Intrinsic hepatic control of plasma albumin concentration

Life Sciences Vol . 6, pp . 2621-2628, 1967 . Printed in Great Britain .

Pergamon Press Ltd .

INTRINSIC HEPATIC CONTROL OF PLASMA ALBUMIN CONCFNTRATION Myron E . Tracht,* Lisa Tallal,** and Diane - G . Tracht Departments of Pathology and Pediatrics, The Mount Sinai Hospital, New York City, and Department of Pathology, Columbia University College of Physicians and Surgeons, New York City (Received 17 May 1967 ; in final form 10 October 1967) The relative stability of normal serum protein levels suggests the presence of effective and sensitive control mechanisms whose nature is as yet obscure .

The present

studies have been undertaken in an effort to characterize the physiological processes regulating plasma albumin concentration, and to develop techniques for studying the control of other plasma protein constituents . Based on the finding of hypoalbuminemia induced by the presence of dextran (1,2), or elevated serum globulin levels (3,4), and upon the rapid return of plasma albumin concentra tion to normal following plasmapheresis, it has been suggested that plasma albumin metabolism is principally responsive to the oncotic pressure of the plasma .

Evidence in support of

such a concept has been reviewed and augmented in the work of Rothschild, et al

(5) .

Previous studies utilizing the intact animal, however, have been hampered by the complexity of protein distribution among body fluid compartments, and, in addition, provide little information regarding the anatomic pathways of plasma protein control . *Present address : Department of Pathology, Holy Name Hospital, Teaneck, New Jersey 07666 . **Present address : Department of Pediatrics, Memorial Center for Cancer and Allied Diseases, 444 East 68th Street, New York City . 2621

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Accordingly, we have used the isolated perfused rat liver in an effort to minimize the effect of alterations in body fluid compartments, and to determine whether the liver is directly responsive to alterations in plasma colloid osmotic pressure, or whether extrahepatic sites exert a regulatory influence on hepatic albumin metabolism . Materials and Methods Sherman strain female albino rats of approximately 300 grams were used both as liver donors and as perfusate blood donors, with the latter bled by cardiac puncture .

All

animals were maintained on Purina lab chow and tap water ad lib, and were fasted for eighteen hours prior to use .

Rat livers

were surgically isolated and perfused by techniques essentially similar to those described by Miller et al (6) . The following three treatment groups were established . 1.

Control group, in which livers were perfused with

pooled whole rat blood, adjusted to a hematocrit of 358 with 0 .858 saline solution . 2.

Plasmapheresis group, in which pooled rat erythrocytes

were suspended in 0 .858 saline solution containing 5 meq/L potassium, with the hematocrit adjusted to 358 . 3.

Dextran group, in which pooled rat erythrocytes were

suspended in 68 Dextran solution (Abbott), containing 5 meq/L potassium, with the hematocrit again adjusted to 358 . In all cases the perfusate volume was approximately 45 cc, and all perfusates contained 1 mg heparin sodium, 20 mg streptomycin and 1000 units penicillin G per 100 ml . After establishment of the perfusion and an equilibration period of 15 minutes, during which the liver became uniformly tan-red, an arteriovenous oxygenation difference became evident,

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and bile flow was well established, 3 ml of an amino acid mixture (Aminosol, Abbott) containing 6 .1 microcuries of DL-Leucine-1-C14, specific activity 0 .32 microcuries per milligram, was added, with mixing to the perfusate .

Two hours

later, the perfusion was discontinued, the liver flushed with 0 .859 saline, blotted dry and carefully weighed, and an aliquot homogenized in four times its volume of distilled water . The erythrocytes of the perfusate were discarded and the perfusate total protein, albumin and globulin concentrations were determined by standard methods (7) .

The perfusate albumin

and globulin were separated by precipitation with 239 sodium sulfate, and the residual albumin was then precipitated by an equal volume of 109 trichloracetic acid .

The precipitate was

washed by the procedure o ¬ Siekevitz (8), and the albumin was subsequently redissolved in 0 .1 N NaOH .

Its specific activity

was then determined by liquid scintillation counting and determination of the protein concentration by the biuret method (7) .

From the known perfusate volume, hematocrit and

albumin concentration, the total perfusate albumin content was calculated .

From this value and the specific activity of

the albumin, the total radioactivity incorporated into perfusate albumin was then calculated . Liver protein was separated from the homogenate by the procedure of Smuckler et al (9), and specific activity and total radioactivity incorporation into liver protein determined as in the case of perfusate albumin . Bile was accumulated in a graduated tube as it drained from a plastic cannula inserted in the common bile duct, and its total volume was measured at the end of the perfusion . Representative portions of the liver were fixed in 101 neutral

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buffered formalin, and examined histologically using hematoxylin and eosin as routine stains . Observations No noteworthy differences were observed among the livers of each of the three treatment groups on gross or microscopic examination .

All showed good preservation of normal hepatic

structure save for rare areas of localized edema and small aggregates of leukocytes, although these changes did not differ in severity among the treatment groups . The data are summarized in Table 1 .

No statistically

significant alteration of bile production was noted as a result of the treatments, although the slight elevation in the plasmapheresis group is conceivably related to the somewhat lower osmotic pressure against which the bile was secreted . Both the plasmapheresis and dextran groups had equivalent diminution in perfusate protein and albumin concentrations . The incorporation of DL-Leucine-l-C14 into serum albumin was approximately doubled in the plasmapheresis group (p<.01), and remained essentially unchanged in the dextran group .

By

contrast, plasmapheresis exekted no significant effect on the incorporation of radioactivity into liver protein, although this was significantly increased in the dextran group (p<.05) Discussion Because of the substitution of protein-depleted media for plasma in the experimental groups, radioactive leucine was administered together with a large quantity of amino acid mixture, in an effort to minimize relative differences in leucine pool size, and to assure adequate substrate for protein synthesis . and by Schurr et al

Using the data presented by Anker (10) (11), it i3 calculated that the leucine

-*Sumbér of perfusions **Mean±Standard Error of the Mean

per gram liver per hour

Bile production, microliters

mg per ml

Perfusate total protein,

mg per ml

Perfusate albumin concentration,

cpm per gram liver per hour

into liver protein,

Incorporation of DL-Leucine-1-C14

cpm per gram liver per hour

into perfusate albumin,

Incorporation of DL-Leucine-1-C14

54±4

65±4 .7

23±1 .6

11,772±772

805±59**

Control (5) *

64±4

17±2 .0

9 .3±1 .2

13,218±922

1,659±165

Plasmapheresis (6)

Treatment Groups,

DL-Leucine -1-C14 into Plasma Albumin and Liver Protein

969±106

Dextran (6)

48±5

16±1 .7

7 .2±1 .0

17,583±1,348

Influence of Perfusate Colloid Osmotic Pressure on Incorporation of

TABLE 1

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pool in the control group may be approximately 178 larger than in the plasmapheresis and dextran groups, assuming a 128 leucine content of rat albumin, and assuming that the liver accounts for total body plasma protein catabolism .

This

degree of dilution does not appear sufficient to account for the observed differences in protein radioactivity, particularly since the difference in perfusate albumin activity is apparent in the comparison of the plasmapheresis and dextran groups, both of which have comparable diminution of endogenous leucine sources . The finding of increased incorporation of radioactive leucine into serum albumin in the presence of hypoproteinemia, and its reversal by the substitution of material with approximately equivalent colloid osmotic pressure is in accord with the findings of Rothschild et al

(5) .

It is of interest

that this influence of dextran is manifest despite the fact that the residual plasma albumin levels were comparable in both the plasmapheresis and dextran groups .

This suggests

that the control of serum albumin levels is indeed responsive to colloid osmotic pressure rather than to the concentration of albumin as a molecular species . A somewhat unexpected finding is the observation that the incorporation of radioactivity into liver protein was not significantly affected by plasmapheresis, but was appreciably increased with the presence of dextran in the perfusate . The possibility exists that this represents plasma albumin whose release into the circulation has been inhibited by the presence of dextran, but whose synthesis may have been stimulated by decreased serum albumin concentrations .

If this

is the case, dextran may be supposed to act on a release

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mechanism rather than on the synthetic mechanisms, while the latter may conceivably be responsive to the concentration of albumin as a molecular species . Summary and Conclusions The present findings suggest that the liver is directly responsive to the oncotic pressure of the medium perfusing it, and that the rate of appearance of radioactive plasma albumin in the circulation is inversely related to the colloid osmotic pressure of the plasma .

It is suggested that

two regulatory mechanisms may be involved, one relating to the actual rate of protein synthesis in the liver and possibly governed either by oncotic pressure or by the concentration of serum albumin as a molecular species, and the other relating to the rate of delivery of protein into the circulation from the liver cell .

The latter mechanism

may be more directly responsive to alterations in colloid osmotic pressure, and may be manifested in the present case as a dextran-induced inhibition of the release of albumin synthesized by the liver . Acknowledgements This work was supported by Grants HE-10347 and AM-10416 from the National Institutes of Health .

The skilled technical

assistance of Mr . Paul B . Hout is gratefully acknowledged . References 1.

JAENIKE, J .R . and WATERHOUSE, C ., Circulation , 11, 1 (1955) .

2.

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BJORNEBOE, M ., and SCHWARTZ, M

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BJORNEBOE, M., Acte Med . Scand . , 123, 393 (1946) .

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ROTHSCHILD, M .A ., ORATZ, M ., WIMER, E ., and SCHREIBER, S .S ., J . Clin . Invest . , 40, 545 (1961) .

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MILLER, L .L ., BLY, C .G ., WATSON, M .L ., and BALE, W .F ., J . Exp . Med ., 94, 431 (1951) .

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OSER, B .L ., ed ., Hawk's Physiological Chemistry , 14th ed ., p . 1082, The Blakiston Division, McGraw-Hill Company, New York (1965) .

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SIEKEVITZ, P ., J . Biol . Chem . , 195, 549 (1952) .

9.

SMUCKLER, E .A ., ISERI, O .A ., and BENDITT, E .P ., J . Exp . Med . , 116, 55 (1962) .

10 .

ANKER, H .S ., in The Plasma Proteins , FRANK W . PUTYAM, ed ., vol . 2, p . 267, Academic Press, New York and London, 1960 .

11 .

SCHURR, P .E ., THOMPSON, H.T ., HENDERSON, L .M ., WILLIAMS, J .N ., and ELVEHJEM, C .A ., J. Biol . Chem ., 182, 39

(1950) .