Trypsin-Binding α-2-Macroglobulin in Patients with Acute Pancreatitis

GASTROENTEROLOGY® Official Publication of the American Gastroenterological Association © COPYRIGHT

VOLUME

1972 THE WILLIAMS

& WILKINS

CO.

March 1972

61

NUMBER

3

TRYPSIN-BINDING a-2-MACROGLOBULIN IN PATIENTS WITH ACUTE PANCREATITIS NAIlEEL

F.

ADHAM, M .D ., BARBARA DYCE, AND

B. J .

HAVERBACK, M.D.

Department of Medicine, University of Southern Californio, Los Angeles, California

We have reported that a 19 S macroglobulin in serum which migrates as an a-2-globulin forms an enzymatically active complex with trypsin and chymotrypsin. The esterolytic activity of the bound enzymes is protected from the inhibitors in serum as well as those from soybean and from pancreas. The purpose of this study was to determine whether a significant difference in serum trypsin-binding activity exists in patients with acute pancreatitis when compared to controls. The serum trypsin-binding activity was determined in 20 control subjects and 12 patients with acute pancreatitis. The mean value for the control group was 114 ± 13 Jlg of trypsin bound per ml of serum and for the pancreatitis group 41 ± 7 Jlg per ml (P < 0.01) . The a-2-macroglobulin content of the sera was determined by an antibody-agar radial diffusion procedure employing a highly purified primary standard of a-2-macroglobulin. The mean value of the 12 normal subjects was 199 ± 24 (2 SEM), and of the 12 pancreatitis patients, 181 ± 30 (2 SEM) . The fact that the serum trypsin-binding activity in acute pancreatitis is markedly reduced with the level of a-2-macroglobulin being unchanged, indicates that in this disease a substance, likely an enzyme, considerably in excess of the normal, is bound to the a -2-macroglobulin. A number of studies have been made searching for the presence of proteolytic enzymes in serum as an aid to the diagnosis of pancreatic disease. Hecent attempts to demonstrate protein-splitting enzymes in the serum of pl:!-tients with acute pancreatitis have yielded variable Received August 27, 1971. Accepted October 27, 1971. Address requests for reprints to: Dr. B. J. Haverback, Department of Medicine; University of Southern California School of Medicine, 2025 Zonal Avenue, Los Angeles, California 90023. This work was supported by the United States Public Health Service Grant AM 04683·11.

results. 1. 2 Nardi and Lees,3 using the synthetic substrate benzoyl-L-arginine amide hydrochloride, observed an increase in the arginine amidase activity of serum in patients with pancreatitis and pancreatic carcinoma. They equated this activity to trypsin and attributed its elevation to an obstructive process in an actively secreting gland. Gullick 4 also reported distinct elevations of benzoyl-Larginine amidase activity in the plasma of patients with pancreatitis and suggested that the plasma benzoyl- L-arginine amide hydrochloride reaction represents blood trypsin activity. Many investigators 365

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ADHAM ETAL .

have attempted to duplicate these results but have been unsuccessful. 5-9 The fact that blood contains many proteolytic enzymes which can hydrolyze the synthetic substrates commonly used for the measurement of trypsin makes the assay of this enzyme in serum difficult and the results open to question. 10 - 14 The measurement of trypsin in serum is further complicated by the presence in serum of a large amount of trypsin inhibitor. This inhibitor has been studied by several investigators and has been shown to behave electrophoretically as an a-l-globulin. 15 - 18 It seemed highly improbable that active trypsin would be found in serum in the presence of large amounts of trypsin inhibitor but Haverback and associates 10 recently have reported a 19 S macroglobulin in serum which migrates electrophoretically as an a-2-globulin and binds trypsin and chymotrypsin. The trypsin (or chymotrypsin) a-2-globulin complex is enzymatically active, but more importantly, the enzyme in this complex is protected from the inhibitors in serum as well as those in soybean, ovalbumin, pancreatic juice and tissue, and bovine parotid gland (Trasylol).lo The a-2-globulin binding protein was isolated and purified from the lipid-poor euglobulin of Cohn Fraction 111-0, and identified as an a-2-macroglobulin closely related to the 19 S glycoproteins. 19 Further studies have shown that the a-2-macroglobulin is separable into five components with differing electrophoretic and enzyme binding properties. 20 This study was designed: (1) to determine the value of measuring the serum trypsin-binding activity (STBA) in acute pancreatitis and other disease states; and (2) to determine whether a change in the level of trypsin bound by a-2-macroglobulin in acute pancreatitis is caused by a change in the a-2-macroglobulin or whether another substance, likely an enzyme, is occupying binding sites. 21

Methods The following materials were used: (1) buffer for trypsin determinations: 0.005 M Tris (hydroxy methyl) aminomethane (pH 8.2) containing 0.04 N NaCI and 0.02 M CaCI 2 ; (2)

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buffer for chymotrypsin determinations: 0.005 Tris (hydroxymethy\) aminomethane (pH 7.8) containing NaCI and 0.005 M CaCI 2 ; (3) the trypsin substrate p-toluene-sulfonylarginine methyl ester (TAME) (Mann Research Laboratories, New York, N.Y.) was prepared in a 0.1 M solution with Tris buffer; (4) the chymotrypsin substrate N-acetylL-tyrosine ethyl ester (ATEE) 1 was prepared in 50% methanol and 50% 0.01 M Tris buffer to give a final concentration of 0.036 M; (5) trypsin 1 (twice-crystallized, salt-free) and chymotrypsin 1 (crystallized three times, saltfree) were prepared in concentrations of 100 mg per 100 ml in 0.005 N HCl containing 0.005 M CaCI 2 • Working standards were diluted to the desired concentration with the same dilutent. (6) Soybean trypsin inhibitor (lyophilized salt-free powder) was prepared in a concentration of 100 mg per 100 ml in normal saline. Subjects. Group A consisted of 20 adult normal subjects all without evident pancreatitis or gastrointestinal disease. Group B consisted of 12 patients with acute pancreatitis. Determination of TAME esterase activity. Serum (0.5 m!) was mixed with 7.0 ml of Tris buffer to which 2.5 ml of the TAME substrate were added and the pH of the final solution adjusted to 8.2 with 0.1 N HC!. The esterolytic activity of the mixture was determined titrimetrically using a pH-Stat automatic titrator (Radiometer Corporation, Copenhagen, Denmark). The titrant was 0.025 N NaOH delivered from a 1-ml syringe. The esterolytic activity was recorded as the millequivalents of titrant delivered per unit time to maintain a constant pH and was converted to micrograms of crystalline trypsin by comparison with a standard curve. 22 , 23 Determination of the A TEE esterase activity. Serum (0.5 ml) was mixed with 4.5 ml of Tris buffer to which 5 ml of the ATEE substrate were added and the pH of the final solution was adjusted to 7.8 with 0.1 N HC!. The ATEE esterase activity of the mixture was determined titrimetrically and converted to micrograms of crystalline chymotrypsin by comparison with a standard curve. Measurement of the serum trypsin-binding activity. The trypsin-binding activity of the serum was measured by adding increasing amounts of trypsin in increments of 5 to 10 /-lg to two sets of tubes each containing 0.1 ml of serum and 1 ml of Tris buffer. The tubes were incubated at 37 C for 30 min after which Tris buffer was added to each tube to bring its total volume to 7.5 m!. To the first set of tubes 2.5 ml of the TAME substrate were added and M

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TRYPSIN-BINDING a-2-MACROGLOBULIN IN PANCREATITIS

March 1972

the tryptic activity was determined titrimetrically. To the second set of tubes, soybean trypsin inhibitor was added after the initial incubation in amounts sufficient to completely inhibit all added crystalline trypsin, and the tubes were allowed to stand for another 45 min before tryptic activity was determined. The trypsin-binding activity is expressed as the number of micrograms of trypsin measurable in 1 ml of serum after the free trypsin is inhibited by' either the naturally occurring serum trypsin inhibitors or the added soybean trypsin inhibitor. Duplicate samples always agreed within 2 to 3%. Determination of the 19 S macroglobulin. The 19 S macroglobulin was determined by analytical ultracentrifugation using the technique described by Steines and Mehl. 2' The quantitative estimation of a-2-macroglobulin in sera. The a-2-macroglobulin concentration was determined by the antibodyagar radial diffusion technique 25 using immunodiffusion plates (Hyland Laboratories, Los Angeles, Calif.) with specific antibody incorporated into the agar and a primary stan-

dard of a-2-macroglobulin (kindly supplied by Dr. John Mehl) to calibrate the immunodiffusion plates.

Results Serum esterolytic activity. The serum TAME-esterase activities in both groups were comparable and were less than 1.4 }.I.g per ml. No ATEE-esterase activity was noted in the sera of either group. Comparison of the serum trypsin binding actiuity in patients with acute pancreatitis and controls. The results illustrated in figure 1 are representative of a typical patient from each group. Note that as increasing amounts of trypsin were added to the serum, increased trypsinlike activity was detected in the mixture until a plateau was reached. At this point all the binding protein in the serum was saturated and thus the plateau represents the maximum serum trypsin binding activity. As more trypsin was added

....---. Recovered tryptic-like activity without Soybean trypsin inhibitor .... _ ._~ Recovered tryptic-like activity with Soybean trypsin inhibitor 24

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FIG. 1. A represents the results from a typical patient of the control group and B represents those from a typical patient of the pancreatitis group. It is noted that as increasing amounts of trypsin were added to the serum an initial increase in the serum tryptic-like activity was recorded which was smaller than that found in similar determinations with trypsin alone. As more trypsin was added the activity curve leveled off and did not increase until enough trypsin had been added to exceed the naturally occurring serum tryptic inhibitory activity. At that point the tryptic activity again increased, but almost as rapidly as it did for trypsin alone. It can be seen that when exogenous soybean trypsin inhibitor is present in serum in addition to the normally occurring a-I-trypsin inhibitor, the marked rise in tryptic activity after the plateau does not occur.

368

ADHAM ETAL.

no further increase m tryptic activity was noted until the naturally occurring serum tryptic inhibitory activity was exceeded. When soybean trypsin mhibitor was added to the serum, no increase in tryptic activity was noted as long as there was enough soybean trypsin inhibitor to neutralize all the free trypsin in the mixture. The STBA of normal subjects and patients with pancreatitis is shown in figure 2. The mean value for the 20 control patients (group A) was 114 ± 13 Ilg per ml (2 SEM) and for the 12 patients with acute pancreatitis (group B) 41 ± 7 Ilg per ml (2 SEM) . The difference between the groups is highly significant (P < 0.01). Ultracentrifugal analysis of macroglobulins. The mean .value for serum macroglobulin determined by ultracentrifugal analysis m 10 of the normal subjects Group A-Control Group B-Acute Pancreatitis Mean - + - - 2 S. E. M.

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2. Comparison of the serum trypsin-binding activity in control patients (group A) and patients with acute pancreatitis (group B). Each dot represents 1 patient. The center horizontal lines repreclose 2 S~M. If the boxes do not overlap. the difference between the mean is statisticaliy significant. FIG.

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Group A- Control Group B- Pancreatitis Mean -- + - 2 S.E.M.

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FIG. 3. Comparison of the serum a-2-macroglobulin content in control patients (group A) and patients with acute pancreatitis (group B) . Each dot represents 1 patient. The center horizontal lines represent the mean of each grouP. and the boxes enclose 2 SEM . When the boxes overlap the difference between the mean is not ~.atistically significant .

was 200 ± 19 (2 SEM) mg per 100 ml. In 12 patients with acute pancreatitis the mean value for serum macroglobulin determined by analytical ultracentrifugation was 208 ± 23 (2 SEM) mg per 100 ml. It is clear that there is no significant difference between these groups. Serum a-2-macroglobulins. Figure 3 shows the serum a-2-macrbglobulin concentration in both groups. The mean value for the 12 control subjects (group A) was 199 ± 24 (2 SEM) mg per 100 ml and for the 12 patients with acute pancreatitis (group B) 181 ± 30 (2 SEM) mg per 100 ml. The difference between the two groups is not significant (P > 0.2). These values are lower than those reported in the literature. 26 This discrepancy is due to the greater purity of the primary standard of the a-2-macroglobulin that we used in our assay. Duration of decreased STBA in acute pancreatitis. The results in table 1 clearly show that the decreased STBA in the

March 1972

TRYPSIN-BINDING a-2-MACROGLOBULIN IN PANCREATITIS

1. Comparison between the admission and discharge serum trypsin-binding activity in 5 patients with acute pancreatitis·

TABLE

Serum t rypsin-binding act ivi ty

Patient

Admission

Discha rge

6 months after discha rge

Condition on discharge

102 80 56 100

All patients asymptomatic; both serum and urine amylase levels were normal

~Ii/ml

S. H. L. D.

R. J . A. W. T.D.

40 34 23 39 30

45 47 30 55 40

• Note that, although the patients were asymptomatic on discharge and had normal serum and urine amylase levels, serum trypsin-binding activity was still very low.

pancreatitis group persisted despite the subsidence of the acute attack. The discharge STBA determined 1 to 2 weeks after the initial attack was low although the patients were asymptomatic and had normal serum amylase and urinary diastase levels. The STBA then gradually rose towards normal levels. STBA in other disease states. STBA was measured in 38 patients with diverse disease states. The mean level was 100 ± 43 (2 SEM) . Three patients, 1 with duodenal ulcer, a 2nd with acromegaly, and a 3rd with cerebrovascular disease had levels of 50, 22, and 48 respectively. Two patients with hereditary pancreatitis had levels of 250 and 200.

Discussion Macroglobulins are serum proteins with high molecular weights and with a sedimentation constant of 17 to 19 S.27 Normally, the total concentration of the macroglobulins determined by ultracentrifugation rarely rises above 4% of the circulating proteins. 28 . 2 9 When the macroglobulins are separated from the serum and examined by electrophoresis, about one-third migrates with a mobility between that of the {3and /,-globulins and this fraction makes up the macroglobulin antibodies (IgM);

369

the remaining two-thirds have the mobility of an a-2-globulin. 27 -3o The many difficulties which beset the immunological determination of the a-2-macroglobulin in serum concerned the difficulty in obtaining a pure a-2macroglobulin preparation. The results of recent studies done in our laboratory31, 32 as well as by others 33 indicate that the normal values for a-2-macroglobulin are in the range of 220 mg per 100 ml of serum. The higher values reported by James et al. 26 probably can be attributed to an impure standard protein preparation. Studies in our labQratory have not confirmed the reported sex difference as there is a considerable overlap between the serum values of a-2-macroglobulin in males and females. 32 The a-2-macroglobulins have no known antibody activity but are capable of forming enzymatically active complexes with trypsin, lO chymotrypsin,lO plasmin,34, 3 5 and thrombin. 36 Of particular interest is the fact that, when these enzymes bind to the a-2-macroglobulin, they lose much of their proteolytic activity but retain most of their estero lytic activity. 10, 35 Also, it would appear that these enzymes are firmly attached to the same binding site of the a-2-macroglobulin. 10, 20 Thus the STBA is influenced not only by the absolute amount of a-2macroglobulin present in serum but also by the availability of free binding sites on the a-2-macroglobulin. In acute pancreatitis a very significant drop in the STBA was noted in all patients studied. Low STBA was also encountered in few patients with various diseases, but it should be noted that low STBA was not uniformly found in one disease state except in acute pancreatitis. The reason for the low values in 3 patients, 1 with duodenal ulcer, a 2nd with acromegaly, and a 3rd with cerebrovascular disease, of a total of 38 patients with diverse disease states, is not known. One cannot be certain that the patient with duodenal ulcer did not have pancreatitis, although his serum amylase value was normal. The highest STBA levels were found in patients with hereditary pancreatitis, and,

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ADHAM ET AL.

although this finding is of great interest, there is no suitable explanation at this time. There are several possible reasons for the observed decreased affinity of the a-2macroblobulin for trypsin in patients with acute pancreatitis: A decreased serum concentration of the binding a-2-macroblogulin. This is unlikely because: (1) the total concentration of the 17 to 19 S macroglobulins determined by analytical ultracentrifugation were similar in both groups (the mean for the pancreatitis group was 208 ± 23 mg per 100 ml (2 SEM) and for the control group 200 ± 19 mg per 100 ml; (2) the serum a-2-macroglobulin concentration determined by antibody agar radial diffusion technique was also comparable in both groups (fig. 3). Another question which is pertinent is whether only a small fraction of the a-2-macroglobulin which binds trypsin is deficient, and therefore the over-all quantity of a-2-macroglobulin would appear to be unchanged or changed only slightly in amount. Pertinent to this point are other studies performed in this laboratory which showed that a-2-macroglobulin can be resolved into five components by preparative polyacrylamide gel electrophoresis. From these studies it was established that the fastest moving band had no ability to bind trypsin but that the four slower moving bands could bind trypsin. 20 Also, the serum a-2-macroglobulin showed more of the slowest moving material, with four less prominent faster moving bands. 20 Thus, it is apparent from these studies that a deficiency of those species of a-2-macroglobulin which bind trypsin readily would have been detected. The binding of trypsin to a-2-macroglobulin appears to be highly irreversible since labeled trypsin did not displace bound trypsin and vice versa. 20 The a-2-macroglobulin prepared by gel filtration had a binding activity of 53 J.l,g of trypsin per mg of a-2-macroglobulin. It was clear that the molar binding ratio with this preparation was between 1 : 1and 2: 1. The reason for this inter-

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mediate ratio is that a-2-macroglobulin prepared by gel filtration contains species of a-2-macroglobulin which bind trypsin but also species which do not bind trypsin. When only the slowest moving species was used, 73 J.l,g of trypsin bound to 1 mg of a-2-macroglbbulin giving a molar binding ratio of 2: 1. 20 A change in the buffering capacity of the blood. The estero lytic activity of the trypsin-a-2-macroglobulin complex was determined by a pH-Stat automatic titration that responds to changes in pH and not to the total amount of acid byproducts produced in the enzyme-substrate reaction. Thus the measurable esterolytic activity in such a system depends on two main factors: (1) the amount of enzyme bound to the a-2-macroglobulin, and (2) the buffering capacity of the serum-substrate mixture. Consequently any increase in the buffering capacity of the blood during the acute attack can falsely give a low STBA. However, this seems unlikely because (1) the decreased STBA persisted long after the subsidence of the acute attack, and (2) the assay system employs a 100-fold dilution of serum which would obviate any possible buffering action by blood. (3) Comparable results were obtained when trypsin-binding activity was measured using the substrate benzoyl-L-arginine-paranitroanilide, a system which is independent of change in pH. An acquired proteinopathy in the setting of acute pancreatitis which leads to an altered or damaged binding site. Underlying this requirement is the assumption that pancreatitis can alter or damage the binding sites on the a-2macroglobulin molecule without affecting its physical and antigenic properties. (The 17-19 S fraction and a- 2-macroglobulin concentration in these patho-· logical sera were determined by ultracentrifugation and immunological assays and were found to be present in normal concentration. 37 Saturation of the binding sites by a substance, likely an enzyme. The a-2macroglobulin is a nonspecific protein carrier for various proteolytic-esterolytic

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TRYPSIN-BINDING 0/-2-MACROGLOBULIN IN PANCREATITIS

enzymes. The stoichiometric combination of this protein with these enzymes is such that the addition of excess trypsin, chymotrypsin, or plasmin to the incubation mixture fails to replace the already bound enzyme. 10, 20, 37, 38 This indicates a very strong noncompetitive binding or a very low dissociation constant of the a-2macroglobulin-enzyme complex. Thus a likely explanation for the decrease in STBA in the sera of patients with acute pancreatitis would be the binding of a substance other than trypsin or chymotrypsin to the a-2-macroglobulin. Although the evidence for this is indirect, studies in our laboratory by Dyce and associates 21 already have shown that an esterolytic enzyme with properties of a kallikrein can be separated from the a-2macroglobulin during its isolation. Studies by Dyce and associates 21 also have shown that this enzyme is not trypsin, chymotrypsin, thrombin, Hageman factor, or first component of complement (C'l-esterase). Our studies have indeed confirmed that the substance occupying the binding sites is not trypsin, chymotrypsin, or thrombin for there is no increase in the esterolytic activity of the serum from patients with acute pancreatitis as compared with normals when TAME or ATEE were used as subRecently Colman and strates. 10, 31 Sherry39 observed that plasma collected in siliconized tubes exhibited no demonstrable arginine esterase activity, whereas specimens collected in glass contain significant activity. This activity was traceable to the activation of a plasma kallikrein by activated Hageman factor. Under the conditions of our experiment, STBA was determined on sera obtained from blood specimens collected in glass tubes. This may well reduce the number of binding sites which exist in the native state of the a-2-macroglobulin molecule. But it should be noted that this would affect both control and pancreatitis sera similarly, and would in no way alter the differences between the two groups unless the enzyme was released, considerably in excess of the normal, prior to blood collection. The nature of, as well as the source

371

of, the substance which binds with the a-2-macroglobulin in acute pancreatitis will be important to ascertain. Interestingly, the decreased STBA in patients with acute pancreatitis, unlike the serum or urine amylase, persisted despite the subsidence of the acute attack and then gradually rose to normal levels. REFERENCES 1. Webster PD, Zieve L: Alterations in serum content of pancreatic enzymes. N Engl J Med 267: 604-607, 654-658, 1962 2. Haverback BJ, Hammond WG, Edmonson HA, et al: Acute pancreatitis. Tice-Harvey Practice of Medicine. Seventh edition. Edited by J Harvey. Hagerstown, WR Prior Company Inc, 1964, p 537-552 3. Nardi GL, Lees GW: Serum trypsin. A new diagnostic test for pancreatic disease. N Engl J Med 258:797-798, 1958 4. Gullick HD: Increased plasma proteolytic activity due to arginine amidase in patients with pancreatitis. N Engl J Med 268:851-857,1963 5. Butin JW, Graham WD: Serum exopeptidase activity in patients with pancreatic disease. Gastroenterology 40:669, 1961 6. Floch MH, Groisser VW: Serum proteolytic enzyme activity in pancreatic disease. N Engl J Med 263:1129-1130, 1960 7. Neimr P, discussion by Nardi GL: Specific polypeptide substrates in enzymatic determination of pancreatic disease. Surgery 46:30-37, 1959 8. Perrier CV, Janowitz HD: The pancreas. Gastroenterology 42:481-496, 1962 9. Groisser VW, Raugh F, Floch M, et al: Serum esterolytic activity in a wide variety of diseases with special reference to pancreatic and liver disease. N Engl J Med 274: 129-133, 1966 10. Haverback BJ, Dyce B, Bundy HF, et al: Protein binding of pancreatic proteolytic enzymes. J Clin Invest 41:972-980,1962 11. Troll W, Sherry S, Wachman J: The action of plasmin on synthetic substrates. J Bioi Chern 208:85-93, 1954 12. Sherry S, Troll W: The action of thrombin on synthetic substrates. J Bioi Chern 208:95-105, 1954 13. Neurath H: Some considerations of multiple specificity of proteolytic enzymes. Ann New York Acad Sci 68:11-24,1957 14. Donaldson VH: Studies on the activation of a serum esterase with ether and its rel/itionship to C'l-esterase. J Clin Invest 40:673-683, 1961 15. Dyce B, Haverback BJ: Serum trypsin inhibitors in the normal and in patients with acute pancreatitis. Am J GastroenteroI34:481-486, 1960

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16. Bundy HF, Mehl JW: Trypsin inhibitors of human serum. I. Standardization mechanism of reaction and normal values. J Clin Invest 37:947955, 1958 17. Shulman NR: Studies on the inhibition of proteolytic enzymes by serum. II. Demonstration that separate proteolytic inhibitors exist; their distinctive properties and the specificity of their action. J Exp Med 95:593- 603, 1952 18. Jacobson K: Electrophoretic demonstration of two trypsin inhibitors in human blood serum. Scand J Clin Lab Invest 5:97-98, 1953 19. Me4l JW, O'Connell W, DeGrott J: Macroglobulin from human plasma which forms enzymatically active compound with trypsin. Science 145:821-822, 1964 20. Saunders R, Dyce BJ, Vannier WE, et al: The separation of alpha-2-macroglobulin into five compounds with differing electrophoretic and enzyme binding properties. J Clin Invest 11: 2376-2383, 1971 21. Dyce B, Wong T, Adham NF, et al : Human plasma kallikrein esterase associated with the alpha-2-macroglobulin binding protein. Clin Res 15: 101, 1967 22. Schwert GW, Neurath H, Kaurman S, et al: The specific esterase activity of trypsin. J Bioi Chern 172:221-239, 1948 23. Neurath H, Schwert GW: The mode of action of the crystalline pancreatic proteolytic enzymes. Chern Rev 46:69-153, 1950 24. Steines WJ, Mehl JW: The elevation of alpha-2macroglobulin and trypsin-binding activity in nephrosis. J Lab Clin Med 67:559- 565, 1966 25. Mancini G, Vaerman JP, Carbonara AO, et al: A single radial diffusion method for the immunological quantitation of proteins, Protides of the Biological Fluids. Edited by H Peters. Proceedings of the 11th Colloquium of Bruges. Elsevier, Amsterdam, 1964, p 370- 373 26. James K, Johnson G, Fudenberg HH: The quantitative estimation of alpha-2-macroglobulin in normal, pathological and cord sera. Clin Chim Acta 14:207-214, 1966 27 . Wallenius G, Trautman R, Kunkel HG, et al:

28. 29. 30. 31. 32.

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Ultracentrifugal studies of major non-lipide electrophoretic components of normal human serum. J Bioi Chern 225:253-267, 1957 Fessel WJ : Clinical analysis of 142 cases with high molecular weight serum proteins. Acta Med Scand 173 (suppI391):5-32, 1962 Ericksen N: Serum macroglobulin levels in relation to age, sex, and disease. J Lab Clin Med 51: 521- 529, 1958 Rosen FS: The macroglobulins. N Engl J Med 267 :491-497,546-550, 1962 Wilding P, Adham NF, Mehl JW, et al : Alpha-2macroglobulin concentrations in human serum. Nature (Lond) 214:1226- 1227, 1967 Adham HF, Wilding P, Mehl JW, et al : Alpha2-macroglobulin levels in human serum. I. The effect of sex and estrogen. II. The role of alpha2-macroglobulin as a carrier protein for insulin. J Lab Clin Med 71 :271- 282,1969 Ganrot PO, Schersten B: Serum alpha-2-macroglobulin concentration and its variation with age and sex. Clin Chim Acta 15:113-120, 1967 Schultze HE, Heimburger N, Heide K, et al: Preparation and characterization of alpha-1trypsin inhibitor and alpha-2-plasmin inhibitor of human serum. Proceedings of the 9th Congress of the European Society of Haematology. Lisbon. S Karger, Basel, 1963, p 1315- 1320 Ganrot PO: Inhibition of plasmin by alpha-2macroglobulin. Clin Chim Acta 16:328-330, 1967 Lanchantin GF, Plasset ML, Friedman JA, et al: Dissociation of the esterolytic and clotting activities of thrombin by trypsin binding macroglobulin . Proc Soc Exp Bioi Med 121 :444-449, 1966 Wilding P, Adham NF, Haverback BJ: The quantitative estimation of macroglobulins in normal and pathological sera. Clin Res 15:130, 1967 Ganrot PO: Interaction of plasmin and trypsin with alpha-2-macroglobulin. Acta Chern Scand 21 :602-608, 1967 Colman RW, Sherry S: A third major arginine esterase enzyme system of human plasma ; its identity with permeability globulins. Fed Proc 26:448, 1967