Characterization of Acetyl-CoA: Lyso-PAF Acetyltransferase of Human Mesangial Cells

Platelet activating factor (PAF) is a potent inflammatory mediator produced by various renal cells and it is implicated in renal pathology. The aim of this study is the characterization of remodeling lyso-PAF acetyltransferase, which is activated under inflammatory conditions, in human mesangial cell. Total membranes of mesangial cells were isolated and enzymatic activity and kinetic parameters were determined by trichloroacetic acid precipitation method. The effect of BSA, divalent cations, EDTA, and various chemicals on the activity of lyso-PAF acetyltransferase was also studied. Various detergents were also tested for the solubilization of the enzyme and only glycerol did not affect its activity. Partial purification of solubilized enzyme preparations of human kidney tissue and mesangial cells was performed on anion exchange column chromatography and native-PAGE electrophoresis and two active fractions were detected

Characterization of the De Novo Biosynthetic Enzyme of Platelet Activating Factor, DDT-Insensitive Cholinephosphotransferase, of Human Mesangial Cells

Platelet activating factor (PAF), a potent inflammatory mediator, is implicated in several proinflammatory/inflammatory diseases such as glomerulonephritis, glomerulosclerosis, atherosclerosis, cancer, allergy, and diabetes. PAF can be produced by several renal cells under appropriate stimuli and it is thought to be implicated in renal diseases. The aim of this study is the characterization of DTT-insensitive cholinephosphotransferase (PAF-CPT) of human mesangial cell (HMC), the main regulatory enzyme of PAF de novo biosynthetic pathway. Microsomal fractions of mesangial cells were isolated and enzymatic activity and kinetic parameters were determined by TLC and in vitro biological test in rabbit washed platelets. The effect of bovine serum albumin (BSA), dithiothreitol (DTT), divalent cations (Mg2+ and Ca2+), EDTA, and various chemicals on the activity of PAF-CPT of HMC was also studied. Moreover, preliminary in vitro tests have been performed with several anti-inflammatory factors such as drugs (simvastatin, IFNa, rupatadine, tinzaparin, and salicylic acid) and bioactive compounds of Mediterranean diet (resveratrol and lipids of olive oil, olive pomace, sea bass “Dicentrarchus labrax,” and gilthead sea bream “Sparus aurata”). The results indicated that the above compounds can influence PAF-CPT activity of HMC

Associations of phase angle with platelet-activating factor metabolism and related dietary factors in healthy volunteers

IntroductionPhase angle (PA) is derived from bioelectrical impedance analysis (BIA). It reflects cell membrane function and decreases in disease. It is affected by inflammation, oxidative stress, and diet. Platelet-activating factor (PAF) is a potent inflammatory lipid mediator. Its levels, along with the activity of its metabolic enzymes, including CDP-choline:1-alkyl-2-acetyl-sn-glycerol-cholinephosphotransferase, acetyl-CoA:lyso-PAF-acetyltransferases, and PAF-AH/Lp-PLA2 are also related to dietary factors, such as the dietary antioxidant capacity (DAC). The aim of the study was to estimate whether the PAF metabolic circuit and related dietary factors are associated with PA in healthy volunteers.MethodsIn healthy subjects, PAF, its metabolic enzyme activity, and erythrocyte fatty acids were measured, while desaturases were estimated. Food-frequency questionnaires and recalls were used, and food groups, macronutrient intake, MedDietScore, and DAC were assessed. Lifestyle and biochemical variables were collected. DXA and BIA measurements were performed.ResultsLp-PLA2 activity was positively associated with PA (rho = 0.651, p < 0.001, total population; rho = 0.780, p < 0.001, women), while PAF levels were negatively associated with PA only in men (partial rho = −0.627, p = 0.012) and inversely related to DAC. Estimated desaturase 6 was inversely associated with PA (rho = −0.404, p = 0.01, total sample). Moreover, the DAC correlated positively with PA (rho = 0.513, p = 0.03, women). All correlations were adjusted for age, body mass index, and sex (if applicable).ConclusionPA is associated with PAF levels and Lp-PLA2 activity in a gender-dependent fashion, indicating the involvement of PAF in cell membrane impairment. The relationship of PA with DAC suggests a protective effect of antioxidants on cellular health, considering that antioxidants may inhibit PAF generation

Lipoprotein-associated phospholipase A2 (Lp-PLA2) activity, platelet-activating factor acetylhydrolase (PAF-AH) in leukocytes and body composition in healthy adults

<p>Abstract</p> <p>Background</p> <p>Lipoprotein-associated phospholipase A₂(Lp-PLA₂) also known as serum platelet activating factor acetylhydrolase (PAF-AH) activity constitutes a novel risk marker for cardiovascular disease. Leukocytes constitute one main cellular source of circulating Lp-PLA₂. The aim of the present study was to evaluate the association of both serum and leukocyte PAF-AH activities with fat distribution and lean tissue. One hundred healthy volunteers without cardiovascular disease history participated in this study (n = 52 men, 44 ± 13 years and n = 48 women, 43 ± 13 years). Body composition was assessed with dual-energy X-ray absorptiometry, while anthropometrical indices were also measured. The activity of Lp-PLA₂and levels of lipid and glycemic parameters were determined in fasting samples.</p> <p>Results</p> <p>Mean Lp-PLA₂activity was 24.8 ± 4.5 and 19.6 ± 5.0 nmol/min/mL in men and women, respectively (P < 0.001). Mean activity of PAF-AH in leukocyte homogenates was 386 ± 127 pmol/min/mg and 292 ± 92 pmol/min/mg in men and women, correspondingly (P < 0.001). In multiple regression models upper and total adiposity measures were positively associated with Lp-PLA₂activity in men after adjusting for LDL-cholesterol, age, smoking, hs-CRP and physical activity, whereas no associations were found with PAF-AH leukocyte homogenates activity. Hierarchical analysis revealed that the variables with the highest explanatory ability of Lp-PLA₂activity in men, were DXA deriving L1–L4 region of interest and arms fat (increase in R²= 0.136, P = 0.005 and increase in R²= 0.118, P = 0.009, respectively), followed by trunk fat and total fat. In women, no association of body composition variables with Lp-PLA₂nor PAF-AH leukocyte homogenates activity was found.</p> <p>Conclusion</p> <p>Lp-PLA₂activity is differentiated across levels of adiposity and topology of adipose tissue, whereas no association was found regarding PAF-AH leukocyte homogenates activity. Our findings suggest that Lp-PLA₂may compensate for the adiposity-associated increases in inflammatory and oxidative burden, in men.</p

Effect of differently fed farmed gilthead sea bream consumption on platelet aggregation and circulating haemostatic markers among apparently healthy adults:A double-blind randomized crossover trial

Fish consumption beneficially affects coagulation markers. Few dietary intervention studies have investigated differently fed farmed fish against these cardio-metabolic risk factors in humans. This double-blind randomized crossover trial evaluated differently fed farmed gilthead sea bream consumption against platelet aggregation and circulating haemostatic markers among apparently healthy adults. Subjects aged 30–65 years, with a body mass index 24.0–31.0 kg/m2, consuming less than 150 g cooked fish per week, were recruited in Attica, Greece. Participants were randomized (n = 38, 1:1) to one of two sequences; consumption of fish fed with fish oil diet (conventional fish, CF)/fish fed with olive pomace-enriched diet (enriched fish, EF) versus EF/CF. The primary outcomes were ex vivo human platelet aggregation and circulating plasminogen activator inhibitor-1 (PAI-1) and P-selectin (sP-selectin) concentrations. EF consumption had no significant effect on platelet sensitivity or haemostatic markers compared to CF. Platelet sensitivity to platelet-activating factor (PAF) decreased after CF consumption during the second period (p p < 0.01 for both). Based on current findings, consumption of enriched farmed gilthead sea bream had no greater effect on coagulation markers in adults compared to the conventionally fed fish

Metabolism and actions of platelet activating factor in human renal tissue and renal cells

Platelet activating factor (PAF) is a potent inflammatory mediator that is produced by a variety of cells. It is biosynthesized either by remodeling pathway from membrane phospholipids either by de novo pathway of glyceryl-ether lipids. Remodeling pathway is activated in inflammatory conditions while de novo pathway produces PAF under physiological conditions. The study of PAF metabolism enzymes is of great important since they are responsible for pathological and physiological PAF levels. Several studies indicated PAF participation in the pathogenesis of many renal diseases. Glomerulosclerosis (GS) is the common pathological feature in most renal diseases and is highly correlated with the progression of renal failure. Although numerous attempts have been made to elucidate the mechanisms underlying glomerulosclerosis, the precise pathogenesis remains undetermined. GS and atherosclerosis are thought to share a common pathogenesis and the term ‘Glomerular atherosclerosis’ has been proposed. Unregulated intracellular lipid accumulation is thought to be critical step in the development of the two diseases and may happen through scavenger receptors or dysregulation of LDL receptors. The aim of the study is 1. To exam the enzyme acetyl-CoA:lyso-PAF acetyltransferase (lyso-PAF AT) of remodeling pathway in human kidney and mesangial cells. In order to do that yhe fraction of total membranes, from human renal tissue and from human mesangial cell line (HMC), was obtained and enzymatic activity was determined by trichloroacetic acid precipitation method. The optimal reaction conditions were estimated for lyso-PAF AT from HMC, more specific optimal temperature was 37οC, optimal pH 7.2-7.4 and concentrations of lyso-PAF and acetyl-CoA 20 μΜ and 200 μΜ respectively. The values of Κm (μΜ) and Vmax (nmol/min/mg) were determined with respect to lyso-PAF 45.2 ± 9.37, 9.37 ± 3.1 and with respect to acetyl-CoA 71.2 ± 15.1, 2.83 ± 0.252, respectively. High concentrations of Mg2+ and Ca2+ partly inhibit enzyme action. EDTA also inhibits the enzyme action and this inhibition is reversible with exogenous addition of Ca2+. BSA, Naf and pefabloc does not influence the activity while inhibition was observed with addition of DTT and mercaptoethanol. Various detergents were tested for the 189 solubilization of enzyme from human kidney and mesangial cells. Only glycerol successfully solubilaze the enzyme without inactivation. Solubilized fractions were successfully purified on HPLC with anion exchange column. Solubilazed fraction from human kidney and active fractions from HPLC from HMC were subjects in native-PAGE. Two active were arisen from both in region of MW 25-30 kDa and 65-80 kDa. 2. To investigate the mechanism of PAF implication in glomerulosclerosis and especially in mesangial cells function. In order to do that the effect of PAF in intracellular lipid accumulation and in activation of the pro-inflammatory transcription factor NF-kB was investigated. A single class of PAF receptor was identified with Kd = 17.5 nM and Βmax = 4.01 pmol/mg of cell protein. Morphological experiments reveal that LDL is able foam cell formation in the presence to cause that PAF (10-6 M). This formation is inhibited by heparin and by BN520121 ahile polyinosic acid did not affect it. In addition PAF (10-6 M) increase levels of LDLr mRNA within 2h (peaked in 12h) even in the presence of excess LDL suggesting that PAF overrides the sterol mediated regulation of LDLr. Lower concentration of PAF (10-9 M) increased the expression of SCr within 2h (peaked at 6h). The effect of various signal transduction inhibitors in PAF action was studied. Moreover PAF (10-9 & 10-6 M) caused NF-kB activation in mesangial cells within 0.5 h and this activation persisted for 24 h. In addition PAF increases mRNA of its receptor possible through activation of NF-kB since promotor 1 of PAFR was detected. This data suggests that PAF may contribute to the progression of renal injury by causing lipid accumulation and activation of pro-inflammatory transcription factor pathways in mesangial cells.Ο Παράγοντας Ενεργοποίησης των αιμοπεταλίων (PAF) είναι ένας ισχυρός φλεγμονώδης παράγοντας λιποειδικής φύσης που παράγεται από πληθώρα διεγερμένων κυττάρων μέσω δυο πορειών. Η εξ’ υπαρχής πορεία (de novo) είναι υπεύθυνη για τα βασικά επίπεδα του PAF ενώ η πορεία ανασχηματισμού (remodeling) ενεργοποιείται σε καταστάσεις φλεγμονής. Η μελέτη των ενζύμων μεταβολισμού του PAF είναι σημαντική αφού αυτά καθορίζουν τα επίπεδα του σε παθοφυσιολογικές καταστάσεις. Πολλές μελέτες, συμπεριλαμβανομένου μελέτες σε πειραματόζωα, καλλιέργειες νεφρικών κυττάρων και κλινικές μελέτες, επισημαίνουν τον κύριο ρόλο του PAF στις νεφρικές παθήσεις. Η σπειραματοσκλήρυνση (GS) είναι το κοινό χαρακτηριστικό των περισσότερων νεφρικών παθήσεων και σχετίζεται άμεσα με τη εξέλιξη αυτών σε νεφρική ανεπάρκεια. Παρόλο τον μεγάλο αριθμό μελετών δεν έχει ακόμα διευκρινιστεί ο ακριβής μηχανισμός πρόκλησης της GS. Τα τελευταία χρόνια μια σειρά μελετών επισημαίνουν ότι η αθηροσκλήρυνση και η σπειραματοσκλήρυνση παρουσιάζουν κοινά ιστολογικά χαρακτηριστικά και έχει προταθεί ο όρος «σπειραματική αθηροσκλήρυνση». Από το εργαστήριο μας έχει προταθεί μηχανισμός εμπλοκής του PAF στην πρόκληση της αθηροσκλήρυνσης και δεδομένου της ομοιότητας ανάμεσα στις δυο νόσους ο μηχανισμός αυτός φαίνεται ότι βρίσκει εφαρμογή και στην GS. Η ανεξέλεγκτη ενδοκυτταρική συσσώρευση λιποειδών στο εσωτερικό των κυττάρων οδηγεί στον σχηματισμό αφρωδών και πιστεύεται ότι παίζει σημαντικό ρόλο στην εξέλιξη των δυο ασθενειών. Η συσσώρευση μπορεί να γίνει τόσο μέσω των εκκαθαριστών υποδοχέων για την τροποποιημένη LDL όσο και μέσω δυσλειτουργίας των υποδοχέων για την μη τροποποιημένη LDL. Σκοπός της εργασίας ήταν να μελετήσει: 1. Το ένζυμο ακετυλο-CoA:λυσο-PAF ακετυλοτρανσφεράση (λυσο-PAF AT), της remodeling πορείας βιοσύνθεσης του PAF, το οποίο ενεργοποιείται σε καταστάσεις φλεγμονής σε ανθρώπινο νεφρικό ιστό και καλλιέργειες μεσαγγειακών κυττάρων. Για το σκοπό αυτό απομονώθηκαν οι ολικές μεμβράνες από νεφρικό ιστό που προέρχεται από νεφρεκτομηθέντες ασθενείς και από καλλιέργειες κυτταρικής σειράς ανθρώπινων μεσαγγειακών κυττάρων (HMC) και μετρήθηκε η δραστικότητα της λυσο-PAF-AT με τη μέθοδο του τριχλωροξικού οξέος. Στην περίπτωση των HMC καθορίστηκαν οι βέλτιστες συνθήκες για τη δράση του ενζύμου συγκεκριμένα : βέλτιστη θερμοκρασία επώασης 37οC, βέλτιστο pH 7.2-7.4, βέλτιστη 187 συγκέντρωση λυσο-PAF και ακετυλο-CoA 20 μΜ και 200 μΜ αντίστοιχα. Στις βέλτιστες συνθήκες υπολογίστηκε το φαινόμενο Κm (μΜ) και Vmax (nmol/min/mg) 45.2 ± 9.37, 9.37 ± 3.1 ως προς τον λυσο-PAF και 71.2 ± 15.1, 2.83 ± 0.252 ως προς το ακετυλο-CoA,, αντίστοιχα. Τα ιόντα Μg 2+ , Ca 2+ σε μεγάλη συγκέντρωση προκαλούν μερική αναστολή του ενζύμου ενώ το EDTA προκαλεί αναστολή που μειώνεται με προσθήκη εξωγενούς Ca 2+. Η BSA, το NaF και το pefabloc δε επηρεάζουν σημαντικά τη δραστικότητα του ενζύμου, ενώ το DTT και η μερκαπτοαιθανόλη το αναστέλλουν. Για τη διαλυτοποίηση του ενζύμου από τις ολικές μεμβράνες τόσο από νεφρικό ιστό όσο και από HMC δοκιμάστηκαν διάφορα απορρυπαντικά. Μόνο η γλυκερόλη κατάφερε να διαλυτοποιήση το ένζυμο χωρίς να το απενεργοποιήσει με απόδοση περίπου 35 %. Το διαλυτοποιημένο κλάσμα καθαρίστηκε περαιτέρω σε στήλη ανιοανταλλαγής (HPLC). Στα κλάσματα της HPLC εντοπίστηκε ενζυμική δραστικότητα. Επίσης πραγματοποιήθηκε μη αποδιατακτική ηλεκτροφόρηση στο διαλυτοποιημένο κλάσμα από το νεφρικό ιστό και στα δραστικά κλάσματα από την HPLC στα HMC και βρέθηκαν δυο περιοχές με δραστικότητα λυσο-PAF AT σε περιοχές μοριακών βαρών 25-30 kDa και 65-80 kDa. 2. Η διερεύνηση της εμπλοκής του PAF στο μηχανισμό πρόκλησης και εξέλιξης της νεφρικής βλάβης και συγκεκριμένα στις λειτουργίες των μεσαγγειακών κυττάρων. Για το σκοπό αυτό χρησιμοποιήθηκε κυτταρική σειρά ανθρώπινων μεσαγγειακών κυττάρων. Ανιχνεύτηκε ένα είδος PAF υποδοχέα με Kd = 17.5 nM και Βmax = 4.01 pmol/mg πρωτεΐνης. Μορφολογικά παρατηρήθηκε ότι η LDL παρουσία αντιοξειδωτικών και PAF 10-6 M μπορεί να σχηματίσει αφρώδη κύτταρα. Η δημιουργία των αφρωδών κυττάρων οφείλεται στις συγκεκριμένες συνθήκες σε δράση των υποδοχέων της LDL αφού αναστέλλεται από την ηπαρίνη, που μπλοκάρει τους LDL υποδοχείς, και από το BN52021 που είναι αναστολέας του PAF, ενώ δεν επηρεάζεται από το πολυϊνοσικό οξύ που μπλοκάρει τους εκκαθαριστές υποδοχείς. Τα παραπάνω ενισχύονται αφού PAF 10-6 Μ προκαλεί αύξηση στα επίπεδα mRNA του υποδοχέα LDL στις 6-12 ώρες επώασης ενώ επιπλέον ο PAF φαίνεται ότι υπερκαλύπτει την επαγόμενη καταστολή στα επίπεδα του mRNA του υποδοχέα LDL από τα υψηλά επίπεδα χοληστερόλης. Επιπλέον ο PAF αυξάνει τα επίπεδα του εκκαθαριστή υποδοχέα SRAI με μέγιστη αύξηση να παρατηρείται σε συγκέντρωση 10-9-10-10 Μ PAF και στις 6-24 ώρες επώασης. Ο ίδιος ο PAF σε συγκέντρωση 10-6-10-11 Μ προκαλεί θετική ρύθμιση του υποδοχέα του σε 6 ώρες επώασης, ενώ και τα δύο είδη υποκινητών, που ελέγχουν τη μεταγραφή του υποδοχέα του PAF, ανιχνεύτηκαν στα μεσαγγειακά κύτταρα. Επιπλέον ο PAF (10-6 και 10-9 Μ) ενεργοποιεί τον μεταγραφικό παράγοντα NF-κΒ μέσα σε 30 λεπτά. Αφού ο υποκινητής 1 περιέχει αλληλουχία δέσμευσής του NF-κΒ είναι πιθανό η θετική ρύθμισή στον υποδοχέα του να γίνεται μέσω του NF-κΒ. Τα στοιχεία αυτά υποδηλώνουν ότι ο PAF παίζει σημαντικό ρόλο στην πρόκληση και εξέλιξη της νεφρικής βλάβης μέσω του σχηματισμού μεσαγγειακών αφρωδών κυττάρων και την ενεργοποίηση του μεταγραφικού παράγοντα NF-κΒ

Exploring Human Metabolome after Wine Intake—A Review

Wine has a rich history dating back to 2200 BC, originally recognized for its medicinal properties. Today, with the aid of advanced technologies like metabolomics and sophisticated analytical techniques, we have gained remarkable insights into the molecular-level changes induced by wine consumption in the human organism. This review embarks on a comprehensive exploration of the alterations in human metabolome associated with wine consumption. A great number of 51 studies from the last 25 years were reviewed; these studies systematically investigated shifts in metabolic profiles within blood, urine, and feces samples, encompassing both short-term and long-term studies of the consumption of wine and wine derivatives. Significant metabolic alterations were observed in a wide variety of metabolites belonging to different compound classes, such as phenolic compounds, lipids, organic acids, and amino acids, among others. Within these classes, both endogenous metabolites as well as diet-related metabolites that exhibited up-regulation or down-regulation following wine consumption were included. The up-regulation of short-chain fatty acids and the down-regulation of sphingomyelins after wine intake, as well as the up-regulation of gut microbial fermentation metabolites like vanillic and syringic acid are some of the most important findings reported in the reviewed literature. Our results confirm the intact passage of certain wine compounds, such as tartaric acid and other wine acids, to the human organism. In an era where the health effects of wine consumption are of growing interest, this review offers a holistic perspective on the metabolic underpinnings of this centuries-old tradition