Biochimica et Biophysica Acta, 491 (1977) 275-285
© Elsevier/North-HollandBiomedicalXPress BBA 37612 ISOLATION OF MYOCARDIAL DEPRESSANT FACTOR FROM PLASMA OF DOGS IN HEMORRHAGIC SHOCK
LEWIS J. GREENE', ROSLYN SHAPANKA,THOMAS M. GLENN and ALLAN M. LEFER Department of Biology, Brookhaven National Laboratory, Upton, N.Y. 11973, Department of Pharmacology, University of South Alabama, College of Medicine, Mobile, Ala. 36688, and Department of Physiology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pa. 19107 (U.S.A.)
(Received August 16th, 1976)
SUMMARY Starting with 3 1 of plasma from dogs in hemorrhagic shock, we have purified the myocardial depressant factor and found that the activity is separate from salts and free amino acids. Moreover, the myocardial depressant factor is present in shock plasma in concentrations of about 1 nmol/ml of plasma. The depressant factor exists as multiple chromatographic forms. The best characterized forms are the anionic forms. A preliminary amino acid composition of the anionic forms has been obtained. These findings should allow more rapid processing of plasma containing high myocardial depressant factor activity to separate the factor and to completely identify this small peptide of great physiologic interest.
INTRODUCTION In 1966, Brand and Lefer [1] reported the presence of a myocardial depressant factor in the circulating blood of cats in hemorrhagic shock. This substance was characterized as a potent negative inotropic agent [1]. Subsequently, the depressant factor was shown to be present in a variety of types of shock including burn [2], cardiogenic [3, 4], endotoxic [5], acute pancreatitis [6] and splanchnic ischemia [7, 8] shock. The factor occurs in humans as well as in a variety of experimental mammals during circulatory shock [9, 10]. Initial investigations indicated that it is water soluble with a molecular weight of approx. 500-1000 [11-13], and is probably a peptide or a glycopeptide [11]. Recently, Wangensteen et al. [14] reported that the myocardial depressant factor activity was due to NaC1 in their preparations. However, NaC1 exerts a significant negative inotropic effect only at concentrations far in excess of that occurring during shock states [18, 19]. Moreover, the myocardial depressant factor activity has been shown to be free of NaCI contamination by other investigators in the United States [15] and elsewhere [4, 8, 15-17]. One of the difficulties in defining the chemical nature of the factor is the fact that it occurs in shock plasma only at very low concentrations (i.e. approximately 1-2 * Present address: Departmentof Biochemistry,Universityde Sao Paulo, Facultadede Medicina de Ribeirao Preto, 14100 Ribeirao Preto, Brazil.
276 ml of plasma are required for a single bioassay). The objective of this study was to develop methods capable of processing 2-31 of shock plasma in order to develop chromatographic systems suitable for isolating the molecular species having myocardial depressant factor activity and to characterize chemical properties of the factor. Achievement of these goals would be of great benefit in understanding the pathophysiology of circulatory shock. METHODS
Preparation of plasma from dogs subjected to hemorrhagic shock Dogs of either sex anesthetized with sodium pentobarbital (30 mg/kg, intravenously) were subjected to a standardized hemorrhage at a mean arterial blood pressure of 40-45 mmHg according to the method of Lefer and Martin [I 2]. The arterial blood pressure was maintained at this hypotensive level for a total duration of 120150 min followed by reinfusion of all shed blood. Blood was maintained in a siliconized lucite reservoir at room temperature for the duration of the oligemic period. Reservoir blood has been shown to contain negligible myocardial depressant factor activity [20]. Arterial blood pressure returned to control values after reinfusion (i.e. about 120-140 mm Hg), and was followed by a post-oligemic period of declining blood pressure. When the arterial blood pressure had fallen to 55 mm Hg, the arterial catheter was opened and blood was collected in polyethylene tubes at 4 °C. Approx. 50-150 ml of blood was collected from each dog. Within 10 rain of collection, blood was centrifuged at 2500 × g and 4 °C for 20 min. The plasma was decanted, stored at --22 °C until deproteinization and deproteinized by trichloroacetic acid.
Preparation of anionic myocardial depressant factor Step l : Trichloroacetic acid-soluble fraction. 300 ml of 50 ~ (w/v) trichloroacetic acid was added with stirring to 2.75 1 of hemorrhagic shock dog plasma held in a water-ice bath at 0-4 °C in a cold room. After 30 min, the precipitate was separated by centrifugation in an International centrifuge at 9000 × g for 20 min at 4 °C. The clear supernatant solution, 2.25 1, was extracted five times each with 400 ml of ether saturated with water at 4 °C. The ether layer was removed by suction and the aqueous phase was evacuated with a water pump for 30 min to remove traces of remaining ether. An aliquot corresponding to 22 ml of plasma was removed for gel filtration on Biogel P-2 and bioassay and the solution remaining was lyophilized. Step 2: Gel filtration on Sephadex G-25 (Fig. 1). Two Sephadex G-25 columns 0.8 × 200cm) connected in series were prepared and developed with 0.2 M pyridine/acetic acid buffer, pH 3.1 (0.2 M pyridine) as described by Greene and Giordano [21]. Lyophilized powder corresponding to the trichloroacetic acid-soluble fraction, derived from approx. 900 ml of hemorrhagic shock dog plasma, was dissolved in 25 ml H20 and the pH was corrected to 3.2 with 50 ~ (w/v) NaOH. The column effluent was monitored by reaction with ninhydrin [22] and by measurement of effluent conductivity at 22 °C. The trichloroacetic acid extract from 2.751 of plasma was processed in three batches and equivalent regions of the column effluent were pooled, evaporated in a rotary evaporator at 40 °C and lyophilized. Elution diagrams obtained for the three runs were essentially identical. 2 ~ of each fraction was used for measurement of the myocardial depressant factor activity.
277
Step 3: Equilibrium and gradient elution chromatography on Dowex 50-X2 (Fig. 2). Fraction 5 (Fig. 1) was dissolved in 12 ml of water and the pH of the solution adjusted from 3.20 to 2.75 with 1 M HCI. The sample was applied to a column of Dowex 50-X2 (1.8 x 60 cm) equilibrated with 0.1 M pyridine/acetic acid buffer, pH 2.81 (0.1 M pyridine), developed at 40 ml/h, 38 °C, and fractions of 12 ml were collected. After elution with 1.62 1 of starting buffer, the column was eluted with a linear gradient prepared from 500 ml each of 0.1 M pyridine/acetic acid buffer, pH 2.81, and 0.2 M pyridine/acetic acid buffer, pH 3.10. A second linear gradient from 0.2 M pyridine/acetic acid buffer, pH 3.1, to 2.0 M pyridine/acetic acid buffer, pH 5.0 (1 1 each), was applied to the column after 875 ml of the first gradient had been used. The effluent corresponding to the fractions indicated at the top of Fig. 2 was pooled, evaporated and lyophilized. 1 0 ~ of each fraction was used for bioassay.
Step 4: Equilibrium chromatography on QAE-Sephadex at pH5.0 (Fig. 3). Fraction 1 (Fig. 2) was dissolved in 5 ml 2 0 ~ aqueous pyridine and applied to a 0.9 × 60cm column of QAE-Sephadex equilibrated and developed with 0.3 M pyridine/acetic acid buffer, pH 5.0 (0.3 M pyridine). Slight shrinkage of the top of the gel bed was observed as the sample was applied. The column was developed as described in the legend to Fig. 3.10 ~ of each fraction was used to detect the depressant factor activity.
Step 5: Equilibrium and step elution chromatography on QAE-Sephadex (Fig. 4). Fraction 5 (Fig. 3) was dissolved in 2 ml of 20 ~ aqueous pyridine and applied to a column of QAE-Sephadex (0.9 × 60 cm) which had been equilibrated in 0.3 M pyridine, pH 8.1. The column was developed as described in the legend to Fig. 4. 20 ~o of each fraction was used to detect the depressant factor activity. Step 6: Analytical gel filtration on Sephadex G-25. Fraction 3 (Fig. 4) was dissolved by 1 ml 0.2 M pyridine/acetic acid buffer (pH 3.1) and applied to a 0.9 × 400 cm column of Sephadex G-25. The column 0.9 × 400 cm was equilibrated and developed with 0.2 M pyridine/acetic acid buffer, pH 3.1 at 5 ml/h, 23 °C and fractions of 2 ml were collected. 20 ~ of each fraction was used to detect the depressant factor activity. Step 7: High voltage paper electrophoresis. Fractions 3 and 4 were combined and subjected to electrophoresis in a Gilson Electrophoretor D apparatus at 2500 V (50 V/cm) for 7 h at pH 2.0 (3.5 ~ formic acid). Each sheet of Whatman 3 MM paper (58 × 68 cm) was washed before use with 125 ml 2 M HC1, with H20 exhaustively, and finally with 125 ml 9 5 ~ ethanol in an apparatus used for descending paper chromatography, and then dried at room temperature. The sample (0.6 ml) was separated into two portions and electrophoresis was carried out twice. The sample was applied to the paper in a 15 cm long band 25 cm from the edge of the paper which would later be immersed in the anode buffer compartment. Marker spots (10 /~1) of sample, HC1Oa-Ala, cysteic acid, were applied on each side of the sample band to be used to locate material after electrophoresis. Peptides were detected in the marker region of the electrophoretogram by reaction with the chloride-tolidine reagent [23] and by reaction with ninhydrin (0.5 ~ in acetone). The paper was cut into horizontal strips on the basis of spots detected by the chlorine-tolidine reagent and eluted with 10 ~ acetic acid. 30 ~ of each fraction was used to detect the depressant factor activity.
278
Bioassay of myocardial depressant factor Samples of unprocessed plasma or column effluents of plasma extracts were assayed for the depressant factor activity on electrically driven isolated papillary muscles taken from the right ventricle of cat hearts according to the method of Lefer et al. [24]. Lyophilized fractions were reconstituted to 10 ml with Krebs-Henseleit solution and added to 10-ml muscle chambers. Developed tension of the isolated papillary muscles were recorded on a Beckman type R oscillographic recorder. Myocardial depressant factor activities were recorded as percent inhibition in developed tension of the papillary muscle at standard conditions of 37 °C and a frequency of stimulation of 1 Hz [24].
Amino acid analysis Samples were hydrolyzed in evacuated sealed tubes with 1 ml of twice distilled constant boiling HC1 for 22h at 110 °C. Amino acid analysis of hydrolysates or samples which had not been hydrolyzed were performed by the method of Spackman et al. [25] on an automatic instrument with provisions for multiple sample application [26]. The instrument was calibrated with standards of 10 nmol of each amino acid. RESULTS
Preparation of anionic myocardial depressant factor The procedure used here for deproteinization of 2.75 1 of hemorrhagic shock plasma is the same as previously described for analytic experiments [19]. Elution of the deproteinized samples on Biogel P-2 in Krebs-Henseleit solution yielded four peaks. The elution profile of the depressant factor activity is very similar to that previously described by Leffler et al. [19] and Lefer and Inge [18] for whole cat and dog plasma. Virtually all the factor activity was recovered in the second large peak (fraction 2) which was eluted between 80 and 100 ml. Only small amounts of sodium were present in this factor peak. The major salt peak was fraction 3 which had only modest depressant factor activity (i.e. only 20 ~ inhibition). Each chromatographic experiment was carried out in two parts. The first, using about 20 ~ of the material was used to test experimental conditions and to locate the myocardial depressant factor activity. All fractions containing the factor activity were then added to the remaining material and the experiment was repeated. The elution diagrams given in Figs. 1-4 represent results obtained from the second preparative run for each chromatographic system. Gelfiltration on Sephadex G-25 (Fig. 1). The trichloroacetic acid solution fraction contains appreciable amounts of ninhydrin-positive material which is eluted over the entire volume of the column but is concentrated in the low molecular weight region. The effluent conductivity profile shows that salts are eluted between 900 and 1100 ml. The dip in the conductivity profile at 870 ml was used as a marker for selecting fractions. The dip does not mean that this area is salt free, but that it is less than the solvent which is equivalent in conductivity to 0.05 M NaCI. Cardiodepressant activity, most pronounced in regions 6 and 7, is due primarily to excessive salt concentration; whereas in regions 4 and 5 the negative inotropic effect is due to the depressant factor activity. This can be shown by measurement of the salt concentration as well as by inspection of the bioassay tracings. In this regard, high salt concentration in-
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Fig. 3. Equilibrium chromatography on QAE-Sephadex of fraction 1 (Fig. 2). The column, 0.9 × 60 cm, was equilibrated and developed with 0.3 M pyridine/acetic acid buffer, pH 5.0, at 22 °C, 14 ml/h, and fractions of 2 ml were collected. - - - , absorbance of 570 nm (ninhydrin reaction after alkaline hydrolysis) ; , effluent pH ; , effluent conductivity. The top panel of the figure shows the effluent that was combined, the fraction number, and the myocardial depressant factor activity.
QAE-Sephadex. Fig. 3 shows that under equilibrium conditions, most of the depressant factor activity (i.e. fraction 5) is retarded by the column, whereas most of the ninhydrin-reactive materials are eluted either in the breakthrough peak (i.e. fraction 1 indicated by the peak of pH and conductivity given in the middle panel of the figure) or in fraction 4. The depressant factor activity in fraction 5 is an appreciable portion of the recovered activity, and only represents a small part of the total ninhydrin-positive material. Further purification of the factor activity separating it from free amino acids was achieved by adsorbing the activity of QAE resin at an alkaline p H and eluting it by reducing the pH (Fig. 4). The myocardial depressant factor activity is eluted after the abrupt change in pH as shown in Fig. 4. Gelfiltration. The fraction containing the factor activity (i.e. fraction 3, Fig. 4) was rechromatographed on Sephadex G-25 under the same conditions as shown in Fig. 1, except that in this experiment an underload analytical column was used. The depressant factor activity was eluted in fractions 3 and 4 between the two major ninhydrin-positive fractions at 220-235 ml effluent volume. Nevertheless, an appreciable enrichment of the depressant factor activity was obtained.
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Fig. 4. Equilibrium and step elution chromatography on QAE-Sephadex of fraction 5 (Fig. 3). The column, 0.9 × 60cm, was equilibrated and developed with 0.3 M pyridine (pH 8.1) at 22 °C, 20 ml/h, and fractions of 4 ml were collected. After 230 ml of effluent had been collected, the column was eluted with 0.2 M pyridine/acetic acid buffer, pH 5.0, for an additional 600 ml followed by 200 ml of 0.3 M pyridine/acetic acid buffer. - - - - , absorbance at 570 nm (ninhydrin reaction after alkaline hydrolysis, bottom panel); - - - - , and effluent pH; (middle panel). The top panel of the figure shows the effluent that was combined, the fraction number, and the myocardial depressant factor activity.
High voltage electrophoresis. The two m o s t active fractions (i.e. fractions 3 a n d 4) were c o m b i n e d a n d s u b m i t t e d to high voltage electrophoresis at p H 2.0 for 7 h. All o f the material detected by the chlorine-tolidine stain reaction was n i n h y d r i n negative indicating t h a t free a m i n o acids were not present in the fractions t h a t h a d depressant factor activity (fractious 3 a n d 8). The area c o r r e s p o n d i n g to fraction 6 was the only one t h a t was fluorescent. The p y r r o l i d o n e carboxylic acid did n o t migrate, b u t all a m i n o acids including cysteic acid migrated off the p a p e r u n d e r these conditions. M y o c a r d i a l d e p r e s s a n t factor activity was detected in two regions o f the electrop h o r e t o g r a m . The m a t e r i a l in fractions 3 and 8 are d e n o t e d A1 a n d A2, respectively. Amino acid composition of the myocardial depressant factor fractions A 1 and A.2 T a b l e I gives the a m i n o acid c o m p o s i t i o n o f A1 and A 2 after elution f r o m the p a p e r TABLE I COMPOSITION OF HYDROLYSATES OF ELECTROPHORETOGRAM MYOCARDIAL DEPRESSANT FACTOR-POSITIVE AREAS Values are expressed as tool amino acid/mol glycine. Amino acid
Glutamic acid Glycine Serine Unidentified amino acid (x)
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Az
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282 and hydrolysis with 6 M HC1. Amino acids not given in the table were present at a level of 0.3 mol per mol glycine or less. No free amino acids above background were detected in samples not submitted to hydrolysis. The unidentified amino acid was eluted from the long column (Beckman PA-28 resin) of the amino acid analyzer after cysteic acid at 55 °C, using pH 3.25 sodium citrate buffer (0.2 M sodium citrate). The unknown amino acid was distinguishable from levulinic acid but not taurine under these conditions. However, when the column was operated at 32 °C, the unidentified amino acid present in the depressant factor acid hydrolysates was separable from taurine. Nevertheless, we have not been able to identify this amino acid at present. It must be emphasized that the ninhydrin color value for aspartic acid has been used to calculate the amount of the unidentified amino acid present in these factor fractions and thus its molar concentration relative to glycine may be in error by as much as 10-30 ~. The interpretation of the analytical data is further complicated by the low levels of peptide recovered from the paper and the presence of glycine and serine as contaminants in paper and common laboratory solvents. On this basis, the amino acid composition proposed below for A1 and A2 must be considered as preliminary: (fraction 3, Fig. 5) AI: X1, Gly~, Glx,, Serl; (fraction 8, Fig. 5) A2: X2, Glyi.
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Fig. 5. Diagrammatic representation of the high voltage paper electrophoretogram of a pool derived from active fractions eluted from an underloaded Sephadex G-25 column (step 6). The asterisks indicate the myocardial depressant factor activity; cross-hatched area indicates material positive to the chlorine-tolidine reagent and negative to ninhydrin. The experimental conditions are given in Methods.
Inotropic activity of free amino acids In an effort to determine whether free amino acids could be contaminating the myocardial depressant factor-positive fractions and contributing to its activity, we assayed the inotropic activity of 20 common amino acids. Although, none of these
283 free amino acids could be expected to exceed 1 mM in any of the depressant factor activity fractions, we assayed each amino acid at 1.0 and 10 mM. At 1 raM, none of the 20 amino acids exerted any significant negative inotropic activity. Glycine, leucine, phenylalanine, and valine exerted modest increases in contractile force of 3--4 ~. At 10 mM, cysteine, histidine and tryptophan exerted moderate negative inotropic effects of 10-20 70. Even if these amino acids were present at a concentration as high as 10 raM, their contribution to the depressant factor activity would be only modest. However, none of these four amino acids was present at these levels in the paper electrophoresis or the Sephadex G-25 analytical fractions having myocardial depressant factor activity. DISCUSSION This report describes chromatographic procedures for the isolation of myocardial depressant factor activity from liter quantities of hemorrhagic shock plasma obtained from dogs. Steps 1 and 2 are trichloroacetic acid precipitation of plasma proteins, followed by gel filtration on Sephadex G-25. These steps effectively separate the factor activity from proteins and salt in plasma and thus are the most significant steps in the preparation of purified depressant factor activity. Much smaller quantities of materials were applied to Dowex-50, QAE-Sephadex column and used for high voltage electrophoresis. The need for large batches of starting material is indicated by the requirement of 1-2 ml of plasma for each bioassay. The amino acid composition and biological activity of purified myocardial depressant factor fractions A1 and A2 taken together with the purification steps indicate that approx. 0.5-1.0 nmol/ml of the factor are present in the blood of animals in hemorrhagic shock. The results show clearly that the factor activity (i.e. measured as a negative inotropic effect on isolated cardiac tissue) exists in trichloroacetic acid-treated, hemorrhagic shock plasma in multiple chromatographic forms. The multiple chromatographic forms which are anionic, neutral and cationic under the conditions employed in these experiments may or may not be chemically related. However, the two anionic forms (i.e. A1 and A2) are similar in amino acid composition. Their anionic properties were used as the basis for their isolation and purification. In general, the behavior of the depressant factor activity with respect to recovery of activity and rechromatography indicate that the factor activity is stable and its behavior is reproducible. The two purified forms of the myocardial depressant factor (i.e. A~ and A2) are clearly distinguishable from free amino acids and salts. We also tested the effects of free amino acids on the contractile force of the isolated cat capillary muscle preparation. None of the 20 common amino acids studied exerted a significant negative inotropic effect (i.e. depress contractile force) comparable to that produced by the factor. These findings are in agreement with those of Gatgounis and Hester [27] obtained in the intact dog. Thus, it is extremely unlikely that free amino acid contamination of the factor samples could account for the depressant factor activity observed in any of the active fractions of our column eluates. The myocardial depressant factor activity was also separated from salt activity in the preparation of the purified factor. Lefer and Inge [18] previously showed that it was eluted prior to salt in the Biogel P-2 column, and that the amount of salt required to elicit a negative inotropic effect comparable to that of the factor is in
284 excess o f 450 mequiv/1. In this study, we also reported that high salt concentrations in the range o f 400-500 mequiv/l depress myocardial contractility in a manner distinctly different from that o f the factor. The myocardial depressant factor depresses developed tension by decreasing active tension without a change in resting tension. High salt concentrations depress developed tension by decreasing active tension and markedly increasing resting tension (i.e. baseline tension). This large increase in baseline tension does not occur with high titers o f the factor in the absence o f high salt concentrations and serves to distinguish this from responses occurring due to the presence o f high a m o u n t s o f salt contamination. The characterization o f the A1 and A2 forms o f the depressant factor indicate the presence o f an acidic amino acid which has not yet been identified. The amino acid composition o f the factor appears to be similar to R D S [28], a substance found in the plasma of shock animals having similar biological properties to the myocardial depressant factor. However, the unknown amino acid in R D S is eluted from the short column o f the amino acid analyzer and thus does not appear to be acidic. Therefore, the two u n k n o w n amino acids do not appear to be identical. The chemical relationship, if any, between the myocardial depressant factor and R D S remains to be determined. ACKNOWLEDGEMENTS We wish to acknowledge Mr. Martin L. Ogletree and Miss Yumi Sakane for their expert technical assistance during this study. This work was supported in part by N.I.H. Research G r a n t No. HL-17745 and No. GM-21235 and by Brookhaven National Laboratory. Research carried out at Brookhaven National L a b o r a t o r y under the auspices o f the U.S, E R D A . REFERENCES 1 Brand, E. D. and Lefer, A. M. (1966) Proc. Soc. Exp. Biol. Med. 122, 200-203 2 Baxter, C. R., Cook, W. A. and Shires, G. T. (1966) Surg. Forum 17, 1-2 3 Glenn, T. M., Lefer, A. M., Martin, J. B., Lovett, W. L., Morris, J. N. and Wangensteen, S. L. (1971) Am. Heart J. 82, 78-85 40kuda, M., Yamada, T. and Hosono, K. (1973) Jap. Circ. J. 37, 1009-1017 5 Wangensteen, S. L., Geissinger, W. T., Lovett, W. L., Glenn, T. M. and Lefer, A. M. (1971) Surgery 67, 410~18 6 Lefer, A. M., Glenn, T. M., Lopez-Rasi, A. M., Kiechel, S. F., Ferguson, W. W. and Wangensteen, S. L. (1971) Clin. Pharmacol. Ther. 12, 506-516 7 Glenn, T. M. and Lefer, A. M. (1970) Circ. Res. 27, 783-797 8 Williams, L. F., Goldberg, A. H , Polansky, B J. and Byrne, J. J. (1969) Surgery 66, 138-144 9 Lovett, W. L., Wangensteen, S. L., Glenn, T. M. and Lefer, A. M. (1971) Surgery 70, 223-230 10 Rosenthal, S. L., Hawley, P. L. and Hakim, A. A. (1972) Surgery 71, 527-536 11 Lefer, A. M. (1970) Fed. Proc. 29, 1836-1847 12 Lefer, A. M. and Martin, J. (1970) Circ. Res. 26, 59-69 13 Lefer, A. M. and Martin, J. (1970) Am. J. Physiol. 218, 1423-1427 14 Wangensteen, S. L., Ramey, W. G., Ferguson, W. W. and Starling, J. R. (1973) J. Trauma 13, 181-194 15 Nagler, A. L. and Levenson, S. M. (1974) Circ. Shock 1,251-264 16 Fisher, W. D., Heimbach, D. W., McArdle, C. S., Maddern, M., Hutcheson, M. M. and Ledingham. McA. (1973) Br. J. Surg. 60, 392-394 17 Lundgren, O., Haglund, U., Isaksson, O. and Abe, T. (1976) Circ. Res. 38, 307-315
285 18 19 20 21 22 23 24 25 26 27 28
Lefer, A. M. and Inge, Jr., T. F. (1973) Proc. Soc. Exp. Biol. Med. 142, 429--433 Leffler, J. N., Litvin, Y., Barenholz, Y. and Lefer, A. M. (1973) Am. J. Physiol. 224, 824-831 Brand, E. D., Cowgill, R. and Lefer, A. M. (1969) J. Trauma 9, 216-226 Greene, L. J. and Giordano, J. S. (1969) J. Biol. Chem. 244, 285-298 Hirs, C. H. W. (1967) Methods Enzymol. 11, 325-329 Pataki, G. (1968) Techniques of Thin Layer Chromatography in Amino Acid and Peptide Chemistry, p. 107, Ann Arbor Science Publishers Inc., Ann Arbor, Mich. l~efer, A. M., Cowgill, R., Marshall, F. F., Hall, L. M. and Brand, E. D. (1967) Am. J. Physiol. 213,492-498 Spackman, D. H., Stein, W. H. and Moore, S. (1958) Anal. Chem. 30, 1190-1206 Alonzo, N. and Hirs, C. H. W. (1968) Anal. Biochem. 23, 272-289 Gatgounis, J. and Hester, W. (1964) Proc. Soc. Exp. Biol. Med. 116, 430--434 Blattberg, B., Frosolono, M. F., Misra, R. P. and Levy, M. N. (1972) J. Reticuloendothel. Soc. 12, 371-386
© Elsevier/North-HollandBiomedicalXPress BBA 37612 ISOLATION OF MYOCARDIAL DEPRESSANT FACTOR FROM PLASMA OF DOGS IN HEMORRHAGIC SHOCK
LEWIS J. GREENE', ROSLYN SHAPANKA,THOMAS M. GLENN and ALLAN M. LEFER Department of Biology, Brookhaven National Laboratory, Upton, N.Y. 11973, Department of Pharmacology, University of South Alabama, College of Medicine, Mobile, Ala. 36688, and Department of Physiology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pa. 19107 (U.S.A.)
(Received August 16th, 1976)
SUMMARY Starting with 3 1 of plasma from dogs in hemorrhagic shock, we have purified the myocardial depressant factor and found that the activity is separate from salts and free amino acids. Moreover, the myocardial depressant factor is present in shock plasma in concentrations of about 1 nmol/ml of plasma. The depressant factor exists as multiple chromatographic forms. The best characterized forms are the anionic forms. A preliminary amino acid composition of the anionic forms has been obtained. These findings should allow more rapid processing of plasma containing high myocardial depressant factor activity to separate the factor and to completely identify this small peptide of great physiologic interest.
INTRODUCTION In 1966, Brand and Lefer [1] reported the presence of a myocardial depressant factor in the circulating blood of cats in hemorrhagic shock. This substance was characterized as a potent negative inotropic agent [1]. Subsequently, the depressant factor was shown to be present in a variety of types of shock including burn [2], cardiogenic [3, 4], endotoxic [5], acute pancreatitis [6] and splanchnic ischemia [7, 8] shock. The factor occurs in humans as well as in a variety of experimental mammals during circulatory shock [9, 10]. Initial investigations indicated that it is water soluble with a molecular weight of approx. 500-1000 [11-13], and is probably a peptide or a glycopeptide [11]. Recently, Wangensteen et al. [14] reported that the myocardial depressant factor activity was due to NaC1 in their preparations. However, NaC1 exerts a significant negative inotropic effect only at concentrations far in excess of that occurring during shock states [18, 19]. Moreover, the myocardial depressant factor activity has been shown to be free of NaCI contamination by other investigators in the United States [15] and elsewhere [4, 8, 15-17]. One of the difficulties in defining the chemical nature of the factor is the fact that it occurs in shock plasma only at very low concentrations (i.e. approximately 1-2 * Present address: Departmentof Biochemistry,Universityde Sao Paulo, Facultadede Medicina de Ribeirao Preto, 14100 Ribeirao Preto, Brazil.
276 ml of plasma are required for a single bioassay). The objective of this study was to develop methods capable of processing 2-31 of shock plasma in order to develop chromatographic systems suitable for isolating the molecular species having myocardial depressant factor activity and to characterize chemical properties of the factor. Achievement of these goals would be of great benefit in understanding the pathophysiology of circulatory shock. METHODS
Preparation of plasma from dogs subjected to hemorrhagic shock Dogs of either sex anesthetized with sodium pentobarbital (30 mg/kg, intravenously) were subjected to a standardized hemorrhage at a mean arterial blood pressure of 40-45 mmHg according to the method of Lefer and Martin [I 2]. The arterial blood pressure was maintained at this hypotensive level for a total duration of 120150 min followed by reinfusion of all shed blood. Blood was maintained in a siliconized lucite reservoir at room temperature for the duration of the oligemic period. Reservoir blood has been shown to contain negligible myocardial depressant factor activity [20]. Arterial blood pressure returned to control values after reinfusion (i.e. about 120-140 mm Hg), and was followed by a post-oligemic period of declining blood pressure. When the arterial blood pressure had fallen to 55 mm Hg, the arterial catheter was opened and blood was collected in polyethylene tubes at 4 °C. Approx. 50-150 ml of blood was collected from each dog. Within 10 rain of collection, blood was centrifuged at 2500 × g and 4 °C for 20 min. The plasma was decanted, stored at --22 °C until deproteinization and deproteinized by trichloroacetic acid.
Preparation of anionic myocardial depressant factor Step l : Trichloroacetic acid-soluble fraction. 300 ml of 50 ~ (w/v) trichloroacetic acid was added with stirring to 2.75 1 of hemorrhagic shock dog plasma held in a water-ice bath at 0-4 °C in a cold room. After 30 min, the precipitate was separated by centrifugation in an International centrifuge at 9000 × g for 20 min at 4 °C. The clear supernatant solution, 2.25 1, was extracted five times each with 400 ml of ether saturated with water at 4 °C. The ether layer was removed by suction and the aqueous phase was evacuated with a water pump for 30 min to remove traces of remaining ether. An aliquot corresponding to 22 ml of plasma was removed for gel filtration on Biogel P-2 and bioassay and the solution remaining was lyophilized. Step 2: Gel filtration on Sephadex G-25 (Fig. 1). Two Sephadex G-25 columns 0.8 × 200cm) connected in series were prepared and developed with 0.2 M pyridine/acetic acid buffer, pH 3.1 (0.2 M pyridine) as described by Greene and Giordano [21]. Lyophilized powder corresponding to the trichloroacetic acid-soluble fraction, derived from approx. 900 ml of hemorrhagic shock dog plasma, was dissolved in 25 ml H20 and the pH was corrected to 3.2 with 50 ~ (w/v) NaOH. The column effluent was monitored by reaction with ninhydrin [22] and by measurement of effluent conductivity at 22 °C. The trichloroacetic acid extract from 2.751 of plasma was processed in three batches and equivalent regions of the column effluent were pooled, evaporated in a rotary evaporator at 40 °C and lyophilized. Elution diagrams obtained for the three runs were essentially identical. 2 ~ of each fraction was used for measurement of the myocardial depressant factor activity.
277
Step 3: Equilibrium and gradient elution chromatography on Dowex 50-X2 (Fig. 2). Fraction 5 (Fig. 1) was dissolved in 12 ml of water and the pH of the solution adjusted from 3.20 to 2.75 with 1 M HCI. The sample was applied to a column of Dowex 50-X2 (1.8 x 60 cm) equilibrated with 0.1 M pyridine/acetic acid buffer, pH 2.81 (0.1 M pyridine), developed at 40 ml/h, 38 °C, and fractions of 12 ml were collected. After elution with 1.62 1 of starting buffer, the column was eluted with a linear gradient prepared from 500 ml each of 0.1 M pyridine/acetic acid buffer, pH 2.81, and 0.2 M pyridine/acetic acid buffer, pH 3.10. A second linear gradient from 0.2 M pyridine/acetic acid buffer, pH 3.1, to 2.0 M pyridine/acetic acid buffer, pH 5.0 (1 1 each), was applied to the column after 875 ml of the first gradient had been used. The effluent corresponding to the fractions indicated at the top of Fig. 2 was pooled, evaporated and lyophilized. 1 0 ~ of each fraction was used for bioassay.
Step 4: Equilibrium chromatography on QAE-Sephadex at pH5.0 (Fig. 3). Fraction 1 (Fig. 2) was dissolved in 5 ml 2 0 ~ aqueous pyridine and applied to a 0.9 × 60cm column of QAE-Sephadex equilibrated and developed with 0.3 M pyridine/acetic acid buffer, pH 5.0 (0.3 M pyridine). Slight shrinkage of the top of the gel bed was observed as the sample was applied. The column was developed as described in the legend to Fig. 3.10 ~ of each fraction was used to detect the depressant factor activity.
Step 5: Equilibrium and step elution chromatography on QAE-Sephadex (Fig. 4). Fraction 5 (Fig. 3) was dissolved in 2 ml of 20 ~ aqueous pyridine and applied to a column of QAE-Sephadex (0.9 × 60 cm) which had been equilibrated in 0.3 M pyridine, pH 8.1. The column was developed as described in the legend to Fig. 4. 20 ~o of each fraction was used to detect the depressant factor activity. Step 6: Analytical gel filtration on Sephadex G-25. Fraction 3 (Fig. 4) was dissolved by 1 ml 0.2 M pyridine/acetic acid buffer (pH 3.1) and applied to a 0.9 × 400 cm column of Sephadex G-25. The column 0.9 × 400 cm was equilibrated and developed with 0.2 M pyridine/acetic acid buffer, pH 3.1 at 5 ml/h, 23 °C and fractions of 2 ml were collected. 20 ~ of each fraction was used to detect the depressant factor activity. Step 7: High voltage paper electrophoresis. Fractions 3 and 4 were combined and subjected to electrophoresis in a Gilson Electrophoretor D apparatus at 2500 V (50 V/cm) for 7 h at pH 2.0 (3.5 ~ formic acid). Each sheet of Whatman 3 MM paper (58 × 68 cm) was washed before use with 125 ml 2 M HC1, with H20 exhaustively, and finally with 125 ml 9 5 ~ ethanol in an apparatus used for descending paper chromatography, and then dried at room temperature. The sample (0.6 ml) was separated into two portions and electrophoresis was carried out twice. The sample was applied to the paper in a 15 cm long band 25 cm from the edge of the paper which would later be immersed in the anode buffer compartment. Marker spots (10 /~1) of sample, HC1Oa-Ala, cysteic acid, were applied on each side of the sample band to be used to locate material after electrophoresis. Peptides were detected in the marker region of the electrophoretogram by reaction with the chloride-tolidine reagent [23] and by reaction with ninhydrin (0.5 ~ in acetone). The paper was cut into horizontal strips on the basis of spots detected by the chlorine-tolidine reagent and eluted with 10 ~ acetic acid. 30 ~ of each fraction was used to detect the depressant factor activity.
278
Bioassay of myocardial depressant factor Samples of unprocessed plasma or column effluents of plasma extracts were assayed for the depressant factor activity on electrically driven isolated papillary muscles taken from the right ventricle of cat hearts according to the method of Lefer et al. [24]. Lyophilized fractions were reconstituted to 10 ml with Krebs-Henseleit solution and added to 10-ml muscle chambers. Developed tension of the isolated papillary muscles were recorded on a Beckman type R oscillographic recorder. Myocardial depressant factor activities were recorded as percent inhibition in developed tension of the papillary muscle at standard conditions of 37 °C and a frequency of stimulation of 1 Hz [24].
Amino acid analysis Samples were hydrolyzed in evacuated sealed tubes with 1 ml of twice distilled constant boiling HC1 for 22h at 110 °C. Amino acid analysis of hydrolysates or samples which had not been hydrolyzed were performed by the method of Spackman et al. [25] on an automatic instrument with provisions for multiple sample application [26]. The instrument was calibrated with standards of 10 nmol of each amino acid. RESULTS
Preparation of anionic myocardial depressant factor The procedure used here for deproteinization of 2.75 1 of hemorrhagic shock plasma is the same as previously described for analytic experiments [19]. Elution of the deproteinized samples on Biogel P-2 in Krebs-Henseleit solution yielded four peaks. The elution profile of the depressant factor activity is very similar to that previously described by Leffler et al. [19] and Lefer and Inge [18] for whole cat and dog plasma. Virtually all the factor activity was recovered in the second large peak (fraction 2) which was eluted between 80 and 100 ml. Only small amounts of sodium were present in this factor peak. The major salt peak was fraction 3 which had only modest depressant factor activity (i.e. only 20 ~ inhibition). Each chromatographic experiment was carried out in two parts. The first, using about 20 ~ of the material was used to test experimental conditions and to locate the myocardial depressant factor activity. All fractions containing the factor activity were then added to the remaining material and the experiment was repeated. The elution diagrams given in Figs. 1-4 represent results obtained from the second preparative run for each chromatographic system. Gelfiltration on Sephadex G-25 (Fig. 1). The trichloroacetic acid solution fraction contains appreciable amounts of ninhydrin-positive material which is eluted over the entire volume of the column but is concentrated in the low molecular weight region. The effluent conductivity profile shows that salts are eluted between 900 and 1100 ml. The dip in the conductivity profile at 870 ml was used as a marker for selecting fractions. The dip does not mean that this area is salt free, but that it is less than the solvent which is equivalent in conductivity to 0.05 M NaCI. Cardiodepressant activity, most pronounced in regions 6 and 7, is due primarily to excessive salt concentration; whereas in regions 4 and 5 the negative inotropic effect is due to the depressant factor activity. This can be shown by measurement of the salt concentration as well as by inspection of the bioassay tracings. In this regard, high salt concentration in-
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Fig. 3. Equilibrium chromatography on QAE-Sephadex of fraction 1 (Fig. 2). The column, 0.9 × 60 cm, was equilibrated and developed with 0.3 M pyridine/acetic acid buffer, pH 5.0, at 22 °C, 14 ml/h, and fractions of 2 ml were collected. - - - , absorbance of 570 nm (ninhydrin reaction after alkaline hydrolysis) ; , effluent pH ; , effluent conductivity. The top panel of the figure shows the effluent that was combined, the fraction number, and the myocardial depressant factor activity.
QAE-Sephadex. Fig. 3 shows that under equilibrium conditions, most of the depressant factor activity (i.e. fraction 5) is retarded by the column, whereas most of the ninhydrin-reactive materials are eluted either in the breakthrough peak (i.e. fraction 1 indicated by the peak of pH and conductivity given in the middle panel of the figure) or in fraction 4. The depressant factor activity in fraction 5 is an appreciable portion of the recovered activity, and only represents a small part of the total ninhydrin-positive material. Further purification of the factor activity separating it from free amino acids was achieved by adsorbing the activity of QAE resin at an alkaline p H and eluting it by reducing the pH (Fig. 4). The myocardial depressant factor activity is eluted after the abrupt change in pH as shown in Fig. 4. Gelfiltration. The fraction containing the factor activity (i.e. fraction 3, Fig. 4) was rechromatographed on Sephadex G-25 under the same conditions as shown in Fig. 1, except that in this experiment an underload analytical column was used. The depressant factor activity was eluted in fractions 3 and 4 between the two major ninhydrin-positive fractions at 220-235 ml effluent volume. Nevertheless, an appreciable enrichment of the depressant factor activity was obtained.
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Fig. 4. Equilibrium and step elution chromatography on QAE-Sephadex of fraction 5 (Fig. 3). The column, 0.9 × 60cm, was equilibrated and developed with 0.3 M pyridine (pH 8.1) at 22 °C, 20 ml/h, and fractions of 4 ml were collected. After 230 ml of effluent had been collected, the column was eluted with 0.2 M pyridine/acetic acid buffer, pH 5.0, for an additional 600 ml followed by 200 ml of 0.3 M pyridine/acetic acid buffer. - - - - , absorbance at 570 nm (ninhydrin reaction after alkaline hydrolysis, bottom panel); - - - - , and effluent pH; (middle panel). The top panel of the figure shows the effluent that was combined, the fraction number, and the myocardial depressant factor activity.
High voltage electrophoresis. The two m o s t active fractions (i.e. fractions 3 a n d 4) were c o m b i n e d a n d s u b m i t t e d to high voltage electrophoresis at p H 2.0 for 7 h. All o f the material detected by the chlorine-tolidine stain reaction was n i n h y d r i n negative indicating t h a t free a m i n o acids were not present in the fractions t h a t h a d depressant factor activity (fractious 3 a n d 8). The area c o r r e s p o n d i n g to fraction 6 was the only one t h a t was fluorescent. The p y r r o l i d o n e carboxylic acid did n o t migrate, b u t all a m i n o acids including cysteic acid migrated off the p a p e r u n d e r these conditions. M y o c a r d i a l d e p r e s s a n t factor activity was detected in two regions o f the electrop h o r e t o g r a m . The m a t e r i a l in fractions 3 and 8 are d e n o t e d A1 a n d A2, respectively. Amino acid composition of the myocardial depressant factor fractions A 1 and A.2 T a b l e I gives the a m i n o acid c o m p o s i t i o n o f A1 and A 2 after elution f r o m the p a p e r TABLE I COMPOSITION OF HYDROLYSATES OF ELECTROPHORETOGRAM MYOCARDIAL DEPRESSANT FACTOR-POSITIVE AREAS Values are expressed as tool amino acid/mol glycine. Amino acid
Glutamic acid Glycine Serine Unidentified amino acid (x)
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Az
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282 and hydrolysis with 6 M HC1. Amino acids not given in the table were present at a level of 0.3 mol per mol glycine or less. No free amino acids above background were detected in samples not submitted to hydrolysis. The unidentified amino acid was eluted from the long column (Beckman PA-28 resin) of the amino acid analyzer after cysteic acid at 55 °C, using pH 3.25 sodium citrate buffer (0.2 M sodium citrate). The unknown amino acid was distinguishable from levulinic acid but not taurine under these conditions. However, when the column was operated at 32 °C, the unidentified amino acid present in the depressant factor acid hydrolysates was separable from taurine. Nevertheless, we have not been able to identify this amino acid at present. It must be emphasized that the ninhydrin color value for aspartic acid has been used to calculate the amount of the unidentified amino acid present in these factor fractions and thus its molar concentration relative to glycine may be in error by as much as 10-30 ~. The interpretation of the analytical data is further complicated by the low levels of peptide recovered from the paper and the presence of glycine and serine as contaminants in paper and common laboratory solvents. On this basis, the amino acid composition proposed below for A1 and A2 must be considered as preliminary: (fraction 3, Fig. 5) AI: X1, Gly~, Glx,, Serl; (fraction 8, Fig. 5) A2: X2, Glyi.
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Fig. 5. Diagrammatic representation of the high voltage paper electrophoretogram of a pool derived from active fractions eluted from an underloaded Sephadex G-25 column (step 6). The asterisks indicate the myocardial depressant factor activity; cross-hatched area indicates material positive to the chlorine-tolidine reagent and negative to ninhydrin. The experimental conditions are given in Methods.
Inotropic activity of free amino acids In an effort to determine whether free amino acids could be contaminating the myocardial depressant factor-positive fractions and contributing to its activity, we assayed the inotropic activity of 20 common amino acids. Although, none of these
283 free amino acids could be expected to exceed 1 mM in any of the depressant factor activity fractions, we assayed each amino acid at 1.0 and 10 mM. At 1 raM, none of the 20 amino acids exerted any significant negative inotropic activity. Glycine, leucine, phenylalanine, and valine exerted modest increases in contractile force of 3--4 ~. At 10 mM, cysteine, histidine and tryptophan exerted moderate negative inotropic effects of 10-20 70. Even if these amino acids were present at a concentration as high as 10 raM, their contribution to the depressant factor activity would be only modest. However, none of these four amino acids was present at these levels in the paper electrophoresis or the Sephadex G-25 analytical fractions having myocardial depressant factor activity. DISCUSSION This report describes chromatographic procedures for the isolation of myocardial depressant factor activity from liter quantities of hemorrhagic shock plasma obtained from dogs. Steps 1 and 2 are trichloroacetic acid precipitation of plasma proteins, followed by gel filtration on Sephadex G-25. These steps effectively separate the factor activity from proteins and salt in plasma and thus are the most significant steps in the preparation of purified depressant factor activity. Much smaller quantities of materials were applied to Dowex-50, QAE-Sephadex column and used for high voltage electrophoresis. The need for large batches of starting material is indicated by the requirement of 1-2 ml of plasma for each bioassay. The amino acid composition and biological activity of purified myocardial depressant factor fractions A1 and A2 taken together with the purification steps indicate that approx. 0.5-1.0 nmol/ml of the factor are present in the blood of animals in hemorrhagic shock. The results show clearly that the factor activity (i.e. measured as a negative inotropic effect on isolated cardiac tissue) exists in trichloroacetic acid-treated, hemorrhagic shock plasma in multiple chromatographic forms. The multiple chromatographic forms which are anionic, neutral and cationic under the conditions employed in these experiments may or may not be chemically related. However, the two anionic forms (i.e. A1 and A2) are similar in amino acid composition. Their anionic properties were used as the basis for their isolation and purification. In general, the behavior of the depressant factor activity with respect to recovery of activity and rechromatography indicate that the factor activity is stable and its behavior is reproducible. The two purified forms of the myocardial depressant factor (i.e. A~ and A2) are clearly distinguishable from free amino acids and salts. We also tested the effects of free amino acids on the contractile force of the isolated cat capillary muscle preparation. None of the 20 common amino acids studied exerted a significant negative inotropic effect (i.e. depress contractile force) comparable to that produced by the factor. These findings are in agreement with those of Gatgounis and Hester [27] obtained in the intact dog. Thus, it is extremely unlikely that free amino acid contamination of the factor samples could account for the depressant factor activity observed in any of the active fractions of our column eluates. The myocardial depressant factor activity was also separated from salt activity in the preparation of the purified factor. Lefer and Inge [18] previously showed that it was eluted prior to salt in the Biogel P-2 column, and that the amount of salt required to elicit a negative inotropic effect comparable to that of the factor is in
284 excess o f 450 mequiv/1. In this study, we also reported that high salt concentrations in the range o f 400-500 mequiv/l depress myocardial contractility in a manner distinctly different from that o f the factor. The myocardial depressant factor depresses developed tension by decreasing active tension without a change in resting tension. High salt concentrations depress developed tension by decreasing active tension and markedly increasing resting tension (i.e. baseline tension). This large increase in baseline tension does not occur with high titers o f the factor in the absence o f high salt concentrations and serves to distinguish this from responses occurring due to the presence o f high a m o u n t s o f salt contamination. The characterization o f the A1 and A2 forms o f the depressant factor indicate the presence o f an acidic amino acid which has not yet been identified. The amino acid composition o f the factor appears to be similar to R D S [28], a substance found in the plasma of shock animals having similar biological properties to the myocardial depressant factor. However, the unknown amino acid in R D S is eluted from the short column o f the amino acid analyzer and thus does not appear to be acidic. Therefore, the two u n k n o w n amino acids do not appear to be identical. The chemical relationship, if any, between the myocardial depressant factor and R D S remains to be determined. ACKNOWLEDGEMENTS We wish to acknowledge Mr. Martin L. Ogletree and Miss Yumi Sakane for their expert technical assistance during this study. This work was supported in part by N.I.H. Research G r a n t No. HL-17745 and No. GM-21235 and by Brookhaven National Laboratory. Research carried out at Brookhaven National L a b o r a t o r y under the auspices o f the U.S, E R D A . REFERENCES 1 Brand, E. D. and Lefer, A. M. (1966) Proc. Soc. Exp. Biol. Med. 122, 200-203 2 Baxter, C. R., Cook, W. A. and Shires, G. T. (1966) Surg. Forum 17, 1-2 3 Glenn, T. M., Lefer, A. M., Martin, J. B., Lovett, W. L., Morris, J. N. and Wangensteen, S. L. (1971) Am. Heart J. 82, 78-85 40kuda, M., Yamada, T. and Hosono, K. (1973) Jap. Circ. J. 37, 1009-1017 5 Wangensteen, S. L., Geissinger, W. T., Lovett, W. L., Glenn, T. M. and Lefer, A. M. (1971) Surgery 67, 410~18 6 Lefer, A. M., Glenn, T. M., Lopez-Rasi, A. M., Kiechel, S. F., Ferguson, W. W. and Wangensteen, S. L. (1971) Clin. Pharmacol. Ther. 12, 506-516 7 Glenn, T. M. and Lefer, A. M. (1970) Circ. Res. 27, 783-797 8 Williams, L. F., Goldberg, A. H , Polansky, B J. and Byrne, J. J. (1969) Surgery 66, 138-144 9 Lovett, W. L., Wangensteen, S. L., Glenn, T. M. and Lefer, A. M. (1971) Surgery 70, 223-230 10 Rosenthal, S. L., Hawley, P. L. and Hakim, A. A. (1972) Surgery 71, 527-536 11 Lefer, A. M. (1970) Fed. Proc. 29, 1836-1847 12 Lefer, A. M. and Martin, J. (1970) Circ. Res. 26, 59-69 13 Lefer, A. M. and Martin, J. (1970) Am. J. Physiol. 218, 1423-1427 14 Wangensteen, S. L., Ramey, W. G., Ferguson, W. W. and Starling, J. R. (1973) J. Trauma 13, 181-194 15 Nagler, A. L. and Levenson, S. M. (1974) Circ. Shock 1,251-264 16 Fisher, W. D., Heimbach, D. W., McArdle, C. S., Maddern, M., Hutcheson, M. M. and Ledingham. McA. (1973) Br. J. Surg. 60, 392-394 17 Lundgren, O., Haglund, U., Isaksson, O. and Abe, T. (1976) Circ. Res. 38, 307-315
285 18 19 20 21 22 23 24 25 26 27 28
Lefer, A. M. and Inge, Jr., T. F. (1973) Proc. Soc. Exp. Biol. Med. 142, 429--433 Leffler, J. N., Litvin, Y., Barenholz, Y. and Lefer, A. M. (1973) Am. J. Physiol. 224, 824-831 Brand, E. D., Cowgill, R. and Lefer, A. M. (1969) J. Trauma 9, 216-226 Greene, L. J. and Giordano, J. S. (1969) J. Biol. Chem. 244, 285-298 Hirs, C. H. W. (1967) Methods Enzymol. 11, 325-329 Pataki, G. (1968) Techniques of Thin Layer Chromatography in Amino Acid and Peptide Chemistry, p. 107, Ann Arbor Science Publishers Inc., Ann Arbor, Mich. l~efer, A. M., Cowgill, R., Marshall, F. F., Hall, L. M. and Brand, E. D. (1967) Am. J. Physiol. 213,492-498 Spackman, D. H., Stein, W. H. and Moore, S. (1958) Anal. Chem. 30, 1190-1206 Alonzo, N. and Hirs, C. H. W. (1968) Anal. Biochem. 23, 272-289 Gatgounis, J. and Hester, W. (1964) Proc. Soc. Exp. Biol. Med. 116, 430--434 Blattberg, B., Frosolono, M. F., Misra, R. P. and Levy, M. N. (1972) J. Reticuloendothel. Soc. 12, 371-386
