h2o, concerning formation - jci

OSMOTICPRESSUREMEASUREMENTSOF SERUMAND ASCITICFLUID IN PATIENTS WITH CIRRHOSIS OF THE LIVER

BURTONGIGES AND HENRYG. KUNKEL

(From the Hospital of The Rockefeller Institute for Medical Research, N'ew York, N. Y.)

(Submitted for publication July 17, 1953; accepted October 7, 1953)

Numerous contributions regarding the patho-genesis of ascites and edema have been made sinceStarling's first description of the balance betweenserum and tissue hydrostatic and osmotic forces.The original concept (1) proposed that, at equi-librium, serum protein osmotic pressure minustissue fluid osmotic pressure equals capillary hy-drostatic pressure minus tissue hydrostatic pres-sure. Direct application of this relationship to theproblem of ascites received relatively little atten-tion until recently when Mankin and Lowell (2)and James (3) employed this approach for theapproximation of the degree of elevation of portalpressure. Further clarification of the applicationsand limitations of this principle to the pressure re-lationships in the formation of ascites representedthe primary purpose of the present study.

The importance of the serum protein osmoticpressure was emphasized in early studies suggest-ing that a critical level existed, approximately 240to 270 mm. H2O, below which edema would oc-cur (4-8) This view was questioned by Kylin,however, who performed plasmapheresis experi-ments in rabbits and was able to obtain very lowserum osmotic pressure values without edema(9). However, when he added salt to the diet,the animals became edematous even at serumosmotic pressure levels near normal. The clinicalcounterpart of these experiments appears in thecases described in the literature (10) in whichedema was absent at serum osmotic pressuresbelow 200 mm.

A similar concept has developed concerning therelationship of the serum osmotic pressure to theformation of ascites. Butt, Snell, and Keys (11)found that ascites occurred most frequently whenthe serum osmotic pressure was below 240 mm.Bjorneboe, Brun, and Raaschou (12) emphasizedthis critical level and concluded that a reducedserum osmotic pressure was the principal factorin the pathogenesis of ascites and that portal hy-pertension played a subordinate role. As they

pointed out, the data were obtained from patientswith cirrhosis following hepatitis. Other investi-gators (13) reached similar conclusions statingthat ascites was probably not due to liver diseasewhen the serum osmotic pressure was greater than300 mm. H20. Armstrong (14) suggested acritical level for the "effective" osmotic pressure(serum osmotic pressure minus ascitic osmoticpressure) below which ascites occurs.

Indirect evidence against the concept of a strictrelationship between serum osmotic pressure andascites formation has been brought forth by severalinvestigators (15, 16). Evidence was obtainedin the present study indicating that the type ofcirrhosis is an important factor to be consideredin evaluating the relationship between ascites andserum osmotic pressure levels.

METHODS

Osmotic pressure measurements were made on serumand ascitic fluid from 46 patients with various types ofcirrhosis of the liver, and from a few patients withChiari's syndrome, idiopathic hypoproteinemia, nephroticsyndrome, and carcinoma of the ovary. The patients weremaintained in a state of equilibrium by a controlled lowsodium diet, and were neither increasing nor decreasingthe amount of ascites. None had sufficient fluid to pro-duce a tightly distended abdomen. Comparisons betweenserum and ascitic fluid were made in 20 patients onsamples obtained simultaneously.

The apparatus 1 used for the measurement of osmoticpressure was patterned after the Hepp osmometer (17),and consisted of an osmotic cell, horizontal capillary, andwater manometer. The osmotic cell was made of twolucite plates (87 by 87 by 19 mm.) held together withperforating screws. The upper plate had a circularhole 50 mm. in diameter, and the lower plate had a holejust large enough to admit the tip of the capillary. Be-tween the plates were a rubber gasket, membrane, anddisc of filter paper moistened with the solution describedbelow. The purpose of the filter paper (Schleicher andSchuell number 604, 50 mm. diameter) was to preventdrying of the membrane, obliterate dead space, and allowrapid transfer of fluid across the membrane. Its use

1Constructed by Mr. C. Galati (4).

257


has been recommended by numerous investigators (2, 18-21). The horizontal capillary was a 1 ml. pipette bentto an L-shape. The tip of the short limb was grounddown to fit tightly into the hole in the lower plate. Theother end was connected to the manometer. The ma-nometer consisted of two long glass tubes filled with waterand mounted vertically on a wooden frame. The tubeswere connected to each other, to the horizontal capillary,and to a levelling bulb which rested on.a balancing chain.The levels of the menisci were read on a millimeter scaleplaced behind them, and the difference between them repre-sented the hydrostatic pressure exerted on the solution inthe horizontal capillary. The meniscus of this latter solu-tion was observed in a horizontal cathetometer.

When the ionic strength of the solution outside themembrane exceeds 0.1 Molar concentration, the osmoticpressure is not affected by small changes in pH, carbondioxide tension, or salt concentration (19, 22, 23). Inthe present experiments, 0.145- M sodium chloride wasused as the outer solution in the filter paper and hori-zontal capillary. Saline has been shown to be as effectiveas an ultrafiltrate of serum, although equilibrium maytake somewhat longer (24). In the early determinations,0.15 Mphosphate buffer was used, but no differences wereencountered. The membranes employed in this studywere mostly the superdense ultrafine filters of Schleicherand Schuell. The diameter was 75 mm., the averagepore size less than 5 mu, and the "T" values 2 exceeded500 minutes. Although these membranes were muchslower than the more permeable "very dense" membranes,many of the latter were found to allow passage of serumproteins and were therefore unsatisfactory. The mem-branes were stored in 1 per cent HCI at 40 C. to preventbacterial contamination. They were washed in salinesolution for several days before being used (25, 26).Membranes handled in this fashion were suitable for sixto eight weeks. In earlier determinations, membraneswere prepared in the laboratory according to the tech-nique of Pierce (27), as modified by Mankin and Lowell(2). It was subsequently decided to use the membranesprepared commercially, however, since special apparatusfor the rigid control of humidity and temperature is usu-ally required to obtain reproducible pore size, thickness,and permeability.

For the actual measurement, 2.0 ml. of the sample weredelivered onto the membrane, and the cell was covered.The manometer was then readj usted frequently untilthe meniscus in the horizontal capillary remained station-ary for a minimum of thirty minutes. The usual lengthof time required to attain equilibrium was two to sixhours. Leakage of protein through the membrane wassuspected if the pressure was not sustained for the re-quired time; it was proven by detecting faint traces ofprotein in the solution in the horizontal capillary. Cor-rection factors for the capillarity of the horizontal capil-

2 "T" value is the time required for 100 ml. of dis-tilled water to flow through 100 sq. cm. of membranearea, at room temperature, under a pressure of 760 mm.Hg (Schleicher and Schuell).

Prsesent No. of -L_ser'ie5 cases * U, *

rip200 300 400 500

EAsciter_~ 0 No ascites

Bjoi'neboe No. of E _Let al. caxes F - R

200 300 400 500Serum OF in nm. H20

FIG. 1. SERUMOSMOTIC PRESSURELEVELS IN 46 PA-TIENTS WITH CIRRHOSIS WITH AND WITHOUT ASCITESILLUSTRATED GRAPHICALLY AND COMPAREDWITH LEVELSFOUNDIN A SPECIAL SERIES REPORTEDFROM DENMARK(12)

lary and the hydrostatic difference between the sampleand the outer solution level were determined by the nethydrostatic pressure required to prevent the movementof the meniscus with saline on both sides of the mem-branes. When the membrane is in place, the same sidemust face upward each time (24, 26). The determina-tions were made at room temperature since, in this typeof osmometer, the osmotic pressure is not altered by minorchanges in temperature (17). The error of the method(2 to 6 per cent) corresponds to that obtained by previousinvestigators whether or not temperature control wasemployed (2, 12, 19-22). This error was obtained bymeasuring samples of a standard 6 per cent albumin solu-tion ten times. The range of results was 330 ± 7 mm.H2O (2 per cent error).

RESULTS

Serum osmotic pressure determinations in vari-ous types of cirrhosis are plotted in Figure 1.Values below 260 millimeters of water are uni-formly associated with ascites. In contrast toBjorneboe's series, however, ascites also occurreddespite serum osmotic pressure levels up to 420mm. of water. In an attempt to explain this ap-parent discrepancy, the cases were divided intothree etiologic groups (Figure 2). In five casesof biliary cirrhosis (four primary and one second-ary), the serum osmotic pressure exceeded 340mm. of water. None of these patients had ascites,although portal hypertension was suggested inthree by the presence of esophageal varices. In-cluded in the classification "cirrhosis of unknownetiology" are patients in whom no history of al-coholism or nutritional deficiencies was obtained.This group consists mostly of young adult femaleswho have been described elsewhere (28). Some

258

OSMOTIC PRESSUREOF SERUMAND ASCITIC FLUID IN CIRRHOSIS

0

0.

.4)

rim.H20

500o

0

0

Sosob9"0

0

Bili Iy

80

3001-

200 _

100-

*Ao:itesoNOazcites

FIG. 2. COMPARISONOF SERUM OSMOTIC PRESSUREMEASUREMENTSIN PATIENTS WITH VARIOUS TYPES OF

CIRRHOSISThe wide spread of values associated with ascites in

patients with alcoholic-nutritional type of cirrhosis isapparent.

of the cases represented cirrhosis following infec-tious hepatitis. There is a rather striking parallelbetween the serum osmotic pressure values ofthese patients with ascites and those describedby Bjmrneboe, Brun, and Raaschou (12). In bothgroups, ascites was present when the serum osmo-

tic pressure was below 260 mm. of water, andabsent if above this level. The patients with al-coholic cirrhosis, however, had ascites at levels ofserum osmotic pressure up to 430 mm. of water.The studies mentioned above (12, 13) were limitedto cases of cirrhosis following hepatitis and didnot include those related to alcoholism. From thepresent data, therefore, it would appear that ascitesin patients with cirrhosis who have a high serum

osmotic pressure is more frequently associatedwith the alcoholic nutritional type. The absence ofa good correlation between serum osmotic pres-

sure and ascites is further emphasized in patientswith nephrosis or idiopathic hypoproteinemia. Intwo such patients, ascites developed at very lowserum osmotic pressure levels and disappearedafter diuresis despite little change in these lowvalues. The findings agree with those of Mun-twyler, Way, Binns, and Myers (10).

Since the difference between serum and ascitic

fluid osmotic pressures is equal to the differencebetween the hydrostatic pressures of these fluidsin accordance with Starling's hypothesis (1, 2,29), it seemed of interest to determine this osmoticdifference in an attempt to evaluate the hydro-static difference. The degree of elevation of thishydrostatic difference is related to the degree ofelevation of the portal pressure because the por-tal bed represents the chief venous system in equi-librium with the ascitic fluid. The hydrostaticdifference thus represents the "effective portalpressure" and not the total portal pressure. Inorder to obtain the total portal pressure the ascitichydrostatic pressure would have to be determinedand added to the "effective portal pressure." Dif-ficulties in evaluating ascitic hydrostatic pressureas well as the realization that other venous chan-nels in addition to the portal system might in-fluence the equilibrium with ascitic fluid, made itpreferable not to deal with the true portal pres-sure in this study. The term "effective portalpressure" is used in quotes because of the manyknown and unknown uncertainties involved in itsindirect estimation from osmotic pressure differ-ences.

Serial determinations of the osmotic pressuredifference between serum and ascitic fluid demon-strated the constancy of this difference in individ-

*3 A

H20 F-Alc. nutrit. cizmhosis --izllAl-lF:l400

Bo0

200

100

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15Case No.

Serum ,H Ascitic o5notic pressureosmotic . Osmotic differencepretZwuAJefetive povtal pr'essure)

FIG. 3. SERUMAND ASCITIC FLUID OSMOTICPRESSURELEVELS IN PATIENTS WITH ASCITES CHARTEDTO SHOWTHE SERuM-AScITIc OSMOTIC PREssURE DIFFERENCE IN-DICATING "EFFECTIVE PORTAL PRESSURE" IN CERTAINCASES

259

BlURTON GIGES AND HENRYG. KUNKEL

ual patients who showed little change in their gen-eral condition while being maintained in a steadystate of protein equilibrium by the use of a con-trolled low sodium intake. In Figure 3, the serumand ascitic osmotic pressure values for 15 patientshave been plotted. In 9 patients with alcoholicnutritional cirrhosis and in 1 patient with Chiari'ssyndrome, the osmotic difference (effective por-tal pressure) exceeded 150 mm. of water. In 2patients with cirrhosis of unknown etiology, it wasapproximately 135 mm. of water. In Case 5, aporta-caval anastomosis was performed becauseof varices and refractory ascites. Postoperatively,the effective portal pressure fell only slightly,which correlated with other evidence of a poorlyfunctioning shunt, namely, persistent ascites anda continued delayed portal circulation time of 31seconds (30). The results in the patient withcarcinoma of the ovary, similar to those obtainedby others (11, 12) indicate a normal effectiveportal pressure.

An important limitation in the use of osmoticpressure measurements for the determination of"effective portal pressure" was found in patientswhere the serum osmotic pressure was close to orlower than the "effective portal pressure." Thissituation, assuming protein equilibrium, wouldbring about a maximum reduction of ascitic fluidosmotic pressure until at very low serum proteinlevels the ascitic fluid would no longer reflect theequilibrium conditions. In the present study itbecame apparent that in those cases where the

M.

h0oM0r

20oF

0oo-

Serumn fflAcitic ounotic pCsuVoamou ^}Osmotic diffcence

pres3t:e .pL,esE:aLectiportal jwewsure)

A B C D

FIG. 4. SERUM-ASCITIc FLUID OSMOTICPREssuRE DIF-FERENCES IN FOUR DIFFERENT TYPES OF CASES OF CIR-RHOSIS WITH AScrrES

The "effective portal pressure" is not measurable intypes B and D because the serum osmotic pressure isrelatively too low to give a significant ascitic osmoticpressure.

ascitic fluid osmotic pressure was less than 15 mm.of water no true evaluation of the hydrostaticforces could be made. Figure 4 shows four typesof cases that illustrate the various relationshipsencountered. In Case A, a high serum osmoticpressure and a sizeable ascitic osmotic pressureyields an elevated value for "effective portal pres-sure." In Case B, the ascitic osmotic pressure istoo low to indicate the exact "effective portal pres-sure," although it is apparent that the value ishigh. Case C illustrates a low serum osmotic pres-sure with a sizable ascitic fluid osmotic pressuregiving a relatively normal "effective portal pres-sure." In Case D, the ascitic fluid osmotic pres-sure is very low and in the presence of the lowserum osmotic pressure little information regard-ing the height of the "effective portal pressure"can be obtained. Examples such as Cases B andD where the exact "effective portal pressure" can-not be determined are frequently encounteredamong patients with cirrhosis of the liver par-ticularly those with unusually low serum osmoticpressure levels.

DISCUSSION

The possibility that the formation of ascites inpatients with the alcoholic type of cirrhosis is re-lated to the elevated "effective portal pressure" issuggested by the data obtained but remains un-proven. Other possibilities acting more specific-ally in these patients than in others, such asadrenal effects causing increased sodium reten-tion, posterior pituitary stimulation causing in-creased water retention, or special local abnormali-ties in lymphatic drainage, must be considered.The high "effective portal pressure" in cirrhosisfound in this study and by Mankin and Lowell(2) is at variance with the concept that there isa generally applicable critical level of effectiveosmotic pressure below which ascites occurs (14).

It has been demonstrated by previous workersthat changes in either serum or ascitic fluid pro-tein are reflected by similar changes in the otherfluid (2, 31, 32) indicating a free exchange ofprotein across the peritoneal membrane. This iscertainly important for calculation of the hydro-static differences from the osmotic pressure differ-ence between serum and ascitic fluid althoughmuch of the equilibrium is established by rapidinterchange of water. The dynamic exchange of

260


protein between ascitic fluid and serum was studiedin this same group of patients by Eisenmengerand Slater (33) employing I 1"1 tagged albuminand y-globulin. A fall in specific activity for theserum and ascitic fluid on a single curve indicatedthe protein equilibrium.

The application of Starling's hypothesis to esti-mate total portal pressure was suggested first byIversen (29), and has been discussed further byMankin and Lowell (2). Since there are severalunknown factors, however, this relationship can-not be used to quantitate the actual portal pressure.The most obvious unknown is the hydrostaticpressure of the ascitic fluid, which may vary de-pending on the amount of abdominal distentionproduced. Although certain standard and repro-ducible methods can be duplicated, the questionmust be raised whether any artificial measure-ment of ascitic hydrostatic pressure accurately de-scribes the pressure exerted at the capillary mem-brane. Conflicting data have appeared in theliterature in regard to the level of ascitic hydro-static pressure (2, 3, 29).

Since there are porta-systemic communicationsin the posterior peritoneum as well as systemicvenous channels, the ascitic fluid is also partiallyin equilibrium with the systemic venous bed. Thecalculated "effective portal pressure" as obtainedby osmotic pressure measurements would be low-ered reflecting the extent of this communication,since the venous hydrostatic pressure is less byvirtue of this connection with the systemic bed.Perhaps a value 0f the "effective portal pressure"particularly in relation to the total portal pressuremeasured directly at operation is that the latter isobtained in the large venous channels, whereas the"effective portal pressure" refers to the pressurein the capillary bed, where the level may be quitedifferent (34). Certainly the term "effective por-tal pressure" as used in this study remains ill-defined. The possibility that variations in thelymphatic system, which is known to transportmost of the protein leaving the peritoneal cavity(35), may influence the osmotic pressure differ-ence between serum and ascitic fluid must be con-sidered. Direct measurement of portal pressureat laparotomy was carried out in five of the pres-ent group of patients. Although a general cor-relation with the serum-ascitic fluid osmotic pres-sure difference appeared to exist, the observations

were too limited to draw any conclusions. Onedefinite instance was encountered in a case ofcarcinoma of the ovary where the osmotic pres-sure difference appeared too great for a normalportal pressure. No explanation was apparentand indicates the probability of further unknownfactors influencing this relationship.

Under equilibrium conditions the ascitic fluidosmotic pressure is primarily governed by theserum osmotic pressure and the hydrostatic forcesof the abdominal venous system and ascitic fluid.It represents a very variable quantity that reflectsthe alterations in the other forces except where theosmotic pressure is less than 15 mm. of water.A certain minimum protein concentration is stillfound in ascitic fluid even when the equilibriumforces are acting in a direction that would reducethe ascitic fluid osmotic pressure to a maximalextent. In patients with ascitic fluid associatedwith peritoneal carcinoma, the serum osmotic pres-sure is usually only slightly reduced and the effec-tive portal pressure is close to normal. Thiswould result in a high osmotic pressure in theascitic fluid if equilibrium conditions prevail, andwould explain the high protein content of suchascitic fluids without involving the concept of anexudate. Similarly in dogs with thoracic inferiorvena cava constriction (31, 36) or constrictivepericarditis (37), the high ascitic protein concen-tration probably reflects this relationship betweenthe serum osmotic pressure and the effective por-tal pressure. Thus, although Starling's hypothesismay not offer the main explanation for the forma-tion of ascites, it nevertheless remains of greatvalue in describing the equilibrium conditions andin accounting for the respective protein concen-trations of serum and ascitic fluid.

SUMMARYANDCONCLUSIONS

1. Osmotic pressure measurements of the serumof patients with cirrhosis of the liver indicated thatascites was associated with levels ranging fromthe low value of 148 mm. H20 to the high valueof 418 mm. H20. The higher levels of serumosmotic pressure were found primarily in patientswith the alcoholic nutritional type of cirrhosis.In other types, the presence or absence of ascitesappeared to be more directly related to a criticalserum osmotic pressure level.

261


2. An attempt at estimation of the "effectiveportal pressure" was made by determination ofthe difference between serum and ascitic fluidosmotic pressures in accordance with the Starlinghypothesis, a steady state being maintained bymeans of a controlled low sodium diet.

3. The "effective portal pressure" could beestimated only if the serum osmotic pressure ex-ceeded it by an amount sufficient to give a sig-nificant ascitic fluid osmotic pressure. This con-ditioning factor limited the application of thesemeasurements. Other limitations are discussed.

4. High values for "effective portal pressure"were obtained in patients with cirrhosis formingascites in the presence of high serum osmotic pres-sure levels.

REFERENCES

1. Starling, E. H., On the absorption of fluids from theconnective tissue spaces. J. Physiol., 1896, 19, 312.

2. Mankin, H., and Lowell, A., Osmotic factors influenc-ing the formation of ascites in patients with cir-rhosis of the liver. J. Clin. Invest., 1948, 27, 145.

3. James, A. H., The mechanism of pleural and asciticeffusions, with a suggested method for the indirectestimation of portal venous pressure. Clin. Sc.,1949, 8, 292.

4. Govaerts, P., Influence de la teneur de serum en albu-mines et en globulines sur la pression osmotique desproteines et sur la formation des cedemes. Bull.Acad. roy. de med. de Belgique, Ser. V, 1927, 7, 356.

5. Schade, H., and Claussen, F., Der onkotische Druckdes Blutplasmas und die Entstehung der renalbedingten 6deme. Ztschr. f. Klin. Med., 1924, 100,363.

6. Iversen, P., and Nakazawa, F., tJber die Biochemiedes Filtrations6dems. Biochem. Ztschr., 1927, 191,307.

7. Barfath, E., Klinische Beitrage zum Verhalten desonkotischen (Kolloidosmotischen) Druckes. I. Deronkotische Druck im serum des arteriellen undvenosen Blutes bei kranken mit und ohne storungenim wasserhaushalt. Ztschr. f. Klin. Med., 1930,114, 702.

8. Kerkhof, A. C., Plasma colloid osmotic pressure as afactor in edema formation and edema absorption.Ann. Int. Med., 1937, 11, 867.

9. Kylin, E., Studien iuber den kolloidosmotischen (on-kotischen) Druck. XXVIII: Mitteilung: Tier-experimentelle Untersuchungen fiber die Bedeutungder Senkung des kolloidosmotischen Druckes furdie Odempathogenese. Arch. f. exper. Path. u.Pharmakol., 1933, 170, 407.

10. Muntwyler, E., Way, C. T., Binns, D., and Myers,V. C., Plasma protein and plasma colloid osmoticpressure in pathological conditions with special ref-

erence to the occurrence of edema. J. aln. Invest.,1933, 12, 495.

11. Butt, H. R., Snell, A. M., and Keys, A., Plasmaprotein in hepatic disease: A study of the colloidosmotic pressure of blood serum and of ascitic fluidin various diseases of the liver. Arch. Int. Med.,1939, 63, 143.

12. Bjorneboe, M., Brun, C., and Raaschou, F., Colloidosmotic pressure in chronic hepatitis. Arch. Int.Med., 1949, 83, 539.

13. Higgins, G., Kelsall, A. R., O'Brien, J. R. P., Stewart,A. M., and Witts, L. J., Ascites in chronic diseaseof the liver. Quart. J. Med., 1947, 16, 263.

14. Armstrong, S. H., Jr., Mechanisms of action of serumalbumin therapy in internal medicine, Am. J. Med.,1948, 4, 390.

15. Balli, E. P., Robson, J. S., Clarke, D., and Hoagland,C. L., Factors influencing ascites in patients withcirrhosis of the liver. J. Clin. Invest., 1945, 24, 316.

16. Patek, A. J., Jr., Mankin, H., Colcher, H., Lowell,A., and Earle, D. P., Jr., The effects of intravenousinjection of concentrated human serum albuminupon blood plasma, ascites and renal functions inthree patients with cirrhosis of the liver. J. Clin.Invest., 1948, 27, 135.

17. Hepp, O., Ein neues Onkometer zur Bestimmungdes kolloidosmotischen Drucks mit gesteigerterMessgenauigheit und vereinfachter Handhabung.Ztschr. f. d. ges. exper. Med., 1936, 99, 709.

18. Bigelow, S. L., and Gemberling, A., Collodion mem-branes. J. Am. Chem. Soc., 1907, 29, 1576.

19. Krogh, A., and Nakazawa, F., Beitrage zur Messungdes Kolloid-osmotischen Druckes in biologischenFliissigkeiten. Biochem. Ztschr., 1927, 188, 241.

20. Turner, A. H., The validity of determinations of thecolloid osmotic pressure of serum. J. Biol. Chem.,1932, 96, 487.

21. Wies, C. H., and Peters, J. P., The osmotic pressureof proteins in whole serum. J. Clin. Invest., 1937,16, 93.

22. Wells, H. S., A modified Krogh osmometer for thedetermination of the osmotic pressure of colloids inbiological fluids (with directions for its use). J.Tenn. Acad. Sc., 1933, 8, 102.

23. Keys, A., The study of colloidal dimensions, thermo-dynamic activity, and the mean molecular weightof the mixed proteins in blood serum. J. Phys.Chem., 1938, 42, 11.

24. Peters, E., and Saslow, G., Performance of the Heppmicro-osmometer. J. Gen Physiol., 1939, 23, 177.

25. Keys, A., and Taylor, H., The determination of thecolloidal osmotic pressure in blood serum and simi-lar fluids. J. Biol. Chem., 1935, 109, 47.

26. Immergut, E., Personal communication.27. Pierce, H. F., Nitrocellulose membranes of graded

permeability. J. Biol. Chem., 1927, 75, 795.28. Kunkel, H. G., Ahrens, E. H., Jr., Eisenmenger, W.

J., Bongiovanni, A. M., and Slater, R. J., Extremehypergammaglobulinemia in young women with

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liver disease of unknown etiology. J. Gin. Invest.,1951, 30, 654.

29. Iversen, P., Untersuchungen fiber die ascites-patho-genese. Klin. Wchnschr., 1928, 7, 2001.

30. Giges, B., and Teschan, P. E., The portal circulationtime in cirrhosis of the liver following portacavalanastomosis. J. Lab. & Gin. Med., 1952, 40, 537.

31. McKee, F. W., Schilling, J. A., Tishkoff, G. H., andHyatt, R. E., Experimental ascites. Effects ofsodium chloride and protein intake on protein me-

tabolism of dogs with constricted inferior vena cava.

Surg., Gynec., & Obst., 1949, 89, 529.32. Kunkel, H. G., Factors in the mechanism of ascites

in Liver Disease, Ciba Foundation Symposium,Philadelphia, Blakiston, 1950, p. 130.

33. Eisenmenger, W. J., and Slater, R. J., Distributionand decay of I111 tagged albumin and gamma glo-

bulin in patients with cirrhosis. J. Clin. Invest.,1953, 32, 564.

34. Landis, E. M., The capillary blood pressure in mam-

malian mesentery as determined by the micro-in-jection method. Am. J. Physiol., 1930, 93, 353.

35. Courtice, F. C., and Steinbeck, A. W., The effects oflymphatic obstruction and of posture on the ab-sorption of protein from the peritoneal cavity.Australian J. Exper. Biol. & Med. Sc., 1951, 29,451.

36. Volwiler, W., Grindlay, J. H., and Bollman, J. L.,The relation of portal vein pressure to the forma-tion of ascitesan experimental study. Gastro-enterology, 1950, 14, 40.

37. Davis, J. O., Lindsay, A. E., and Southworth, J. L.,Mechanisms of fluid and electrolyte retention inexperimental preparations in dogs. I. Acute andchronic pericarditis. Bull. Johns Hopkins Hosp.,1952,90, 64.

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