Hyaluronan fragments produced during tissue injury: A signal amplifying the inflammatory response.

Inflammation is a complex mechanism that plays a key role during diseases. Dynamic features of the extracellular matrix (ECM), in particular, during phases of tissue inflammation, have long been appreciated, and a great deal of several investigations has focused on the effects of ECM derivatives on cell function. It has been well defined that during inflammatory and tissue injury, ECM components were degraded. ECM degradation direct consequence is the loss of cell homeostasis, while a further consequence is the generation of fragments from larger precursor molecules. These bio-functional ECM shred defined matrikines as capable of playing different actions, especially when they function as powerful initiators, able to prime the inflammatory mechanism. Non-sulphated glycosaminoglycan hyaluronan (HA) is the major component of the ECM that undergoes specific modulation during tissue damage and inflammation. HA fragments at very low molecular weight are produced as a result of HA depolymerization. Several evidence has considered the plausibility that HA breakdown products play a modulatory action in the sequential stages of inflammation, although the effective mechanism of these HA derivative compounds act is not completely defined. This review will focus on the pro-inflammatory effects of HA fragments in recent years obtained by in vitro investigations.

Evidence for embryonic haemoglobins from Sparus aurata under normal and hypoxic conditions.

Teleost haemoglobins vary in polymorphisms and primary structure, although display similar functional properties. Key amino acids for Root effect (a reduction in oxygen-carrying capacity and loss of cooperativity with declining pH) are conserved throughout fish evolution. For the first time, we cloned and characterised Sparus aurata L. embryonic globin chains (eα1, eα2, eß). We also studied haemoglobins (eHbI, eHbII) behaviour in normal and low-oxygen conditions. Several amino acids in fry globins are different in chemical type (e.g. polar → non-polar and vice versa), compared to adult globins. His55α1, crucial for Root effect, is substituted by Ala in fry, presumably enhancing oxygen capture, transport and reducing the dependence of Root effect from pH. Phylogenetic trees demonstrate that eα1 globin diversified more recently than eα2; moreover, eα1, eα2 and eß globins evolved earlier than adult α and ß globins. In low-oxygen conditions, fry haemoglobins display the same behaviour of the adult haemoglobins (probably, embryonic and adult-type I Hbs display a higher oxygen affinity than type II Hbs, operating through a rapid cycle of heme-Fe auto-oxidation/reduction). Therefore, based on our results and on the comparison with adult haemoglobins, we hypothesise that embryonic haemoglobins have evolved to better adapt fry to variable habitats. We studied Sparus aurata for its economical relevance in Mediterranean aquaculture. The information we provide can help understand Sparus aurata behaviour in the wild and in rearing conditions. Further studies with functional assays will deepen the knowledge on the molecular mechanisms of fry haemoglobin physiology.

Serglycin is involved in inflammatory response in articular mouse chondrocytes.

Serglycin is expressed by a variety of cell types and mediates different functions in both normal and pathological conditions by interacting with different biological molecules, such as the CD44 receptor. Many studies suggest that serglycin has a crucial role in inflammatory response, but there are limited data on the functions of this proteoglycan in chondrocytes. In this study we investigated the effect of serglycin knockdown induced by a specific serglycin small interfering RNA (SRGN siRNA) in normal mouse chondrocytes stimulated with lipopolysaccharide (LPS). LPS administration in normal chondrocytes increased the expression of serglycin mRNA and related protein and the production of the pro-inflammatory mediators TNF-alpha, IL-1beta, IL-6, iNOS and MMP-9, through NF-kB activation. In addition, a marked increased expression of CD44 after LPS stimulation was observed. Notably, the CD44 expression and the inflammatory response were significantly reduced by SRGN siRNA treatment in LPS treated chondrocytes. Similar results were obtained in normal mouse chondrocytes exposed to LPS, using a specific blocking antibody against CD44. These results indicate that serglycin produced in LPS-induced inflammation in normal mouse chondrocytes is able to modulate inflammation by interacting with CD44 receptor, suggesting a possible key role in the cartilage inflammation.

Micronized palmitoylethanolamide reduces joint pain and glial cell activation.

OBJECTIVE AND DESIGN: Temporomandibular disorder (TMD) is a common painful condition in the temporomandibular joint (TMJ). Joint inflammation is believed to be a chief cause of pain in patients with TMD, through the release of pro-inflammatory cytokines that induce peripheral sensitization of nerve terminals followed by microglial stimulation. MATERIALS AND SUBJECT: TMJ was induced in rats with the injection of complete Freund's adjuvant (CFA) emulsion into the left TMJ capsule. TREATMENT: The present study would assess the effects of micronized palmitoylethanolamide (m-PEA) on glial activation and trigeminal hypersensitivity. METHODS: Ten mg/kg m-PEA or corresponding vehicle was administered 1 h after CFA and mechanical allodynia and edema were evaluated at 24 and 72 h after CFA injection. RESULTS: CFA-injected animals showed TMJ edema and ipsilateral mechanical allodynia accompanied by a robust growth in GFAP protein-positive satellite glial cells and activation of resident macrophages in the TG. Moreover, m-PEA administration significantly reduced the degree of TMJ damage and pain, macrophage activation in TG and up-regulation of Iba1. CONCLUSIONS: The results confirm that m-PEA could represent a novel approach for monitoring pain during trigeminal nerve sensitization.

2-pentadecyl-2-oxazoline: Identification in coffee, synthesis and activity in a rat model of carrageenan-induced hindpaw inflammation.

N-acylethanolamines (NAEs) comprise a family of bioactive lipid molecules present in animal and plant tissues, with N-palmitoylethanolamine (PEA) having received much attention owing to its anti-inflammatory, analgesic and neuroprotective activities. 2-Pentadecyl-2-oxazoline (PEA-OXA), the oxazoline of PEA, reportedly modulates activity of N-acylethanolamine-hydrolyzing acid amidase (NAAA), which catabolizes PEA. Because PEA is produced on demand and exerts pleiotropic effects on non-neuronal cells implicated in neuroinflammation, modulating the specific amidases for NAEs (NAAA in particular) could be a way to preserve PEA role in maintaining cellular homeostasis through its rapid on-demand synthesis and equally rapid degradation. This study provides the first description of PEA-OXA in both green and roasted coffee beans and Moka infusions, and its synthesis. In an established model of carrageenan (CAR)-induced rat paw inflammation, PEA-OXA was orally active in limiting histological damage and thermal hyperalgesia 6h after CAR intraplantar injection in the right hindpaw and the accumulation of infiltrating inflammatory cells. PEA-OXA appeared to be more potent compared to ultramicronized PEA given orally at the same dose (10mg/kg). PEA-OXA markedly reduced also the increase in hindpaw myeloperoxidase activity, an index of polymorphonuclear cell accumulation in inflammatory tissues. NAAA modulators like PEA-OXA may serve to maximize availability of NAEs (e.g. PEA) while providing for recycling of the NAE components for further resynthesis.

Emerging drugs for acute lung injury.

INTRODUCTION: Acute respiratory distress syndromes (ARDS) are devastating disorders of overwhelming pulmonary inflammation and hypoxemia, resulting in high morbidity and mortality. AREAS COVERED: The main pharmacological treatment strategies have focused on the attempted inhibition of excessive inflammation or the manipulation of the resulting physiological derangement causing respiratory failure. Additionally, such interventions may allow reduced occurence mechanical ventilation injury. Despite promising preclinical and small clinical studies, almost all therapies have been shown to be unsuccessful in large-scale randomized controlled trials. The evidence for pharmacological treatment for ARDS is reviewed. Potential future treatments are also presented. EXPERT OPINION: We suggest for future clinical trials addressing prevention and early intervention to attenuate lung injury and progression to respiratory failure.

Protective effect of polyphenols in an inflammatory process associated with experimental pulmonary fibrosis in mice.

Polyphenols have been described to have a wide range of biological activities, and many reports, published during recent years, have highlighted the beneficial effects of phenolic compounds, illustrating their promising role as therapeutic tools in several acute and chronic disorders. The purpose of study was to evaluate, in an already-assessed model of lung injury caused by bleomycin (BLM) administration, the role of resveratrol and quercetin, as well as to explore the potential beneficial properties of a mango leaf extract, rich in mangiferin, and a grape leaf extract, rich in dihydroquercetin (DHQ), on the same model. Mice were subjected to intra-tracheal administration of BLM, and polyphenols were administered by oral route immediately after BLM instillation and daily for 7 d. Treatment with resveratrol, mangiferin, quercetin and DHQ inhibited oedema formation and body weight loss, as well as ameliorated polymorphonuclear infiltration into the lung tissue and reduced the number of inflammatory cells in bronchoalveolar lavage fluid. Moreover, polyphenols suppressed inducible nitric oxide synthase expression, and prevented oxidative and nitroxidative lung injury, as shown by the reduced nitrotyrosine and poly (ADP-ribose) polymerase levels. The degree of apoptosis, as evaluated by Bid and Bcl-2 balance, was also suppressed after polyphenol treatment. Finally, these natural products down-regulated cyclo-oxygenase-2, extracellular signal-regulated kinase phosphorylated expression and reduced NF-κBp65 translocation. Our findings confirmed the anti-inflammatory effects of resveratrol and quercetin in BLM-induced lung damage, and highlight, for the first time, the protective properties of exogenous administration of mangiferin and DHQ on experimental pulmonary fibrosis.

Micronized/ultramicronized palmitoylethanolamide displays superior oral efficacy compared to nonmicronized palmitoylethanolamide in a rat model of inflammatory pain.

BACKGROUND: The fatty acid amide palmitoylethanolamide (PEA) has been studied extensively for its anti-inflammatory and neuroprotective actions. The lipidic nature and large particle size of PEA in the native state may limit its solubility and bioavailability when given orally, however. Micronized formulations of a drug enhance its rate of dissolution and reduce variability of absorption when orally administered. The present study was thus designed to evaluate the oral anti-inflammatory efficacy of micronized/ultramicronized versus nonmicronized PEA formulations. METHODS: Micronized/ultramicronized PEA was produced by the air-jet milling technique, and the various PEA preparations were subjected to physicochemical characterization to determine particle size distribution and purity. Each PEA formulation was then assessed for its anti-inflammatory effects when given orally in the carrageenan-induced rat paw model of inflammation, a well-established paradigm of edema formation and thermal hyperalgesia. RESULTS: Intraplantar injection of carrageenan into the right hind paw led to a marked accumulation of infiltrating inflammatory cells and increased myeloperoxidase activity. Both parameters were significantly decreased by orally given micronized PEA (PEA-m; 10 mg/kg) or ultramicronized PEA (PEA-um; 10 mg/kg), but not nonmicronized PeaPure (10 mg/kg). Further, carrageenan-induced paw edema and thermal hyperalgesia were markedly and significantly reduced by oral treatment with micronized PEA-m and ultramicronized PEA-um at each time point compared to nonmicronized PeaPure. However, when given by the intraperitoneal route, all PEA formulations proved effective. CONCLUSIONS: These findings illustrate the superior anti-inflammatory action exerted by orally administered, micronized PEA-m and ultramicronized PEA-um, versus that of nonmicronized PeaPure, in the rat paw carrageenan model of inflammatory pain.

Hydrogen sulfide-releasing cyclooxygenase inhibitor ATB-346 enhances motor function and reduces cortical lesion volume following traumatic brain injury in mice.

BACKGROUND: Traumatic brain injury (TBI) induces secondary injury mechanisms, including dynamic interplay between ischemic, inflammatory and cytotoxic processes. We recently reported that administration of ATB-346 (2-(6-methoxynapthalen- 2-yl)-propionic acid 4-thiocarbamoyl-phenyl ester), a hydrogen sulfide-releasing cyclooxygenase inhibitor, showed marked beneficial effects in an animal model of spinal cord injury, significantly enhancing recovery of motor function and reducing the secondary inflammation and tissue injury. METHODS: Here we evaluated the neuroprotective potential of ATB-346, a hydrogen sulfide-releasing derivative of naproxen, using the controlled cortical impact (CCI) injury model in mice, one of the most common models of TBI. Moreover, the aim of the present study was to carefully investigate molecular pathways and subtypes of glial cells involved in the protective effect of ATB-346 on inflammatory reaction associated with an experimental model of TBI. In these studies, TBI was induced in mice by CCI and mice were orally administered ATB-346, naproxen (both at 30 µmol/kg) or vehicle (dimethylsulfoxide:1% carboxymethylcellulose [5:95] suspension) one and six hours after brain trauma and once daily for 10 days. RESULTS: Results revealed that ATB-346 attenuated TBI-induced brain edema, suppressed TBI-induced neural cell death and improved neurological function. ATB-346 also significantly reduced the severity of inflammation and restored neurotrophic factors that characterized the secondary events of TBI. CONCLUSIONS: These data demonstrate that ATB-346 can be efficacious in a TBI animal model by reducing the secondary inflammation and tissue injury. Therefore, ATB-346 could represent an interesting approach for the management of secondary damage following CNS diseases, counteracting behavioral changes and inflammatory process.

Pelagia noctiluca (Scyphozoa) crude venom injection elicits oxidative stress and inflammatory response in rats.

Cnidarian toxins represent a rich source of biologically active compounds. Since they may act via oxidative stress events, the aim of the present study was to verify whether crude venom, extracted from the jellyfish Pelagia noctiluca, elicits inflammation and oxidative stress processes, known to be mediated by Reactive Oxygen Species (ROS) production, in rats. In a first set of experiments, the animals were injected with crude venom (at three different doses 6, 30 and 60 µg/kg, suspended in saline solution, i.v.) to test the mortality and possible blood pressure changes. In a second set of experiments, to confirm that Pelagia noctiluca crude venom enhances ROS formation and may contribute to the pathophysiology of inflammation, crude venom-injected animals (30 µg/kg) were also treated with tempol, a powerful antioxidant (100 mg/kg i.p., 30 and 60 min after crude venom). Administration of tempol after crude venom challenge, caused a significant reduction of each parameter related to inflammation. The potential effect of Pelagia noctiluca crude venom in the systemic inflammation process has been here demonstrated, adding novel information about its biological activity.

Short Road Transport and Slaughter Stress Affects the Expression Profile of Serotonin Receptors, Adrenocortical, and Hematochemical Responses in Horses.

Horse transport is considered a cause of stress in animals and is known to affect the 5-HT concentrations in both the brain and other tissues. The aim of this research was to investigate the effect of horse transportation and slaughter stress on plasma serotonin's concentration and the expression levels of the related 5-HT1B and 5-HT2A receptors in PBMCs. Furthermore, the IL-12 levels and a variety of blood parameters, including triglycerides, total cholesterol, glucose, aspartate aminotransferase, creatine phosphokinase, lactate dehydrogenase, were also considered. This research was carried out on 32 horses submitted to short road transport of 40 km to slaughter. Blood samples were collected in baseline conditions (T0) and 24 h later, after they were slaughtered (T1). The results showed a significantly increased expression of 5-HT1B and 5-HT2A receptors and a significantly decreased expression of IL-12 in PBMCs at T1 vs. T0. Furthermore, a significant increase in cortisol and glucose concentrations, and LDH activity was observed at T1. In contrast, a significantly lower circulating 5-HT concentration was observed at T1 vs. T0. These results indicate that the stress induced by transport and slaughter stimuli led to the serotoninergic system's activation, suggesting that the expression of serotonin receptors could be used as a pivotal marker of stress, with potential applications for the improvement of elective protocols to observe the guidelines relating to transported horses.

Bergamot Byproducts: A Sustainable Source to Counteract Inflammation.

Chronic inflammation is the result of an acute inflammatory response that fails to eliminate the pathogenic agent or heal the tissue injury. The consequence of this failure lays the foundations to the onset of several chronic ailments, including skin disorders, respiratory and neurodegenerative diseases, metabolic syndrome, and, eventually, cancer. In this context, the long-term use of synthetic anti-inflammatory drugs to treat chronic illnesses cannot be tolerated by patients owing to the severe side effects. Based on this, the need for novel agents endowed with anti-inflammatory effects prompted to search potential candidates also within the plant kingdom, being recognized as a source of molecules currently employed in several therapeutical areas. Indeed, the ever-growing evidence on the anti-inflammatory properties of dietary polyphenols traced the route towards the study of flavonoid-rich sources, such as Citrus bergamia (bergamot) and its derivatives. Interestingly, the recent paradigm of the circular economy has promoted the valorization of Citrus fruit waste and, in regard to bergamot, it brought to light new evidence corroborating the anti-inflammatory potential of bergamot byproducts, thus increasing the scientific knowledge in this field. Therefore, this review aims to gather the latest literature supporting the beneficial role of both bergamot derivatives and waste products in different models of inflammatory-based diseases, thus highlighting the great potentiality of a waste re-evaluation perspective.

Heavy Metals Bioaccumulation in Mytilus galloprovincialis and Tapes decussatus from Faro Lake (Messina), Italy.

The aim of this study was to evaluate the potential bioaccumulation of arsenic (As), copper (Cu), zinc (Zn), cadmium (Cd), lead (Pb) and mercury (Hg) in the haemolymph and corpus of Mytilus galloprovincialis and Tapes decussatus from Lake Faro. The lake is particularly prone to the accumulation of substances that are potentially toxic to aquatic organisms, due to the input of pollutants from urban and agricultural sources and the low rate of water exchange. The combination of saltwater from the Tyrrhenian Sea, the Strait of Messina and freshwater from hilly aquifers has created brackish conditions in the lake, resulting in an area of high commercial shellfish productivity. As, Cd, Cu, Pb and Zn were determined using a single quadrupole inductively coupled plasma mass spectrometer; Hg was determined using a direct mercury analyser (DMA-80). Physicochemical parameters of the water from Lake Faro were also performed. Statistical analysis was carried out using GraphPad Prism 9.0 (GraphPad Software, Inc., Boston, MA, USA) and Shapiro-Wilk normality was applied. Concentrations of Cd, Hg and Pb below the permitted MRLs in Mytilus galloprovincialis and Tapes decussatus used as ''biological indicators'' show that Lake Faro is not at risk of contamination by these pollutants and, moreover, is free of health problems for the consumer based on regulatory limits.

Surgical Site Infiltration with Comfort-in Device and Traditional Syringe in Dogs Undergoing Regional Mastectomy: Evaluation of Intra- and Postoperative Pain and Oxidative Stress.

The surgical site infiltration of a local anesthetic is defined as the direct injection of a drug. This study aimed to compare the effects of surgical site infiltration with 4 mg kg-1 lidocaine using a Comfort-in device and traditional syringe on oxidative status and intra- and postoperative pain in dogs undergoing regional mastectomy. Sixty adult female dogs divided into C (Comfort-in device), S (traditional syringe), and CTR (control) groups received 2 µg kg-1 dexmedetomidine and 4 mg kg-1 tramadol IM, 5 mg kg-1 tiletamine/zolazepam IV, and isoflurane. The physiological and anesthesiological parameters were measured. The assessment of intra- and postoperative responses to the surgical stimulus was performed using a cumulative pain scale (CPS score of 0-4) and the Colorado Pain Scale (CSU-CAPS score of 0-4). The hematological and biochemical parameters and inflammatory oxidative status were measured. The CPS scores showed no significant differences between the C and S groups (p = 0.236), while the comparison between the CTR, C, and S groups, respectively, showed a significant difference (p < 0.001). The postoperative analgesia scores were significantly lower in the C group compared to those of the S and CTR groups (p < 0.001). In the C group, no subject received rescue analgesia during the intra- and postoperative periods. The level of oxidative inflammatory stress was lower in group C than those in S and CTR groups, and no side effects were observed in all the groups.

Effect of surgery on oxidative stress and endogenous tocopherol concentrations in juvenile female dogs.

BACKGROUND: Surgery such as ovariectomy causes an inflammatory and oxidative stress. This study was designed to evaluate endogenous tocopherol levels in response to surgical oxidative stress induced by abdominal surgery (ovariectomy) in thirty-two juvenile female dogs. The dogs received meloxicam before surgery (0.2 mg/kg SC) and after surgery (0.1 mg/kg OS every 24 h), 0.03 mg/kg of atropine sulfate (IM), and propofol 4 mg/kg intravenously (IV). General anesthesia was maintained with sevoflurane. Physiological, hematological and biochemical parameters, malondialdehyde (MDA) and α-, δ-, γ-tocopherols were evaluated at baseline, 36 and 48 h after surgery. RESULTS: The physiological parameters remained within normal ranges. Blood glucose concentration increased, while the albumin levels decreased after surgery. Rescue analgesia was not required. MDA levels increased above the baseline at 36 and 48 h after surgery (P < 0.001). The α-, δ-, and γ-tocopherol concentrations decreased from baseline at 36 and 48 h after surgery (P < 0.001). CONCLUSIONS: Surgery in juvenile female dogs revealed oxidative, increased MDA concentrations, reduced tocopherol levels, and had a clinically insignificant influence on homeostasis.

Oxidative stress in relation to serotonin under general anaesthesia in dogs undergoing ovariectomy.

Abdominal surgery such as ovariectomy is a traumatic event that can cause oxidative stress. The aim of the present study was to evaluate the concentration of serotonin in relation to ovariectomy-induced oxidative stress in dogs undergoing general anesthesia. Thirty-two female dogs, under general anesthesia, received meloxicam before surgery (0.2 mgkg-1 SC) and after surgery (0.1 mgkg-1 OS every 24 h). The physiological, hematological, and biochemical parameters: glycemia, aspartate transaminase (AST), alanine aminotransferase (ALT), total protein, albumin and BUN were evaluated. Oxidative stress was determined by malondialdehyde (MDA) assay, catalase (CAT), superoxide dismutase (SOD), myeloperoxidase (MPO) and butyrylcholinesterase (BuChe) at baseline, 36 and 48 h after the last administration of meloxicam. Serotonin (5-HT) concentration was also evaluated at baseline, 36 and 48 h after the last administration of meloxicam. Responses to surgical stimulus were evaluated. Physiological and hematological parameters they fell within the normal ranges for anesthetized dogs. Glycemia increased, albumin levels decreased after surgery. No rescue analgesia was required. MDA and 5-HT concentrations significantly increased from the baseline at 36 and 48 h after surgery (p < .001). 5-HT levels could be used as an indicator for oxidative stress induced by surgery and it might be employed for objectively quantifying the well-being of the surgical patient.

Occurrence and health risk assessment of mineral composition and aflatoxin M1 in cow milk samples from different areas of Sicily, Italy.

This study aimed to determine 16 mineral elements (Cd, Pb, As, Na, Mg, Al, Ca, K, Cr, Mn, Fe, Ni, Cu, Zn and Se) using inductively coupled plasma mass spectrometry (ICP-MS) and a direct mercury analyzer (DMA-80) for Hg evaluation. Aflatoxin M1 was determined by high-performance liquid chromatography with fluorescence detection (HPLC-FLD) in cow milk samples. This research considered 180 milk samples, 20 by province (Palermo, Catania, Messina), collected for a period of three years (2020-2022) to assess the potential risks for consumer, the safety status and nutritional quality related to mineral intake by consuming of milk. All samples showed a Pb concentration below the limit reported by European Regulation 915/2023. Cadmium and Hg concentrations were below the Limit Of Quantification (LOQ) in all samples analyzed. The milk samples analyzed proved to be a good source of Ca (up to 44.5 % of the dietary reference values), with well percentages also for Na (up to 7.6 %), K (up to 23.1 %) and Mg (up to 11.1 %). Regarding trace elements, the results reported that chromium requires attention; its value was always higher than 168.8 % in all samples analyzed. Levels of arsenic and lead were up to 20.2 % and up 7.1 % respectively. Aflatoxin M1 concentrations were below the limit of detection (< 0,009 mcg/kg) in all milk analyzed. Therefore, further studies are needed to safeguard consumer health, the quality of the product and to assess the state of animal health.

Adaptive Response of Estrogen-Iron Axis in Pregnant Purebred Spanish Mares of Different Age.

The maintenance of iron (Fe) homeostasis is vital for the physiological function along life. In sexually mature humans and experimental animals, estrogens downregulate hepcidin (Hpc) expression, in order to improve the intestinal absorption and to mobilize Fe stores for maternal erythropoietic expansion and placental development. However, changes of these mechanisms related to regulation of Hpc on the availability of Fe during gestation with advancing age in mares, remain unknown. The objective of this study was to evaluate the interrelationships between serum Fe, Ferritin (Ferr) and Hpc with estrone (E1) and estradiol-17ß (E2) concentrations in pregnant mares of different ages. Blood samples were taken from 40 pregnant Spanish Purebred mares belonging to four different age groups, 10 subjects for each group: 4 to 6 years, 7 to 9 years, 10 to 12 years, and >12 years were used in this study. Fe concentrations of 4 to 6 and 7 to 9 years groups were higher (P < .01) than 10 to 12 and >12 years groups. Ferr concentrations of 4 to 6 years group were higher (P < .01) than other groups. Hpc concentrations increased and E1 decreased (P < .01) in >12 years group compared to other age groups. E2 concentrations of 7 to 9, 10 to 12 and >12 years groups were higher (P < .01) than those of 4 to 6 years group; 7 to 9 years group had higher E2 concentrations (P < .01) than >12 years group. Fe and Ferr were negatively correlated with Hpc (r = -0.81 and r = -0.67, respectively). E1 and E2 were negatively correlated with Fe (r = -0.23 and r = -0.11, respectively). E2 was positively correlated with Hpc (r = 0.78). In pregnant Spanish Purebred mare, the increase of estrogens, according to the more efficient iron status in response to Hpc inhibition and consequent mobilization of circulating and iron reserve, shows the existence of "estrogen-iron axis" in young mares. Nevertheless, these mechanisms are reversed in old mares, suggesting a less efficient iron metabolism with advancing age. It is hoped that new investigations are needed to understand in depth and clarify further the complex metabolic and hormonal mechanisms involved also in equine species.