2293
the different types of glycosaminglycans in the bronchial mucus and in the aetiology o£ the disease remain to be investigated. Acknowledgment I would like to thank Dr. E. Nikolaou for helpful suggestions and discussions during the course of this investigation. References I. F.S. WUSTEMAN,
Experientia
28
887-888
2. A.L. HORWITZ and R.G. CRYSTAL,
(1972)
J.Clin. Invest.
56 1312-1318
(1975). 3. P.M. KRAEMER,
Biochemistry
10 1445-1451
4. R.J. RICHARDS and R.G. MORRIS, 5. C. FRIMAN~ Acta 33
(1973)
(1977).
D. DEGAND, A. BOERSMA and C. RICHET, Clin.Chim.Acta
443-454
(1971).
7. S. SAHU and N.S. LYNN, Biochem. J. 173 8. R.S.
(1971) 12 441-451
P.E. HELLSTROM, M. JUVANI and H. RISKA, Clin.Chim.
76 357-361
6. R. HAVEZ,
Life Sci.
565-568
IRWIN and H.M. THOMAS, Am. Rev.Resp. Dis.
(1979)
108 955-959
(1973). 9. D
HEINEGARD,
Chem. Scr. 4
199-201
10
T
11
C P. TSIGANOS and H. MUIR,
(1973)
BITTER and H.M. MUIR, Analyt.Biochem. Biochem.J.
4 330-334
113 879-883
(1962) (1969)
12
T A. SCOTT and E.H. MELVIN, Analyt.Chem.
13
C A. ANTONOPOULOS,
14
M T.LOPEZ-VIDRIERO and L. REID, Br.Med. Bull.
34 63-74
15
D A. THEOCHARIS,
(1985)
16
C A. ANTONOPOULOS,
Acta Chem. Scand.
Int.J.Biochem.
In the press
(1978)
1-19
(1964).
C.A. ANTONOPOULOS, A. BJELLE and L-A. FRANSSON,
Biochm. Biophys.Acta 18. D.A. THEOCHARIS,
215
522-526
(1970).
Ph.D.Thesis of University of Patras
19. H. SAITO, T. YAMAGATA and S.J. SUZUKI, J.Biol.Chem. 1536-1542
(1953)
(1962)
S. GARDELL, J.A. SZIRMAI and E.R. TYSSONK,
Biochim. Biophys.Acta 83 17. A. ANSETH,
25 1656-1661
16 1521
(1977) 243
(1968).
20. R. HATA and Y. NAGAI, Analyt. Biochem.
45 462-468
(1972)
Pergamon Press
GLYCOSAMINOGLYCANS IN THE BRONCHIAL MUCUS OF PATIENTS WITH CHRONIC BRONCHITIS AND MUCOID IMPACTION OF THE BRONCHUS Dimitrios A. Theocharis Laboratory of Biological Chemistry,School of Health University of Patras,Patras, Greece.
Sciences,
(Received in final form April 3, 1985) Summary Glycosaminoglycans were isolated from mucus of patients with chronic bronchitis and mucoid impaction of the bronchus,whose contents were approximately 56 ~moles and 80 ~moles of hexosamine per g of dry weight of mucus respectively. Electrophoretic and chemical characterization and enzymatic susceptibility demonstrated that the glycosaminoglycans in mucus from bothgroups of the patients contained hyaluronic acid as the main constituent, with undersulphated chondroitin as a minor component. In addition,in mucus from the patient with mucoid impaction of the bronchus chondroitin sulphate a n d h e p a r a n sulphate or heparan sulphate-like substance were identified. It is known that all major glycosaminoglycan types are found in lung tissue or lung fibroblasts from various species (I-4). Little is known about the glycosaminoglycans present in pleural fluids from patients with various pulmonary diseases other than mesothelioma (5,6). Only a few biochemical investigations on the respiratory mucus glycosaminoglycans have been published. Hyaluronic acid is present in the pulmenary secretions of patients with asthma (7). The present study reports the isolation and characterization of various types of glycosaminoglycans from bronchial mucus of patients with chronic bronchitis and one case of a patient with mucoid impaction of the bronchus. Materials and methods Seven patients of either sex (aged 30-65 yr) with chronic bronchitis which were on medication for bronchitis, and one woman (age 42 yr ) with mucoid impaction of the bronchus were used for this study under the direction of Dr. E.Nikolaou. The patients were studied during a stable phase of the disease and were hospitalized at the time of the study to avoid as far as possible variations in environmental factors. Particularly in the case of the patient with mucoid impaction of the bronchus, the following information is given: G.P a 42 year-old,housewife had been admitted because of a sudden onset of a severe cough, which subsided only after excretion of great quantities of mucus casts, which were plugs themselves. The length of the excreted mucus material was between 3 and 15 cm. There was no history of previous drug or irritant substances exposure. Complete and 0024-3205/85 $3.00 + .00 Copyright (c) 1985 Pergamon Press Ltd.
2288
Glycosaminoglycans in Human Bronchial Mucus
Vol. 36, No. 24, 1985
detailed radiological,microbiological (sputum cultures for Aspergillus, Mucobacterium tuberculosis and other aerobic or anaerobic microorganisms),cytological and parasitological studies were negative, as were serological studies for the detection of antifungal antibodies and other examinations. The episode was not repeated and she is in excelent health, with no medication whatever for an observation period of 18 months. We believe that our mucoid impaction case cannot be correlated with any of the diseases reported by Irwin and Thomas (8), namely asthma, chronic bronchitis, cystic fibrosis and allergic bronchopulmonary aspergillosis. Materials and chemicals. Sephadex G-100 was purchased from Pharmacia Fin-e Chemicals ( Uppsala, Sweden). ECTEOLA-cellulose (ET-11) and cellulose powder (CF-11) were Whatman (W and R.Balston Ltd.,U.K.). Papain twice recrystallized 16-40 U/ mg of protein, testicular hyaluronidase and chondroitinase ABC were obtained from Sigma Chemicals Co.,St.Louis,MO,USA. All other reagents were of analytical grade,except for glucosaminehydrochloride, galactosamine hydrochloride, glucuronolactone and carbazole which were of reagent grade. Analytical methods. Uronic acid was determined by an automated modification (9) of the carbazole reaction (10) with glucuronolactone as standard. Glucosamine and galactosamine were determined by using a Beckman amino acid analyser (11). Hexoses were determined by the anthrone method (12),with galactose as standard and sulphate was determined by a modification of the benzidine method (13). Isolation of glycosaminoglycans as ethanolic precipitate. Mucus from each patient separately was collected as described by LopezVidriero and Reid (14) and then immediately mixed with 10 vol. of acetone and after standing overnight at 4°C,the mixture was centrifuged washed once with acetone and dried in air. Known amounts of dry mucus were exhaustively digested with papain and then the glycosaminoglycans were precipitated by addition of 4 volumes of ethanol as described previously (15). After standing for at least 8 hr in the cold,the precipitate formed was collected by centrifugation and dried in air. Each sample was dissolved in a known volume of water. Aliquots of individual samples were used for chemical analysis and the remainders were pooled in two groups (group A: the patients with chronic bronchitis and B: the patient with mucoid impaction of the bronchus). Groups A and B were used for further purification and characterization of the glycosaminoglycans. Fractionation of glycosaminoglycans. Samples from each group were fractionated by the cetylpyridinium chloride ( CPC )-- cellulose chromatographic procedure (16). The columns were eluted with two volumes of I% CPC-0.005M-Na2SO~ , 0.3M-NaCI, 0.5M-NaCI and 1.5M-NaCI; all salt solutions contained 0.05% CPC. The I% CPC fractions were first freed from CPC by extraction with isoamyl alcohol and then chromatographed on Epichlorohydrin Triethanolamine (ECTEOLA)-cellulose columns (I~). The columns were eluted with two bed vol. of 0.02M-HCI and 2M-NaCI. Each fraction from both chromatographies were concentrated by dialysis through Amicon UM-2 membranes and then analysed. The 2M-NaCI fractions contained no hexosamine. The recovery of hexosamine from combined chromatography was 94-95%. Enzymatic treatments. Samples of glycosaminoglycans were subjected to hyaluronidase digestion as described elsewhere (18), or to
Vol. 36, No. 24, 1 9 8 5
Glycosaminoglycansin Human Bronchial Mucus
2289
chondroitinase ABC digestion as described by the method of Saito et ai.,(19). The digests were chromatographed on a Sephadex G-100 column and the eluted fractions were analysed for uronic acid. Gel chromatography. Samples of glycosaminoglycans were c h r o m a t ~ graphed on columns (150 X 0.65 cm) packed with Sephadex G-100, equilibrated and eluted with 0.2M-sodium acetate pH 6.5. Fractions of 1.5 ml were collected and analysed for uronic acid. Electrophoresis. Samples of glycosaminoglycans from variousSractions were subjected to electrophoresis on cellulose acetate (20) and the strips were stained with 0.5% Toluidine blue in 15% of ethanol. Results and Discussion Analyses for determination of the chemical composition of the isolated macromolecular hexosamine-containing material from mucus of patients with chronic bronchitis and one patient with mucoid impaction of the bronchus,were carried out on known amounts of dry material, which was previously exhaustively digested with papain. The results from these analyses are shown in Table I. TABLE I Chemical analysis of the isolated glycosaminoglycans from mucus of patients with chronic bronchitis and mucoid impaction of the bronchus. The results are expressed as mg of each compound per g dry weight of mucus. Patients Compounds
Chronic bronchitis mean ± SD
with Mucoid impaction of the bronchus
Total hexosamine(T.HexN)
10.8 _+ 2.35
14.2
Uronic acid
10.1 -+ 1.77
15.1
0.4 + 0.09
0.7
Hexoses GlcN/GalN*(molar ratio)
13.1 -+ 0.4
8.1
S04/T.HexN
0.02
0.16
(molar ratio)
_+ 0 . 0 0 3
* GlcN:Glucosamine and GalN:Galactosamine. The mucus from the patient witb mucoid impaction of the bronchus contained greater amounts of uronic acid and hexoses than that from the patients with chronic bronchitis and different molar ratios of glucosamine to galactosamine and sulphate to total hexosamine. Gel chromatography of the isolated macromolecular material from mucus of patients with chronic bronchitis revealed two peaks,I and II (Fig. IA), which when subjected to hyaluronidase digestion and rechromatographed,disappeared completely. In contrast,gel chromatography of the respective isolated macromolecular material from the patient with mucoid impaction of the bronchus revealed three peaks I,II and III. Peaks I and II disappeared completely when rechromatographed after digestion with hyaluronidaseand chondroitinase
2290
Glycosaminoglycans in Human Bronchial Mucus
ABC, w h i l e p e a k ABC d i g e s t i o n .
Vol. 36, No. 24, 1985
III was r e s i s t a n t to h y a l u r o n i d a s e and This p e a k (III) r e p r e s e n t s 9% of total
chondroitase hexosamine.
I
25
A
2O --.
15
¢J
f
¢~ 25
~ 0
f
I
20
B
5 0 F RA C
ON
NO
~/~
FIGURE I. Sephadex G-100 gel chromatography of glycosaminoglycans isolated from human bronchial mucus of patients withchronic bronchitis (A) and patient with mucoid impaction of the bronchus (B); before (~ o) and after hyaluronidase or chondroitinase ABC digestion (= ~). Vo, V t ~dicate void and total volume of the column respectively. The aminosugar in this peak was identified as glucosamine. The fractions of this peak were pooled,concentrated and then electrophoresed on a cellulose acetate strip (Fig.2,1ane RF). One spot was observed with an electrophoretic mobility similar to heparan sulphate standard. In order to identify and determine the proportion of each type of hexosamine-containing material, fractionation on CPC-cellulose and ECTEOLA-cellulose columns was performed on known amounts from
Vol. 36, No. 24, 1 9 8 5
Glycosaminoglycansin Human Bronchial Mucus
2291
each group of patients. The results of this fractionation are shown in Table II. The fractionation of the hexosamine-containing material from patients with chronic bronchitis indicated that, 93% of the total hexosamine was eluted in the 0.3M-NaCI fraction. The analytical TABLE II Chemical composition of glycosaminoglycans purified on combined chromatography (details see in text). The results are expressed as ~moles/g of dry weight of mucus; figures in brackets refer to percentage of total hexosamine. Group of patients with
Fraction
Chronic bronchitis(A)
Uronic acid
GaIN
0.3M-NaCI
51.8
0
0.SM-NaCI
4.2
1.5M-NaCL
0
4.0(7) 0
Mucoid impaction 0.3M-NaCI
67.6
of the
0.5M-NaCI
13.5
5.9(7)
1.5M-NaCI
3.3
3.5(4)
bronchus
(B)
0
GIcN
S04/T.HexN (molar ratio)
52.1(93)
0
traces
0.3
0
0
68.0(80)
0
7.5(9)
0.8
0
0.9
data of this fraction were typical of hyaluronic acid i.e.the hexosamine and uronic acid occur in equimolar amounts, the hexosamine was identified as glucosamine, no sulphate was found in the fraction. The electrophoretic mobility was the same as the standard of the hyaluronic acid (Fig.2,1ane IA). The remainder hexosamine (7%) was eluted in 0.5M-NaCI. The aminosugar was identified as galactosamine. The fraction had a low sulphate content (molar ratio S04/ /hexosamine= 0.3). The electrophoretic mobility was slower than the chondroitin sulphate standard (Fig.2,1ane 2A). The respective fractionation of the hexosamine-containing material from the patient with mucoid impaction of the bronchus, revealed that there are quantitative and qualitative differences between the patients of the two groups. The fractionation of the glycosaminoglycans from mucus of the patient with mucoid impaction of the bronchus, indicate that, 80% of total hexosamine was eluted in 0.3M-NaCI fraction. The analytical data of this fraction were typical of hyaluronic acid as in the previous case. In contrast, 16% of total hexosamine was eluted in 0.5M-NaCI fraction where the 7% was identified as galactosamine and the remainder 9% identified as glucosamine. The fraction had a sulphate content higher than the respective fraction in the previous case (molar ratio SO4/hexosamine=0.8). Upon electrophoresis of this fraction two spots were observed (Fig.2,1ane 2B). One of these spots had electrophoretic mobility similar to heparan sulphate and the other spot was slower than the chondroitin sulphate as in the case of the 0.5M-NaCI fraction from patients with chronic bronchitis. A little proportion of hexosamine (about 4%) was eluted in 1.5M-NaCI fraction. It was identified as galactosamine with molar ratio S04/hexosamine=0.9. The electrophoretic mobility was the same as
2292
Glycosaminoglycans in Human Bronchial Mucus
chondroitin
sulphate
standard
(Fig.2,1ane
Vol. 36, No. 24, 1985
3B).
On the basis of these analytical and electrophoretic data it may be concluded with safety that, the degraded substances by hyaluronidase in bronchial mucus of the chronic bronchitic patients (Fig. IA) are hyaluronic acid as the main constituent and undersulphated chondroitin. In bronchial mucus from the patient with
Chondroitin
Heparin--nD sulphate-b
mmm qUlP
Heparan s u l p h a t e - ~ Hyaluronic acid--~
origin--~
.........................
G St IA 2A FIGURE
I B 2B 3B RF 2.
Electrophoretic pattern of glycosaminoglycans in various fractions from the combined chromatography. IA, 2A, IB, 2B, 3B indicate the 0.3M-NaCI, 0.5M-NaCI, 0.3M-NaCI, 0.5M-NaCI, and 1.5M-NaCI fractions as it appears in Table 2. RF: Resistant fractions (Fig.l, peak III) to hyaluronidase and chondroitinase ABC digestion. St: Standards of various types of glycosaminoglycans mucoid impaction of the bronchus,the analytical and electrophoretic data and the behaviour of the glycosaminoglycan types to hyaluronidase and chondroitinase ABC digestion (Fig. IB) suggest that, there are, in addition,chondroitin sulphate and heparan sulphateor a heparan sulphate-like substance. The presence of various types of glycosaminoglycans in lung ~tissue or lung fibroblasts have been reported by Other investigators, concerning different species (I-4). Results similar to those found in patients with chronic bronchitis have also been obtained by SAHU and LYNN (7) whose found that, hyaluronic acid was the only glycosaminoglycan present in the pulmonary secretion of patients with asthma. The case of a patient with mucoid impaction of the bronchus shows particular interest,because the presence of heparan sulphate and chondroitin sulphate may be specific for this particular disease and prove to be of clinical significance. It is obviously, difficult to propose the glycosaminoglycan pattern found in a single patient, as a criterion for differential diagnosis. Nevertheless, we feel that this is a significant observation and the study of more cases could be valuable. The mechanism of accumulation and the role of
Vol. 36, No. 24, 1 9 8 5
Glycosaminoglycansin Human Bronchial Mucus
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