The protective effects of PEA can be traced back in literature to 1943.
The American bacteriologists Coburn and Moore demonstrated in that year that feeding of dried egg yolk to underprivileged children living in poor parts of New York city prevented the recurrence of rheumatic fever in spite of repeated attacks of hemolytic streptococcal infection. In a subsequent study for two years, 30 children at a convalescent rheumatic home received four egg yolks daily. No other change in diet was made and no antibacterial drugs were given. 22 of these children contracted 24 proved group-A streptococcal infections (based on serology), but none showed clinical evidence of rheumatic recurrences. This was in sharp contrast to the previous experience in the convalescent home where rheumatic recurrences had been seen each year.
Subsequently, Coburn and colleagues were also the first to report in 1954 a phospholipid fraction prepared from egg yolk that showed antiallergic activity in an assay in the guinea pig.
The antiallergic factor of egg yolk was subsequently purified by Long and Martin in 1956 such a way that it was clear that this factor showed a biological and chemical similarity to a preparation obtained earlier in 1950 from arachis (peanut) and what appeared to be a closely related substance from “vegetable lecithin.”
1957 was the birth year of PEA. Kuehl and other employees of MSD reported to have succeeded in isolating a crystalline anti-inflammatory factor from soybean lecithine and they identified it S-(2-hydroxyethyl)-palmitamide.
They isolated the compound also from a phospholipid fraction of egg yolk and from hexane-extracted peanut meal. The products obtained was subsequently tested by in a local passive joint anaphylaxis assay in the guinea pig.
When applying their isolation procedure to soybean lecithin, they obtained a partially purified fraction from which the homogeneous factor was obtained by crystallization from cyclohexane. The crystalline material, had a melting point of 98-99′, and was described as neutral, optically inactive, and possessed the formula C18H3702N.
Hydrolysis of the factor resulted in palmitic acid and ethanolamine and thus the compound was identified as N-(2-hydroythyl)-palmitamide. In order to close the circle of isolation and identification, they were able to synthesize the compound by refluxing ethanolamine with palmitic acid according to a well-known procedure described in the chemical literature of that time. They further analyzed the anti-inflammatory activity of a series of derivatives of PEA and could prove that the basic moiety of the molecule was responsible for its anti-inflammatory activity. The nature of the acid group appeared to them to be of no importance because in addition to ethanolamine itself, N-(2-hydroxyethyl)-lauramide, S- (2-hydroxyethyl) -salicylamide and N- (2-hydroxy- ethyl)-acetamide were all potent anti-inflammatory properties. These pharmacological properties of the ethanolamine-derivates appeared to be quite specific since other homologs did not show a biological response in the assay.
The protective effects of PEA ‘avant la lettre’ in Streptococci infections
Coburn presented a review of his work and his hypothesis that eggs contained an important protective factor against infection, especially in rheumatic fever, in 1960 in the Lancet, and argued that:
(a) inadequate nutrition is part of a poor environment;
(b) rheumatic-fever children usually lack sufficient eggs in their diets;
(c) the escape from poverty is followed by an increase in the consumption of eggs and a decrease in the incidence of rheumatic fever;
(d) supplementation of children’s diets with egg yolk or certain fractions thereof is followed by decreased rheumatic susceptibility; and
(e) there is a fraction of egg yolk which in extremely small amounts has been found to have high antiallergic activity in laboratory animals.
Coburn described his field studies in some detail, and we summarize these here.
Field study # 1, n= 89: rheumatic boys and girls living at home in New York City all received food supplements, and no prophylactic drugs were given. 60 children had supplements (including eggs) during winter and spring months, and 29 served as ” controls “.
The clinical results were:
Of the 29 children on their normal diet (many deficiencies) 11 had a recurrence.
Of the 35 children, whose normal diet was reinforced daily with two eggs, a quart of milk, meat, butter, halibut-liver oil, 3 had a recurrence.
Of the 25 children, whose normal diet was reinforced only with powdered egg yolk (equivalent to six eggs daily), only 1 had a recurrence.
Field study nr # 2: n= 56. A two-year study was made of the effect of giving egg-yolk powder (equivalent of four egg yolks daily) to rheumatic children for three to four weeks after they developed haemolytic (group A) streptococcal pharyngitis. No medication was given during this period. Of 28 receiving this supplement, only 1 showed fresh rheumatic activity, whereas among 28 ” controls “, receiving no eggs, 10 children had fresh rheumatic activity.
Field study nr #3: n =40. A one-year study in which about 40 rheumatic children (many dietary deficiencies) received a daily supplement of only the protein fraction from four egg yolks was discontinued because of many rheumatic recurrences.
Field study #4: n= 45. A four-year study (Chicago, period 1952-56) the normal (deficient) diet of 45 rheumatic children was reinforced with egg-yolk alcohol-soluble material (A.S.M.; Wilson Laboratories). No other changes in their inadequate diets were made; no sulphonamides, antibiotics, or other significant drugs were administered. On the average, 35 highly susceptible rheumatic children received this supplement throughout the school year, September to July. The equivalent of 3 egg yolks was consumed, in the form of an elixir taken twice daily. All but one of these rheumatic children were under fifteen years of age. During the four years 45 children, considered highly susceptible rheumatic subjects, were given this material.
Result: a minimum of 17 attacks was expected among them after streptococcal infections, but only 5 occurred.
Coburn concluded: “The data obtained under these various conditions, both in New York and a decade later in Chicago, were found to be statistically significant.” However, he himself pointed out that all studies had methodological weaknesses.
Coburn pointed out various experimental findings in that time reporting that there is at least one anti-inflammatory substance in egg-yolk alcohol-soluble material, which was not present in the protein or acetone-soluble material. The anti-inflammatory activity was confirmed by different groups, for instance by measuring joint and skin lesions in either the Arthus or tuberculin reaction. Different models were all support for these observations, such as the reverse Arthus reaction were the anti-inflammatory effects of egg-yolk phospholipid fractions became clear. The anti-inflammatory compound clearly was part of the lipid fraction of the egg and not the protein-water fraction.
Already in 1965 the anti-inflammatory activity of PEA seemed to be widely known in the scientific community, as Bachur, from the Laboratory of Clinical Biochemistry and Experimental Therapeutics Branch, National Heart Institute, National Institutes of Health, Bethesda, Maryland, and colleagues refer extensively to the findings of Kuhl et al (1957) and wrote:
“Kuehl et al. had previously reported the isolation of palmitoylethanolamide, as a naturally occurring anti-inflammatory agent, from egg yolks. Pallmitoylethanolamide was known to occur in nature and to have pharmacological activity.” (Bachur et al, 1965).
The group of Bachur analyzed the content of PEA and found it to be present in several tissues of the rat and guinea pig. The amounts found in liver were quite variable, but PEA was consistently found in brain, liver, and muscle and was not detected in the other tissues examined. The method used was described as sensitive to 0.025 pg levels, 0.005 pg per gram wet weight, could have been detected with their methodology, according to their saying.
In 1973, the modifying effects of PEA on immunological reactions were well established; Perlik et all summarized:
“It has been shown that t N-(2-hydroxyethyl)-palmitamide (PEA) can decrease the intensity of several inflammatory and immunological processes.” However, between 1958 and 1969 the interest in this compound apparently decreased, as the authors stated:
“Recently the interest on biological properties of PEA has been revived because of its capacity to increase nonspecific tolerance to several bacterial toxins.”
In the period 1969- 1979 the results a variety of clinical trials were published, in total 5 trials in adults and one trial in children; all double-blind and placebo-controlled.
By the end of the 60s of last century, a branded PEA entered the market, Impulsin, produced by Spofa, a Check pharmaceutical industry. They apparently produced 300 mg tablets.
In 1969, the first results were published: results of a prophylactic trial with Impulsin in children’s institutions showed a reduced incidence of respiratory infections.
In 1974, the results of two new large scale double blind field trials in 1345 volunteers in two separate communities, were reported to evaluate the pro- phylactic efficacy of Impulsin in upper respiratory tract infections.
In 1979, Epps et al described the accumulation of NAE’s in infarcted myocardium, and they pointed out this accumulation might be of physiological importance because of its reported pharmacological activities of which the documented anti-inflammatory activity could be of special significance.
Long-chain N-acylethanolamines were found by them at levels of 400-500 nmol per g tissue in the infarcted areas of canine myocardium 24 hours after coronary artery ligation. Peripheral infarct areas also contained substantial amounts (200 nmol/g) while apparently, normal heart muscle contained very little (< 10 nmol/g). The amide-linked fatty acids were mainly 16:0, 18:0, 18:l and 18:2.
Epps et al was the first to find an accumulation of NAE’s under pathological conditions. The explanation they gave for this fact was: “This accumulation may be a side effect of the degenerative changes induced by ischemia or it may signify a response of myocardial tissue to injury directed at minimizing damage and promoting survival.”
In 1990 Schmidt presented a thorough review of the lipids belonging to the family that can be obtained from the N-acylethanolamine phospholipids by sequential degradation include glycerophospho(N-acyl)ethanolamine and N-acylethanolamine (NAE). The latter term, as he pointed out, describes a group of lipid amides that have been designated in different ways. Ss an example he presented N-palmitoylethanolamine or N-(2-hydroxyethyl)palmitamide or palmitoyl-ethanolamide. He supported the previous idea of the biological significance PEA accumulation in infarcted tissue and called it ” a defense mechanism aimed at minimizing the areas of irreversible injury and the ultimate size of the infarct.”
Schmidt also pointed out that accumulation of PEA and related lipids could be found in other models as protection against noxe, such as brain ischemia. He further discussed the mechanism of action and highlighted the membrane stabilizing effects of the NAE family of lipids. Discussing the protective effect of PEA in cardiac ischemia models:
The recovery of myocardial cells from ischemic injury depends on the survival of mitochondrial functions. Because these functions depend on the integrity of the inner membrane and because long-chain N-acylethanolamines are generated during myocardial infarction, it was of interest to test their effects on cardiac mitochondria. Collaborative studies with D. R. Pfeiffer and his colleagues indicated that NAE can inhibit the development of increased inner membrane permeability in mitochondria of both rat heart and liver produced by calcium-releasing agents. Rat heart mitochondria can neither synthesize nor degrade NAE and it was, therefore, possible to determine the effects of NAE structure and concentration. N-Oleoylethanolamine was found to be most effective in preventing the nonspecific permeability increase assayed and Its effect was concentration dependent.
Higher NAE levels inhibited energy-dependent Cal+ accumulation, maximal rates of succinate oxidation, and the development of membrane potential. Half-maximal effects for these activities occurred at about 120 PM.
The data reviewed by Smidt indicated for him that NAE prevents swelling and Ca2+ release in ischaemic tissue, without affecting lipid metabolism. Thus, its stabilizing effects on mitochondrial membranes according to him were most likely due to physical interactions. Thus, he further contributed to the general idea at that time that PEA worked via an unspecific mechanism, increasing resistance against bacteria and viruses and against noxe such as ischemia and toxins. He thought in the case of ischemia such interactions could also occur with myocardial sarcolemma and other membranes, strengthening his original hypothesis that NAE generation by the transacylation-phosphodiesterase pathway occurs in infarct tissue as a defense mechanism aimed at minimizing irreversible injury and infarct size.
Apart from the physicochemical inhibition of calcium influx, Schmidt also discussed other membrane effects of NAEs. He summarized studies done of the behavior of N-oleoylethanolamine in liposomes and unilamellar vesicles and concluded NAEs play also a role in influencing membrane stability. last but not least, he pointed out that relatively low levels of NAE in ischemic myocardium might help to stimulate myocardial contractility by increasing the rate of Ca 2+ flux across the sarcoplasmic reticulum. He further referred to studies where N-palmiitoyl- and N-stearoylethanolamines were shown to inhibition transport through the veratrine-activated fast sodium channels.
In summary, the NAEs according to the review of Schmidt had the following properties
Pharmacological Effects of palmitoylethanolamide
(a) Anti-inflammatory, antianaphylactic, and antiserotonin activity.
NAEs, the saturated fatty amides, such as palmitoylethanolamide, as well as the polyunsaturated forms, like 2-AG and anandamide play an important physiological role in the modulation of hyperactive immune-reactions.
In Celiac disease, an autoimmune disorder of the small intestine, caused by a reaction to gliadin, a gluten protein found in wheat, anandamide and PEA concentrations were significantly elevated (100% and 90%, respectively) in active celiac patients as were the CB1 receptors in the active inflammatory state. The levels returned to normal after remission with a gluten-free diet.
In an elegant study on the anti-inflammatory and proapoptotic activities of anandamide, it was shown that it can inhibit tumor necrosis factor-α-induced NF-κB activation by direct inhibition of the I κB kinase (83). The NF-κB inhibitory activity of anandamide was independent of CB1 and CB2 activation in TNFα-stimulated 5.1 and A549 cell lines. Structure–activity relationships were examined and it was found that analogs with saturated fatty acyl groups were more active than unsaturated analogs. When the ethanolamide group was replaced with a vanillyl group, a potent inhibition of TNFα-induced NF-κB-dependent transcription was observed.
Recently, the levels of endocannabinoids (ECs) in samples of cerebrospinal fluid from MS patients were compared with samples from healthy subjects and significant differences were observed (72). In a group of 26 relapsing patients vs. 25 healthy controls, increased anandamide concentrations, but not 2-AG, were seen. In the brains of mice with experimental autoimmune encephalomyelitis, a preclinical model of MS, similar results were reported. Peripheral lymphocytes from these patients also had elevated anandamide concentrations suggesting its possible involvement in inflammation. The authors believe that other aspects of MS may also be affected by anandamide. In a separate commentary (73), they point out that endocan-nabinoid manipulation offers a “unique opportunity to modify neurodegeneration and neuroinflammation”.
The transcription factor, peroxisome proliferator-activated receptor-alpha, has been proposed as the receptor-mediating PEA’s anti-inflammatory activity. N-Stearoyl ethanolamine like PEA does not activate CB1 and is the subject of a recent paper describing its anti-inflammatory properties (76). the effect was not reduced by CB1 or CB2 antagonists but was inhibited by capsazepine, a transient receptor potential vanilloid receptor 1 antagonist. They also reported that FAAH is an in vivo regulator of the concentrations of N-stearoyl ethanolamine. These findings suggest that increasing the levels of saturated fatty acid ethanolamides may result in anti-inflammatory actions.
In two models of colonic inflammation, 2,4-dinitrobenzene sulfonic acid (DNBS) and oral administration of dextran sulfate sodium, the involvement of the CB1 receptor was studied as protection against proinflammatory responses (80). Comparing the effects in CB1-deficient mice (CB1(−/−)) with those in wild-type littermates (CB1(+/+)), a greater degree of inflammation was seen in the receptor-deficient mice. It was also reported that genetic deletion of fatty acid amide hydrolase resulted in protection against DNBS-induced colitis
Evidence has been presented that 2-AG may function as an endogenous inhibitor of cyclooxygenase-2 (COX-2) thereby resulting in a protective effect on neurons that are exposed to harmful insults such as those due to inflammation (82).
Massa, G. Marsicano, H. Hermann, A. Cannich, K. Monory, B. F. Cravatt, G. L. Ferri, A. Sibaev, M. Storr, and B. Lutz. The endogenous cannabinoid system protects against colonic inflammation. J. Clin. Invest. 113(8):1202–1209 (2004).
Sancho, M. A. Calzado, V . Di Marzo, G. Appendino, and E. Munoz. Anandamide inhibits nuclear factor-kappaB activation through a cannabinoid receptor-independent pathway. Mol. Pharmacol. 63(2):429–438 (2003).
D’Argenio, S. Petrosino, C. Gianfrani, M. Valenti, G. Scaglione, I. Grandone, S. Nigam, I. Sorrentini, G. Mazzarella, and V . Di Marzo. Overactivity of the intestinal endocannabinoid system in celiac disease and in methotrexate-treated rats. J. Mol. Med. (Berlin, Germany). 85(5):523–530 (2007).
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