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Naunyn-Schmiedeberg's Arch. PharmacoL 306, 301- 304 (1979)

Pharmacology 9 by $pringer-Verlag 1979

Short Communication

A Material with Opioid Activity in Bovine Milk and Milk Products V, Brand and H. Teschemacher Department of Neuropharmacology, Max-Planck-Institut fiir Psychiatric, Kraepelinstrasse 2, D-8000 Miinchen 40, Federal Republic of Germany

Summary. Chloroform-methanol extracts of lyophi-

lized milk, of commercially available dried milk or baby food and of casein digests were tested for opioid activity on the guinea-pig ileum longitudinal muscle-myenteric plexus preparation. Compounds with opioid activity which proved to be resistant to peptidases - were detected in certain batches of baby food, casein digest, and cow milk in considerably varying amounts. Key words: Endorphins - Opiate receptor ligands Pronase-resistant opioids - Milk - Baby food.

out at room temperature. 1 g of residue, 1 g of commercially available dried cow milk (Nestle), 1 g of baby food obtained from dried milk (Milupa, Alete, Humana, Hipp) or I g of enzymatic casein digest (Serva, Heidelberg) were mixed with 1 g of silicic acid. To these mixtures 10 ml of chloroform-methanol (2/1 ; v/v) were added and the suspensions filtered through a Schleicher-SchfilI Filter No. 595-I/2. The residues were washed with 5 ml chloroform-methanol and the combined filtrates were evaporated to dryness. The residues, hereafter referred to as "crude extracts", were either a) taken up in distilled water and tested for opioid activity, or b) dissolved in chloroformmethanol (9/1; v/v) for chromatographic characterization, or c) dissolved in butanol prior to further purification,

Testing of Extracts for Opioid Activity by"Bioassay and Radioreceptor Assay. Crude or partially purified extracts were tested for opioid

Introduction

In recent years a number of opioid peptides, called endorphins, have been detected in brain, pituitary and other tissues (for review see Teschemacher, 1978). In addition, substances have been found in brain (Gintzler et al., 1976), blood (Pert et al., 1976; Schulz et al., 1977) and urine (Schulz and Wfister, /977) which, although interacting with opiate receptors, appear to be different from endorphins in being resistant to pronase (Wiister et al., 1978). In view of the possibility that such pronase-resistant opioids found in the blood might be able to pass from blood into milk, we undertook a search for these compounds in bovine milk and its products. Methods Extraction Procedure. Fresh bovine milk from a local cattle farm (Munich University Research Farm Oberschleissheim) was frozen on dry ice and then lyophilized. The following procedures were carried

Send offprint requests" to H. Teschemacher's present address: Pharmakologisches Institut der Justus Liebig-Universit~it Giessen, Frankfurter Strasse 107, D-6300 Lahn-Giessen 1, Federal Republic of Germany

activity in the electrically stimulated guinea-pig ileum longitudinal muscle-myenteric plexus preparation, hereafter referred to as the "guinea-pig ileum preparation". Preparation, mounting and electrical stimulation (frequency: 0.1 Hz; pulses : 60 V, 0.5 ms) were carried out as described by Kosterlitz et al. (1970) and Schulz and Goldstein (1972). Opioid activity was determined in terms of inhibition of electrically induced contractions of the guinea-pig ileum preparation, as far as this effect proved antagonizable by the specific opiate antagonist naloxone. Opioid activity of extracts was also tested in an opiate receptor binding assay according to the method of Pert and Snyder (/973). For quantitative evaluation, the opioid effects of the extracts in both assays were compared with the opioid effects of normorphine tested under identical conditions.

Characterization. Crude extracts were taken up into chloroformmethanol (9/i; v/v) and loaded onto a column (I.D. 1 cm, length 10cm) packed with controlled pore glass (CPG-10-75, Serva, Heidelberg). Elution was performed with chloroform-methanol mixtures (25ml of each) with increasing proportion of methanol (90/10, 80/20, 65/35, 40/60, 0/100; v/v) and the eluates evaporated to dryness. For thin layer chromatography (TLC), the residues were taken up into chloroform-methanol-ammonia (0/30/5; v/v/v), as described by Pert et al. (1976) and applied to silica gel G plates (Merck, Darmstadt). After exposure of plates to this solvent system sections of the silica gel were removed and extracted with chloroformmethanol (65/35; v/v). The resulting suspensions were centrifuged and the supernatants evaporated to dryness and then dissolved in water for subsequent determination of opioid activity. For testing sensitivity to pronase, casein and dried milk extracts were incubated, together with pronase (Pronase P, Serva, Heidelberg, at a concentration of 20 o1"45 ~g/ml) at 37~ for 30 rain to 2 h in a Krebs-Ringer solution previously bubbled with O2/CO2, 95/5% (Schulz and Goldstein, 1972). Incubations under identical conditions

0028-1298/79/0306/0301 / $01. O0

302 were performed with the extracts or the enzymes separately, as controls. Incubation was terminated by heating the solutions at 95~ f o r / 0 rain. After centrifugation, the supernatants were tested on the guinea-pig ileum preparation for opioid activity.

Naunyn-Schmiedeberg's Arch. Pharmacol, 306 (1979)

A

Results

Opioid Activities of Extracts from Bovine Milk and Milk Products In M a y / 9 7 7 , when this study was started, chloroformmethanol extracts of some out of a series of batches of baby food, casein digest or fresh bovine milk were observed to contain compounds with opioid activity. Their opioid effects in the guinea-pig ileum are shown in Fig. 1. The inhibition of the contractions of t h e guinea-pig ileum preparation upon addition of the extracts to the organ bath was blocked or abolished by the specific opiate antagonist (-)-naloxone. The inhibitory effect, however, was not antagonizable by the inactive stereo-isomer of (-)-naloxone, (+)-naloxone (not shown here). In some cases, the onset of effect proved rather slow (Fig. 1 A), whereas in other cases the wash-out time proved very long (Fig. 1 B). Extracts of human milk (collected in a pilot study in May / 977) also displayed opioid activity. All extracts which displayed opioid activity in the guinea-pig ileum behaved like opiates in opiate receptor binding assays in inhibiting the binding of 3H-naloxone to opiate receptors in a rat brain homogenate. Parallel lines were obtained by log-probit analysis for both the inhibition of 3H-naloxone binding by the extracts and by normorphine (these results will be published in detail elsewhere). In pilot experiments, after intraventricular injection of partially purified extracts in rats, naloxoneantagonizable analgesia and catatonia were observed as well as exophthalmus and Straub tail phenomena.

Characteristics of Opioids Extracted from Bovine Milk and Milk Products Chromatographic characterization using TLC was performed with that type of casein digest (Serva, C / T L G 16300), or with that batch of baby food (Milupa, Heilnahrung), which had been observed to elicit opioid effects on the guinea-pig ileum. At least two materials with opioid activity were found in both the extract from casein digest (RF-values: 0.45; 0.86) and the baby food extract (RF-values: 0.28; 0.85). The opioid activity displayed by the chloroformmethanol extracts of milk and milk products proved to be pronase-resistant. However, as found in pilot experiments, opioid activity proved sensitive to heating for 10min at 400~ under nitrogen. Preliminary mass-

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(in this case a pepsin hydrolysate of s-casein). They reported the loss of opioid activity after pronase treatment. On the other hand, pronase-resistant materials with opioid activity have been described which, however, differ from the opioid material found in the milk and its constituents. Gintzler et al. (1976) reported a pronaseresistant material, which behaved like an opiate in opiate receptor binding assays (Blume et al., 1977), but which elicited an inhibitory effect on the guinea-pig ileum which was not antagonized by the specific opiate antagonist naloxone (Gintzler et al., 1976). Marzullo and Friedhoff (1977) reported that heavy metal ions, such as copper, display opioid activity in opiate receptor binding assays; however, an inhibitory effect of a copper-glutathione complex on the guinea-pig ileum was not reversed by the specific opiate antagonist naloxone (Marzullo and Friedhoff, 1977). Moreover, our casein extract lost its opioid activity upon heating for 10min at 400~ under nitrogen, which should exclude a heavy metal ion as the active principle. The reasons for the highly varying amounts of the pronase-resistant opioids in the milk and in milk products remain to be clarified. Inconsistencies in the extraction or assay techniques can be excluded by the fact that opioid activity in the baby food and in the casein digest was at constant level throughout the investigation period. Acknowledgement.The authors wish to express their gratitude to Ms. Ingeborg Haarmann for expert technical assistance and to Drs. G. Bretschneider and L. Furthmayr/J. Meyer for generous supplies of dried milk and fresh bovine milk respectively. Further, the authors are indebted to Drs. A. E. Jacobson for the gift of (+)-naloxone, M. J. Millan and H. Osborne for stylistic revision of the paper, Ch. Gramsch for performance of the RIA and Ch. Meitinger for helpful cooperation.

References Blume, A. J., Shorr, J., Finberg, J. P. M., Spector, S. : Binding of the endogenous non-peptide morphine-like compound to opiate I:eceptors. Proc. Natl. Acad. Sci. U,S.A. 74, 4927-493/ (1977) Gintzler, A. R., Levy, A., Spector, S.: Antibodies as a means of isolating and characterizing biologically active substances: Presence of a non-peptide, morphine-like compound in the

304 central nervous system. Proc. Natl. Acad. Sci. U.S.A. 73, 21322136 (1976) H/511t, V., Przewlocki, R., Herz, A.: Radioimmunoassay of flendorphin, Basal and stimulated levels in extracted rat plasma. Naunyn-Schmiedeberg's Arch. Pharmacol. 303, 171 - 174 (1978) Kosterlitz, H. W., Lydon, R. J., Watt, A. J. : The effects of adrenaline, noradrenaline and isoprenaline on inhibitory c~- and /3adrenoceptors in the longitudinal muscle of the guinea pig ileum. Brit. J. Pharmacol. 39, 398-413 (1970) Marz-ullo, G., Friedhoff, A. J. : An inhibitor of opiate receptor binding from human erythrocytes identified as a glutathionecopper complex. Life Sci. 21, 1559-1568 (1977) Pert, C. B., Pert, A., Tallman, J. F. : Isolation of a novel endogenous opiate analgesic from human blood. Proc. Natl. Acad. Sci. U.S.A. 73, 2226-2230 (1976) Pert, C. B., Snyder, S. H. : Opiate receptor: Demonstration in nervous tissue. Science 179, 1011 - 1014 (1973) Schulz, R., Goldstein, A. : Inactivity of narcotic glucuronides as analgesics and on guinea pig ileum. J. Pharmacol. exp. Tiler. 183, 404-410 (1972) Schulz, R., Wfister, M. : Renal excretion of endogenous opioids. Europ. J. Pharmacol.. 43, 383-384 (1977)

Naunyn-Schmiedeberg's Arch. Pharmacol. 306 (1979) Schulz, R., Wtister, M., Herz, A. : Detection of a long acting endogenous opioid in blood and small intestine. Life Sci. 21, 105-116 (1977) Teschemacher, H. : Endogenous ligands of opiate receptors (endorphins). In: Development in Opiate Research (A. Herz, ed.), pp. 67-151. New York: Marcel Dekker 1978 Wajda, I. J., Neidle, A., Ehrenpreis, S., Manigault, I. : Properties and distribution of morphine-like substances. In: Opiates and endogenous opioid peptides. (H. W. Kosterlitz, ed.), pp. 129-136. Amsterdam: Elsevier/North-Holland Biomedical Press, 1976 Wfister, M., Loth, P., Schulz, R. : Characterization of opiate-like materials in blood and urine. In: Advances in biochemical psychopharmacology. (E. Costa and M. Trabucchi, eds.), pp. 313-319. New York: Raven Press 1978 Zioudrou, C., Klee, W. A. : Exorphins-peptides with opioid activity derived from e-casein and wheat gluten. In: Characteristics and function of opioids. (J. van Ree and L. Terenius, eds.), pp. 2 4 3 244. Amsterdam: Elsevier/North-Holland Biomedical Press 1978

Received October 25, 1978/Accepted January 30, 1979

A material with opioid activity in bovine milk and milk products.

Naunyn-Schrniedeberg's Archivesof Naunyn-Schmiedeberg's Arch. PharmacoL 306, 301- 304 (1979) Pharmacology 9 by $pringer-Verlag 1979 Short Communic...
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