protein-losing enteropathy - from bmj and acp · intestinal protein loss the available techniques...

J. clin. Path., 24, Suppl. (Roy. Coll. Path.), 5,45-54

Protein-losing enteropathyA. S. TAVILL

From the Division of Metabolism, Clinical Research Centre, Northwick Park Hospital, Harrow, Middlesex

A large mass of evidence has been accumulated overthe past 10 years which has conclusively demon-strated that the gastrointestinal tract plays a role incatabolism of plasma proteins (Wetterfors, 1965).An increased rate of catabolism resulting from ex-cessive loss of plasma proteins into the gastro-intestinal lumen is associated with hypoprotein-aemia when the rate of synthesis is unable to com-pensate for this loss. This is the situation in a largenumber of gastrointestinal disorders (Citrin, Sterling,and Halsted, 1957; Schwartz and Thomsen, 1957;Gordon, 1959; Jeffries, Holman, and Sleisenger,1962; Jarnum, 1963; Waldman, 1966; Waldmann,Wochner, and Strober, 1969).

Qualitative detection of serum proteins in thegastrointestinal secretions by electrophoretic andimmunological techniques (Gullberg and Olhagen,1959; Holman, Nickel, and Sleisenger, 1959;Barandun, Aebersold, Bianchi, Kluthe, Muralt,Poretti, and Riva, 1960) confirms that intact proteinmolecules pass across the gastrointestinal mucosafrom plasma into lumen in health. However, they arethen subjected to the normal digestive processes andsubsequently reabsorbed as their constituent aminoacids. Thus, apart from the difficulties of ensuringcomplete recovery of secretions, the rapid digestiveand absorptive process invalidates the above tech-niques for quantification of normal and abnormallosses of serum proteins into the gut lumen. By thesame reasoning nitrogen balance studies may fail todetect loss of proteins (Jarnum, 1963) unless theabnormal process involves the large intestine.

Disorders Associated with Excessive GastrointestinalProtein Loss

The mechanism of protein loss is understood in onlya minority of the associated disorders. In thiscategory are two groups of disease processes. In thefirst group the process results in ulceration of anarea of gastrointestinal mucosa with resultant exuda-tion of plasma and interstitial fluid. This is probablythe explanation in diffuse chronic gastritis, acutegastroenteritis, shigella dysentery, regional granulo-matous enteritis, and ulcerative colitis. Secondary

ulceration of neoplastic lesions may have a similareffect.The second group is composed of diseases

associated with obstruction to intestinal lymphatics.This may result in loss of protein-rich and lympho-cyte-rich fluid into the intestinal lumen. Primaryinvolvement occurs in intestinal lymphangiectasia,Whipple's disease, retroperitoneal fibrosis, andmalignant lymphomas affecting the mesentericlymphatics. Defects in lymph drainage via the mainlymphatic channels may also occur secondary to asevere increase in central venous pressure. This maybe the case in constrictive pericarditis, tricuspidincompetence, and thrombosis of the superior venacava.

In the vast majority of gastrointestinal disordersassociated with abnormal protein loss the patho-physiology is unknown. The main diseases in thiscategory are giant gastric rugal hypertrophy, gluten-induced enteropathy, tropical sprue, allergic gastro-enteropathy, intestinal parasitic infestation (such asgiardiasis and hookworm), and diffuse colonic poly-posis. In these disorders, the pathology may bedistinctive and the segment of the gastrointestinaltract responsible for the abnormal loss may belocalized. In other disorders the intestinal proteinloss seems to be generalized; certain dysgamma-globulinaemias, the carcinoid syndrome, the neph-rotic syndrome, amyloidosis, scleroderma, systemiclupus erythematosus, angioneurotic oedema, andpost-cytotoxic therapy are but a few of the disordersin this category. A comprehensive list of all therecorded protein-losing enteropathies is provided bySchultze and Heremans (1966) and Waldmann(1970).An interesting feature of the normal transport of

proteins from plasma to gut lumen, which persistsin all forms of protein-losing enteropathy, is theapparent lack of selectivity with regard to molecularsize of the proteins leaking into the intestinal lumen.The fraction of the intravascular pool which leaksinto the lumen in both health and disease seems tobe the same for a variety of different sized proteins:albumin, transferrin, IgG, IgA, IgM, fibrinogen,caeruloplasmin, and lipoproteins (Waldmann, 1966).

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Thus, any selectivity of the capillary basementmembrane appears to be absent and plasma proteinspass through in bulk in the same concentration asthey exist in plasma. In contrast, endogenous frac-tional catabolic rates are widely different for theseproteins, and although these rates also bear nosystematic relationship to molecular size, they indi-cate the existence of a variety of mechanisms inaddition to that of gut loss for the normal degrada-tion of plasma proteins. A very different situationprevails in the nephrotic syndrome, where selectivityaccording to molecular size may be maintained. Asimilar investigative approach as that relating dis-ordered function to ultrastructural changes in theglomerulus may prove equally valuable for thegastrointestinal mucosa.

Detection and Measurement of Abnormal Gastro-intestinal Protein Loss

The available techniques for the detection andquantification of plasma protein loss in the gututilize a variety of radioisotopically labelled macro-molecules. The ideal macromolecule would bealbumin, since this is the predominant protein to belost and contributes most to the reduction in totalplasma protein levels. The ideal label would satisfythe following requirements.

CRITERIA FOR AN IDEAL LABEL1 The labelling procedure or the presence ofattached label should not alter the metabolic be-

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haviour of the protein. Its survival should be thesame as that of unlabelled, native protein.

2 The label, once attached, should not be elutedfrom the protein, except as part of the normaldegradation of the protein molecule.

3 There should be no loss of label into the gutlumen in any form, other than as the intact labelledprotein. This includes isotope released by catabolismof the protein in sites other than the gastrointestinaltract. Failure to satisfy this condition would resultin an overestimate of the gastrointestinal contribu-tion to overall catabolism.

4 Once excreted into the gut lumen there shouldbe no reabsorption of the isotope, either as intactlabelled macromolecule or as free isotope releasedby digestive degradation of the macromolecule.Failure to satisfy this condition would result in anunderestimate of protein loss in the gut.

5 Ideally, the isotope should be excretedminimally in the urine so as to reduce the risk oferror due to urinary contamination of the stools.

6 Finally, the isotope should be freely available,reasonably priced and easily measured in the stools.The labelling procedure should be simple and thereshould be no radiation hazard to the patient.A material complying with all these conditions

would permit calculation of enteric loss of protein byexpressing stool radioactivity as a percentage ofplasma radioactivity, ie, a clearance as fractionalrate of loss of the intravascular pool per day. How-ever, it would be ideal if the overall fractionalcatabolic rate of the protein could also be deter-

Fig. 1 Schematic diagram of theideal properties ofan isotopicallylabelled protein to be usedformeasurement of enteric protein lossand measurement offractionalcatabolic rate. The macromoleculeis indicated by the open symbol,the isotope by a shaded symbol.(This notation is used in all theFigures.) Albumin is used as anideal protein. There is no dis-crimination between labelled andunlabelled albumin for catabolismor for passage into the gut. Isotopeis released quantitatively into theurine following catabolism. Thereis no excessive uptake of labelledprotein or free label by the reticulo-endothelial system. There is noexcretion offree label into the gut.Although there may be absorptionofprotein digestive products fromthe gut, released isotope should notbe reabsorbed but should appearquantitatively in the faeces.

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Protein-losing enteropathy

mined. In the case of an isotope which is rapidlycleared by the kidney following its release from de-graded protein, the overall fractional catabolic ratemay be calculated as a percentage of the intra-vascular pool both from its rate of disappearancefrom the plasma and its rate of appearance in theurine. In order to be able to make both calculationswith the same labelled macromolecule, condition 5may have to be sacrificed (Fig. 1). Suffice it to say,that none of the labelled preparations availablesatisfy all these requirements. It may be of somevalue in this type of review to list these techniquesand to evaluate their relative merits.

TECHNIQUES

I Radioiodinated plasma proteins (Fig. 2)Providing that care is taken with the preliminaryprotein fractionation procedure and with the degreeand evenness of the iodination procedure (McFar-lane, 1956 and 1958), 131I- or 1251-labelled proteinsbehave identically to native proteins (Campbell,Cuthbertson, Matthews, and McFarlane, 1956;Cohen, Holloway, Matthews, and McFarlane, 1956;Bennhold and Kallee, 1959). Radioactive iodineliberated by endogenous catabolism of intravascularprotein is excreted in the urine providing that thethyroid gland is blocked with stable iodide. Evalua-tion of the semi-logarithmic plot of plasma radio-activity and excreted urine radioactivity gives ameasure of the intravascular and total exchangeablepool, the fractional catabolic rate, and by inference

in steady-state conditions the synthetic rate of theprotein. The labelling procedure is simple and thereis no risk to the patient. Thus, criteria 1, 2, and 6(above) are satisfied. Unfortunately, radioactiveiodide released by endogenous catabolism is notexclusively excreted by the kidneys; it appears inappreciable amounts as iodide actively secreted bythe salivary, gastric, and small intestinal glands. Inaddition, both this iodide and 1311 or 1251 releasedfrom labelled protein which may have leaked intothe intestinal lumen is rapidly reabsorbed furtherdown the intestine and is predominantly excreted inthe urine. The total effect of this recycling of iodideis to produce a low stool radioactivity and an under-estimate of enteric protein loss (Kerr, Du Bois, andHolt, 1967). Attempts have been made to overcomethese limitations to requirements 3 and 4. Measure-ment of protein-bound activity by direct intubationof accessible parts of the gastrointestinal tract maygive an indication of localized protein loss (Citrin etal, 1957). By design, such studies are short-term andmay give a quantitative overestimate of daily lossesby this route (Tavill, 1970). Attempts have beenmade to prevent reabsorption of released, luminalradioactive iodide by the administration of an anion-exchange resin, Amberlite IRA 400 (Jeejeebhoy andCoghill, 1961). However, in addition to the fact thatthe resin is inefficient at trapping all the 1311 liberated(Freeman and Gordon, 1964), it has been shown thatall the radioactivity measured in the faeces could beaccounted for by the salivary excretion of radio-active iodide (Jones and Morgan, 1963). Such

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Fig. 2 Radioactive iodine-labelledalbumin as a means of measuringcatabolism and enteric loss ofprotein. Its limitations for thelatter result from the reabsorptionoffree isotope. Amberlite resin willbind both iodide released fromalbumin and free iodide activelysecreted into the gastrointestinaltract.

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observations invalidate radioactive iodine-labelledplasma proteins for direct quantification of entericloss of protein. However, they remain the onlyreliable means for measurement of endogenouscatabolism. In all the other techniques to be evalu-ated, correlation has been sought between gastro-intestinal protein loss (exogenous catabolism) andendogenous catabolic rates measured with 1311_ orI 5I-labelled proteins. Such studies (Jarnum, 1963;Waldmann, 1966; Waldmann et al, 1969) haveshown that gastrointestinal protein loss is associatedwith reduced body pool sizes, a shortened albuminsurvival, and with the steady state synthetic rateswhich were normal or slightly raised. Direct measure-ment of the albumin synthetic rate using the(14C)carbonate technique in certain patients withgastrointestinal protein loss has demonstrated anincreased rate of hepatic protein synthesis (Tavill,Craigie, and Rosenoer, 1968; Wochner, Weissmann,Waldmann, Houston, and Berlin, 1968).

Radioactive iodine-labelled protein studies areuseful therefore only in distinguishing betweenhypercatabolism and diminished synthesis. They areof little value in detecting or quantifying loss ofprotein into the gastrointestinal tract.

II 131I-labelled polyvinylpyrrolidineGordon (1959) introduced 1311-polyvinylpyrrolidoneas a substance to detect abnormal permeability tomacromolecules in the digestive tract. It is a syntheticpolymer with an average molecular weight of 40,000which is unaffected by digestive enzymes and is onlyminimally absorbed from the gut lumen. Followingintravenous injection normal subjects excrete0-1 -6% (mean 0-8 %) of the dose in the stools in fourdays. A similar range has been found by Dawson,Williams, and Williams (1961). In patients withgastrointestinal protein loss 2 9-32-5% of the dosemay appear in the stools in four days. Althoughthere appears to be a clear-cut distinction betweennormals and abnormals, there are a number ofserious limitations which preclude the use of 131I1polyvinylpyrrolidone in the quantification of theprotein loss. The preparation is unstable on standing.Between 6% and 56% of its radioactivity is dialyz-able. An inverse correlation is found between thesize of the dialyzable fraction and the magnitude ofthe faecal 131I output (Jarnum, 1963). In addition,variable amounts (between 10% and 68%) of the131I label that enters the gastrointestinal tract areabsorbed (Gordon, 1959; Jarnum, 1961). It is not anormal metabolite and consequently disappearsrapidly from the plasma, being taken up in thereticuloendothelial system within 48 hours of injec-tion. No significant correlation exists between theexcreted activity and the plasma albumin concentra-

A. S. Tavill

tion or the fractional catabolic rate (Jarnum,Westergaard, Yssing, and Jensen, 1968). It thus pro-vides no information about endogenous proteincatabolism and no quantitative assessment of proteinloss. Its place seems to be limited to that of a simplescreening test.

III 5lCR-labelled proteins (Fig. 3)(51Cr)albumin was first introduced by Waldmann in1961 as a means of quantifying gastrointestinalprotein loss. More recently he and his coworkershave reviewed their accumulated experience with thiscompound (Waldmann et al, 1969). A total of 230studies were carried out in 180 subjects: 50 controlswith normal serum albumin levels and no evidence ofgastrointestinal disease and 130 patients with hypo-albuminaemia which could not be explained by pro-teinuria or liver disease. Repeated 61Cr studies wereperformed on 25 patients; and 70 patients had com-bined (51Cr)albumin and (1251)albumin studies.

Carrier-free 51CrC14 of high specific activity isincubated with albumin at pH 5-5 for 30 minutes.Excess 51Cr is removed on an Amberlite MB-1 resincolumn, previously washed with a relatively largevolume of unlabelled albumin solution. The efficiencyof labelling varies between 23 and 50%, and 85-96%of the radioactivity of the final preparation is pre-cipitable with phosphotungstic acid (PTA). Of theprotein-bound activity, 95% migrates with albuminon electrophoresis and 2-3% as f-globulin. The(51Cr)albumin (10-30 ,uCi) is administered intra-venously. For screening purposes, the percentage ofthe administered dose appearing in the first fourdays' stool is measured. However, this does not takeadvantage of the relatively long survival of (61Cr)-albumin in the serum. A more meaningful expressionof stool loss as a clearance in percentage of theplasma pool lost per day can be calculated from thethird to 12th day of the study. To allow for intestinaltransit time the stool loss should be related to theprevailing radioactivity in the preceding 24 hours.

Following oral administration of (5'Cr)albumin,93-99% of the dose is recovered in the stools.Requirement 4 (above) is therefore satisfied. Therewould be no significant reabsorption of excreted51Cr. The metabolic half-life in the plasma in normalsubjects ranges from 3 5 to 11 days (mean 7 days).This is much shorter than that of a preparation ofradioiodinated albumin administered simultaneouslyto normal controls (13-20 days). A preparation ofalbumin doubly labelled with 125I and 6lCr gave ati of 17 days for the former and five days for thelatter isotope. This clearly indicates that theshortened survival of (51Cr)albumin is not due todenaturation of the protein but is rather due to elu-tion of the label. This was also shown by Kerr et al

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(1967) using (1251)albumin heavily labelled with 61Cr.Elution of the label is associated with labelling offl-globulins, in particular transferrin. In fact the tiof the exponential part of the plasma curve afterintravenous (51Cr)albumin approaches the mean tiof transferrin of 8-7 days (Jarnum and Lassen, 1961).The transfer of label from (56Cr)albumin to /3-globu-lins can be minimized by the addition of ferricchloride to labelled serum in vitro (Van Tongerenand Majoor, 1966).The shortened metabolic survival of (51Cr)-

albumin, although not the result of denaturation,precludes its use for measurement of rates of catabol-ism or synthesis. However, it remains valid tocalculate protein loss or clearance into the gut sincethis makes the assumption only that the specificactivity of the protein lost is the same as that of theplasma. Direct validation of this assumption hasbeen provided by comparison of plasma albuminwith albumin leaked into the pleural fluid of a

patient with pleural effusion and into the urine of a

patient with nephrotic syndrome. The specific radio-activities were similar in both instances. Likewise,the specific activity of both 125J.- and 51Cr-labelledalbumin was closely similar in plasma, bile, andgastric juice of the experimental dog (Kerr et al,1967).

Stool excretion in normal subjects varies between0 1 and 0 7 % of the injected dose in four days. Stoolclearance of 51Cr is 0-2-1-6% (mean 0 64%) of theplasma pool per day, representing 2-15% (mean6 4%) of the overall catabolism of albumin (Wald-

Fig. 3 Chromium-Si labelled7 albumin for the measurement of

catabolism and enteric protein loss.Its limitations for the former result

000 °O from the affinity of 51Cr for other-94°gD plasma proteins. Its rate of disap-

pearance from the plasma reflects* *lX a heterogeneous catabolic process.

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mann et al, 1969). Of the patients with hypo-albuminaemia, those with a normal (1 25I)albuminfractional catabolic rate excreted less than 0 7% ofthe dose in four days. Thus, no false negatives weredetected. The remainder excreted 09-35% of theinjected dose in four days. All those with an excretionin excess of 2% of the dose had a significantly raisedfractional (1'25I)albumin catabolic rate. Thus, nofalse positives were found. Clearances in 20 of thesepatients with abnormal excretions demonstrated aloss of 2-60% of the intravascular pool per day. Inaddition, in patients in whom appropriate therapywas employed there was complete return of anabnormal 51Cr loss to the normal range.The properties of (51Cr)albumin may be sum-

marized in relation to the criteria listed above for anideal label.

1 The labelling procedure does not appear toalter the metabolic behaviour of the protein, butsince its survival can be measured only by the per-sistence of label, elution produces effectively thesame result.

2 Elution of label and reattachment to otherplasma proteins, in particular transferrin, precludesits use in the determination of the endogenouscatabolism of any individual protein.

3 In spite of multiple labelling, the 51Cr excretedinto the bowel is protein-bound. Since the ratio ofconcentrations of proteins in the plasma is similar tothe ratio of proteins appearing in the gut lumen, it isstill possible to express loss as a clearance or ratiobetween excreted label and total intravascular

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activity. Whether one makes the calculation fromindividual ratios of stool and plasma activity(Waldmann et al, 1969) or from total stool loss as apercentage of the integrated plasma radioactivitycurve (Van Tongeren and Reichert, 1966) makeslittle difference. The clearance appears to remainconstant after equilibration. The use of the clearancemethod enables determination of the fraction ofoverall breakdown that occurs by loss into the gut;it minimizes errors due to variation in stool transittime, and avoids errors due to variable distribution,degradation, and excretion by the kidneys.4 There is no significant reabsorption of label

excreted into the gut lumen. In this respect it isconsiderably superior to radioactive iodine.

5 As with (1311) or (1251)albumin, the appearanceof 5'Cr in the urine during the post-equilibrationperiod reflects endogenous degradation of labelledproteins. Precautions are necessary during theclearance study to avoid contamination of faeceswith urine.

6 The isotope is easily counted in plasma andstools. Its peak energy permits discrimination from125I but not from 1311. Since 5'Cr is eluted fromlabelled albumin in vitro there is little point in under-taking any laboratory labelling procedure. Intra-venously injected 5'CrC13 will bind avidly to plasmaproteins in vivo. The disappearance rate of 51Cr fromplasma following such an intravenous injection isidentical with that of labelled albumin in vitro or

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whole serum (Van Tongeren and Majoor, 1966).A close correlation between the clearance of 61Cr

and both serum albumin level and fractionalcatabolic rate has been found in those patients inwhom hypoalbuminaemia is not associated withdiminished synthesis (Waldmann et al, 1969). Thus,providing that endogenous catabolism is inde-pendently assessed with radioactive iodine-labelledserum albumin, the fraction of overall breakdownthat results from loss into the gut can be assessedwith 51Cr. This is because the same fractional rateof clearance exists for all the plasma proteins labelledin vivo. In this way it is possible to assess abnormalloss and also to provide some estimate of the gastro-intestinal role in normal catabolism. Figures ofaround 10% of overall catabolism in normal subjectsagree well with estimates made by other acceptablemethods of investigation.

IV 05NB-labelled albumin (Fig. 4)Albumin can be labelled electrolytically with95niobium and the unreacted isotope removed bydialysis or by passage through DEAE-cellulose.Such a preparation has been used for the study ofgastrointestinal protein loss (Jeejeebhoy, Singh,Mani, and Sanjana, 1965; Jeejeebhoy, Jarnum,Singh, Nadkami, and Westergaard, 1968) in 18control patients and five patients with protein-losingenteropathy. Although some of the control patientshad low serum albumin levels all had either a normal

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Fig. 4 The use of 95Nb-labelledalbumin for measurement ofcatabolism and enteric loss ofprotein. Reservations about itsvalue for the former lie in therapid rate ofplasma disappearanceofsome preparations.

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(51Cr)albumin test or a normal catabolic ratemeasured with (1311)albumin. The five patients withprotein-losing enteropathy had been judged ab-normal on the basis of an elevated catabolic rate andan abnormal -"Fe iron dextran or 51Cr-labelledalbumin test.

Studies in vitro on the (95Nb)albumin preparationshow that 77-85% is non-dialysable and 54-72% isprecipitable with trichloracetic acid, while only7-31 % is non-dialysable against a protein-containingsolution. The latter may be improved by secondtreatment on a DEAE-cellulose column. This resultsin a preparation which on electrophoresis or DEAE-Sephadex A50 chromatography shows radioactivityexclusively confined to the albumin peak.

Criteria of acceptability are based on the com-parative behaviour of (95Nb)albumin and (131I)-albumin in vivo. Comparison of plasma disappear-ance curves expressed as ratios of fractional catabolicrates calculated by graphical analysis (Matthews,1957) show good agreement between the two prepara-tions. In controls, for the first six days, 95Nb rangesfrom 0-1-7-0% (mean 2-0%) of the injected dosewhile the faecal clearance expressed as a percentageof the intravascular (95Nb)albumin pool is 0-3-2-2%(mean 1-2 %) per day. In patients with provenenteric protein loss the faecal excretion of 95Nb in-creases to 7-31 % of the injected dose in six days,giving a clearance of 3-33% of the intravascular poolper day.

In all groups of patients the urinary excretion is

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low compared to that of 131I or 1251 in studies usingradioactive iodine albumin. The urinary clearancecannot therefore be used to calculate endogenouscatabolic rates. However, it is still possible to esti-mate faecal clearance as a percentage of the overallcatabolic rate. A value of about 12% of the totalcompares well with figures obtained using othertechniques.

Providing that care is taken in the labelling processto remove excess 95Nb and to purify further theprotein by DEAE chromatography, a labelled pro-tein can be prepared which has a similar metabolicbehaviour to (131I)albumin. There is no evidence thatnon-protein-bound 95Nb normally passes into thegastrointestinal lumen, and no evidence for signi-ficant reabsorption. The dialysability of the prepara-tion does not seem to alter over a period of 10 daysand does not appear to affect the faecal excretion of95Nb providing this is expressed as a clearance ofthe intravascular (95Nb) albumin pool. Urine con-tamination of faeces may be a source of experimentalerror. The isotope has the advantage of reasonablylong physical half-life and its peak energy makes iteasy to discriminate from the other isotopes incommon use for this purpose.

V 5"Fe-labelled iron dextran (Fig. 5)Andersen and Jamum (1966) introduced 59Fe-labelled iron dextran as a means of overcoming theerrors due to urine contamination of faeces whichare present with the 51Cr method. No radioactivity

j Fig. 5 The use of 59Fe-irondextran for quantification of entericprotein loss. Not being a protein,

(( IA its rate of disappearance from theplasma is very rapid owing to

ao uptake by the reticuloendothelial'"o ; system. However, urine loss ofoQa 0o0o isotope does not occur andfaecal17A - output of isotope can be expressedv_J- as a clearance from the plasma

during a short-term study.

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appears in the urine following intravenous injectionof this preparation. Although these earlier studiesindicate a variable recovery of an orally administereddose of (59Fe) iron dextran in the faeces (51-105 %),there is clear separation of the faecal loss of anintravenously injected preparation in normals andpatients with enteric protein loss (0-2-07% against1 0-1388%). Stools are collected from the time ofinjection until the appearance of a carmine markerswallowed four days after the injection.

Iron dextran has a molecular weight spectrumbetween 50,000 and 250,000 and it is speculated thatits faecal clearance might be related to the entericloss of plasma proteins with a similar molecularweight range. Its plasma disappearance rate on theother hand is very different to that of any plasmaprotein. It disappears from the plasma with a ti of16-21 hours. Nevertheless, its plasma clearance intothe faeces is 0 to 0-8% (mean 0 4%) of the plasmapool per day which compares well with that of 51Cradministered as 61CrC13 (Jarnum et al, 1968). It ispossible to discriminate 59Fe in the presence of l3lJ,125, or 56Cr; this is a distinct advantage over thelatter isotope which can be measured in the presenceof 125J only. In a definitive study in 21 patients withprotein-losing gastroenteropathy, Jarnum et al (1968)demonstrated a clearance of (59Fe) iron dextranranging between 2-0 and 32-6% (mean 11-3 %) of theintravascular pool per day. There is a significantnegative correlation between serum albumin andfaecal 59Fe clearance and a significant positive cor-relation between fractional catabolic rate and faecal

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69Fe clearance. This is in spite of the fact that inthree patients who received simultaneous "Fe-labelled iron dextran and 51CrCI3 the clearance of59Fe was less than that of "ICr in every case. In fact,from the regression coefficient relating faecal 59Feclearance to fractional catabolic rate it is calculatedthat a four-fold increase in the latter would be re-flected by only a two-fold increase in clearance. Thus,although the evidence points to bulk loss of macro-molecules through the intestinal mucosa there maybe some retention of leaked-out 59Fe or possiblyeven some reabsorption through diseased areas.

Since iron dextran is a synthetic polymer and nota protein it can give no information about overallprotein catabolism. However, although it is rapidlytaken up by the reticuloendothelial system, its rateof clearance into the gut is useful in the detection ofincreased enteric protein loss. Its special advantageslie in the ease of counting in the presence of otherisotopes, its absence in the urine, and the fact thatonly a short-term study is necessary.

VI 6 7Cu-labelled caeruloplasmin (Fig. 6)It has been pointed out by Waldmann, Morell,Wochner, Strober, and Sternlieb (1967) that caerulo-plasmin labelled with 67Cu possesses many of thequalities of the ideal label for the detection of gastro-intestinal protein loss. It is a plasma protein (mole-cular weight 140,000) containing eight copper atomsas an intrinsic part of the molecule. Although thecopper atoms can be exchanged in vitro this does notoccur in vivo. Introduction of 67Cu into the molecule

CO Fig. 6 The use of 67Cu-labelledcaeruloplasmin for measurement ofcatabolism and enteric loss ofprotein. Although urine excretionis minimal, its rate ofdisappearancefrom the plasma gives a measuresof catabolic rate. Providing that

° o0 some correction for biliary extre-'Do °o tion of 67CU is made, the faecalao 0 c0 output of isotope reflects the

clearance of labelled caeruloplasminfrom the plasma into the gutlumen.

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in vitro does not alter the metabolic behaviour of theprotein (Sternlieb, Morell, Tucker, Greene, andScheinberg, 1961). Radioactive non-protein boundcopper is not actively secreted into gut lumen and isrelatively poorly absorbed. Urine loss is minimal. Itcan therefore be used for estimation of overallcatabolism (and in the steady state also synthesis)and for the measurement of the role of the gastro-intestinal tract in normal and abnormal metabolism.

Preparation and labelling of a pure caeruloplasminis an arduous task. The final preparation gives a

single line on Ouchterlony immunodiffusion againstanti-whole human serum, and over 98% of the 67CUis precipitable with phosphotungstic acid.The plasma ti averages 6-1 days and the fractional

catabolic rate of 17-8% of the intravascular pool perday compares well with a figure of 21 % for 1251_labelled caeruloplasmin. Clearance into the stool, ex-pressed as the ratio of counts in the stool per 24hours to counts in the intravascular pool 24 hoursearlier, ranges from 1 9 to 3 9% of the intravascularpool per day. This accounts for only 11-22% of thenormal total catabolism. The use of multicompart-mental analysis corrects for loss of some unbound67CU into the bowel released from catabolizedcaeruloplasmin and brings this figure down to lessthan 10% of overall catabolism.

In patients with lymphangiectasia and protein lossthe plasma ti may be reduced to 3-1 days, giving afractional catabolic rate of 32'4% ofthe intravascularpool per day. The gastrointestinal clearance of 67Cuunder these circumstances is increased to 25'5% ofthe intravascular pool per day, accounting for 75%of total catabolism. Thus, there is good correlationbetween fractional catabolic rate and stool loss.

It is clear that (67Cu) caeruloplasmin studies can

give precise quantification of both enteric loss andoverall catabolism. However, the preparation isdifficult and expensive to produce and the isotope

has an inconveniently short half-life of 62 hours. Onthe other hand, it has given a very accurate indica-tion of the role of the gastrointestinal tract in normalcatabolism and may be of special value in patho-logical states where other techniques give contradic-tory results.

Summary

The suspicion of protein-losing enteropathy shouldbe aroused in any hypoproteinaemic situation inwhich no obvious cause for diminished proteinsynthesis or excessive loss by other routes is evident.It is necessary to exclude liver disease, protein mal-nutrition, and the nephrotic syndrome in the firstinstance. The most difficult situation may arise insteatorrhoeic states, many of which can be associatedwith excessive protein loss in addition to diminishedsynthesis consequent upon impaired digestion or

absorption. Such conditions may be diagnosed bymeans of specific tests of absorption, histologicalexamination of mucosal biopsies, intestinal bacterio-logy and parasitology, pancreatic function tests, anda variety of radiological techniques, includingbarium studies and lymphangiography.The situation may still remain where it is im-

possible to distinguish between diminished synthesisand excessive degradation. This requires investiga-tion with radioactive iodine-labelled albumin. If thisshows a diminished survival of labelled protein, thenexcessive degradation is the explanation for thehypoproteinaemia. However, it gives no clue as tothe cause of hypercatabolism and other specific testsfor enteric protein loss are required. The advantagesand disadvantages of the available techniques are

summarized in Table I. It seems that for routine use

intravenous 51CrCJ3 is the best available technique,and that stool clearance should be expressed as a

ratio of faecal to total plasma radioactivity.

Technique Measurement of Pool Quantification of Urine Excretion CommentSizes and Endogenous Enteric Loss as of IsotopeCatabolism Fraction of Intra-

vascular Pool clearedper Day

I Radioactive iodine labelled proteins Yes No Yes For detection of hypercatabol-ism; to be used in conjunctionwith other tests

II (5"1I)polyvinylpyrrolidone No No Yes Can be used as a screening testIII (5'Cr)albumin No Yes Yes Valuable. Can be simplified by

using in vivo labelling with CrCI,IV (95Nb)albumin Possible, with care Yes Yes May prove valuable for routine

useV ("9Fe)iron dextran No No No Can be used as a rapid screening

test. Easy to discriminate fromisotopes

VI (67Cu)caeruloplasmin Yes Yes Minimal Too costly for routine use

Table I An assessment of the relative merits of the available methods for measuring enteric loss ofplasma protein

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