Distribution of an Analgesic Palmitoylethanolamide and Other N-Acylethanolamines in Human Placental Membranes


 Human amniotic and amniochorionic membranes (AM, ACM) are the most often used grafts accelerating wound healing due to their anti-inflammatory, anti-microbial, anti-fibrotic, and analgesic properties. We assessed the distribution of endogenous fatty acid amides N-acylethanolamines (NAEs): palmitoylethanolamide (PEA), oleoylethanolamide (OEA) and anandamide (AEA) in placental tissues, as they could participate in wound healing properties of AM/ACM grafts. Ten placentas were collected after caesarean delivery. NAEs were detected using ultra-high-performance liquid chromatography-tandem mass spectrometry analysis in fresh samples of AM, ACM, placental disc, umbilical cord, umbilical serum and vernix caseosa), and decontaminated samples of AM and ACM. NAEs were present in all studied tissue types, with mean concentrations in fresh tissues ranging: 76 – 350 (PEA); 31 – 220 (OEA); 7 – 30 ng/g (AEA). The highest mean concentrations were found in AM (PEA: 350 ng/g) or placenta (OEA: 220 ng/g; AEA: 30 ng/g), respectively. Low levels of NAEs were found in serum and vernix. Decontamination of AM, but not ACM, induced a significant (3 – 3.5-fold) increase in the levels of NAEs. PEA is the first compound with direct analgesic effect detected in AM and ACM. We thus propose NAEs, especially PEA, as one of the possible factors responsible for the anti-hyperalgesic, anti-inflammatory and neuroprotective effects of AM/ACM grafts, stimulating their wound healing effect. The increase of NAE levels in AM and ACM after tissue decontamination indicates that tissue processing may play an important role in maintaining the analgesic effect.


Introduction
Profound wound healing e ciency and relatively high availability of the source tissue, the placenta, make the amniotic membrane (AM) and amniochorionic membrane (ACM) ones of the most widely used grafts worldwide. Their wound healing effect is given by the interplay of anti-in ammatory, anti-microbial, and anti-brotic components [1,2]. The presence of numerous growth factors, cytokines and other bioactive proteins in AM/ACM has been experimentally proven [1][2][3][4][5] and their therapeutic e cacy has been repeatedly con rmed at the clinical level in ophthalmology, surgery, and wound healing [6][7][8][9].
The application of an AM/ACM graft on the wound leads to the signi cant pain relief [10][11][12]. To date, no speci c substances directly responsible for the analgesic effect of these grafts have been reported [13,14]. The pain-relieving effect of AM/ACM is usually explained by the mechanical protection of exposed nerve endings after a tight adherence of the graft to the wound surface, hydration of the wound bed and the presence of anti-in ammatory and anti-scarring components which may alleviate nociception indirectly [13][14][15]. However, it seems unlikely that such a strong and generalized analgesic effect is invoked without major contribution from a speci c compound [13,14]. As speci c proteins have been determined to be responsible for the anti-in ammatory and anti-microbial properties of AM, we hypothesized that a similar mechanism could be involved in the analgesic effect of AM/ACM. Therefore, we were interested in establishing whether some known substances that have analgesic properties are present in placental tissue, and thus explain the substantial pain relief generally reported in relation to application of AM/ACM. After an extensive search for potential candidates we shortlisted a group of endogenous bioactive lipid-related signalling molecules N-acylethanolamines (NAEs), in which particularly palmitoylethanolamide (PEA) has been shown to have profound analgesic and antinociceptive effect [16,17].
PEA was rst isolated from egg yolk, soybeans, and peanuts where its anti-anaphylactic activity was also reported [18,19]. Later on, anti-in ammatory, neuroprotective, analgesic and anti-nociceptive effects of PEA were shown in experimental animal studies [16,20,21,27,38,39], and con rmed in human clinical trials [40,41]. It has been reported in rodent models that PEA also inhibits lung, liver and retinal brosis [42][43][44]. PEA has also been shown to have a positive effect on viral respiratory infections [38,45], and was recently proposed as a promising nutraceutical in COVID-19 infection [46]. The anti-hyperalgesic effect of PEA has been utilized in the treatment of peripheral neuropathy and chronic pain, e.g. sciatic pain or pain from carpal tunnel syndrome [40,47,48], and its e ciency is independent of the etiopathogenesis of pain [47].
Binding of PEA to the PPAR-α receptor leads to attenuation of nociceptive and in ammatory responses, as con rmed in PPAR-α null mice [55,56]. The activation of PPAR-α can aid in the regeneration of mice peripheral nerves at the level of axon repair [57], mediated via satellite glial cells [57,58]. PPARα activation downregulates nuclear factor kB (NF-kB) followed by the decrease of proin ammatory proteins, such as inducible NO synthase (iNOS), cyclo-oxygenase-2 (COX2), tumour necrosis factor-α (TNFα) or interleukin 1 and 6 or prostaglandin E2 (PGE2) [41,59], which all can contribute to the anti-in ammatory properties of AM/ACM grafts. PEA also stimulates macrophages to remove invading bacteria and apoptotic neutrophils [60,61].
To date, of the NAEs lipid mediators, only AEA has been reported in frozen placental tissues [25,62]. However, its concentrations were not measured in isolated AM or ACM [63], which are the most important placenta related tissues used for grafting. We therefore designed our study to determine whether NAEs are present in placental tissues and whether their concentrations can explain the analgesic effect of the AM dressing.

Discussion
To date, the anti-in ammatory, analgesic, and neuroprotective properties of AM and ACM have been associated with various proteins discovered in these tissues [2,5]. Although the effect of AM on pain relief was mainly attributed to wound dressing and anti-in ammatory effects, it has been suggested that such rapid action in pain relief is mediated by hitherto unde ned analgesic mediators [13]. As far as we know, this is the rst study to demonstrate the presence of direct-acting analgesic compounds in placental membranes.
We established reference concentrations of PEA, OEA and AEA in all tested tissues, including AM and PL, by averaging values from the central and peripheral part of placenta, which did not differ in their statistical signi cance (Supplementary Table S4). An observed trend in the distribution of NAEs through all measured specimens (PEA > OEA > AEA) is in accordance with previously published results [22,25,53].
From placental specimens, NAEs have been previously detected in umbilical plasma and amniotic uid [25,63], and AEA concentration has been measured in unseparated frozen placental membranes and placenta, where higher levels of AEA were found [63]. Lower concentrations of AEA in amniotic and amniochorionic membranes compared to placental samples were also detected in our experiments. The same trend was observed for OEA. Opposite values (i.e. lower levels in placental samples relative to AM/ACM) were found for PEA. This observation may re ect a different function of PEA compared to OEA and AEA in placental tissue. To date, of the NAEs present in the placenta, only the function of AEA, which stimulates oxytocin levels in human placenta at term, has been elucidated [62].
Our results show that the 24-hour decontamination procedure leads to a signi cant increase in NAE concentration in AM (about 3-fold) and a slightly smaller (not signi cant) elevation in ACM samples. This shows that particular steps of AM/ACM processing, such as decontamination, can affect the amount of these bioactive substances in the tissue prepared for grafting. We additionally observed a comparable increase in NAE concentration in AM samples stored in two additional solutions (standard cell culture medium or saline) and under different conditions (Supplementary Table S5), which suggests that the enrichment of NAE in tissue samples is more likely to be affected by the length of storage than by the composition of storage solution.
A similar observation; increase of AEA concentrations with increasing tissue processing time was previously observed by Marczylo et al. 2009 [63]. The authors also found elevated AEA levels after storage at -80°C, which increased with the number of freeze-thaw cycles [63]. This indicates that overall NAE levels may be affected more by tissue degradation allowing a release of tissue lipids from cells than by continuing synthesis of NAEs by surviving cells, which is balanced by cellular enzymatic degradation of these highly lipophilic, sparingly soluble compounds [64]. In any case, our results indicate that decontamination, as a basal step in AM/ACM processing, in uences the quantity of NAEs in prepared grafts.
PEA and AEA are the most interesting of the NAEs studied here because their anti-in ammatory, analgesic, anti-brotic and neuroprotective properties [17,27,40] strongly overlap similar features described in AM/ACM [2,3,6,65,66]. Thus, they may be at least partially responsible for the positive effects associated with AM/ACM in the healing process. It is known that PEA operates primarily through PPAR-α receptor, which is expressed also in the skin [53], the site where AM/ACM grafts are applied. It has been shown that activators of PPAR-α receptor promote differentiation of human epidermal keratinocytes [67]. Furthermore, PPAR-α expression, which is restricted under normal physiological conditions to basal epithelium [68,69], is upregulated by the entire epithelial layer at the edge of the wound during wound healing [69,70]. We propose that PEA, released from AM after grafting may operate via this mechanism, and/or down-modulate mast cell activation [20], or regulate other blood-borne leukocytes (basophiles, macrophages) recruited to the injury site [27]. We suggest that the analgesic features of AM/ACM are mainly related to PEA, but anti-in ammatory and wound healing activities are linked to AEA [54].
Interestingly, Bauer et al. suggested PPAR-γ, which is activated by AEA (but not by PEA or OEA) [71], is the main target of amniotic membrane-mediated anti-in ammatory and antiapoptotic effects on macrophages of mice corneas [72].
Regarding clinical effect of NAEs, it has to be taken into a consideration that their overall wound healing effects depends on their half-life duration in tissue, which is contingent on the ratio between synthesis and degradation rates [53,73]. The observed differences in NAEs concentration in samples from individual subjects are likely to re ect current state of health of donors, such as the presence of obesity, stress, or in ammation [53]. On the other hand, the concentration ratios between tested tissue types (AM, ACM, etc.) from a single donor remain roughly constant.
In this work we detected PEA, AEA, OEA in placental tissues and determined their basal concentrations levels. To our knowledge these are the rst directly acting analgesic compounds detected in placenta. It is plausible that other endogenous substances participate in the analgesic effect of placental derivative after grafting, however, they have yet to be determined. We propose that analgesic, anti-in ammatory and neuroprotective properties of PEA and AEA are involved in analgesic and anti-in ammatory activity of AM/ACM membrane, and substantially contribute to their wound healing effect. Our data indicate that NAEs levels (in AM, ACM) increase after a 24-hour decontamination period in antibiotic solution, suggesting that the tissue processing may play an important role in maintaining or modulating the analgesic effect of the tissue prepared for grafting. and tissue decontamination solution BASE 128 (Alchimia srl, Italy) were used as purchased. Water was prepared using a Milli-Q integral system (Merck Millipore, Burlington, MA, USA).

Material
The study was approved by the Ethics Committees of the General Teaching Hospital and the First Faculty of Medicine of Charles University, Prague, Czech Republic and adhered to the tenets set out in the Declaration of Helsinki for research involving human subjects. Human placentas were obtained after a caesarean section delivery in Motol University Hospital (Prague, Czech Republic), and the General University Hospital (Prague, Czech Republic) from donors with a normal pregnancy who signed a written informed consent. Only healthy donors (mean age 36 years), screened for hepatitis B and C, syphilis and HIV were involved. The placentas with evident pathologies or visible injuries, such as hematomas, were excluded.
Specimens were prepared from ten fresh placentas (P1 -P10), which were processed within two hours after retrieval. The tissue was repeatedly rinsed by physiological solution and blood clots were removed. Then the placental membranes (AM, ACM) were manually separated from the residual placental tissue, dissected, transferred to a mesh support (Sanatyl, Tylex Letovice, Czech Republic) for a better manipulation with tissue and divided into 4 cm 2 samples [6,65]. AM and ACM were processed as either fresh tissue (set 1) or tissue was decontaminated (AM-d, ACM-d) with BASE 128 for 24 h at 4-8°C (set 2), according to standard decontamination procedures used for placental membranes in clinical practice [6,65]. As concentrations of some proteins are not homogeneously distributed within placenta and AM [74], we collected samples from two different areas of placental disc (PL): from the area near the umbilical cord (AM1, PL1) and from the area in the periphery of the placenta (AM2, PL2). Specimens of approximately 0.125 cm 3 were prepared from placental disc (after removing AM) and from umbilical cord (UC). All tissue types were prepared in triplicates. Where possible, a respective vernix caseosa (vernix, VX) sample was collected from healthy newborns immediately after full term delivery and frozen at -80°C until processing.
In addition, umbilical serum (US) samples were prepared from each placenta. Umbilical cord blood was collected from fresh placenta immediately upon reception, precipitated for 2-3 hours at room temperature (RT), centrifuged (3 000 g, 15 min, 4°C) and stored at -80°C until NAEs measurement (two months at maximum). A list of all samples is given in Table 1.

Specimen preparation for N-acylethanolamines detection
For NAEs analysis, all tissue samples (fresh and decontaminated) were washed in saline and homogenised mechanically by scissors for 120 seconds in 1 ml of cold acetonitrile [75]. To all homogenates, 10 µl (1 µg/ml) of PEA-d 4 internal standard solution was added, and these were then allowed to shake at 4°C and 800 rpm for 22-24 hours. Insoluble material was removed by centrifugation (20 min, 15 000 g, 4°C); the collected extracts (900 µl) and remaining pellets were stored at -80°C until NAEs detection (up to two months). The extracted material (pellets) was dried in an evacuated centrifuge (Refrigerated CentriVAp Concentrator, Labconco Corporation, Kansas City, MO, US) and weighed (analytical balance).
Puri cation of the extracts The extracts were evaporated to dryness in a vacuum centrifuge set at 0°C (Refrigerated CentriVAp Concentrator, Labconco) and re-dissolved in 1 ml of 30% (v/v) methanol/Milli-Q water. All extracts were puri ed by a slightly modi ed solid-phase extraction method [25] using a vacuum manifold (Agilent Technologies). The ow rate of solvent through the cartridge was pressure controlled to approximately 1 ml/min. Oasis HLB 1 cc, 30 mg cartridge (Waters) was washed using 1 ml of 100% methanol and preconditioned using 1 ml 30% (v/v) methanol. The extract was placed onto the cartridge. The cartridge was washed with 1 ml of 30% methanol, and the analytes were eluted with 100% acetonitrile to 2ml Eppendorf tube. Umbilical serum (0.25-0.5 ml) were spun at 13 000 g, enriched with 10 µl of PEA-d 4 (1 µg/ml) standard solution, made up to 1 ml with deionised water, vortexed and puri ed, as described above.
Liquid chromatography -mass spectrometry analysis The ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC/MS) system was composed of the ExionLC UHPLC AD chromatography system and the QTRAP 6500 + mass spectrometer (both Sciex, Foster City, CA, USA) equipped with electrospray ionisation (ESI) ion source. The analysis was performed with VanGuard Acquity UPLC BEH C18 pre-column (2.1×5 mm, particle size 1.7 µm) connected to the analytical column Acquity UPLC BEH C18 (2.1×50 mm, particle size 1.7 µm) (both Waters). The temperature of the column and the autosampler were set at 40°C and 5°C, respectively. and PEA calibration curves spiked with internal standards PEA-d4 (10 µl of 1 µg/ml) were performed. The calibration curve was constructed for the analyte's relative signal intensity (for the area of the analyte peak divided by the peak area of the internal standard). Peaks integration, calibration curve construction and evaluation of analyte concentration were made using Analyst 1.6.3 (Sciex, Darmstadt, Germany). For tissues and vernix, the concentration was recalculated to the weight of extracted material.

Statistical analysis
Each type of sample obtained from one placenta was analysed in triplicate. The resulting mean + SD was always calculated from a minimum of 6 mean values calculated for each triplicate. The signi cance of differences in NAEs' concentrations between the fresh (control) and decontaminated samples of AM/ACM and placenta (PL1, PL2) were tested by Wilcoxon test. The descriptive statistics for each data set was calculated using R package [76]. The outliers were identi ed using the interquartile range (IQR) criterion. Outlier values were excluded from the Wilcoxon test. Only data with a p-value of ≤ 0.05 were considered statistically signi cant.

Declarations
Data availability: The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.