27. Takcbayi~shi.S.. (;robe. II . von B;~ssca~t/.D. H.. and rliormnn. 11. Ultrastruc- prepnratlon.) tural a\pects of veihel .llteratlon\ In homoc!\t~nur~a.V~rcha\r'r Arch. Aht. .A 33 Wong. P. W. K.. Scliworl. V . and Komro~er.(i. M.: The b~os!nthc\~\of P;~thol Anat . 1154- 4 (1071). c)st:~thlonrnc In pntlcntc s~thhornoc!\tinur~,~. Ped~at.Rer . 2- 149 (196x1. 28. T:~ndler. H. T.. I:rlandson. R. A,, and Wbnder. E. 1.. Rihol1;lvln and mou\c 34 'The authors would l~kcto thank K. Curleq, N. Becker, and k.Jaros~u\ for thcir hepatic cell structure and funct~on.hmer. J. Pnthol.. 52 69 (1968). techn~cal:Is\l\tancc and Dr\. B. Chomet. Orville T B:llle!. and Mar) B. 29. Ti~nikawa, L.: llltrastructurnl A\pect\ of the I iver and Its I)~sordcr\(Igaker Buschman for the~ruggehtlona In the ~ntcrpretation\of braln and Iner electron Shoin. I.td.. Tok!o. 1968). micrograph\. 30. Uhlendorl, B. W.. Concrl!. E. R.. and Mudd. S. H.: tlomoc!st~nur~a:Studle, In 35 Th~sstud! &a\ \upported b! Publrc Health Servlcc Kese;~rch Grant no. KO1 tissue culture. Pediat. Kes.. 7: 645 (1073). N5OX532NlN and a grant from the Illino~\Department of Mental llealth. 31. Wong. P. W. K.. and Fresco. R.: T~\suec!\tathlon~nc in mice trei~tedw~th 36 Requests for reprint\ should be i~ddressed to: Paul W. K. Wong. M.D.. cystelne and homoser~ne.Pedlat. Res . 6 172 (1972) Department of Ped~atric.Prcsh!tcr~an-St. Luke's Medical Center. Rush 32. Wong. P. W. K.. Justice. P.. Weiss. E.. Hruby. M.. and Dlarnond. F.. A fa~n~ly Med~calSchool. Chicago, Ill. 60612 (USA). u~thfour caxs of 5.10-meth!lcnetetrah!droI'oIate rcducta\e def~clenc) (In 37. Accepted for puhlicatlon December IX. 1975.

Copqrlght O 1976 International Pediatric Research Foundat~on.Inc Prirrred irl L'..S.4

Pediat. Res. 10: 609-613 (1976) Calcium cystic fibrosis mucin

Effects of Calcium on Intestinal Mucin: Implications for Cystic Fibrosis

J. F. FORSTNER " AND G. Ci. FORSTNER

Rr.vc~archIt~srrr~rre. 771e Ho.~pital/i)r Sic.k Cltildreti. Toronto. Ot~tario,Canada

Extract whether Ca changes the physical properties of purified mucin macromolecules of intestinal or bronchial origin. A major feature of the diseaw cvstic fibrosis i\ the excessite Over the past few years a native goblet cell mucin has been concentration of mucus within ducts and glands of mucous- purified in our laboratory from rat small intestine (9, 11). It producing organs. Some mucous \ecretion\ a150 show an elelation in resembles mucins purified by others from several human and calcium concentration. LGng purified rat intestinal goblet cell animal organs (I, 4, 6, 17-19). in that it is a polydisperse viscous mucin as a model mucin, we hale inlestigated the effect of glycoprotein rich in carbohydrate. containing high quantities of millimolar additions i 1 25 mM) of Ca< I, on the phj\ical properties serine, threonine. and proline within the peptide core. of the mucin. I\otonicity of incubation media was prelerted in order We have used rat goblet cell mucin as a model for the to mimic in vivo conditions. CaCl, (&I5 mM) caused a 1533% investigation of Ca-mucin interaction. Previously we showed that decrease in lircositj, no change in electrophoretic mobilit\ in rat mucin binds Cn ions. especially under conditions of low ionic acrylamide gels, and a 20 30% decrease in solubility of the mucin. strength. but that during binding Ca caused on14 minor physical k 4 Solubility changes were retersed by the addition of 0 1 I20 mM) changes in the mucin (8). In the present study we have measured to incubation\. lnsolubilitj war also produced in incubation5 of the effects of Ca upon some of the phys~calproperties of mucin in mucin with a mixture of soluble intestinal contents I haCI washings). isotonic media in order to mimic more closely it1 vi~~ocond~tiorla. These findings strongly suggest that the mucin became 5maller and Our results indicate that Ca may alter the three-dimensional more dense as calcium was added, a proce5, most probably achieved architecture of mucin so as to increase its densit) and exclude by loss of intramolecular water. water. It is postulated that this mechanism may be involved in the formation of mucous plugs in cystic fibrosis. Speculation

It is hypothesized that elevated concentrations of calcium within glycoprotein secretions of patient with cystic fibrosis may substanti- ally increase the density and insolubility of mucins, promoting the The preparation of goblet cell mucin (GCM) from rat intestine. formation of mucous "plugs." radioactive labeling of the mucin using precursor [l'Cljglucosa- mine. the techniques of polyacrylamide disc gel electrophoresis. analytic ultracentrifugation, and measurement of solubility of the Mucous secretions of patients with cystic fibrosis are excessive mucin have been described in detail in earlier publications (8, 9, in amount in salivary glands, bronchi, intestine, gall bladder, and I I). Minor modifications and specific details are included as cervix (7, 12, 14). The calcium concentration in man) of these appropriate under Results. secretions is also high, ranging from "slightly elevated" to over Intestinal washings, when required for solubilitl experiments, 100-fold in excess of normal (12). It has been hypothesized that Ca were collected by washing the entire small intestine of each of 10 may contribute to the formation of mucous "plugs" in glands and rats with approximately 20 ml 0.15 M NaCI. The washings were ducts by causing polymerization, aggregation. or gel formation of pooled. centrifuged at 4" at 30,000 x g for 15 min to remove otherwise normal mucin macromolecules. A few studies have particulate material, and the suernatant solutions saved. The shown that Ca is responsible for aggregation of small salivary soluble supernatant fluid was stored frozen in a dilute state or else proteins (4. 5. 13). but no studies have been performed to discover concentrated isotonically 10-fold by an Amicon Dia-Flo ultrafilter 610 FORSTNER AND FORSTNER and then stored frozen as a concentrate. Both dilute and concen- trated washing solutions were thawed immediately before use in solubility experiments with goblet cell mucin. The water used in the preparation of all solutions was rendered ion free as described earlier (8). Chloride salts of various cations (Ba, Zn, Mn, Mg) were made using anhydrous powders (oven dried to constant welght) of the chloride salts. The usual buffers in solubility studies included 0.01 M Tris-HCI in 0.14 M NaCI, pH 7.4, or 0.155 M Tris-HCI, pH 7.4. In a few instances the buffer 'C' used was 0.15 M Hepes, pH 7.4. and the results were identical with those obtained usine.. Tris or Tris-NaCI buffers. Viscosity of goblet cell mucin was measured in a Cannon- Ubbelohde semimicro capillar) viscometer at 20" as described earlier (8) and in a Brookfield Synchrolectric model LVT rotary viscometer over a wide range of shear rates (22-230 sec '). Units of viscosity were centistokes and centipoises for capillary and

rotary viscometry. respectively, and were converted into units of viscosity "number" by the following equation: qsp/c = -!+ c. where 7, is the viscosity of the solvent, q is the mucin viscosity in that solvent, and c is the concentration of goblet cell mucin in grams, dry weight per 100 ml. Viscosity of the mucin at zero shear was obtained from viscosity data over a range of shear rates extrapolated to zero.

FFFhCT OF Ca ON VISCOSITY OF GOHLtT CE1.L MUClN The viscosity of mucin in 0.i55 M Tris-HCI, pH 7.4, was examined over a range of mucin concentration and shear rate values using both capillary and rotary viscometry (Fig. 1). In the absence of Ca the viscosit, varied with both shear rate and mucin concentration. In general. the viscosity was higher at low shear Fig. 2. Polyacryl;imidc disc gel electrophoresis of mucin. Goblet cell rates (- - ) and underwent a sharp increase at concentrations of mucin above 0.5 g/ 100 ml. at which visible gelation began. At the mucin (32 pg dry wt) was mixed with hromphenol blue dye in 60%)sucrose higher shear rates of capillary viscometry (--) gelation was not with or without CaCI, (X mM) in a final volume of 0.05 ml. Sample\ were visible. In both cases the addition of CaCI, (5-25 mM) caused a applied to 4% acrylamide gels made in 0.15 M Tris-borate huffer, pH 8.6, decrease in viscosity, which was approximately 15-l8%, for Lero with or without the addition of 8 mM CaCI,. Gels were run for 55 niin at 2 shear and 33% for high shear conditions in 10 mM CaC1, (Fig. 1). mA/tuhe and subsequently stained with periodic acid-Schiff reagcnt. Ingel At the higher concentrations of mucin, Ca appeared to retard gel I there was no Ca in the mucin sumplc or the gel; in gel 2 there was CaCI, formation at zero shear. in the gel and the buffer during electrophoresis, and In gel 3 there wah Under no conditions of goblet cell niucin concentration. temper- CaCI, in the mucin sample, the gel. and the huffer during electrophoresis. ature variation (20"- 37"), shear rate, or CaCI, concentration (1 -50 mM) did Ca cause an elevation in viscosity suggestive of gelation or polymerization of the macromolecule.

POLYhCKYLAM1L)E DISC GEL. ELtCTKOPHORESIS In an attempt to rule out dissociation by Ca, which rnight have accounted for the lowering of viscosity, goblet cell mucin was subjected to electrophoresis in 4% polyacrylamide gels at pH 8.6 in the presence and absence of CaCI, in the gel and buffer systems (Fig. 2). As described earlier (9). the basic pattern consisted of one broad periodic acid-Schiff-positive band near the origin and two faint bands below. In the presence nf C;ICI ,(3, 8, :lnd 25 mM), the mucin did not undergo any detectable change in band widths. intensity of stain, or mobility.

SEDIMENTATION Vt.I.OCITY ST111)ItS 02 0.4 06 08 rnucln conc (~/IOO m~) Goblet cell mucin (2.5 mg dry wt/ml) was subjected to boundary Fig. I. Viscosity ofgohlet cell mucin. Rotary viscometry was performed ultracentrifugation in 0.01 M Tris-HCI-0.14 M NaCl buffer, pH over a range of shear rates (22 230 sec ') extrapolated to give zero shear 7.4, containing 0, 1. 5, and I0 mM CaCI,. In the absence of Ca, the rate values for viscosity (0 0).q,,/c refers to viscosity number. Schlieren optical pattern consisted of a single peak with a leading Capillary viscometry values (Wpm) were obtained using a Cannon- shoulder (previously published (10)). No change in pattern was Uhhelohde sem~microviscomcter. + and - refer to the presence or observed in the presence of CaCI,. Thus, there was no indication, absence. respectively, of 10 mM CaCI, in the buffer solvent which was by either gel electrophoresis or sedimentation pattern, that Ca had either 0.155 M Tris-HCI, pH 7.4, or 0.01 M Tris-HCI-0.14 M NaCI, pH caused the mucin to dissociate into lighter or smaller fragments. 7.4. Each v~scosity value was the average of triplicate determinations It was observed, however, that the addition of CaCI, to GCM which did not exceed a range of more than +0.05 viscosity number samples caused a decrease (18% a1 10 mM CaCI,) in the units. measurable concentration of GCM, as determined from Schlieren Ca-MUCIN INTERACTION IN Ct

peak areas (Table I. colunltr 3).This happened despite the original addition of exactly 2.5 mg niucin/ml to each cell, and uas associated with a small but significant increase in calculated ,\ ,,, ,, values (colut~tr4). It was suspected that some of the mucin ma\ have aggregated with CaCI, and sediniented rapidly to the bottoni of the ultracentrifuge cell before the attainment of maximuni rotor speed. Thus only the remaining soluble mucin was detected in Schlieren tracings. By comparing test samples with control GCM samples (no added CaC1,) having the same GCM concentration. .v,,,,, values were seen to be identical (Table 2). Thus the remaining MOLARITY 0 01 0 155 0 30 soluble GCM in Ca-containing cells possessed sedimentation 4 a , properties characteristic of the intact control mucin. Since Ca 0 0 08 0 16 0 24 appeared to have primar~lyaffected GCM solubility, separkite IONIC STRENGTH (1/2 cr2) experiments were conducted to examine solubilit! more directl!. Fig. 3. Soluhilit) of goblet cell mucin. Radioact~vclq lahcled ("C) rnucln bas ~ncubatedin Tr~s-HC'Ibuffers of increas~ngmolarit! for 30 min SO1 CIBIL ITY at room temperaturc. Each incubation conta~ned0.67 nlg dr! \\t of rnucin. bout i001.000 cpm, and wa\ 0.2 rnl ~nvolume. After centrifugation at A soluble mixture of mucin (8.33 nig dry wt/ml) in 0.01 M 12.500 r g for 5 mln, rad~oactivityin the pcllet and supernatant solutions Tris-HCI-0.14 M NaCI. pH 7.4, became visibly insoluble at 4. 25. and 37" after the addition of CaCI, (final concentration 10 mM). was determined and solubilit! baa calculated a\ a percentage. Ionic strength was determ~ncd from the formula 0.5 c~5Each value Because of the scarcity of mucin, however. and the subjectivit! of represents the mean of values obtained from thrcc separate incuhat~ons. visual inspection for solubility, a quantitative microcentrifugation tach vcrtic;~lhar represents the total range ol'valuea obtained. Kadioactiv- technique (8) was employed. Briell!, this method involved the 114 determination uas performed on dupl~catesample5 b) \c~nt~llation incubation of '"C-radioactively labeled mucin (0.25 mg) in isotonic counting in Bray's aoIut1011.Totill recovery of rnuc~nradio:~ctiv~ty in pellet buffer to which CaCI, was added. followed by centrifugat~tion of plus supernatant was 100"/r + 2'4. aliquots (0.06 ml) in microhematocrit capillary tubes. Solubility was calculated as a percentage of control (Ca-free) samples by measuring the radioactivity of supernatant and pellet fractions.

Table I. Sedimetrtutiotr ~,eloc,itj.of ,goblet ct.11 ttlucitr (6C'M)'

GCM added, GCM meaaured, CaCl,, mM mg/rn! mg/ml Yo "0, w - Control 0 2.5 2.5 1 1.95 0 10 20 30 Test I 2.5 2.5 (0) 1 1.96 CaC12 jm~) EDTA (ITIM) Control 0 2.18 2.18 12.20 Test 5 2.5 2.IX(13) 12.17 Fig. 4. Solubility of mucin with CLIand LDTA. "C-Labeled goblet cell Control 0 2.05 2.05 12.54 mucin (0.25 mg) was incubated in 0.01 M Tris-HCI-0.14 M NaCI, pH 7.4, Test 10 2.5 2.05 (18) 12.52 containing increasing amounts of CaCI, and solubility determined as descrihed in the legend to F~gurc4. The effect of Ca is shown on the left. ' CaCI, and

In subsequent experiments the mucin was incubated in isotonic intestinal lumen. It seems likely, therefore, that excessive calciur buffer containing 15 mM CaCI, to which increasing Na, EDTA if present within cystic fibrosis mucous secretions, could substar. was added (Fig. 4, right). With increasing EDTA, the solubilit? of tially increase the density and reduce the solubility of mucinc mucin was gradually restored to about 90%' of control levels. Whether other divalent cations play an important physiologic rol Efjcect ofCa on M~icinin lr~ie.stinalL~it~lirral Washir~,qs. In order in altering mucins is open to conjecture. Only calcium is likely t, to find out whether intestinal mucin would respond to Ca in the be present in sufficient concentration extracellularly to have ar, same fashion within a more physiologic environment, the following appreciable effect. experiment was performed. 14C-Labeled goblet cell mucin was Decreased hydration and size, increased density, and loss of incubated with or without CaCI, for 30 min at 37" in either dilute solubility indicate that the mucin sheet may become more granular or 10-fold concentrated intestinal washings. Figure 5 indicates that and less gelatinous in the presence of calcium. These changes nu\ in each case Ca caused a progressive drop in solubility to explain the "hyperpermeability" described by Gibson et al. (12) for approximately 50% of control values. Thus goblet cell mucin cystic fibrosis gastrointestinal mucosa in ~,ivo,and for salivary became partly insoluble with Ca in a medium containing isotonic mucous films in vitro. Increased granularity might interfere with NaCl and normal soluble luminal contents. which are presumed to the spreading quality of the mucin over thc epithelial surface, include bacterial products, digested food products, pancreatic and possibly decreasing its uniformity and leaving denuded hyper- other enzymes, bile salts, cellular debris, and other proteins, lipids, permeable areas of intestinal surface. glycoproteins, and ions. The significance of our findings lies in the fact that mucinc. Specificity of Ca Ions. It was of interest to determine whether normally account for the viscosity of bronchial and intestinal the solubility of mucin was affected by other divalent or monoval- secretions, and are major components of obstructive plugs in clstic ent cations. As seen in Table 2, the Ca effect was arbitrarily desig- fibrosis (14). Previous authors (5, 14) have shown that small nated as 1.00 for 25 mM CaCI,, and the effectiveness of other molecular weight glycoproteins in saliva are aggregated by addi- cations related to it. Zn. Mn, and Mg were all found to be slighly tional calcium, and that the aggregation is reversed by EDTA. more effective, and Ba slightly less effective than Ca. Tris-HCI Warton and Blomfeld (22) have described hydroxyapatite crystals and NaCl were much less effective. Thus monovalent cations had in saliva of cystic fibrosis. Although these components may very little effect, but all of the divalent cations tested were effec- contribute to the turbidity which is characteristic of cystic fibrosis tive and there did not seem to be a high degree of specificity for saliva (7. 13), they bear little resemblance to highly glycosylated Ca. mucin macromolecules. Whether rat intestinal mucin is approprl- ate as a model for human intestinal or bronchial niucin has not DISCUSSION been unequivocally demonstrated. Rat mucin does, however. possess most of the features characteristic (9) of intact epithelial Our results indicate that calcium, in concentrations known to mucins isolated from many organs, including a bronchial mucin obtain in some secretions in cystic fibrosis (13). crucially modifies from cystic fibrosis patients (19). the behavior of a model epithelial rnucin under isotonic conditions, In addition to the implications for cystic fibrosis, the present making it denser, less viscous, and more insoluble. The fact that study raises certain questions relevant to the normal function of viscosity fell but the molecule clearly became heavier, as indicated mucins. It has been hypothesized, for example, that mucous by its solubility characteristics, strongly suggests that the rnucin provides protective "waterproofing" for epithelial surfaces (16, became smaller and more dense as calcium was added, a process 20). Anatomically, the mucous blanket in the intestine occupies a most probably achieved by loss of intramolecular water. Insolubil- zone through which all solutes destined for absorption must ity, which might be taken as the most dramatic and advanced penetrate. Therefore, agents such as Ca ions which alter mucous evidence of this process, was not seen in hypotonic media (8). structure might be expected to influence water or solute transport. Clearly, therefore, the divalent cation effect required a near This may be analogous to the effects of Ca upon ion fluxes through isotonic background, and these conditions are precisely those artificial phospholipid membranes (15) or collagen membranes (2), which are encountered in secretions in vivo (12). Insolubility was where Ca produces a "tightening" of structure and neutralization also not limited to simple test tube conditions. but could be of membrane negative charge. demonstrated in the presence of complex organlc contents of the The interaction of Ca and intestinal mucin may also have physiologic significance within normal goblet cells of the intestine. Warner and Coleman (21) have shown recently by electron probe analysis that goblet cells contain a major share of the total intestina! Ca. The reason for this distribution is not clear. but in view of the present work, it is possible that Ca helps to keep mucin dense or compact within the secretory granules of the cell. Once released into the lumen, the rnucin would then be diluted, the Ca removed, and the mucin would swell and spread over the mucosal surface.

SUMMARY

Millimolar additions (8-25 mM) of CaC1, to rat intestinal goblet cell mucin in isotonic buffer media caused a 15-33% de- crease in viscosity, no change in electrophoretic mobility, and a 20-30% decrease in solubility of the mucin. If elevated Ca con- Fig. 5. Solubility of mucin in intestinal washings. Intestinal washings centrations similarly affect the solubility of human mucins, they were obtained from 10 rats using 20 ml 0.15 M NaCl/rat. The washings could contribute to mucous "plug" formation in cystic fibrosis. were centrifuged at 30,000 x g for 30 min to remove particulate matter and the supernatant material either stored or concentrated isoton~cally10-fold, REFERENCES AND NOTES and then stofed at 20°. incubation of mucin in the intestinal washing solutions was carried out as described in the legend of Figure 5 with I. Allen, A,, and Snary. D.: The structure and function of gastrlc mucous. Gut. 13. increasing CaCI, additions. Values represent the average (and total range 666 (1972). 2. Bartolini. A.. Gliozzi. A,, and R~chardson,I. W.. Electrolytes control llows of of values) of three incubations. C and D refer to solubility in concentrated water and sucrose through collagen membranes. J. Membrane Biol., 13. 283 or dilute intestinal washings. (1973). HEMOGLOBIN SC

3. Bella. A,, and Kim, Y. S.: Rat small intestinal mucin: Isolation and characterira- 585 (1967). tion of a water-soluble mucin fraction. Arch. B~ochem.Biophys., 150. 679 14. Jakowska, S.: Cystic Fibrosis and Related Human and Animal D~seases,p. 162 (1972). Gordon and Breach Scient~ficPublishing Co.. New York). 4. Bettelheim, F. A.: Light-scattering studies of bovine submaxillary mucin. 15. Joos, R. W, and Carr. C. W.: The binding of calcium in mlxtures of Blochim. Biophys. Acta. 236: 702 (1971). phospholipids. Proc. Soc. Exp. Biol. Med., 124: 1268 (1967). 5. Boat, T. F.. Wlesman. U. N., and Pallavicini, J. C.: Purificat~onand propertlea of 16. Negus, V. Mucus. Proc. Roy. Sac. Med., 60: 75 (1967). the calcium-precipitable proteln In submaxlllary saliva of normal and cystlc 17. Pigman, W., and Herp. A,: Aspects biochlmiques et physiologiques des fibrosis subjects. Pediat. Res., 8- 531 (1974). glycoproteldes des secretions muqueuaes. Ann. Anat. Pathol., 17. 227 (1972). 6. Buddecke. E.: In: A. Gottschalk M~scellaneousglycoproteins In glycoproteins. 18. Roberts. G. P.: Isolation and character~zationof glycoproteins from sputum. Eur. thelr composition, structure and function, p. 558 (Elseb~erPublishing Co., J. Biochem., 50: 265 (1974). Amsterdam, 1966). 19. Roussel. P., Lamblin, G., and Degand, P.: Heterogeneity of the carbohydrate 7. di Sant'Agnese, P. A,, and Talamo, R. C: Pathogenesis and physiopathology of chams of sulfated bronchial glycoproteins ~solatedfrom a patlent suffering cystic fibrosis of the pancreas. N. Engl. J. Med., 277: 1287 (1967). from cystic fibrosis. J. Biol. Chem., 250: 21 14 (1975). 8. Forstner. J. F., and Forstner, G G.: Calcium binding to intestinal goblet cell 20. Taylor. M. The origins and funct~onsof nasal mucus. Laryngoscope. 34: 612 mucln Biochim. Biophys. Acta. 386: 283 (1975). (1974). 9. Forstner, J. F., Jabhal, I.. and Forstner, G G.: Goblet cell mucin of rat small 21. Warner. R. R., and Coleman, J. R.: Electron probe analysis of calcium transport intestine: Chemical and physical character~zation.Can. J. Biochem., 51. 1154 by small intestme. J. Cell Biol., 64: 54 (1975) (1973). 22. Warton, K. L., and Blomficld. J.: Hydroxyapat~tein the pathogenesis of cyst~c 10. Forstner, J. F., and Manery, J. F.: Calcium binding by human erythrocyte fibrosis. Brit. Med. J,3. 570 (1971). membranes. B~ochem.J.. 124: 563 (1971) 23. The authors are indebted to the Medical Research Counc~lof Canada and the I I. Forstner. J. F , Taichman. N., Kaln~ns,V., and Forstner. G. G.: Intest~nalgoblet Canadian Cystic Fibrosis Foundation for financial support, to Dr. Dav~dKells cell mucus isolation and ~dent~ficationby immunofluorescence of a goblet cell of the Department of Biochemistry. University of Toronto, for performing glycoproteln. J. Cell Sci.. 12. 585 (1973). analyt~cultracentrifugation studies, and to Mrs. Cecilia Feng for technical 12. Gibson, L. E., Matthews. W. J.. Minihan. P. T., and Pattl, J. A,: Relating assistance. mucous, calc~umand sweat in a new concept of cystic fibrosis. Ped~atrics.48. 24. Requests for reprints should be addressed to: J. F. Forstner, M.D., Research 695 (1971). Institute, The Hoap~talfor S~ckChildren, 555 University Ave., Toronto. 13. Gugler. E. C., Pallavicinl, J. C.. Swerdlow, H.. and di Sant'Agnese, P.: The role Ontario M5G 1x8 (Canada). of calcium In submax~llarysal~vo of patients with cystic fibrosis. J. Ped~at..71: 25. Accepted for publication December 18. 1975.

Copyright @ 1976 lnternat~onalPediatric Research Foundation, Inc Prinred in U.S.A.

~ediat.Res. 10: 61 3-620 (1976) Erythrocytes sickle cell anemia hemoglobin a-thalassemia

Sickle Cell Syndromes. I. Hemoglobin SC-a-Thalassemia

GEORGE R. HONIG,'5" UNSAL GUNAY, R. GEORGE MASON. LOYDA N. VIDA, AND CHRISTINE FERENC

Univerriry Illinors Srckle Cell Cetrler and Deparrnzerzr of Pediatrics, Ihe Abraham Lirrcoln School of Medicine, Llniversiry of Illinois Medical Center, Chicago, Illinois, LISA

Extract months typical findings of mild hemoglobin H disease appeared, with HbH making up 6.5% of the total hemoglobin. Hematologic and globin synthesis studies were performed in a black P~ericznfzrnil~~ ..J in which the genes fnr n-thz!zssen?i- 2nd Speculation hemoglobins (Hb) S and C were segregating. The following distribution of these abnormalities was found: father, sickle cell The presence of CY-thalassemia in the proband of this study trait + n-thalassemia; mother, HbC trait + n-tbalassemia, proposi- appeared to modify her HbSC disease so as to reduce its clinical tus, HbSC + a-thalassemia; older sibling, n-thalassemia trait; severity as well as its pathologic potential. The ameliorative effect of and younger sibling, hemoglobin H disease. n-thalassemia would appear to be related to a reduction in the The child with HbSC-0-thalassemia demonstrated more setere intracellular hemoglobin concentration in the patient's erythrocytes, anemia and a more hemolytic picture than is typical of HbSC but other factors may also be responsible for these changes. Further disease. Her erythrocytes exhibited decreased osmotic fragility in study of genetically modified sickle hemoglobinopathy syndromes comparison with HbSC erythrocytes, but had an indistinguishable may ultimately aid in the development of effective means for the oxygen equilibrium curve and 2,3-diphosphoglycerate (2,3-DPG) treatment of these disorders. level. Erythrocyte sickling in the patient, however, was significantly reduced, with less than 35% sickle forms observed at nearly com- plete oxygen desaturation. The clinical expression of sickling disorders may vary widely, The sibling with hemoglobin H disease exhibited 26% Bart's even among individuals having an apparently identical form of (7,)hemoglobin at birth, a level comparable with that seen in in- sickle cell disease. This variability has stimulated investigation of fants with HbH disease in Far Eastern populations. At age 5 the role of genetic as well as nongenetic factors in the hematologic