Stabilization of methionine-enkephalin in human and rat blood

Life Sciences, Vol. 39, pp. 21-28 Printed in the U.S.A.

Pergamon Press

STABILIZATION OF METHIONINE-ENKEPHALIN IN HUMAN AND RAT BLOOD Vincent J. Aloyo I, Shaker A. Mousa 2 and Glen R, Van Loon 3 Veterans Administration Medical Center and Department of Medicine University of Kentucky, Lexington, KY 40511 (Received in final form April ii, 1986) SUMMARY Methods of preventing the degradation of 3H-methlonlne-enkephalln (3H-ME) in human blood both at 37°C and under conditions of immediate cooling were examined. We found that, contrary to previous suggestions, use of aprotlnln (with or without immediate cooling) was ineffective in preventing the degradation of 3H-ME in blood. Thus, previous reports on the circulating levels of ME which relied on such procedures to stabilize the ME may have reported artlfactually low values. However, we found that citric acid effectively prevents 3H-ME breakdown in both human and rat blood. Thus, we propose the use of citric acid, mixed with blood immediately upon collection, as an effective method for the stabilization of ME in blood. The oplold peptlde, methionlne-enkephalln (ME) is secreted into peripheral blood from the adrenal medulla (1,2) and perhaps from sympathetic nerve endings (3). The circulating levels of ME may vary as a function of the normal or pathophysiological state of the organism (4-7). Since ME is rapidly degraded in blood (8-10), accurate measurement of ME levels depends upon the prevention of such breakdown. Earlier studies have recommended the use of aprotinln (I) and the use of ice-chilled syringes (7) for the prevention of ME degradation in blood. However, in preliminary experiments we found that aprotlnin was not effective in preventing ME degradation. Thus, we examined other compounds for their ability to limit the degradation of ME in blood. We found that citric acid effectively prevented ME degradation in blood and stabilized blood ME levels. Materials and Methods Effects of immediate cooling on enkephalln degradation. Time course of de~radatlon. Human blood (2ml) was collected in Ice-chilled syringes containing heparln (14 unlts/ml) and immediately added to 0.22mi of ice cold cocktail containing aprotlnln (FBA Pharmaceuticals) and ethylenedlamlnetetraacetic acid (EDTA) plus either HPLC purified [tyrosyl-3, 5-3H]enkephalln(5-L-methlonine) (New England Nuclear, 39.5Ci/mmole, 55.8

Present Address: IDepartment of Pharmacology, Medical College of PA, Philadelphia, PA 19129; 2Dupont, Biomedical Department, North Billerlca, MA 01862; 3To whom correspondence should be addressed.

0024-3205/86 $3.00 + .00 Copyright (c) 1986 Pergamon Journals Ltd.

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pmole/ml,3H-ME) or [tyrosyl-3,5-3H]enkephalln(5-L-leuclne) (New England Nuclear, 43.6Ci/mmole, 50.5 pmole/ml,3H-LE). One aliquot was immediately centrifuged at 30,000xg for 20 mln at 4°C. Other allquots were allowed to stand in an icewater bath for 40, 80 or 140 mln before centrlfugatlon. Immediately following centrifugation, the plasma was acidified with HCI (0.55N, final concentration). The 3H-LE or 3H-ME was separated from its metabolltes by both polystyrene bead chromatography and thin layer chromatography (TLC). Survey of several inhibitors. Subsequent experiments were performed to test the ability of aprotinln, EDTA, citric acid or bestatin (Sigma Chemical Co.) either alone or in various combinations to stabilize 3H-ME in blood subjected to immediate cooling. Blood, collected as described above, was added to the ice-cold cocktail containing the inhibltors plus 3H-ME and immediately placed on ice. After 30 min, the sample was centrifuged as described above and the plasma obtained was acidified.lmmedlately. The 3H-ME was separated from its metabolltes by polystyrene bead chromatography. De~radatlon of 3H-ME at 37°C. Breakdown in rat plasma. Male rats (Sprague-Dawley, 300g) were decapitated and mixed arterial and venous blood was collected in heparin (200 unlts/ml) and centrifuged at 6000xg for 20 min at 4°C. Allquots of plasma were combined with saline or test compound and prewarmed at 37°C for 5 mln before the addition of 3H-ME (39.5 Ci/mmole, 16 pmoles/ml). The incubation was continued for various times before the reaction was stopped by acidifying allquots of the reaction mixture with HC1 (0.5N final concentration). The aliquots were kept on ice until the 3H-ME was separated from its metabolltes by polystyrene bead chromatography. Breakdown in human plasma. Plasma was prepared from human blood collected in heparin (14 unlts/ml) by centrlfugatlon at 6000xg for 20 mln at 4°C. Allquots of plasma were combined with saline or test compound and prewarmed at 37°C for 5 mln before the addition of 3H-ME. Breakdown was determined exactly as described for rat plasma. Breakdown in human blood. Human blood, collected in heparin (14 units/ml), was immediately combined with saline or test compound. Aliquots were prewarmed at 37°C for 5 mln before the addition of 3H-ME and then incubated at 37°C for various times. To stop the degradation, a 200 ul aliquot of reaction mixture was rapidly added to 800 ul of Ice-cold saline containing the peptldase inhlbltors, bestatin (20 uM), thiorphan (Peninsula, luM) and Captopril (Squibb, luM). The sample was centrifuged at 15,000xg for 15 sec and the supernatants were immediately acidified with HCI (0.SN final concentration). This "stop" procedure was completed within 45 sec. 3H-ME was separated from its metabolltes by polystrene bead chromatography. Separation of enkephalins from their metabolites. Pol~styrene bead chromatography. Polystyrene beads were used to separate the 3H-enkephallns from their metabolltes according to the procedure of Vogel and Altsteln (12). The acidified sample was applied to a sillconlzed glass column containing 80 mg of resin (Porapak Q, Waters Associates) which had been prevloulsy washed with 95% ethanol and then equilibrated with water. The 3}{metabolites were eluted with 4xl ml water washes and subsequently the intact 3H-enkephalin was eluted with 4xl ml 95% ethanol washes. The water eluate and the ethanol eluate were separately subjected to liquid scintillation counting by using Multlsol (Isolab). Counting efficiency was determined by the addition of internal standard ([3H]-water or [3HI-toluene, New England Nuclear). The total recovery of tritium from the column was calculated for each sample and In all cases was greater than 95%. The performance of the columns was monitored

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by separatel~ applying and eluting purified [3H]-L-tyrosine (New England Nuclear) or 3H_ME to the polystyrene bead columns. In agreement wlth Vogel and Altsteln (121, it was determined that the water washes eluted 97.5% of the 3H-tyroslne and 8.4% of the 3H-ME and the ethanol washes eluted 2.5% of the 3H-tyrosine and 90% of the 3H-HE. Furthermore, the presence or absence of plasma proteins did not affect the elution profile (data not shown). The results presented below are corrected based on these recovery values. Thin layer 9hromato~raph~. The enkephallns were also separated from their metabolltes by two different TLC systems (12,13). Allquots of the acidified samples were mixed wlth a cold carrier cocktail contalning3tyrosine , t y r o s y l - g l y c l n e , t y r o s y l - g l y c y l - g l y c i n e and e i t h e r LE f o r H - L E - c o n t a l n i n g samples or ME p l u s HE s u l f o x l d e f o r 3H-ME c o n t a i n i n g s a m p l e s . The samples were s p o t t e d on s i l l c a g e l TLC p l a t e s (Merck; s i l l c a g e l 601. The M E - c o n t a i n i n g samples were d e v e l o p e d i n s o l v e n t I c o n s i s t i n g of c h l o r o f o r m : m e t h a n o l : a c e t i c a c i d : w a t e r ( 4 5 : 3 0 : 6 : 9 , v / v / . L E - c o n t a i n i n g samples were i n d e p e n d e n t l y d e v e l o p e d i n s o l v e n t I o r i n s o l v e n t I I c o n s i s t i n g of ethylacetate:isopropanol:5% acetic acid (2:2:l,v/vl. A f t e r d r y i n g , the samples were v i s u a l i z e d w i t h n t n h y d r i n s p r a y . The s p o t s c o r r e s p o n d i n g to a u t h e n t i c LE or ME p l u s ME s u l f o x i d e were s c r a p e d from t h e p l a t e and the amount of 3H was d e t e r m i n e d by l i q u i d s c i n t i l l a t i o n c o u n t i n g . S i n c e ME s p o n t a n e o u s l y o x i d i z e s to i t s s u l f o x i d e d u r i n g h a n d l i n g ( 1 1 , 1 4 / , t h e t r i t i u m r e p o r t e d as 3H-ME i s the combined r a d i o a c t i v i t y c o n t a i n e d i n t h e ME and ME s u l f o x t d e s p o t s .

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FIG. I Effect of immediate coolin~ on enkephalln breakdown in human blood. Human blood, collected in Ice-chilled syringes, was combined wlth aprotinln (175 KU/ml) and EDTA (2.4 mM) and either 3H-ME (A) or 3H-LE(B) and i m m e d i a t e l y p l a c e d i n an i c e - w a t e r m i x t u r e f o r t h e t i m e s indicated. The r e c o v e r y of i n t a c t e n k e p h a l i n was d e t e r m i n e d by p o l y s t y r e n e bead chromatography ( O ) o r by TLC i n s o l v e n t s y s t e m I ( • ) or I I ( [] I as d e s c r i b e d i n t h e Methods S e c t i o n .

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Results Effects of immediate cooling. In the presence of aprotlnin plus EDTA, both 3H-ME and 3H-LE were substantially degraded by whole human blood even though the samples were immediately placed in the cold (Fig.l). Figure IA shows that even with immediate centrifugation at 4°C, only 50 to 60% of the 3H-ME was recovered intact. Further incubation on ice resulted in a greatly reduced rate of degradation such that by 140 mln only a further 10% was degraded. For 3H-LE also, immediate cooling and processing allowed the recovery of 50 to 60% of the 3H-LE (Fig. IB) and longer incubation on ice resulted in only a small amount of further degradation (Fig. IB). For both 3H-LE and 3H-ME, the recovery obtained by the TLC procedures agreed well with that obtained by use of the polystyrene bead chromatography. In the absence of inhibitors, 40% of the 3H-ME added to freshly collected human blood was lost although the warm blood was placed immediately on ice and maintained there for 30 min (Table I). Aprotinln plus EDTA failed to improve the recovery of 3H-ME. Inclusion of the aminopeptidase inhibitor, bestatin, in the cocktail containing aprotonln plus EDTA increased the recovery of 3H-ME to 93%. Citric acid either alone or with bestatin increased the recovery of 3H-ME to greater than 98% (Table i). TABLE 1 Effect of Immediate Cooling on 3H-ME Degradation in Human Blood TREATMENT No Inhibitor Aprotlnin plus EDTA Aprotinln and EDTA plus Bestatln Citric Acid Citric Acid plus Bestatln

PERCENT 3H-ME REMAINING 60 61 93 98 99

Human blood was collected in ice-chilled syringes and immediately added to ice-cold cocktail containing inhibitor and =H-ME and then held on ice for 30 min. After preparation and acidification of the plasma, the 3H-ME was separated from its metabolites by polystyrene bead chromatography. The inhlbltors used were aprotinin (175 KU/ml), EDTA (2.4, mM), bestatin (88 uM) and citric acid (23 mM). Degradation of 3H-ME at 37°C. In the absence of inhlbitors, 3H-ME was rapidly degraded at 37=C in rat plasma, with less than 50% remaining after I min (Fig.2). Addition of aprotinin plus EDTA was ineffective in protecting the 3H-ME. The potent and selective aminopeptldase inhibitor, bestatln (15), decreased the rate of 3H-ME degradation so that 50% remained after 5 min. Citric acid alone and citric acid plus bestatin were equally effective in reducing the rate of 3H-ME breakdown such that reater than 90% remained after 15 min. Further incubation of rat plasma with H, ME plus citric acid alone resulted in a recovery of over 90% of the 3H-ME after 30 min (Fig.2).

~

The breakdown of 3H-ME in human blood or plasma at 37°C was also very rapid (50% remaining after 4 min or 2.5 min in human blood or plasma, respectively) (Fig.3). Aprotlnin plus EDTA was only minimally effective in increasing the recovery of 3H-ME (Fig.3). However, citric acid plus bestatin effectively protected the 3H-ME such that greater than 95% remained after 27 min at 37°C.

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Discussion We have demonstrated that ME is rapidly degraded by both human and rat blood at physiological temperature. The rate of ME breakdown in rat plasma is approximately twice as fast as that in human plasma (Flg.2,3), consistent with the suggestion of Hambrook et al (I0). Furthermore, we have demonstrated that aprotlnln plus EDTA is of little value in protecting ME from degradation by blood either at 37°C or with immediate cooling. These results corroborate those of Clement-Jones et al. (II) who reported that about 75% of the exogenously added ME was recovered when human plasma was in~edlately frozen in the presence or absence of aprotlnln. This lack of protection by immediate cooling or even immediate freezing may reflect, at least in part, the tlme required for the warm, freshly ~oilected

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Degradation in rat plasma at 37°C. Each I ml aliquot of rat plasma was mixed with 145 ul of either saline ( O ) , aprotlnln (175 KU) plus EDTA (I mM) (~)), citric acid, (23 mM) ( O ) , bestatln (88uM) ( D ) or citric acid (23 mM), plus bestatin (88 um) ( ~ ) . The mixture was prewarmed at 37°C for 5 mln before the addition of 3H-ME (16 pmoles/ml) and the incubation continued for the times indicated. The reaction was stopped and the percent of intact 3H-ME remaining at each time point was determined as described in the Methods section.

-t

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blood to cool. As shown in Figures 2 and 3, only a few minutes at 37°C is required to degrade 50% of the 3H-ME. Thus, even though the blood is placed on Ice immediately, during the time It is cooling the enkephallns are being degraded. In support of this suggestion, Flg.l shows that for both 3H-ME and 3H-LE little degradation occurs after the blood has presumably cooled, e.g. between 40 and 140 min. on ice. The rapid loss of another oplold peptide,~ -endorphln, from blood has been reported to be due to Its binding (or uptake) to blood cells (16). However, it is unlikely that in our experiments the rapid loss of 3H-ME, in whole blood Is due to a similar mechanism. First, the rate of loss of 3H-ME at 37°C is similar for whole blood and cell-free plasma. Indeed, the rate of degradation appears to be slightly faster in plasma than whole blood (Fig.3). Furthermore, in the experiments designed to study the effects of immediate cooling, the total

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FIG.3 Inhibition of 3H-ME degradation In human blood or plasma at 37°C. Human plasma (A) or whole blood (B) was combined wlth either 145 ul

per ml of s a l i n e ( O ) , a p r o t l n i n (175 KU/ml) p l u s EDTA (ImM) ( O ) or c i t r i c acid (23 mM) p l u s b e s t a t l n (88 uM) ( ~ ) . The m i x t u r e was prewarmed at 37°C for 5 mln before the addition of 3H-ME and the incubation continued for the times indicated. The reaction was stopped and the percent of intact 3H-ME remaining at each time point was determined as described in the Methods section.

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amount of tritium associated with the blood cell pellet was found to be approximately 10%, independent of the percentage of 3H-ME recovered intact (data not shown). Thus, it is likely that the tritium associated with the blood cell pellet is related to trapped plasma. The primary cleavage of ME in blood appears to be at the tyrosine-glyclne amide bond (8,10) suggesting the action of an aminopeptldase. Our results are consistent with this suggestion since the selective amlnopeptidase inhibitor, bestatin (15), either alone (Fig.2) or in combination with aprotinln plus EDTA (Table I) reduces the rate of ME degradation. The failure of bestatln to completely inhibit ME breakdown suggests that either insufficient bestatln was employed or that peptldases other than aminopeptldases are active in blood and plasma. Although the K i of bestatln for plasma amlnopeptldase(s) has not been determined, the concentration of bestatln employed in this study, 88 uM, is 88 times the reported ICb0 for the soluble amlnopeptldase isolated from rat brain (17). Thus, this concentration should be sufficient. An alternative explanation that enkephalin may be degraded by C-terminal cleavage in blood has been suggested by Hogue-Angelettl and Roda (9). An amlnopeptldase isolated from human blood was characterized by ColettiPreviero et al (18). They note that EDTA is not an inhibitor of this enzyme suggesting that the addition of EDTA to blood samples would not help stabilize ME. Our results studying the effect of aprotlnin plus EDTA on ME degradation, support this suggestion. Furthermore, they (18) found that this amlnopeptldase, which has an optimal pH of 8, is denatured at pH 5.5. Since the normal pH of human blood of 7.4 is decreased to pH 5.7 by the addition of 23 mM citric acid, citric acid may protect ME from degradation in our experiments by acidic denaturation of aminopeptidase (5) and perhaps other peptldases. Another possibility is that since citric acid is a metal ion chelator, it may inhibit the metalopeptldases responsible for enkephalln degradation (19) by removing their essential metal group. Even though citric acid is effective in stabllizing ME in blood and plasma, care must be taken to ensure that it is added during blood collection. Acidification after obtaining plasma is too late since even a 15 sec. delay results in measurable loss of ME (Fig.2). Immediate cooling of blood (Table I) or even freezing (11), with or without aprotinln, does not fully protect exogenous ME from degradation. Thus, previous studies which relied upon such procedures to stabilize ME during blood collection (4,7) may have reported artifactually low values for clrculatlng ME. The problem of obtalning the true concentration of endogenous ME in plasma is further complicated since prior in vivo drug treatment may increase the rate of ME degradation (20). However, we have shown that citric acid effectively protects ME in blood and plasma. Citric acid has two practical advantages as a stabilizer of ME in blood; first, it is inexpensive relative to more specific peptidase inhlbitors such as bestatln and second, since citric acid is itself an anticoagulant, no other anticoagulant is required during blood collection. We propose that a cocktail containing citric acid be placed directly in the collection syringe to ensure rapid mixing of the citric acid with the blood to stabilize ME levels. Alternatively, blood may be directly dripped into the citric acid containing cocktail with continuous mixing. We also suggest the inclusion of aprotlnln to prevent the metabolism of large precursor peptides to ME. Acknowledgements The a u t h o r s a r e v e r y g r a t e f u l t o D a r l e n e Spino f o r t h e e x p e r t t y p i n g of t h i s m a n u s c r i p t . T h i s s t u d y was s u p p o r t e d by g r a n t s from t h e U n i v e r s i t y of Kentucky Tobacco and H e a l t h R e s e a r c h I n s t i t u t e and by t h e V e t e r a n s A d m i n i s t r a t i o n .

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