Sialic acid and its relation to chronic bronchitis

CLINICA CHIMICA ACTA

VOL. 2 (1957)

SIALIC

ACID

AND

ITS

RIZLATION

TO CHRONIC

491

BRONCHITIS

In chronic bronchitis, the normal secretion of mucus in the bronchiai passages is increased considerably. The increased viscosity of the bronchial secretion in chronic respiratory diseases (bronchitis, bronchiectasis and asthma) has been attributed (ARMSTRONG AND WHITEI, WHITE, ELMES AND WALSH*) mainly to leucocytes entangled in fibrils of deoxyribonucleoprotein when the sputum is purulent, and leucocytes embedded in mucoprotein when it is mucoid. The sputa from chronic bronchitis is generally of the latter type. Sialic acid (or neuraminic acid) is now recognised as an important carbohydrate moiety present in many mucoproteins and constitutes as much as 17 ‘A of bovine submaxillary gland mucoprotein (GOTTSCHALK“). In diseases of many kinds (acute tuberculosis, sub-acute bacterial endocarditis, advanced cancer, and others) the sera of the patients has been found to contain 2-3 times more sialic acid than normal sera (WINZLER~). The sialic acid was believed to be associated in each case with the serum glycoprotein fraction. Sialic acid is known also to occur in gangliosides (compounds related to cerebrosides) and these have been found to accumulate in the brains of patients suffering from Tay-Sach’s disease (KLENK AND SCHUMANN~). The present communication describes a comparison of normal saliva and sputa from patients suffering from chronic bronchitis, with particular reference to sialic acid. EXPERIMENTAL Source of sputa Through the kindness of Dr. L. E. HOUGHTON, Harefield Hospital, Middlesex, sputa of patients suffering from chronic bronchitis was sent to us from a special unit for the treatment of chronic chest diseases set up at Benenden Hospital, Kent. The normal saliva was obtained from healthy research workers in the Chemistry Department, The University, Birmingham. Initial treatment of materials The sputum samples were each diluted with an equal volume of distilled water and dialysed at 2’ for 5 days against several changes of distilled water. The flocculent precipitates that separated in the dialysis bags were centrifuged off and freeze-dried. The soluble material present in each supernatant was also recovered by freeze-drying. Table I shows the yields of soluble and insoluble materials from bronchitis sputa and normal control salivae. As a rule the yields of solid materials per ml of original samples were higher in the bronchitis samples than in the controls, although there were a few exceptional

cases.

Kefrrrnresp. 496

T. ANZAI,

49”

4

s *h

ic *9 IO 11 JL 13 14 15 16 77 18 I9 LO

K. c H. ?V. L. B. E. A. H. E. UT. v. E. c. E. A. II. w. T. T. G.

&“,c .

S. A. BARKER,

56 .58 59 jr LO .55 52 61 58 59 42 57 52 41 57 SL 36 46

I .

F. R. R. T. R. B. s. \4:. 1. J. D. H. v. P. G.

I-e(-) +

-

hl. STACEY

55-o 6.5 60.0

42.63 L.IT

2j.0

5.13

3b.o 8.5 12.j 80.0 7j.0

.__

35.0 32.5 1j.O 12.j

I.24

8.20

8.48 1.07 7.35 6.35 6.93 4.04 X5.92 1,74

2 (x957)

3.13 9.20 5.56 I.35 1.61 8.07 11.02

3.67 1.70 4.‘4 5.4i 2.00 4.79

5

3.19 8.34 71.97 13.99 29.55

.SS.O 55.0

I .96

0.38 I.10

34.0 44.0 35.0

z.29 I .04 I.47

0.85 0.74 0.68

0.66 I.83

0.56 I.97

12.5 ICI.0 12.j

+ _-

VOL.

12.0 5.

0.50

4.38 6.16 7.23 0.86

Controls 2

3 24 Zj

26 ‘7 28

P. H. S. A. B. Ji.&c;. ;. 3. P. H. B. \v. P. M. G.

30 31 35 ~6 ‘7 25 24

42.0

30.0

I .+2

* Same patient

remonstration

of blood grouppolysacch~~~des

A small portion (cu. 5 mg) of the soluble material of sample No I, was dissolved in physiological saline (IO ml) and the solution heated in a boiling water bath for IO min. Absorption of anti-A serum (for 30 min) with the clear supematant prevented the serum from agglutinating group A red cells, but absorption of anti-B serum with the supematant did not prevent the anti-B serum from agglutinating group B red cells. It was therefore deduced that patient No. I was a group A secretor. The control sample No. 2 behaved in the same way, and was therefore also from a group A secretor. When a portion of the soluble material of control sample No. 3 was treated in the same way it prevented neither anti-A nor anti-B serum from agglutinaiing the corresponding red cells and was therefore from a non-secretor, who was later shown to be group A. With other patients the Lewis blood group type indicated whether they were non-secretors Le(a+) or secretors Le(a-) (see Table I). Determination of mcleic

acid

content

Soluble fractions were dissolved in water (0.2 mgjml) and the ultraviolet absorption determined in the range 22~~380 rnp. From the absorbancies at 260 m,u the rn~~~rn~rn possible nucleic acid contents were calculated on the assumption that pure References p. 496

SIALIC ACID AND CHRONIC BRONCHITIS

VOL. 2 (1957)

nucleic acid would have an extinction

of 1.0 when the concentration

493 was 0.05 mg/ml

and the cell thickness I cm: Several samples showed a peak absorption at 275-280 m,u, indicating that an appreciable part of this absorption was due to protein. As can be seen from Table II, there was no significant difference in nucleic acid contents between the pathological samples and the controls. Infrared

spectra

The infrared spectra of the soluble fractions of Nos. 6, IO, and 20 were almost identical over the whole of the range 700-3500 cm- I. The insoluble fractions of the same samples showed the same similarity amongst themselves, except that the insoluble fraction of No. 20 showed a strong absorption (probably due to the C=O stretching of a -COOH group) at 1730 cm-l not shown by the others or by the soluble fractions. The most prominent features of all the above spectra were the very strong broad absorption peaks shown at ca. 1650 cm-l and 1550 cm-‘, which were probably due to the C=O and N-H of the peptide links of the proteins. The sialic acid-rich mucoprotein fraction (see later) of soluble No. IO showed a spectrum little different from that of soluble No. IO. The infrared spectrum of N-acetyl sialic acid itself showed prominent absorption peaks at 1730 cm-1 (C=O of -COOH group), 1660 cm-l (C=O of N-acetyl group) and 1535 cm-l (N-H of N-acetyl group). Its complex spectra in the “finger-print” region (700-1000 cm-l) should prove extremely useful for its characterisation. Comparison of the spectra of corresponding fractions of No. 20 and No. 3 showed negligible differences except the extra peak at 1730 cm-1 in No. 20 insoluble reported above. It is concluded that infrared spectra are relatively insensitive to the difference between bronchial normal saliva, probably because the major portion of both is protein. Determination

sputum and

of sialic acid

Sialic acid contents of soluble and insoluble fractions were estimated using the standard Cu + +-Bial’s reagent described by SVENNERHOLM~. The reagent was calibrated against authentic N-acetyl sialic acid, and the differences in absorbancies at 575 rnp and 680 rnp were found linear with respect to the amount of N-acetyl sialic acid up to 75 ,ug. As can be seen from Table II, the sialic acid contents of bronchitic samples were far higher than those of the normal salivae. Sample No. I was an exceptional case and the authors are inclined to think that the patient might have been in the convalescent stage when the sputum was collected. In every case the complete spectrum was measured in the range 320-800 rnp. With two exceptions, all the samples showed a single absorption peak at 575 rnp which was typical of sialic acid. The exceptions were the insoluble fractions of No. 6, which showed an absorption peak at 460 m,u as well as 575 rnp, and No. 13, which showed a strong absorption peak at 460 m,u but none at 575 m,u. Isolation of mucoprotein fraction from spa&m

No. IO

The soluble fraction (IOO mg) of No. IO was dissolved in distilled water (I ml). Dilute hydrochloric acid was added until the cloudy precipitate just dissolved on the acid side of its isoelectric zone. The mucoprotein fraction was then precipitated by dilution with a few volumes of distilled water. The precipitate was redissolved in water and the procedure repeated. The precipitate was again dissolved in water, and

l&frmces p.

qg6

r. AXZAI, S. A. BARKER,

VOL. 2 (1957)

M. STACEY

T.tBLElI

0.017 I.120 0.102 O.lCjl O.Oj” 0.310

~60, 280

0.355 0.381 O.ljfl 0.160 0.083 O.L()O 0.201 O.ObL$ 0.280 0_3,
375 1..?00

0.

260 0.12

0.OI.j

0.3.i

0.000

o.oy

0.003

O.LCj

0.000

2jO

o.rj

0.00-j

270

0.28

0.01

260, 280 280 275 '7.5

I

both the precipitate and the supernatant solution recovered from the fractionation were dialysed and freeze-dried. The sialic acid content of the mucoprotein fraction (10.2 mg) was g.6%, while that of the fraction (84.6 mg) recovered from the supernatant was 2.68%. Since the sialic acid content of the starting material was 3.45 ‘/& the mucoprotein fraction (10.2 'A) contained 28.4 “/*of the total sialic acid. Hydrolysis 7 ofmucoprotein

fraction

The mucoprotein fraction (8.5 mg) in water (I ml) was dialysed against 0.01 N H,SO, for 3 days at o” with exchange of the outer solution twice a day. The sialic acid was then split off from the mucoprotein fraction by heating the dialysed solution at So” for I hour and recovered by dialysis. The hydrolysis of the residual mucoprotein with 0.01 N H,SO, was repeated, and more sialic acid recovered. The combined sialic acid fractions were neutralised with barium hydroxide to pH 6.0 and freeze-dried. The material remaining after the two hydrolyses with 0.01 N H&O, was submitted to further hydrolysis with z N H&GO, at 100" for 4 hours. After neutralising with barium carbonate, the hydrolysate was concentrated. Paper chromatographic analysis7 of the 0.01 N H,SO, hydrolysate in n-butanolR~~fmwes

.b.

496

VOL. 2 (1957)

SIALIC ACID ASD CHKOSIC BRONCHITIS

495

acetic acid-water (4 : I : 5), n-butanol-p~idine-water (9 : 5 : S), sec-butanol-acetoneacetic acid-water (3 : 3 : 1.5 : z.5), and set-butanol-acetic acid-water (4 : I : 5), Showed a single component when sprayed with orcinol-trichloracetic acids which had an RF identical with that of N-acetyl sialic acid. A rapid and convenient method of identification of N-acetyl sislic acid in the 0.01 N H&O, hydrolysate was by paper ionophoresis in acetate buffer, pH 5.0 at 800 V for 3 hours on Whatman No. 3 paper. under these conditions N-acetyl sialic acid was found to migrate as an acid with a mobility 0.61 that of glucuronic acid. Paper chromatographic and ionophoretic analysis of the 2 N H,SO, hydrolysate showed the presence of galactose, fucose and hexosamine. Further identification of the hexosamine showed it to be galactosamine. DISCUSSIOX In X950, RosEQ showed that a labile factor in the mucoid secretions of the human respiratory tract was able to inhibit viral hemaggl~ltination in high dilution. Certain mucoprote~nslo exhibit this same property but lose this capacity upon treatment with enzymes (e.g. those present in the influenza virus or Vibrio cholera) which split off sialic acid. These mucoproteins appear to act competitively, since there is evidence” that the influenza virus is adsorbed on the red cells by virtue of the presence c.f sialic acid in certain “receptor areas” on the surface of the cells. In chronic bronchitis the excessive production of mucoproteins containing sialic acid may therefore be part of the body’s general defence mechanism. FLOREY la has suggested that mucin production is stimulated by local mechanical and chemical irritation. He pictures the continuously moving layer of tracheal and bronchial mucus as acting both as a mechanical barrier and as a site for the adsorption of, for example, an invading influenza virus, thus attempting to prevent the infection of the sensitive cells of the trachea and bronchi. Unfortunately for the patient, the virus is able to counter-attack with an enzyme which liberates sialic acid from the mucoproteins and thus free itself.

ACKNoWLEDGE.MENTS The authors wish to thank Dr. I_. E. HOUGHTON and his colleagues for samples of bronchitis sputum, the Birmingham Accident Hospital for gifts of anti-A and anti-B serum, and Drs. BLIX AND SVENNERHOLMfor gifts of sialic acid. One of us (T.A.) wishes to thank the Uritish Council for financial assistance.

Samples of sputum from chronic bronchitis patients have been shown to contain up to five times the amount of sialic acid present in normal saliva. The actual content per ml of sputum was much greater. The sialic acid was shown to be associated with the mucoprotein

fraction.

D’apres des prdlevements, le crachat des malades atteints de bronchite chronique contient jusqu’k cinq fois plus d’acide sialique que la salive normale. La teneur reelle par ml de crachat est beaucoup plus @levee. 11 est demontre que l’acide sialique est associe Zi la fraction mucoprot~ique. R+Wirct%p. 496

T. ANZAI,

496

Sputummuster SialinsSure Sputum

S. A. BARKER,

chronischer

Bronchitispatienten

auf als im normalen

Speichel enthalten

war

vie1 hiiher.

Die SialinsZure

VOL.

M. STACEY

wiesen

(1957)

bis zu fiinfmal

ist. Der wirkliche

war mit

2

mehr

Gehalt

pro ml

der Mucoproteinfraktion

ver-

bunden. PE3IOME &,06bI

MOKPOTbI

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I Mn.MOKPOTbI

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YCTaHOBAeHO,

@aK~&feii.

HEFI
R. E. L. L. R.

H. A. F. H.

67 (1954)

105

J. WINZLER, Methods ofBiochemical Analysis, 2 (1955) 279. KLENK AND E. SCHUMANN,BBY., 7jB(1942) 1632. SVENNERHOLM, Arkiv Kemi, IO (1cg.57) 57'7. SVENNERHOLM, Acta Sot. Med. Upsalienszs, 61 (1956) 7.5. KLEVSTRAND AND A. SORDAL, Acta Chem. Scmnd., 3 (1950) 1310. 31. HOSE, Federation PYOC., 9 (1950) 390. GOTTSCHALK, Biochenz. J., 61 (1955) 298. hf. BURNET, Ann. Rev. Microbial., 6 (19.51) LZ). FLOREY, Proc. Roy. Sm. (London), 14.7 (Igj_j) 117,

Keceived

August

moth,

1957