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CLINICAL MEASUREMENT OF
To a large extent, the clinical isotopic study of plasma-protein (PP) metabolism has been limited to measuring the distribution kinetics and metabo lism of the principal PP components. Radioiodinated proteins have been the principal tracer substances employed, and their use has promoted greatly our knowledge of PP metabolism in health and disease. During the past few years, more efforts have been directed at developing clinical isotopic methods to measure quantitatively the synthesis of PP. These efforts were largely motivated by the recognition of certain shortcomings and limitations in the radio iodinated-protein methods, particularly in studying protein synthesis in a changing metabolic system. The present article is an attempt to appraise the radioisotopic methods which are currently available for measuring PP synthesis in man. The principal aim is to present the theoretical and conceptional principles on which these methods are based.
METABOLIC SYSTEM OF PLASMA PROTEINS The physiological metabolic system of PP is gen erally considered to be composed of the following components:
- Synthesis sites. Plasma albumin and fibrinogen are exclusively formed in the liver (1—3). The greater part of alpha- and beta-globulins are syn thesized in the liver. It has been estimated that 10% of alpha1-globulin, 25 % of alpha2-globulin and 50% of beta-globulin are formed extrahepatically (4). Most, if not all, of the gamma-globulin is formed by the lymphocytic and plasma cells (5). Present evidence indicates that the free intracellu lar amino acids are the immediate precursors of the same acids incorporated into protein (6). The time necessary for the completion of intracellular pro tein synthesis is quite short—3 mm in the case of mouse serum albumin (7). Because very little PP produced in the liver is stored there, newly formed protein emerges rapidly in the venous blood or hepatic lymph. Liver-produced PP appears in the plasma within 15—30 mm in various animal species (3—14). This contrasts with the much slower trans
fer of new gamma-grobulin from extravascular syn thesis sites to plasma by the lymphatic system (15).
- Intravascular plasma pool. The plasma-protein pool constitutes a well-mixed compartment that can be adequately sampled during the course of a radiotracer experiment without seriously alter ing its size or disturbing the equilibrium of the system.
- Extravascular interstitial protein pool. Because
PP passes outward across the capillary wall into the
interstitial tissue spaces, the distribution of extra.- vascular protein is a function of capillary permeabil ity. Capillaries of the liver, spleen and intestines exhibit high permeability in contrast to low values found for capillaries of muscle and skin (16,17). It is generally believed that protein flow between blood and lymph is an unidirectional process (18—
- in which proteins circulate into the highly rami fled tissue spaces and small lymphatics and thence to plasma (21).
4. Breakdown sites. The exact site of PP catabo
lism is not known. However, various theoretical and experimental considerations suggest that the catabolic sites must be functionally close to the intravascular pool (22—29). The products of protein catabolism are amino acids that can re-enter the precursor pool for reutilization in the synthesis of various tissue and blood proteins (30).
PP METABOLISM STUDY BY TRACER TECHNIQUES
A complete kinetic description of the metabolism
of a specific plasma protein should include estimates of the total exchangeable protein, its partitioning into intravascular and extravascular compartments and the rates of intercompartmental exchange. The rates of synthesis and catabolism of the protein should also be given. Tracer experiments can be directed towards either the anabolic or catabolic components of PP metab olism.
Received March 9, 1967; accepted Oct. 19, 1967. C Present address: Faculty of Medicine, University of Alexandria, U.A.R.
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PLASMA-PROTEIN SYNTHESIS
Hassan K. Awwad* and E. James Potchen
The Edward Mallinckrodt Institute of Radiology, Washington University School of Medicine, St. Louis, Mo.
AWWAD AND POTCHEN
scription of the prevailing physiologic processes in the unsteady metabolic system. This is particularly critical when the feedback and homeostatic regu latory mechanisms have to be considered (32).
METHODS OF EVALUATING PLASMA-PROTEIN SYNTHESIS RATE Direct method. The use of labeled amino acids for measuring synthesis of PP depends on the precursor product relationships defined by Zilversmit (33) and Reiner (34). These methods are applicable only in the case of liver-produced PP as will be shown below. When a labeled amino acid is injected, the absolute rate of synthesis of a specific protein product is given by synthesis rate = f/ta@ gm/hr
Radiotracer study of PP synthesis involves the in troduction of a labeled amino acid into the pre cursor pool. Some method is then needed to evaluate the specific activity of the intracellular amino-acid pool as well as the product protein. The realization of these two measurements and their use in the cal culation of the absolute synthesis rate of protein will be discussed later. In the most commonly used catabolic-type radio tracer study an aliquot of a preformed plasma pro tein is labeled in vitro with radioiodine; it is then reintroduced into plasma, and its disappearance is recorded for a given time. The distribution of the labeled protein and its catabolic rate can then be deduced. Both anabolic and catabolic types of radiotracer studies are technically feasible in clinical applica tions. However, each has its own specific merits and limits of applicability. Studies of synthesis require the existence of a single well-defined precursor pool for the specific plasma protein. Albumin and fibrinogen, for ex ample, are formed exclusively in the liver and are therefore particularly suitable for synthesis studies. Gamma-globulin, on the other hand, is synthesized
by the widely distributed lymphocytic cells and the
precursor pool is too diffuse for the effective use of anabolic tracer methods.
A catabolic study can give the kinetics of dis
tribution of the labeled protein as well as its catabolic rate. The synthesis rate can be equated to that of catabolism in an equilibrated system. A study of synthesis, on the other hand, gives the anabolic rate directly but does not indicate the distribution of protein. Catabolic studies which last 2 or 3 weeks require the assumption of a steady state. A study of syn thesis, in contrast, can be eompleted in a few hours and an unsteady state can be tolerated because the metabolic activities are unlikely to alter dramatically during the course of a few hours. This method is therefore applicable during the expansion or dis integration of the protein system before the full activation of feedback and homeostatic factors alter the metabolic state to a new equilibrium. Such dis equilibrium states are of considerable interest from the standpoint of understanding basic physiologic functions as well as of clarifying mechanisms in volved in the changing protein system. Attempts have been made recently to extend the mathematical formulation of the catabolic studies with radioiodine-labeled proteins beyond the steady state (31,32). The difficulties that such an ap proach encounters are not only in the mathematical formulations, but also in the adequate physical de
in which f is the fraction of the injected radioactiv ity appearing in protein in the course of t hours, a is the mass in grams of the particular amino @ acid residue in 1 gm protein and is the mean specific activity of the intracellular free amino acid at the site of synthesis expressed as a fraction of the injected radioactivity per gram amino acid. The value of f in Expression 1 is given by
f aE = s(t)+kf s(b)dbI 1 0
(2)
where 5(t) is the specific activity of the employed amino acid in protein at time t, k is the fraction of labeled protein formed at time b and lost in the interval t-b through diffusion into the extravascular @ space or catabolism, b is any time between 0 b @ t and p is the total mass of intravascular protein. In tracer synthetic studies lasting only a few hours no correction for the return of labeled protein from the extravascular space is needed. Moreover re utilization of the labeled amino acid released from the breakdown of labeled proteins can be neglected in such short-term studies (35). The value of f in Eq. 2 can be determined di rectly by specific-activity measurements of the cir culating protein, provided that the amino-acid pre cursor used is not readily converted into other amino acids so that the radioactivity in protein remains associated with one amino acid only. However, the metabolism of most labeled amino acids used in clinical and experimental studies results in secondary labeling of other amino acids, e.g., labeling of serine from injected 14C-glycine (36) or labeling of cys teine and cystine after the injection of 355_ or 75Se.- labeled methionine (37,38). The usual practice, therefore, involves the hydrolysis of the purified plasma protein and the isolation of the amino acid
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AWWAD AND POTCHEN
proved to be of practical use and will be discussed where a(t) in both expressions represents the in in detail. stantaneous specific activity of A. The ratio B4/C Precursor-product relationships circumventing the is given by necessity of measuring the specific activity of the pre- B4 k B @@@ cursor pool: The general scheme. Let A be a pre- (t0,t1 ) k C m 10) @ cursor compound whose pool size at time t is given @@,t1) (t0,t1) n by A(t). Consider two products, B and C, which The last expression can be written in an alternative are exclusively formed from A in one and only one way: metabolic system, and let A, B and C possess a During the interval (t0,t1). common chemical site, 5, which can be effectively labeled. If kb and k@are, respectively, the two rate Radioactivity entering B constants of the two reactions A -@ B and A -* C Radioactivity entering C expressed as fractions of A per unit of time, then ( lOa) the masses of the products B and C formed from A Mass of A entering (or converted into) B during a time interval t0,t1 are given, respectively, by Mass of A entering (or converted into) C
ft1 or @@@ B(t@,t1) = nkb J A(t) dt (5) R d B to Mass of A entering B = a loactivity entermg Radioactivity entenng C and C(t0,t1) = mk@f@1 A(t) dt (6) X Mass of A entering C ( lOb) to If B is a liver-produced PP and A is a precursor where n and m are, respectively, the ratios of the amino acid which can be labeled in a specific site, number of grams of B and C to the number of grams the problem of measuring protein synthesis will re of A in B or C in the event that precursor A is solve itself into finding a product C that is exclu incorporated intact in B or C. Alternatively, if the sively formed in the liver to which the radioactive products are simple chemical modifications convert- marker can be transferred by a specific metabolic ing A into the two products with preservation of the reaction and whose synthesis can be measured by specific locus s, then n and M represent, respec- some relatively simple independent method. Exam tively, the ratios of 1 gm of the product to the mass ples of this method are as follows. of A in grams necessary to synthesize 1 gm of the 1. The arginine urea system : Urea synthesis in product. the liver proceeds according to the following reac It is noted that Expressions 5 and 6 permit the lions: possibility that the pool A is variable and is hence CO. + ammonia + omithine— Citrulline given as a function of time. This contrasts with the expression for the glycine pool size where it is as- Citrulline + ammonia Arginine sumed to be constant.. Argmme.. (^) + arginase. ________ (^) ‘Urea+ ornithine.- The ratio of the masses of the products formed during to,t1 is given by These reactions take place exclusively in the liver. Arginine represents precursor A in the general (7) scheme whereas the carbon atom in the 6-guanido position of arginine corresponds to the specific locus If a radioactive precursor A4 labeled in the S which can be substituted by 14C and which is the specific locus s is introduced into the reaction sys- origin of urea carbon. Products B and C in this tem, radioactivity will eventually appear in B and system are, respectively, a liver-produced PP (e.g., C. During the time interval t@,t1,the radioactivity albumin or fibrinogen) and urea. B4 appearing in B is given as A specialized form of Relation lOb can be written: ti Arginine guanido C entering albumin B(t@,,t1) = kb f A(t) a(t) dt. (8). - @ to — Radioactivity entenng albumin ,.*
. -.. - - Radioactivity entering urea Similarly the radioactivity C4 appearing m C is given by x Guanido C used in urea synthesis. ti Dividing both sides of this equation by the mass of C4(t@,,t1)= k@f A(t) a(t) dt (9) guanidine carbon in the arginine of the total serum to albumin we obtain in a given time interval:
B(t0,t1) —nkb C(t0,t1) —mk
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CLINICAL MEASUREMENT OF PLASMA-PROTEIN SYNTHESIS
Fractional synthesis of protein
= Urea synthesis (gm) urea carbon x Spec act of quamdo C in arginine of protein Total activity associated with urea
Urea excreted in urine during the interval = Urea in body water at t — Urea in body water at t0.
It was soon realized that such an estimate is not accurate in view of certain pecularities of urea me tabolism (46). Endogenous decomposition of urea, a phenomenon that was demonstrated in the forties (47,48) can lead to an underestimate of urea syn thesis if calculated from the above formula. Most of the degradation of urea takes place through the urease activity of micro-organisms (49—52)although some urease activity is also found in the liver, kid ney and gastric mucosa (49,53,54). Endogenous degradation can be minimized by the administration of neomycin (46). Extrahepatic synthesis of urea isanothersourceoferror.Arginaseactivityhasbeen demonstrated in the skin (55), brain (56) and intestinal coliform bacteria (57). Protein-synthesis measurement then will be in error in proportion to the extent of extrahepatic urea synthesis. In an attempt to circumvent these difficulties, Mc Farlane derived the fractional synthetic rate of urea from the disappearance curve of injected 13C-urea, whose slope k gives the fractional catabolic rate which can be equated to that of synthesis in an equi librated urea system (58). The disappearance curve of endogenously labeled 14C-urea following the ad ministration of 14C-carbonate or ‘4C-arginine cannot be used to derive the fractional catabolic rate. The continued synthesis of urea from hepatic arginine and the demonstration of the occurrence of signifi cant reutilization of the 14C-label in the synthesis of both urea and protein (58) will result in an under estimation of urea degradation. The situation is even worse if 15N-urea is used as an indicator of urea catabolism (58). It would seem, therefore, that the best estimate of urea degradation is that derived from the disappearance of intravenously injected 13C-urea. To convert the urea fractional synthetic rate into mass of urea, k has to be multiplied by the urea pool size. The estimate of the pool size, however, has its own difficulties. One of these is that urea concentra tions within the different compartments of body water might differ from that of plasma (60,61 ). A second difficulty is the recent demonstration of the existence of a renal urea pool not exchangeable with total-body urea and accounting for 2—15% of the total pool (46). In view of these considerations, an expression for protein synthesis can be derived by rewriting Eq. 12 as follows:
Fractional rate of synthesis of protein
= Fractional rate of synthesis of urea x (13)
(12)
The arginine-urea system was first used in the study of PP metabolism by Swick (44). It could be demonstrated that urea carbon emerging from the liver of rats fed for several days on a diet cotitain ing constant amounts of Ca14CO3 could be used as an indicator of the specific activity of guanido carbon of free hepatic arginine. McFarlane (45) substi tuted a single injection of 14C-carbonate for the
continuous feeding procedure of Swick with the
advantage that only a minute fraction of the ‘4C-label is incorporated as guanidine carbon in liver-pro duced PP, whereas the feeding procedure will result in extensivelabelingof bloodand tissueproteins. Reeve and co-workers used a single intravenous in jection of 6-14C-arginine with the advantage of a higher utilization efficiency in protein synthesis (35). The carbonate method has a lower utilization effi ciency, yet there is preferential labeling of liver produced PP because 6-14C-arginine is exclusively formed in the liver (39). The actual procedure takes 4—6hr and involves separation of albumin or fibrinogen from 4—6 se quential plasma samples, protein hydrolysis and iso lation of arginine on an ion-exchange resin. The specffic activity of guanido carbon of arginine is determined after its conversion into CO2 by incuba tion with arginase (39). Urea carbon specific activity is alsodeterminedasCO2liberatedthroughtheac tion of urease (46). From these specific-activity measurements the total activity associated with urea can be calculated. This included both the urea ex creted in urine during a given time interval as well as urea retained in body water. The specific activity of guanido carbon of arginine in protein should be corrected for the fraction of labeled protein lost by diffusion and catabolism. This loss can be estimated by the injection of the corresponding radioiodinated human protein simul taneously with the 14C-label. Alternatively an ap proximate correction can be introduced by increas ing the apparent synthetic rate by 10% for both albumin and fibrinogen when the interval used is 3.5—4.4 hr and by 15% for intervals between 5. and 6.6 hr (39). This correction does not take into consideration individual variations of these values. The main difficulty of the urea-arginine method is the estimation of urea synthesis used in expression (12). In the original procedures of Reeve (35) and McFarlane (39) urea synthesized in the interval to,tl was equated with
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CLINICAL MEASUREMENT OF PLASMA-PROTEIN SYNTHESIS
serum albumin specific activity as a function of time after the preliminary equilibrium period. The fundamental premise of this method is the assumption of equality of the serum albumin specific activity with the average specific activity of body albumin so that the fall of the latter's specific activity is a function of the rate of formation of new Un labeled albumin. More rigorous mathematical de scription of the kinetics of iodoalbumin (20,65), however, shows a net transfer of labeled albumin from the extravascular space back to plasma where it is catabolized. The existence of specific-activity differences between plasma and interstitial albumin would affect the slope of the serum-albumin specific activity curve unrelated to synthesis of unlabeled albumin. Moreover, a knowledge of the relative pool size of the plasma and extravascular protein and the changes that may occur during the period of meas urement are required. The advantage of the method, however, is that it gives estimates of both the cata bolic and anabolic components of the metabolism of the labeled protein with the use of a single tracer.
CONCLUSION The theoretical rationale and methodology for the clinical assessment of plasma-protein synthesis has been discussed. The catabolic-anabolic balance methods are the simplest to perform on a routine basis, but they suffer from the necessity of assuming a steady state and prolonged observation time which cause rapid or short-term alterations in plasma protein anabolism to be obscured. The indirect meth ods relying on precursor-product relationship which avoid the necessity of measuring the specific activity of the precursor pool are the most practical means of determining plasma-protein synthesis on a short term basis. Of these techniques, the arginine-urea system advocated by McFarlane currently provides the most reliable assessment of albumin and fi brinogen biosynthesis, notwithstanding the limita ton of extrahepatic urea formation. The proposed cyst(e)ine-sulfate system offers advantages over the current methods from both a technical and theoreti cal point of view. However, the site of endogenous sulfate formation must be clearly defined before this method can achieve routine clinical application.
ACKNOWLEDGMENT This project was supported in part by U.S. Atomic En ergy Commission, Contract AT(30-1) 3442 under which this manuscript becomes Document NYO 3442-20.
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