Neurochemical Research, Vol. 28, No. 2, February 2003 (© 2003), pp. 177–185
Erucamide as a Modulator of Water Balance: New Function of a Fatty Acid Amide* Anders Hamberger1,3 and Gunnar Stenhagen2 (Accepted September 3, 2002)
The aim of this study was to isolate a compound from blood plasma that inhibits intestinal diarrhea and that appears also to regulate fluid volumes in other organs. The isolation procedure included lipid extraction, liquid chromatography, and gas chromatography. The active substance was identified by mass spectrometry as erucamide (MW 337 Da). The biological effect was reproduced with authentic erucamide. Erucamide is a fatty acid amide, such as oleamide and anandamide, which modulate other physiological functions in a receptor-mediated fashion. All the exact biological functions of erucamide are as yet to be defined, but it is already known to stimulate angiogenesis. Erucamide concentrations were determined in body organs from the pig. The blood plasma level was 3 ng/g, and those of lung, kidney, liver, and brain were 12, 2.5, 1.0, and 0.5 ng/g, respectively. Erucamide was below detection level in the intestine, but is known to be present in the cerebrospinal fluid. In the rat, 3H-erucamide was accumulated in vivo into lung, liver, and spleen and in vitro into lung, liver, brain, and intestine. The in vitro uptake was time and temperature dependent, but not saturable.
KEY WORDS: Erucamide; fatty acid amides; intestine; diarrhea; dose-response.
INTRODUCTION
understood. Key structures in brain water transport are the capillary endothelium, choroid plexus, ependyma, and astrocytes, with each having specific functions, receptors, and water channels (1–3). Modulators of water transport are often related to a multiorgan axis; that is, they may be activated by events also outside the brain. Examples of such modulators are the atrial natriuretic peptides, cardiac hormones that are elevated in heart failure (4) and appear to attenuate certain forms of brain edema (5–7). Other regulating compounds are still unidentified, such as the endogenous ouabain-like factor (EOLF), closely related to the endogenous digitalis-like factor (EDLF) (8,9). EOLF is present in blood plasma (10), milk (11), and cerebrospinal fluid (CSF) (12,13). Its activity in the CSF increases upon expansion of the extracellular fluid volume, and it also has the capacity to suppress CSF production (14). EOLF has been classified variously as a peptide, a steroid, and a fatty acid (15). Another
The identification of regulating factors, which are produced in the body in response to a disturbed balance in metabolism, ions, or fluids, may provide a new class of agents for therapeutic purposes. There is an intense search for neuroprotective compounds, and some of the pathology is similar in a large group of neurological disorders. An example of this is brain edema, where the mechanisms of development are only partly * Special issue dedicated to Dr. Anders Hamberger. 1 Department of Anatomy and Cell Biology, University of Göteborg, Göteborg, Sweden. 2 Department of Organic Chemistry, Chalmers University of Technology, Göteborg, Sweden. 3 Address reprint requests to: Anders Hamberger, MD, Department of Anatomy and Cell Biology, University of Göteborg P.O. Box 420, S-405 30 Göteborg, Sweden. Tel: ⫹46-31-773 33 68; Fax: ⫹46-31-41 28 05; E-mail:
[email protected]
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178 compound, also protective against neuronal injury, but apparently unrelated to the EOLF, is anandamide (16). This compound belongs to a relatively new group of lipoid neuromodulators, the fatty acid amides, which are produced in the brain in response to certain functional disorders in the CNS, such as severe pain and sleep deprivation (17). This study was initiated by our findings that intracerebroventricular injections of a partly purified fraction of pig blood plasma inhibited the transport of Na⫹ from blood plasma to the CSF (unpublished data); transport of Na⫹ reflects the rate of formation of CSF (18,19). Furthermore, we found that the plasma fraction interacted with transport of neurotransmitter amino acids into brain tissues (unpublished data). Such a blood plasma fraction had been partly purified by Lönnroth and Lange (20–23) and was classified by them as an antisecretory factor (AF), because it inhibits diarrhea. This group also cloned a protein and showed that the biological activity was due to a small peptide segment (24). We decided to investigate whether additional information could be obtained by further purification of the AF from pig blood and examine its functions in the CNS. Using the inhibition of cholera toxin–induced diarrhea as a biological assay to monitor the purification steps, we now have obtained a water-insoluble fatty acid amide—erucamide (MW 337 Da)—having the designated antidiarrheic properties. Proper identification was further confirmed by showing that authentic, commercially available erucamide exhibits the biological activity. In addition to having a function similar to that of the AF peptide (24), erucamide has been shown to be an activator of angiogenesis in injured organs (25,26). The exact mechanisms of action of erucamide, its precise biological function, as well as its potential as a neuroprotective modulator are yet to be detailed.
EXPERIMENTAL PROCEDURE Protocols of Purification Extraction from Blood. Plasma was prepared from blood of adult pigs and delivered in plastic bottles with refrigerated transport (6 ⫻ 25 L, Ellco Food AB, Kävlinge, Sweden). Preparation of the plasma water phase was carried out at 4°C. Prior to the addition of agarose (Sepharose 6B, Pharmacia LKB Biotechnology, Stockholm, Sweden), 1.5 L per 25 L plasma, the plasma was filtered through fine-meshed gauze. To mix, the bottles were turned upside down every 20 min for 2 h. The agarose was gravity sedimented overnight, and the plasma supernatant was discarded. The agarose was washed 7–8 times by filling the bottles with phosphate buffered saline (PBS, 0.15 M NaCl, 0.05 M NaPO4, pH 7.2, containing 0.05 M glucose, 0.013 M tri-Na-citrate, 0.0002 M Na2S2O3 and 2.5 ⫻ 10⫺7 M phenyl
Hamberger and Stenhagen methyl sulfon fluoride [Sigma Chemical Co, St. Louis, MO, USA]). After mixing, the agarose was sedimented again. The agarose was then eluted with 5 L 10% methyl ␣-glucopyranoside (Sigma Chemical Co, St. Louis, MO, USA) in 5% MeOH, 95% H2O, and the resulting eluate was diluted with 4 vol. PBS. Lipids were extracted from this eluate with solid phase cartridges (100 mg ⫻ 6 ml Isolute ENV⫹, IST, Mid Glamorgan, UK), each cartridge was used for approximately 3 L eluate and was then eluted with 30 ml methanol. One vol. water and 2 vol. chloroform were added and mixed with the methanol eluate. The chloroform phase was collected and evaporated under vacuum. The dry material was then dissolved in 2% isopropanol in hexane for liquid chromatography (LC) on a Diol column (Lich Diol 10 m, 12 ⫻ 250 mm, Jones Chromatography, Mid Glamorgan, UK). The column was eluted isocratically with 2% isopropanol in hexane, at 3 ml/min. The eluate was monitored at 218 nm with a variable wavelength UV detector (Model 2050, Varian AB, Solna, Sweden). In a few experiments, fractions were dried and redissolved in 95% methanol for LC on a C18 column. The column was eluted with a linear gradient, from 95% methanol in water to 100% methanol during 40 min. Fractions of eluates from the Diol and C18 columns were evaporated to dryness and dissolved in 5–20 l chloroform to provide samples for tests of biological activity and for mass spectrometry (MS). Extraction of Erucamide from Body Organs. The method for extraction of erucamide from body organs was modified from that of Arai et al. for anandamide extraction (27). Fifty-gram samples of brains, lungs, kidneys, and livers were obtained from pigs with a body weight of 35– 40 kg. Each organ sample was dispersed in 100 ml water in the glass container of a food processor (MX 2050, Braun GmbH, Kronberg, Germany). The knife was rotated for 1 min at high speed. Water blanks, homogenates, and blood plasma were transferred to glass bottles containing 2 vol. acetone and 4 vol. toluene. Trace amounts of 3H-erucamide (13–14 3H, spec. act. 50 Ci/mmol, Moravek Biochemicals Inc, Brea, CA, USA) were added for calibration and monitoring of the purification steps. The bottles were closed with Teflon caps and were then shaken and briefly sonicated for a total of 10–15 min. They were then left overnight in the cold. The clear, upper, organic phase was collected and centrifuged for 10 min at 1500 g, when required and evaporated to dryness. The residue was dissolved in 250 l 5% ethanol in hexane for solid phase extraction (Isolute Diol, 500 mg, IST, Mid Glamorgan, UK). The cartrideges were preconditioned with 3 ml ethanol and 3 ml hexane. The applied material was washed with 3 ml hexane and eluted with 5% ethanol in hexane in 1.5-ml fractions. The third or fourth fraction, which contained most of the 3H-erucamide radioactivity, was evaporated to dryness. It was again dissolved in 250 l 5% ethanol in hexane, and the same procedure was performed, now with a NH2 cartridge (Isolute NH2, 500 mg, IST, Mid Glamorgan, UK). Again, erucamide radioactivity was concentrated in the third or fourth fraction. Recovery of erucamide was 35%– 40%, as calculated from radioactivity. The fraction was evaporated to dryness and redissolved in 10 l chloroform. Mass Spectrometry. A method was developed for determination of the content of erucamide in extracts from blood plasma and body organs. Gas chromatography–mass spectrometry (GC-MS) was employed with full scan (23–550 Da at 880 Da/s) on a Finnigan TSQ 700 (Thermoquest, San Jose, USA) instrument with a HP5890 gas chromatograph (Agilent Technologies, Palo Alto, USA) and a nonpolar DB1-XLB column (30 m long ⫻ 0.25 mm (ID); J & W Scientific, Folsom, USA) with helium as carrier gas. All GC analyses injections were made in the split-less mode at 250°C with the column held at 50°C and the temperature programmed at 10°C/min to 320°C. The
Erucamide as a Modulator of Water Balance: New Function of a Fatty Acid Amide erucamide peak in the mass chromatogram was integrated. It has the molecular ion m/z 337 (⫾0.5) and the retention time of 23.4 –23.7 min. An almost linear curve was obtained for the integrated areas versus standard amounts of erucamide (10–500 ng). For compound identification, GC-MS was also performed with a magnetic sector instrument (VG 70-70E, Micromass, England) with an HP5890 gas chromatograph (Agilent Technologies, Palo Alto, USA) and the same nonpolar DB1-XLB column. Mass spectra were recorded at 70 eV electron energy and 5 kV acceleration voltage. Scan rates were 300 Da/s from 20 to 600 Da. Mass calibration was performed with a reference voltage from the magnetic field. MS with direct probe was also performed on this instrument. Mass spectra were compared with reference spectra from databases (Wiley and NIST). Mass spectra were also obtained with fast atom bombardment (FAB/LSIMS) on a magnetic sector instrument (ZabSpec, Micromass, England). Conditions: Matrix: glycerol or 3-nitrobensyl alcohol; primary beam Cs⫹ at 30 kV; Mass range 2000–100 Da at a scan time of 2 s/decade. Mass calibration was performed with glycerol cluster ions. Authentic Fatty Acids and Fatty Acid Amides. Erucamide, erucic acid, arachidonic acid, and oleamide were purchased from SigmaAldrich (Stockholm, Sweden). Anandamide was purchased from ICN (Labora Chemicon, Sollentuna, Sweden).
Biological Studies Assay System for Biological Activity. Aliquots during all steps of the purification protocols were monitored for their ability to inhibit induced fluid accumulation in a closed intestinal segment (28). Sprague-Dawley rats, weighing 250 g, were lightly anesthetized with ether. The intestine was surgically exposed to allow localization of the jejunum. An approximately 100-mm jejunal segment was isolated with two silk ligatures. Fluid hypersecretion was induced by injection of 3 g cholera toxin (List Biological laboratories, Inc., Campbell, CA, USA) into the lumen of the intestinal loop. Muscles and skin were closed and the animals woke up from anaesthesia within a few minutes. The rats were anesthetized and sacrificed by cervical dislocation 5 h later. The intestinal segment was cut just outside the ligatures and its length and weight were determined. The weight of intestinal tissue per se at various loop lengths was measured in separate experiments. Samples of fractions from the plasma purification protocols were evaporated, redissolved in PBS containing 1% dimethyl sulphoxide (DMSO, BDH Chemicals, Poole, Great Britain), and sonicated prior to their IV administration to the rat, 1 min before the local injection of cholera toxin. The inhibition of hypersecretion caused by a test sample was expressed as the percent decreased fluid accumulation with reference to the amount of fluid in the intestinal loop of animals, which received only IV PBS. All experimental protocols were approved by the animal ethics committee of the University of Göteborg. Turnover of 3H-Erucamide. Male Sprague-Dawley rats, weighing 250 g, were anesthetized with ether and injected IV with 4 Ci (0.6 ng) 3H-erucamide, dissolved in 0.5 ml PBS, containing 1% DMSO. The rats were again anesthetized and sacrificed after 2, 5, 10, 30, and 130 min. A 0.5-ml blood sample from the heart was taken into heparinized tubes before the perfusion and centrifuged immediately. The rats were then perfused transcardially with 250 ml PBS. The pelleted blood cells, the blood plasma, and samples from brain, intestine, kidney, liver, lung, omentum, and spleen (wet weight 10–60 mg) were placed in 1 ml tissue solubilizer (Soluene-100, Packard instrument Co, Downers Grove, IL, USA) and left overnight.
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Ten milliliters scintillation fluid (Ultima Gold, Packard Bioscience B.V., Groningen, Holland) was added, and radioactivity was determined in a liquid scintillation counter (1215 Rackbeta, Wallac Oy, Turku, Finland). Separate series of organ samples were processed to determine the metabolism of the 3H-erucamide. Pieces (1–10 g) of liver, lung, and spleen were homogenized in water. Two vol. of acetone and 4 vol. of toluene were added and thoroughly mixed (27). The organic phase was collected and evaporated to dryness. It was then redissolved in 1 ml 2% isopropanol in hexane for LC on a Diol column, as described above. The eluated fractions, one of which contained most of the erucamide, were monitored with respect to radioactivity. Five hundred microliters of the organic phase was mixed with 10 ml scintillation fluid, and the radioactivity was measured as described above. Uptake of 3H-Erucamide into Tissue Slices. Samples of liver, intestine, lung, spleen, and brain (hippocampus) were dissected from 250 g Sprague-Dawley rats, which had been anesthetized with ether and sacrificed by transcardial perfusion with 250 ml physiological saline. The samples were placed in ice-cold balanced saline-glucose medium (122 mM NaCl, 3 mM KCl, 1.2 mM Mg2SO4, 1.2 mM CaCl2, 0.4 mM KH2PO4, 25 mM NaHCO3 and 10 mM glucose) and rat blood plasma, 10 l/ml. The medium was gassed with O2/CO2 (95/5). Tissue slices (0.4 mm) from the organs were prepared with a McIlwain tissue chopper (Betlehem Trading, Göteborg, Sweden). Three tissue slices per organ were transferred to a covered glass beaker containing 2 ml medium, 1% DMSO, and 0.3 Ci/ml 3H-erucamide. Erucamide was included at 0.6–120 M in different experiments. Incubation was carried out for 5, 10, or 20 min at 37°C in a slowly shaking water bath during gassing with the O 2 /CO 2 mixture. In a temperature-controlled series, slices were also incubated on ice. After incubation, the slices were removed from the medium for determination of wet weight. They were then transferred to a mixture of 0.5 ml H2O, 1 ml acetone and sonicated. Two milliliters toluene was added and the mixture was sonicated again. The organic phase was collected, and samples were taken for determination of radioactivity.
RESULTS Trial and Error. A number of protocols were tested in the attempt to purify a biologically active fraction. The plasma fraction with affinity for agarose was employed empirically (29). This was then used for gel filtration (BioGel P-200, Bio-Rad Laboratories, Hercules, CA, USA). Biological activity was recovered in a UV absorbing (280 nm) peak with a molecular weight of approximately 60 kD, based on time of elution. This material was then used for gel electrophoresis. The approach yielded little success, but we did obtain indications that the biological activity was associated with compounds of a molecular weight lower than 10 kD. This was confirmed by ultrafiltration experiments (Diaflo YM10, Amicon Division, Beverly, MA, USA), in which the biological activity was recovered both in the filtrate and the retained material; denaturation of the latter with methanol yielded more low molecular weight material with biological activity, suggesting dissolution
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Fig. 1. Elution profile of inhibitory activity against time and UV absorption (218 nm) on a Diol column. Mobile phase: hexane, containing 2% isopropanol. Solid line: UV absorption; dashed line: inhibition of the fluid accumulation in the intestinal loop.
of an active–inactive molecular complex. Various columns for separations of proteins/peptides were tested (C4-C18, Resource Q, HIC); however, organic solvents were always required for elution of the biological activity. The lipophilic properties of the active fraction was confirmed by methanol-water (50/50,v/v)/chlorform partition. Attempts to separate the plasma lipids on silica columns with hexane-isopropanol-acetone-water produced several peaks with biological activity.
Hamberger and Stenhagen Isolation of Erucamide from Blood Plasma. In the finally adopted protocol, lipids were extracted and separated on a Diol column, which was then eluted isocratically with 2% isopropanol in hexane. Most of the biological activity eluted after approximately 50 min, but a small amount eluted within the first 10 min (Fig. 1). Little UV absorption (218–225 nm) was associated with the 50 min fraction. GC separation provided only one peak, at approximately 27.6 min (Fig. 2). This peak was identified as erucamide by comparison with mass spectra from databases (Fig. 3). The 50-min fraction from the Diol column was occasionally purified with a C18 column (a 95%–100% ethanol gradient) but repeated reapplication to the Diol column proved to be more efficient. Monitoring of Fractions for Biological Activity and Specificity of Erucamide. The indicated function of erucamide, isolated from pig blood plasma, was reproduced with commercially obtained, authentic erucamide (Fig. 4). Both the isolated material and the authenticated compound exhibited a bell-shaped dose response curve, which had previously also been observed with partially purified fractions. The bell shape gave problems during the preparative work, in that a negative result could be due to either an absence or below detectable levels of the compound or an excess of biological activity.
Fig. 2. Gas chromatography of the fractions from the Diol column with the highest biological activity (see Fig. 1) on a nonpolar DB1-XLB column with helium as carrier gas. Erucamide eluted at 27.6 min with very little contamination.
Erucamide as a Modulator of Water Balance: New Function of a Fatty Acid Amide
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Fig. 3. Mass spectrum of the 27.6-min peak obtained with gas chormatography (see Fig. 2).
After the examination of the biological activity with erucamide, a series of other lipids were tested at a range of concentrations. Neither the fatty acids erucic acid, arachidonic acid, or oleic acid, nor the fatty acid amides oleamide and anandamide, had any appreciable activity. Although high concentrations of erucic acid showed a biological activity, similar to erucamide, it was found that this was due to a contamination of the commercial product with erucamide. Concentration of Erucamide in Body Organs. In a limited series (n ⫽ 5), the concentration of erucamide was determined in blood plasma and organs of the pig. The plasma level was 2.9 ⫾ 0.63 ng/g (M ⫾ SEM) tis-
Fig. 4. Dose response for synthesized erucamide, with respect to inhibition of cholera toxin–induced fluid accumulation in the intestinal loop. Maximal inhibition was a single dose of 5 ⫻ 10⫺11 g/kg. Means ⫾ SEM for 4 –5 animals at each dose level.
sue and a similar concentration was recorded for the kidney, 2.4 ⫾ 1.07 ng/g. The concentration in the lungs was several times higher, 11.8 ⫾ 5 ng/g, while liver, 1.0 ⫾ 0.17 ng/g, and particularly brain, 0.4 ⫾ 0.03 ng/g, had lower concentrations than plasma. The concentration of erucamide in the small intestine was below detection. Lipids were extracted, purified by solid phase extraction, and separated on GC. The 27.6-min peak was integrated. The observation of high levels of erucamide in lungs and low levels in brain became more striking when related to lipid phosphorus instead of wet weight. Brain, lung, liver, and kidney from the pig had concentrations of lipid phosphorus of 3.0, 0.02, 0.04, and 0.02 mol/g, respectively (n ⫽ 3). Turnover of 3H-Erucamide. The radioactivity in blood plasma declined rapidly; that is, it was measurable only during the first 5 min after an IV injection of 3 H-erucamide (Fig. 5). Pelleted red blood cells contained only 4%–5% of the radioactivity in plasma at 2 min, and only 1%–2% of the plasma radioactivity at 5 min, all with reference to wet weight. The time courses of organ radioactivity showed an early peak for the lungs, that is, after 5 min, while the peak of radioactivity for liver and spleen occurred at 10 min (see Fig. 5). At 2 min after injection, 90% of the radioactivity was in erucamide in both liver, lung, and spleen. After 10 min, the radioactivity in erucamide declined to 80% in the liver, while remaining over 90% in lung and spleen. At
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Hamberger and Stenhagen DISCUSSION
Fig. 5. Radioactivity in blood plasma (䊊), liver (〫), lung (ⵧ), spleen (䉭), kidney (䉱), intestine (⫺), skeletal muscle (⫻), and omentum (䊉). Time after an IV injection of 4 Ci 3H-erucamide (n ⫽ 4).
130 min, 20% of the radioactivity in the liver remained in erucamide, compared to 70% for lung and spleen. Under the conditions employed, brain, kidney, gastric ventricle, small intestine, skeletal muscle, omentum, and heart had no appreciable radioactivity during the entire time period. Uptake of 3H-Erucamide into Organ Slices. The time course of uptake of 3H-erucamide into the brain (hippocampus), lung, liver, and intestine is shown in Fig. 6. The uptake was temperature sensitive, reduced by approximately 90% at 0– 4°C during 20 min. Using a single concentration of erucamide, the relative rates of uptake among the organs did not differ appreciably and no saturation of 3H-erucamide incorporation could be demonstrated.
Fig. 6. Time course of incorporation of erucamide in vitro. Radioactivity in the acetone-toluene (1:2) extracts from 0.4-mm tissue slices of lung (䉭), liver (䊏), brain (䉱), and intestine (ⵧ) after incubation at 37°C (n ⫽ 3). The concentration of erucamide was 6 M and 3H-erucamide was added at 10 nCi/ml.
This is a report on the isolation, from pig blood plasma, of a previously unidentified inhibitor of induced diarrhea. Our early observations led to the hypothesis that the biological activity was associated with a fairly small, methanol-soluble molecule. Purification procedures included separation by agarose affinity and isolation of the lipid fraction. This was followed by LC on a Diol column and GC on a nonpolar capillary column. Finally, the identity of erucamide, a long-chain fatty acid amide (MW 337 Da), was determined by MS and the biological effect was confirmed with authentic erucamide. The mechanisms by which erucamide exerts this newly uncovered biological action, as well as its earlier known function as an angiogenic factor (25,26), remain to be elucidated. The ability of adipose tissue, particularly from the omentum, to stimulate new blood vessel formation has been recognized for a century by surgeons treating myocardial and cerebral infarctions. Wakamatsu et al. (25) isolated erucamide from the omentum and showed that synthesized erucamide possesses angiogenic activity. Mitchell et al. (26) showed that a sustained release of erucamide from a polymer matrix has a dose-dependent angiogenic effect on skeletal muscle, regenerating after an injury. A dose range of 30 g to 300 g had no effect different from the controls, whereas a dose of 3 g erucamide did significantly stimulate angiogenesis when compared to the appropiate controls (26). It is now evident that erucamide displays a bell-shaped dose response curve both with respect to its angiogenicity and in terms of its action reported in this paper. The AF peptide also displays a bell-shaped dose response curve (23). Many dose response curves elicited by lipid or peptide actions exhibit such a bell shape (30–33). Nevertheless, there does not appear to exist an obvious commonality in mechanisms between the angiogenic effect of erucamide and its antidiarrheal action. Still, both effects may be connected biologically because injured tissues, besides having an impaired circulation, also often are edematous immediately after a traumatic incidence. Correction of the water content combined with repair of the circulation certainly will have a synergistic effect on the regenerative process. Fatty acid amides have received considerable interest as a new class of bioactive lipids (34) and appear to serve a variety of functions within and outside the CNS (35,36). Oleamide, a fatty acid amide, similar to, but slightly smaller than erucamide, induces physiological sleep without the side effects of CNS depressants (37– 40). Anandamide, another fatty acid amide
Erucamide as a Modulator of Water Balance: New Function of a Fatty Acid Amide also modulates sleep analogous to oleamide (39) and demonstrates analgesic (41) properties. Both these compounds may mediate some of their action via specific receptors. Anandamide has cannabinoid effects, by binding to the CB1 and CB2 receptors (41,42); on the other hand an analgesic effect of oleamide may occur without receptor binding (43,44). Another property, common to oleamide and anandamide, is that their physiological actions in one animal can be passively transferred to a second, recipient animal. Administration of CSF or blood plasma from sleep-deprived dogs causes sleep in recipient dogs (38). Oleamide and anandamide also have other poperties in common, such as blocking gap junctions, typical of CNS astroglia, a property not shared by erucamide (45,46). Both oleamide and anandamide are synthesized periventricularly (42). Fatty acid amide hydrolase (FAAH), the enzyme that degrades many fatty acid amides (47), including erucamide, is enriched in the choroid plexus (48). Inhibition of FAAH represents one way to increase levels of fatty acid amides (49,50). Fatty acid amides accumulate in the CSF in response to the appropriate physiological stimulus, for example, sleep deprivation (51) and pain stimulation (42). There is no information on the production site for erucamide or how its level in the CSF (37) is modulated. It is noteworthy that not only FAAH, but also the AF peptide, are high in the choroid plexus (52). The concentration of erucamide in pig blood plasma was determined at 8.6 ⫻ 10⫺9 M. An administered dose to a rat of 5 ⫻ 10⫺11 mol/kg body weight gave maximal inhibition of cholera toxin–induced intestinal fluid hypersecretion. This dose would represent only a marginal increase of erucamide if evenly distributed in the body. However, the administered erucamide may be initially very high in the blood. Furthermore, the plasma levels in a pig may be orders of magnitude higher than in a laboratory rodent because these animals are almost continuously subjected to diarrhea-inducing feeds. Also, the equilibrium between protein-bound and free erucamide in plasma is unknown. The relatively very low erucamide concentration in the brain was unexpected, because erucamide is known to be present in the CSF (37) and other fatty acid amides are formed in the brain (42). The highest erucamide concentration was found in the lungs and the highest uptake of 3H-erucamide was in tissue slices from the lungs. Although a time- and temperaturedependent uptake of erucamide was demonstrated in thin tissue slices also from liver, brain, and intestine, we were unable to demonstrate a saturable uptake. Such
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uptake, as well as time and temperature dependency, for both anandamide and oleamide, has been shown in primary cultures of neurons, glia, and other cells (53–55). Those criteria denote an uptake mediated by a protein carrier. However, the virtual water insolubility of fatty acid amides make them less suitable for the methodology employed to study water-soluble neurotransmitters, such as amino acids (56). The in vivo turnover of labeled erucamide showed a rapid incorporation of radioactivity in the lungs (5 min), compared with liver, brain, and intestine (10 min). Even though the bolus of radioactivity reaches the lungs before other organs, the delay is not likely to account for the 5-min difference. The decay of total radioactivity was rapid in the lungs, but the FAAH activity appeared to be considerably higher in the liver than in the other organs. In nature, rapeseed is the principal source for erucic acid, the fatty acid parent compound for erucamide. In oils extracted from different varieties of rapeseed, erucic acid makes up 0.5%–50% of the total fatty acid content. Oils low in erucic acid are suitable for human consumption, but high erucic acid rapeseed oils that are used to synthesize erucamide on an industrial scale cause a variety of lesions in the heart (57). Erucamide, as well as oleamide, stearamide, and oleic acid, are used as additives to plastics, textiles, rubber, and lubricant oils. Erucamide is valued for its ability to infer superior antistatic, antisticking, and lubricant properties to materials (58). The abundance of erucamide and similar compounds in the environment requires special precautions when determining nanogram quantities in biological samples (59). In conclusion, indications of neuroactive properties of a previously discovered antidiarreal peptide stimulated to the isolation of erucamide from pig blood. Erucamide showed also a high potency and bell-shaped dose response curve for inhibition of induced diarrhea. The mechanisms of action and possible effects of erucamide on other organs remain to be studied. However, the potential of erucamide is strengthened by its previously shown angiogenicity and by being member of a family of lipoid neuromodulators, the function of which was largely discovered during the 1990s. ACKNOWLEDGMENTS The authors gratefully acknowledge Anita Palm and Britta Nyström for their endurance in the laboratory over the years; Mei Ding, who spent most of her graduate time on the project in search of a link between the biological activity and brain function; and Kenneth Haglid, who helped carry the project into the lipid phase.
184 We also thank Pam Fredman and Jan-Erik Månsson for determinations of the phosphorus content of organ lipids. The collaboration, support, and advice from Christina Hall, Gunnar Westman, Charlotta Damberg, Jonas Bergquist, Göran Oresten, Bernd Hamprecht, and Gianfrancesco Goracci have helped the project over critical steps. Finally, we appreciate Nico van Gelder’s continuous encouragement and hard work with the report. The study was supported by the Swedish MRC (grant nr 00164).
Hamberger and Stenhagen
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