Clearance of Circulating Radiochromated Albumin and Erythrocytes by the Gastrointestinal Tract of Normal Subjects

Vol. 52, No.1 Printed in U.S.A.

GASTROENTEROLOGY

Copyright ©

1967 by The Williams

& Wilkins Co.

CLEARANCE OF CIRCULATING RADIOCHROMATED ALBUMIN AND ERYTHROCYTES BY THE GASTROINTESTINAL TRACT OF NORMAL SUBJECTS WARREN

L.

BEEKEN,

M.D.

Department of Medicine, Hitchcock Clinic, Hitchcock Foundation, and Dartmouth Medical School, Hanover, New Hampshire

absorbed from the gut lumen. 2 , 7 Unfortunately, these particular assets are not usually fully exploited, because fecal isotope losses have been related either to the total isotope dose or to serum radioactivity before equilibrium has been reached between intravascular and extravascular pools. As illustrated in figure 1, after intravenous administration of 5lCr-albumin, intravascular radioactivity rapidly declines in a curvilinear fashion suggesting a multi compartmental distribution similar to that observed with l3lI-albumin. Studies during the distribution period reflect primarily the course of circulating intravascular albumin, approximately 150 g, rather than egress from the total equilibrated albumin pool which is in excess of 300 g.8 Although some investigators have advocated the principles of clearance techniques for quantifying enteric. protein 10ss,9, 10 data from studies utilizing these concepts in normal subjects are insufficient to establish upper limits of normal clearance values. In view of the increasing interest in the gut as a site of protein degradation and the paucity of data in normal subjects, it seemed appropriate to reexamine the problem of albumin entry into the gut and to contrast it with the rate at which erythrocytes enter the lumen. Accordingly, 20 studies of enteric clearance of 5lCr-albumin and 20 of 5lCr-erythrocyte entry were conducted in 19 healthy subjects, utilizing the principle of the clearance technique and taking into account the distribution characteristics of serum albumin.

With the definition and elucidation of protein-losing enteropathies, measurement of protein loss into the gastrointestinal tract has become a commonly employed technique for the study of hypoalbuminemic states and a variety of circulatory and gastrointestinal disorders. A number of isotopically labeled proteins have been employed for these measurements, and all have their particular disadvantages. Albumin labeled with trivalent chromium-51 is one of the most satisfactory compounds currently available. The physicochemical characteristics of 51Cr-albumin have been studied in a number of ways which indicate at least some degree of heterogeneity,1-3 and the biological half-life of the labeled protein is short due both to sequestration within liver and spleenl, 2 and to liberal urinary excretion of isotope soon after administration. 2 Furthermore, there is evidence to suggest that 51Cr eluted or freed from albumin by catabolism may label other circulating proteins. 4 ,5 However, 51Cr-albumin has several major advantages in that freely circulating chromium is, for the most part, promptly excreted in the urine 2 , 6 and is neither secreted into nor Received June 30, 1966. Accepted August 30, 1966. Presented in part at the combined meetings of the American Gastroenterological Association and Canadian Association of Gastroenterology, Montreal, Quebec, Canada, May 29, 1965. Address requests for reprints to: Dr. Warren L. Beeken, Department of Medicine, University of Vermont College of Medicine, Burlington, Vermont 05401. The author is grateful for the helpful advice of Dr. Franklin G. Ebaugh, Jr. and the untiring technical assistance of Misses Martha Benson and Dorothy Sears.

Methods Subjects studied. Paid volunteers were selected from healthy hospital personnel, medical stu35

36

BEEKEN

Vol. 52, No.1

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DECAY CURVE CR" - ALBUMIN 5

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FIG. 1. Diagrammatic representation of conventional method of estimating gastrointestinal protein loss. Note that the stool collection period occurs during the curvilinear distribution period of the plasma radioactivity plot. Fecal radioactivity usually related to administered radioactivity.

dents, and physicians without histories of anemia or gastrointestinal bleeding. Subject ages ranged from 22 to 45 years with a mean of 28 years. Pre study benzidine dihydrochloride tests on stool specimens, serial hematocrit, and serum albumin values were negative or within normal limits. Each subject had at least two separate studies, one to quantify erythrocyte egress and another to measure albumin loss, at intervals sufficiently separated to allow blood radioactivity to reach background levels. 5lGr-Erythrocyte studies. Erythrocytes were labeled according to the method of Ebaugh and co-workers,l1 utilizing hexavalent OlCr. Approximately 12 ml of the subject's blood were anticoagulated with 2.5 ml of acid-citrate-dextrose solution (Squibb). Fifty to 100 p..c of N a2 5l CrO. were added, and the solution was incubated at room temperature for 1 hr with periodic gentle swirling. Approximately 10 ml were then administered intravenously to the donor and the study conducted as illustrated in figure 2. After administration of the labeled red cells, 3 or more days elapsed to permit urinary excretion of unbound isotope. Complete collections of 4-day stool specimens were then made, and radioactivity was

measured in weighed aliquots of diluted feces. Microhematocrits and radioactivity levels were determined on blood samples drawn at the beginning and at the end of the 4-day collection period. OlGr-Albumin studies. The same principles were used in measuring albumin loss and are illustrated in figure 3. Fifty to 100 p..c of 51Cr_ albumin (kindly supplied by Dr. Gordon Lindenblad, Squibb Institute for Medical Research, New Brunswick, N. J.) were administered intravenously, and 4 to 7 days elapsed before the 4-day stool collections were begun. Thus, intravascular 51Cr-albumin was in equilibrium with the total circulating pool at this time and could enter the gut not only from the intravascular space but also from extravascular sites adjacent to the lumen. Radioactivity of homogenized stool collections was monitored, and integrated mean serum radioactivity levels during collection periods were calculated from serial determinations of serum radioactivity. Serum albumin was measured at the beginning and end of the fecal collections. Stool collections. Complete 4-day stool specimens were collected in tared I-gal paint cans

CLEARANCE OF ALBUMIN AND ERYTHROCYTES

January 1967

containing 100 ml of 70% alcohol. The cans were fitted to portable commode chairs to facilitate collection. After dilution with tap

DECAY CURVE CR

51

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FIG. 2. Diagrammatic representation of "Crerythrocyte clearance study.

water to a consistency which enabled automatic pipetting, the specimens were weighed and homogenized for 3 min in a Red Devil paint shaker. Aliquots of approximately 4 ml were transferred immediately to tared counting bottles and weighed prior to radioactivity monitoring. Additional aliquots were centrifuged, and the supernatant fluid was frozen for polyethylene glycol determinations (see below). Radioactivity monitoring. Radioactivity in samples of 1 to 4 ml of whole blood and serum was measured in a scintillation well counter (Nuclear Chicago Model C-120-1) for periods sufficient to insure counting errors of less than 5%. Differences in counting geometry for various sample volumes were corrected to 4 ml by appropriate predetermined factors. Weighed aliquots of fecal solutions approximating 4 ml were similarly monitored. Radioactivity levels of some fecal samples were so low that random counting errors at times exceeded 10% despite counting times in excess of 80 min. Polyethylene glycol (PEG) determination. An attempt was made to provide correction factors for fecal collections not precisely reflecting 4-day excretion because of diarrhea, constipation, or missed collections, by having each subject ingest a constant daily dose of the non-

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FIG. 3. Diagrammatic representation of 51Cr-albumin clearance study. Note that clearance rates are determined after intravascular-extravascular distribution has reached equilibrium.

BEEKEN

38 TABLE

Subj ect

- -1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 7 16 17 18 19

51CrAlbumin loss

Equivalent

gjday

ml/day

,

serum

loss

ml/day

27.20

0.30 0.86 0.18 3.56" 0.75 0. 19 0.20 0.31 0.14 0.36 1.52" 0.91 0. 20 0.70 0.67 0.53 0.28 0.17 1.09 0.05

0.65 2.05 0.44 7.921.58 0.44 0.45 0.78 0.32 0.82 3.49" 2.10 0.43 1.54 1. 83 1. 23 0.69 0.41 2.37 0.12

33.58 12.21

0.44 0.30

1.01 0.70

23.88 52.11 37.23 24.47 17 .69 17.35 25.75 30 .03 21.40 39.00 28.60 40.30 18.80 55.70 46.25 54.70 43.90

1.80

34.60 85.60"

1.73 0.63

61Cr_ Equivalent Erythrocyte whole blood loss loss

ntl/day

1.19 2.53 1. 73 1. 22 0.91 0.95 1.25 1.44 1.13 2.20 1.60 1.97 1.15 3. 12 2.22 2.74 2.37 4.79" 1.33

Mean SD

1. 51Cl'-Albumin and 51Cr-erythrocyte clearance values

a Values greater than 2 SD from uncorrected mean; not included in calculation of mean, SD .

abso rbable marker PEG (kindly supplied as "Carbowax 4000" by the Union Carbide Chemical Company, New York, N. Y.) throughout the study and measuring PEG in stool specimens. The daily PEG dose was from 2 to 4 g in water, taken at intervals throughout the day. Aliquots of diluted, homogenized 4-day stool specimens were centrifuged, and PEG in supernatant fluids was quantified by a modification of the method of Hyden ." Standard curves were derived from stool supernatant fluids containing known quantities of PEG. To 1 ml of stool sllpernatant fluid, 10 m1 of distilled water, 1 ml of 10% barium chloride, 2 ml of 0.3 N barium hydroxide, and 2 m1 of 5% zinc sulfate were added in rapid succession. The sllspension was thoroughly mixed, allowed to stand for 10 min, and fi ltered. Four milliliters of water and 5 ml of 30% trichloracetic acid were added to 1 ml of protein-free filtrate, and turbidity was measured after 60 min in a spectrophotometer at 650 mIL. Calculations. ·'Cr-erythrocyte and ·'Cr-albumin clearances were calculated as follows:

Vol. 52, No. 1

Milliters of erythrocytes cleared/day radioactivity in stool/day mean radioactivity/milliliter of whole blood X hematocrit

(1)

Grams of albumin cleared/day radioactivity in stool/day mean radioactivity/milliliter of serum

(2)

X grams of albumin/milliliter of serum Since serum per se was not labeled in either study, erythrocyte clearance may not have reflected precisely losses of whole blood; similarly, albumin clearance could not necessarily be equated to serum egress. However, volumes of whole blood and serum containing amounts of radioactivity equivalent to fecal radioactivity were related by t he following equations: Milliliters of whole blood cleared/ day radioactivity in stool/ day (3) mean radioactivity/milliliter of whole blood Milliliters of serum cleared/day radioactivity in stool/day mean radioactivity/milliliter of serum

(4)

PEG correction factors grams of PEG ingested/4 days grams of PEG in 4-day stool Values obtained from equations 1 through 4 were multiplied by the appropriate PEG factor to obtain "PEG-corrected" values for erythrocyte, equivalent whole blood, albumin, and equivalent serum clearances.

Results and Discussion

The results of gastroenteric clearance of 51Cr-albumin and 51Cr-erythrocytes are presented in table 1. The mean erythrocyte entry of 0.44 ml and equivalent whole blood loss of 1.01 ml per day correspond quite well to results reported by other investigatorsY. 13-19 The results of daily albumin and equivalent serum clearance of 1.73 g and 33.58 mI, respectively, approximate values suggested by Stanley,t° i.e., 2 to 2.5 g of albumin, 30 to 60 ml of serum, and they

January 1967

CLEARANCE OF ALBUMIN AND ERYTHROCYTES

are slightly higher than the figures of Waldmann's studies,9 which were initiated prior to establishment of compartmental equilibrium. The results are also ,consistent with studies utilizing95 Nb-albumin which indicate that albumin catabolism within the gut accounts for no more than 12.6%20 (approximately 1.8 g) of the total daily albumin turnover. In contrast, clearance of albumin and equivalent serum entry are distinctly lower than the results of measurements utilizing 13lI-albumin as a tracer.21.22 The shortcomings of iodinated protein have recently been emphasized ;7. 9 namely, the isotope is absorbed after intraluminal proteolysis and circulating unbound radioiodine is secreted in saliva, gastric juice, and bile. The use of iodinebinding resins fails to circumvent these difficulties. 23 The short biological halflife of 51Cr-albumin (mean, 7 days this study) and the rapid fall of serum radioactivity during the equilibrium period, however, render the radiochromated protein unsuitable for turnover studies. The clearances in this study should be considered maximum for normal subjects, because intestinal transit time was assumed to be negligible. Thus, fecal isotope losses may well have reflected entry into the gut during a period of somewhat higher serum radioactivity values than those used for the clearance calculation. If this were so, the denominator in equation 2 would be larger and calculated clearance values lower. Clearance studies conducted after equilibrium, when the serum radioactivity curve is linear and its slope minimal, are less subject to transit time errors than are clearances conducted during distribution. The use of accurate stool markers might further help to overcome transit errors in part, but, since protein may enter the gut from numerous levels, even an ideal marker technique could not eliminate completely this source of error. The contrast between the rates of erythrocyte clearance and albumin clearance is striking and suggests logically that the protein is transferred from multiple compartments adj acent to the gut lumen or in a selective fashion from the vasculature,

39

or both, rather than from a single openended vascular space as in the case of the red cell. The data suggest also that less than 2 g of albumin enter the gut daily, thus accounting for about 10% of the total daily albumin catabolism in normal man. Table 2 shows the PEG factors and PEG-corrected values. Mean PEG factors were considerably in excess of 1, and mean recovery of the marker approximated 70% of that ingested. Thus PEG-corrected values are probably spurious. Furthermore, uncorrected erythrocyte and whole blood losses were remarkably consistent with values previously reported, supporting further the contention that PEG-corrected values were too high. The low PEG recoveries could have been due to failure of the subjects to take the compound, to delayed intestinal transit of PEG, to absorption from the lumen, or to inadequate sensitivity of the chemical method as used for measuring PEG. It is noteworthy that incomplete fecal recovery of ingested PEG has also been reported in animal investigations. 24 Chronic toxicity studies in animals have failed to show gross or microscopic evidence that oral PEG produces lesions which might accelerate enteric loss of blood or protein ;25 hence, it is unlikely that the marker itself influenced the results of these investigations. Summary

Gastrointestinal clearances of 5ICr-albumin and 5lCr-erythrocytes were measured in 19 normal subjects in 40 separate studies. Albumin clearances were conducted after equilibrium had been established between intravascular and extravascular compartments. Daily albumin clearance through the gut averaged 1.73 g, and mean daily erythrocyte clearance was 0.44 ml, equivalent to 33.58 ml of serum and 1.01 ml of whole blood, respectively. Attempts to derive correction factors for incomplete stool collections by the use of daily polyethylene glycol ingestion and fecal assay were unsuccessful because of incomplete recovery of the marker in fecal specimens.

40

BEEKEN TABLE

2. 5lCr-Albumin and 6lCr-erythrocyte clearance values-"PEG-corrected"

Subject

PEG factor

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 7 16 17 18 19

1.03 1.91 1.95 2.96 7 .52" 0.79 2.24 1.02 0 .85 1.18 1.34 0 .63 2 . 19 1.25 1.04 0.73 0 .84 0 .87 1.80 0.87

Mean

1.34 0 .63

SD

Vol. 52, No.1

• Values greater than 2

" Cr-Albumin loss

Equivalent serum loss

g/day

ml/day

1.23 4.81 3.38 3.43 6.83" 0.72 2.66 1.40 0.96 2.46 2.03 1.19 2.38 3.70 2.17 1.99 2.00 1.57 8 . 19" 1.17

24.55 99.27 72 .60 72.41 133.03" 13.73 57.67 30.63 18.10 46.05

2.18 1.06 SD

PEG factor

nCr-Erythrocyte loss

Equivalent whole blood loss

"'l/day

ml/day

25 .39 41.12 69 .59 48.10 39.80 37.00 30.20 154. 17" 23.80

1. 24 1.47 2 . 12 1.23 1.24 0.96 1.00 1.14 0.96 1.25 0 .81 1.33 1.04 1. 96 1. 75 0.94 1.48 0.95 1.08 0.97

0 .37 1.27 0 .38 4.39" 0.93 0.18 0 . 20 0.35 0.14 0.46 1.23 1.21 0.24 1.37 1.17 0.50 0.42 0.16 1.18 0.05

0.81 3.02 0 .92 9.75" 1. 96 0.42 0 .45 0.88 0 .31 1.03 2.83 2.79 0 .51 3.0l 3. 20 1.16 1.02 0.39 2.57 0.12

43.80 22.46

1.25 0.34

0.62 0.47

1.44 1.10

38.29

from uncorrected mean; not included in calculation of mean,

The rapid clearance of albumin in contrast to erythrocytes suggests that albumin enters the gut lumen in a selective fashion or from multiple compartments, or bot h, rather than from a simple open-ended vascular space. REFERENCES 1. Wetterfors, J. 1965. Albumin. Acta Med. Scand. 177 : Suppl. 430: 24-29. 2. Mabry, C. C., R. H. Greenlaw, and W. D. D eVore. 1965. Measurement of gastrointestinal loss of plasma albumin: a clinical and laboratory evaluation of "chromium labeled albumin. J. Nuc!. Med. 6: 93-108. 3. Anghileri, L. J. 1964. A study of the stability of chromium-51 labeled serum albumin. J. Nuc!. Med. 5: 216-217. 4. Guillen, R. T., and M. L. Peterson. 1964. The fate of trivalent chromium in normal man and in patients with protein-losing enteropathy. J . Lab. Clin. Med. 64: 865. 5. Hopkins, L. L., and K. Schwarz. 1964. Chromium (III) binding to serum proteins,

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January 1967

CLEARANCE OF ALBUMIN AND ERYTHROCYTES

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spectrometeric technique. J. Lab. Clin. Med. 53: 777-788. Jeejeebhoy, K. N., B. Singh, R. S. Mini,and S. M. Sanjana. 1965. The use of Nb""-labeled albumin in the study of gastrointestinal protein loss, p. 61-67. In J. Kobelt, P. Vesin, H. Diggelmann and S. Barandun [eds.], Physiology and pathophysiology of plasma protein metabolism. Grune and Stratton, Inc., New York. Wetterfors, J., R. Goldberg, S. O. Liljedahl, L. O. Plantin, G. Birke, and B. Olhagen. 1960. Role of the stomach and small intestine in albumin breakdown. Acta Med. Scand. 168: 347-363. Wetterfors, J. 1964. The normal passage of serum albumin into the gastrointestinal tract and its role in the catabolism of albumin. Acta Med. Scand. 176: 787-799. Freeman, T., and A. H. Gordon. 1964. Human and rat intestine as a site of catabolism of albumin? p. 226-228. In H. Peeters [ed.], Protids of the biological fluids, Vol. 11. American Elsevier Publishing Company, Inc., New York. Corbet, J. L., J. F. D. Greenhalgh, P. E. Gwynn, and D. Walker. 1958. Excretion of chromium sesquioxide and polyethylene glycol by dairy cows. Brit. J. Nutr. 12: 226-276. Smyth, H. F., C. P. Carpenter, and C. S. Weil. 1950. The chronic oral toxicology of the polyethylene glycols. J. Amer. Pharm. Assn. 44: 27-30.