‘Dynorphin’ in plasma: enzymatic artifact and authentic immunoreactivity

Regulator), Peptides, 8 (1984) 131-140 Elsevier

131

R P T 00267

'Dynorphin' in plasma: enzymatic artifact and authentic immunoreactivity Trevor A. Howlett a.,, Janet Walker b, G.M. Besser a and Lesley H. Rees b Departments of ~ Endocrinology and h Chemical Endocrinology, St, Bartholomew's Hospital, London ECIA 7BE, U.K. (Received 6 October 1983; revised manuscript received 13 December 1983; accepted for publication 3 January 1984)

Summaff The potent opioid peptide dynorphin (DYN) is found in posterior pituitary vasopressinergic neurones and in adrenal medullary cells suggesting that secretion into plasma is likely. We have developed a sensitive radioimmunoassay in order to study plasma DYN in man. It transpired that extraction prior to assay was essential since unextracted plasma caused gross and non-parallel inhibition of binding of tracer. Plasma extracted using leached silica glass (Vycor) caused inhibition of tracer binding which diluted in parallel to synthetic DYN suggesting the presence of substantial amounts of DYN-like immunoreactivity (irDYN) in plasma. Further investigation however demonstrated that this irDYN was artifactual and caused by enz~cmatic degradation of tracer. Although use of Seppak C18 cartridges resulted in reliable extraction of synthetic porcine DYN from acidified plasma, we have not detected irDYN in any plasma so far studied using this technique. However, extraction of non-acidified plasma using our antibody coupled to Sepharose CNBractivated 4B followed by gel filtration chromatography demonstrated a single peak of irDYN of molecular size similar to DYN. These data suggested that a small amount of a DYN-like peptide does circulate in human plasma although this is not identical to porcine DYN(1-17). The implication of our results for the measurement of other similar peptides in plasma is discussed. opioid; dynorphin; enzymes; radioimmunoassay

* To whom all correspondence should be sent. 0167-0115/84/$03.00 © 1984 Elsevier Science Publishers B.V.

132 Introduction

The heptadecapeptide dynorphin A (DYN) [1] is arguably the most potent endogenous opioid yet isolated, being 700 times more potent than the related pentapeptide leucine enkephalin in the guinea pig ileum bioassay [2]. With the other C-terminally extended Leu-enkephalins derived from the pro-dynorphin precursor [3] it is localised within the secretory granules of the neurohypophyseal vasopressinergic neurones [4,5] and the chromaffin cells of the bovine adrenal medulla [6,7] and has been demonstrated to be released from both these tissues in vitro [6,8,9]. Considerable amounts of irDYN have also been described in a number of human phaeochromocytomas [10,11] although release has not yet been demonstrated. It therefore seems likely that DYN must be released into plasma and its high opioid potency would suggest that distant effects are a possibility. IrDYN has been reported in stressed rat plasma in the p m o l / m l range [12] although it was noted that this immunoreactivity was proving difficult to characterise and was unaltered by hypophysectomy [13]. Acid acetone extracts of human plasma have also been shown to contain irDYN following gel filtration chromatography although most of this irDYN was of larger molecular size than porcine DYN(1-17) [14]. We have therefore developed a sensitive, specific radioimmunoassay (RIA) for DYN in order to confirm the existence of irDYN in human plasma and to study its physiology.

Materials and Methods

Radioimmunoassay DYN(1-13), the fragment initially sequenced [2], was conjugated to bovine thyroglobulin using glutaraldehyde and the conjugate used to immunise New Zealand White rabbits at six weekly intervals. Three out of four rabbits produced a usable antiserum of which one (Karis 5/82) was chosen for use in RIA at a final dilution of 1/15 000. 125I-labelled DYN(1-13) was prepared using chloramine T [15] and purified by loading onto a small (3 × 0.5 cm) column containing ODS-silica and eluting the iodinated peptide with increasing concentrations of methanol in 1% trifluoracetic acid. Satisfactory DYN tracer (specific activity 250/~Ci/nmoi peptide) eluted at approximately 51% methanol and was stored in aliquots at - 7 0 ° C in 0.25 M acetic acid plus 0.25% (w/v) human serum albumin. Synthetic porcine DYN(1-17) was used as assay standard once the full structure had been elucidated [1]. The incubation mixture consisted of 200/.tl of a dilution of standard or sample, 50 ~i antiserum (initial dilution 1/2500) and 50/~1 tracer all in 150 mM phosphate buffer (pH 7.4) plus 0.25% (w/v) human serum albumin, 0.1% Triton X-100, and phenol red as indicator. Triton X-100 is necessary to prevent excessive adsorption of peptide to the walls of test tubes [16]. After overnight incubation at 4°C free and bound tracer were separated using cold (4°C) dextran-coated charcoal. Assay sensitivity was 4 fmol/assay tube using both DYN(1-17) and (1-13) as standard. The antibody recognises the mid portion of the DYN sequence showing

133 100% crossreactivity with the fragments DYN(1-12), (1-11) and (1-10), 30% with DYN(1-9) and 15% with DYN(1-8). Crossreactivity was < 0.01% with DYN(1-6), Leu- and Met-enkephalin, a-neo-endorphin and all other peptides tested.

Plasma collection Blood was collected into cooled lithium heparin tubes, with or without 0.5 ml aprotinin (Trasylol C~20 000 K I U / m l ) per 10 ml blood, and immediately centrifuged at 4°C. Acidification, if required, was achieved by addition of 750/~1 glycine-HCl (1.6 g glycine/100 ml N HCI) per 5 ml plasma [17] and samples were either processed immediately or flash frozen on solid CO 2 and stored at - 2 0 ° C . Vycor extraction Extraction with leached silica glass (Vycor) was performed using a modification of the method used in this laboratory for the RIA of ACTH [18], /3-LPH [19], /3-endorphin [20] and somatostatin [21]. 150 mg Vycor was added to 5 ml of non-acidified plasma without aprotinin and the sample rotated for 30 min at 4°C. Following washes with 3 ml distilled water and 2 ml 1 M HCI, the peptide was eluted with 1 ml 60% acetone and evaporated to dryness under a constant stream of nitrogen at 60°C. The sample was reconstituted in 500 #1 assay buffer and after pH adjustment if necessary, using 1 M NaOH, 400/~1 was taken for doubling dilutions in the assay. Synthetic DYN was extracted in parallel from normal horse serum (500 fmol DYN per ml of horse serum) to determine recovery. In subsequent experiments plasma was extracted following addition of aprotinin or acidification and plasma already extracted once was re-extracted by the same procedure, again with or without addition of aprotinin or acidification. Other reconstituted samples were placed in a boiling water bath for 10 min to destroy enzyme activity prior to assay. Further assays were performed following the addition of aprotinin (10 ml/100 ml) to assay buffer. In all cases synthetic DYN in horse serum was treated in the same manner as a control. In order to demonstrate integrity of the tracer, plasma extracts were incubated for 3 days with antiserum and tracer as in standard RIA before being pooled, acidified and subjected to gel filtration chromatography (see below). Seppak extraction Extraction with 'Seppak C18' cartridges was performed by a modification of the method used in this laboratory for Met-enkephalin RIA [17]. Briefly, 5 ml acidified plasma were loaded onto the cartridge and, following washes with 0.9% N a C I / I % formic acid and 1% formic acid, eluted with 2 ml 80% m e t h a n o l / l % formic acid. The eluate was evaporated under a stream of nitrogen at 60°C and any remaining moisture and excess acid removed by placing under vacuum for 1 h in the presence of NaOH pellets prior to reconstitution with buffer and assay as above. Synthetic DYN (250 fmol/ml plasma) was extracted from pooled Seppak-stripped human plasma as control. Affinity gel extraction Antibody was precipitated from 0.5 ml of neat DYN antiserum by addition of

134 saturated ammonium sulphate to a final concentration of 40% (previously shown to precipitate the majority of DYN-binding antibody). Following extensive dialysis against 0.1 M N a H C O 3 plus 0.5 M NaC1 (pH 8) the antibody was coupled to Sepharose CNBr-activated 4B according to manufacturer's instructions. 1 g Sepharose was reconstituted (to 3.5 ml of gel), mixed with the dialysate and additional 0.1 M N a H C O 3 and 0.5 M NaCI to a total volume of 10 ml and gently rotated overnight at 4°C. Unbound protein was removed by washing with the bicarbonate buffer and the affinity gel stored at 4°C. Incorporation rate of antiserum into the gel was 95%. For extraction, 750 ~1 gel (representing approximately 100/~1 of neat antiserum) was placed on a glass wool filter in a small polypropylene column at room temperature. Plasma was collected with the addition of aprotinin as above and dripped through the gel at a rate of approximately 0.5 m l / m i n extracting total volumes of up to 60 ml plasma. The column was washed with several column volumes of 0.1 M Tris-HC1, pH 8.5, and 0.5 M NaCI and the peptide bound to antibody was then eluted by sudden lowering of pH using 0.1 M sodium acetate plus 0.5 M NaCI, pH 4. The first 1.5 ml of the acid eluate were then subjected to gel filtration chromatography without further processing. Synthetic DYN(1-17) in both plasma and assay buffer was used to confirm DYN-binding and elution from the gel.

Gel filtration chromatography Gel filtration chromatography was performed using a Sephadex G-25 1 x 95 cm column eluted under dissociating conditions with 1% formic acid containing 1 g/1 polypep [22] at a flow rate of 1.8 ml/h. Fractions were evaporated to dryness at room temperature under vacuum and redissolved in assay buffer prior to RIA. Materials DYN(1-17), (1-13), (1-8) and (1-6) were obtained from Universal Biologicals Cambridge and remaining DYN fragments from Peninsula Laboratories Inc. Vycor glass was obtained from Corning Glass, New York, normal horse serum from Wellcome Laboratories and aprotinin (Trasylol *) from Bayer U.K. Limited.

Experimental Results Unextracted plasma assay Addition of even a few microlitres of unextracted plasma to the RIA mixture caused gross non-parallel inhibition of tracer binding indicating that an extracted RIA was essential. Vycor plasma extracts Synthetic DYN(1-17) and (1-13) were reliably extracted by Vycor with a recovery of approximately 50%. Extracts of plasma from normal subjects caused marked inhibition of tracer binding diluting in parallel to DYN(1-17) standard in

135

Doubling dilutions of samples i

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Fig. 1. Dynorphin assay of plasma extracts using Vycor glass. Effects of volume, re-extraction, addition of aprotinin (Trasylol ®) and acidification of plasma.

assay., initially suggesting substantial amounts (up to 150 fmol/ml plasma) of irDYN in human plasma (Fig. 1). However, further experiments revealed that this apparent irDYN was unrelated to volume of plasma extracted and identical amounts Doubling dilutions oT samples 50

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Fig. 2. Dynorphin assay of plasma extracts using Vycor glass. Effects of assay incubation time, aprotinin (Trasylol ~) in assay buffer and heat inactivation of enzymatic activity.

136 were obtained on re-extraction of the same plasma. Addition of aprotinin to the assay buffer or to plasma prior to extraction or re-extraction reduced this apparent irDYN and acidification of plasma abolished it completely, although none of these manoeuvres altered overall recovery of synthetic DYN. Boiling of plasma extracts prior to assay abolished this artifactual effect but also reduced recovery of synthetic DYN by 50%. The degree of inhibition of tracer binding was proportional to the time of incubation of the RIA mixture (Fig. 2) and examination of the 1231DYN(1-13) tracer by chromatography after 3 days incubation confirmed that the majority of the tracer had been broken down to smaller fragments (Fig. 3). Chromatography of Vycor plasma extracts showed that the apparent irDYN eluted in the void volume. We therefore conclude that this 'irDYN' is artifactual and due to enzymatic tracer degradation by (an) enzyme(s) extracted from plasma by the Vycor glass.

Seppak extracts DYN(1-17) and (1-13) were again reliably extracted from plasma by Seppak cartridges with a recovery rate of between 30 and 40%. Recovery rate was constant over the range of concentrations of synthetic DYN tested (30-500 fmol/ml plasma). However, using this technique to extract plasma we have not demonstrated irDYN in any sample so far studied despite a calculated detection limit of 6 f m o l / m l plasma. We have studied samples from normal individuals studied basally, during dehydration and hypertonic saline infusion and during insulin induced hypoglycaemia, and samples from patients with a wide variety of endocrine diseases including phaeochromocytomas, and adrenal and jugular vein samples obtained during diagnostic catheter venous sampling. We have also examined rat plasma and found no apparent irDYN by this technique in contrast to previous reports [12,13]. 1125 DYN (1-13)*

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137

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DYN is, however, rapidly degraded in blood - of 1.25 pmol DYN(l-17) standard added to 10 ml whole blood and treated as a normal sample only 20% was recovered despite the precautions described. This represents a loss of approximately 50% of peptide compared to extracted standard in the assay. Therefore the possibility that DYN may be secreted into plasma by the pituitary or adrenal and be rapidly degraded, or that small amounts of DYN may exist in peripheral plasma, cannot be excluded. Affinity gel extraction

The affinity column was able to extract 85% and 95% of synthetic DYN(l-17) from plasma and assay buffer respectively. After acid elution followed by direct assay 80% of the bound peptide was recovered. Sephadex G-25 chromatography of normal subjects’ plasma extracted by this method revealed a single peak of irDYN of apparent molecular size compatible with a DYN-like peptide and clearly separated from the salts of the affinity gel elution buffer (Fig. 4). This peak does not coelute with any of the synthetic DYN fragments available but runs closest to DYN(l-9), however since DYN(l-17) is significantly retarded in the system precise characterisation is not possible by this technique. The amount of irDYN extracted has varied from subject to subject ranging up to 15 fmol/ml and being undetectable in some individuals. Such levels might therefore have been expected to be detected by Seppak extraction.

Discussion Tracer degradation by plasma enzymes has long been recognised as a potential source of artifact in unextracted plasma RIAs [23] and is part of the rationale for selecting an extracted RIA. Vycor glass readily absorbs many small and intermediate sized peptides but would not be expected to extract proteins of the size of enzymes. We have, however, clearly demonstrated that plasma contains a DYN-degrading

138 peptidase which is extracted by this system and is resistant to the various acidification and dessication steps in the procedure. This situation is similar to that reported by Goldstein [24] who described an acid and heat stable endopeptidase extracted from Escherichia coli resulting in apparent irDYN and suggested that the presence of any peptide in this organism should not be reported until tracer integrity had been demonstrated. Our data suggest that the same considerations should apply to investigation of both DYN and many other labile peptides in human plasma even when employing extracted RIAs. The consistent parallelism of our extracts to standard in assay confirms that this parallelism alone is not sufficient to confirm coidentity. Chromatographic characterisation of both the apparent immunoreactivity and of tracer after incubation to demonstrate integrity are both essential. Seppak plasma extracts do not seem subject to the same enzymic artifact but we have not yet been able to demonstrate irDYN in human or rat plasma by this technique. It therefore seems likely to us that previous brief reports of irDYN in rat plasma in p m o l / m l quantities [12,13] may well represent the enzymic artifact that we have demonstrated. In support of this, Spampinato and Goldstein [25] have recently published more extensive studies of rat plasma and confirm that DYN tracer is rapidly degraded by unextracted rat plasma in assay unless a combination of potent enzyme inhibitors is employed in the assay buffer. Using such inhibitors they suggested the presence of irDYN of higher molecular size in rat plasma. These workers also subsequently used Seppak cartridges to extract rat plasma and suggested the presence of authentic irDYN in amounts up to 10 fmol/ml. This is close to our calculated detection limit of 6 fmol/ml and since they employed a different elution system our results are probably not contradictory. The relationship to the large molecular size irDYN in human plasma described by Boarder et al. [14] is more difficult to assess since Seppak C18 might well not extract peptides of this size [17] and a different antibody was employed in their RIA. However we note that our peptidase is acetone soluble and that acid/acetone was used by Boarder et al. for plasma extraction. These data therefore suggest that, if DYN is indeed secreted into plasma it is rapidly degraded either in vivo in blood or during sample collection, or else is present in concentrations below the sensitivity of our RIA. The extraction of significant amounts of irDYN of similar molecular size to porcine DYN by the affinity gel is therefore somewhat surprising and two hypotheses can be proposed to explain these data. Thus, human plasma irDYN, although of similar molecular size to porcine DYN(1-17) may have a different structure and therefore behave differently in the Seppak system. Alternatively, human plasma may actually contain larger molecular weight forms of DYN precursors which would not be extracted by Seppak nor recognised by our RIA. This latter situation would be analogous to Met-enkephalin where tryptic digestion of column fractions after chromatography of whole plasma generates large amounts of Met-enkephalin from large molecular weight precursors which themselves show low crossreactivity with the antiserum employed in the Met-enkephalin RIA [26,27]. A similar dynorphin precursor might be slowly metabolised by plasma peptidases to release the immunoreactive DYN peptides. Under normal circumstances such DYN peptides would, as we have shown, be rapidly degraded to yet smaller fragments again not

139

detected by our RIA. However in the presence of our affinity gel these immunoreactive DYN peptides would bind to our solid phase antibody and therefore be protected from further enzymatic degradation and subsequently be detectable in assay. Whatever the explanation however it seems clear from our data that human plasma does contain a small amount of authentic DYN-like immunoreactivity. Further definition of this presumed peptide will require higher resolution separation techniques such as HPLC and the very small quantities present obvious difficulties in the development of such methods.

Acknowledgements Dr. T.A. Howlett is a Medical Research Council Training Fellow. This work was supported by the Joint Research Board of St. Bartholomew's Hospital and the Peel Medical Research Trust. We thank Dr. A. Goldstein for his gift of his antiserum 'Lucia' during the development of our assay.

References 1 Goldstein, A., Fischli, W., Lowney, L.I., Hunkapiller, M. and Hood, L., Porcine pituitary dynorphin. Complete amino acid sequence of the biologically active heptadecapeptide, Proc. Natl. Acad. Sci. U.S.A. 78 (1981) 7219-7223. 2 Gqldstein, A., Tachibana, S., Lowney, L.I., Hunkapiller, M. and Hood, L., Dynorphin (1-13), An extraordinarily potent opioid peptide, Proc. Natl. Acad. Sci. U.S.A. 76 (1979) 6666-6670. 3 Kakidani, H., Furutani, Y., Takahashi, H., Noda, M., Morimoto, Y., Hirose, T., Asai, M., Inayayama, S., Nakanishi, S. and Numa, S., Cloning and sequence analysis of cDNA for porcine fl-neo-endorphin/dynorphin precursor, Nature, 298 (1982) 245-249. 4 Watson, S.J., Akil, H., Fischli, W., Goldstein, A., Zimmerman, E., Nilaver, G. and van Wimersma Greidanus, T.B., Dynorphin and vasopressin: Common localisation in magnocellular neurones, Science, 216 (1982) 85-87. 5 Molineaux, C.J. and Cox, B.M., Subcellular localisation of immunoreactive dynorphin and vasopressin in rat pituitary and hypothalamus, Life Sci., 31 (1982) 1765-1768. 6 Day, D., Denis, D., Barabe, J., St-Pierre, S. and Lemaire, S., Dynorphin in bovine adrenal medulla. I. Detection in glandular and cellular extracts and secretion from isolated chromaffin cells, Int. J. Peptide Protein Res., 19 (1982) 10-17. 7 Denis, D., Day, D. and Lemaire, S., Dynorphin in bovine adrenal medulla. I1. Isolation partial characterisation and biological activity of two distinct molecules, Int. J. Peptide Protein Res., 19 (1982) 18-24. 8 Seizinger, B.R., Maysinger, D., Holh, V., Grimm, C. and Herz, A., Concomitant neonatal development and in vitro release of dynorphin and a-neo-endorphin, Life Sci., 31 (1982) 1757-1760. 9 Dumont, M., Day, D. and Lemaire, S., Distinct distribution of immunoreactive dynorphin and leucine enkephalin in various populations of isolated adrenal chromaffin cells, Life Sci., 32 (1983) 287-294. 10 Yoshimasa, T., Nakao, K., Oki, S., Tanaka, I., Nakai, Y. and Imura, H., Presence of dynorphin-like immunoreactivity in phaeochromocytomas, J. Clin. Endocrinol. Metab., 53 (1983) 213-214. 11 Suda, T., Tozawa, F., Tachibana, S., Demura, H., Shizume, K., Sasaki, A., Mouri, T. and Miura, Y., Multiple forms of immunoreactive dynorphin in human pituitary and phaeochromocytoma, Life Sci., 32 (1983) 865-870.

140 12 Millan, M.J., Tsang, Y.F., Przewkocki, R., Holh, V. and Herz, A., The influence of foot-shock stress upon brain, pituitary and spinal cord pools of immunoreactive dynorphin in rats, Neurosci. Lett,, 24 (1981) 75-79. 13 Hollt, V., Haarmann, I., Seizinger, B.R. and Herz, A., Levels of dynorphin (1-13) immunoreactivity in rat neurointermediate pituitaries are concomitantly altered with those of leucine enkephalin and vasopressin in response to various endocrine manipulations, Neuroendocrinology, 33 (1981) 333-339. 14 Boarder, M.R., Erdelyi, E. and Barchas, J.D., Opioid peptides in human plasma: evidence for muhiple forms, J. Clin. Endocrinol. Metab., 54 (1982) 715-720. 15 Hunter, W.H. and Greenwood, F.C., Preparation of iodine 131 labelled human growth hormone of high specific activity, Nature, 194 (1962) 495-496. 16 Ho, W.K.K., Cox, B.M., Chavkin, C. and Goldstein, A., Opioid peptide dynorphin (1-13): adsorptive losses and potency estimates, Neuropeptides, 1 (1980) 143-152. 17 Clement-Jones, V., Lowry, P.J., Rees, L.H. and Besser, G.M., Development of a specific extracted radioimmunoassay for met-enkephalin in human plasma and cerebrospinal fluid. J. Endocrinol., 86 (1980) 231-243. 18 Rees, L.H., Cook, D., Kendall, J.W., Allen, C., Kramer, R.M., Ratcliffe, J.G. and Knight, R.A., A radioimmunoassay for rat plasma ACTH, Endocrinology, 89 (1971) 254 261. 19 Jeffcoate, W.J., Rees, L.H., Lowry, P.J. and Besser, G.M., A specific radioimmunoassay for human fl-lipotrophin, J. Clin. Endocrinol. Metab., 47 (1978) 160-167. 20 Jeffcoate, W.J., Rees, L.H., McLoughlin, L., Ratter, S.J., Hope, J. and Besser, G.M., fl-endorphin in human cerebrospinal fluid, Lancet, 2 (1978) 119-121. 21 Penman, E., Wass, J.A.H., Lund, A., Lowry, P.J., Stewart, J., Dawson, A.M., Besser, G.M. and Rees, L.H., Development and validation of a specific radioimmunoassay for somatostatin in human plasma, Ann. Clin. Biochem., 16 (1979) 15-25. 22 Ratter, S.J., Lowry, P.J., Besser, G.M. and Rees, L.H., Chromatographic characterisation of adrenocorticotrophin in human plasma, J. Endocrinol., 85 (1980) 359 369. 23 Chard, T., The radioimmunoassay of oxytocin and vasopressin, J. Endocrinol., 58 (1973) 143-160, 24 Goldstein, A., 'Immunoreactive dynorphin' in Escherichia coli: tracer degradation by a heat-stable endopeptidase, Life Sci., 31 (1982) 2267-2270. 25 Spampinato, S. and Goldstein, A., Immunoreactive dynorphin in rat tissues and plasma, Neuropeptides, 3 (1983) 193-212. 26 Smith, R., Grossman, A., Gaillard, R., Clement-Jones, V., Ratter, S., Mallinson, J., Lowry, P.J., Besser, G.M. and Rees, L.H., Studies on circulating met-enkephalin and fl-endorphin-normal subjects and patients with renal and adrenal disease, Clin. Endocrinol,, 15 (1981) 291-300. 27 Medbak, S., Mason, D.F.J. and Rees, L.H., Chlorpropamide-ethanol induced met-enkephalin secretion in dogs: release mechanisms and biochemical characterisation, Regul. Peptides, 7 (1983) 195-206.