Platelet-Activating Factor (PAF) Antagonists in Foods: A Study of Lipids with PAF or Anti-PAF-like Activity in Cow’s Milk and Yogurt Smaragdi Antonopoulou, † Charis E. Semidalas, ‡ Stamatis Koussissis, ‡ and Constantinos A. Demopoulos* ,† Department of Chemistry, University of Athens, Panepistimioupolis 15771, Athens, Greece, and School of Food Technology and Nutrition, Technological Educational Institute of Athens, Agiou Spyridonos, Egaleo, Greece Yogurt, a food with indisputable nutritional value, is also used for therapeutic purposes. Milk derivatives are blamed for some pathological effects of yogurt noted in selected subjects such as sensitive newborns or infants. In this study, we investigated the probable existence of platelet- activating factor (PAF) and lipids inhibiting PAF action in raw and incubated milk and yogurt. Detection of these substances may explain the controversial properties of these milk products. The in vitro biological study of lipids in washed platelets showed little production of PAF in incubated milk (0.3-0.8 ng/100 mL sample) although the concentration of PAF in milk fat remained constant during the majority of the incubation time. Yogurt lipids of intermediate polarity presented stronger inhibitory activity against PAF than lipids corresponded to raw or incubated milk. Our data demonstrated that Streptococcus thermophilus and Lactobacillus bulgaricus biosynthesize important quantities of PAF inhibitors, whereas random contamination of milk leads to the production of small amounts of PAF and PAF inhibitors. Keywords: Platelet activating factor (PAF); PAF inhibitors; lipids; yogurt; milk bacteria INTRODUCTION Platelet activating factor (PAF; 1-O-alkyl-2-acetyl-sn- glycero-3-phosphocholine) is one of the most potent inflammatory phospholipid mediators (Demopoulos et al., 1979) that is synthesized by and that acts on most proinflammatory cells (Hanahan and Kumar, 1987). The role of PAF in modulation of the immune response has been well documentated, but it also exhibits profound effects on the physiology and pathology of lung, kidney, blood vessels, heart, and digestive tract, including lather gastric ulcers and intestinal damage (Koltai et al., 1990). Two metabolic steps are involved in the biosynthesis of PAF. The action of phospholipase A 2 on membrane alkyl-acyl phosphorylcholine lipids results in the pro- duction of lyso-PAF, and acetylation of the lyso- compound by an acetyltransferase yields the biologically active molecule. In addition to mammals, a lot of bacteria, including pathogenic ones, are able to produce PAF with exogenous lyso-PAF (Denizot et al., 1989). The antibacterial action of fermented milk and lactic drinks is known to cure mild cases of diarrhea in patients who have consumed pathogenic bacteria, and yogurt has been used since ancient times to prevent diarrhea and intestinal problems. Yogurt is also very effective against lactose intolerance, and many studies since 1973 have shown an antitumor activity of this product. It is believed that all these actions are due to the live bacteria of the yogurt, mainly Lactobacillus species, and to the highly digestible nutrients that cause a normalizing of the intestinal microflora and an increase in the Bifidobacterium population (Yukushi et al., 1992). Denizot and Benveniste (1989) detected PAF and large amounts of immediate PAF precursors, such as alkyl-acylglycerophosphocholine and lyso-PAF, in com- mercial cheese and yogurt samples. The presence of PAF in these products (1 ng/100 g of sample) is less than the necessary amount for any pathological effects in humans (10 ng/kg). However, considering the immu- noregulation effects of PAF and the putative effect of yogurt on the immune response (De Simone et al., 1986), we believe that a relation between these two properties could be postulated. It remains to be ascertained whether or not the presence of PAF in dairy products can explain some of the pathological effects of milk derivatives in selected subjects, such as sensitive new- borns or infants. In this work, which is part of a study to investigate the relation between PAF and foods (Koussisis et al., 1993, 1994), lipid fractions of cow’s milk and yogurt were tested for PAF or anti-PAF like activity in washed rabbit platelets. The presence of these substances was related to the random infection and the increasing bacterial population in the dairy samples. MATERIALS AND METHODS Solvents and Reagents. All solvents used were of ana- lytical grade and were purchased from Merck (Darmstadt, Germany). Standard lipids were purchased from Sigma (St. Louis, MO), Merck, or Serva (Heidelberg, Germany). Semi- synthetic PAF was synthesized in our laboratories (Demopou- los et al., 1979). Thin-Layer Chromatography (TLC) Plates. The 20 × 20-cm glass TLC plates were coated with silica gel G and acti- vated by heating at 130 °C for 30 min. The thickness of the TLC plates was between 0.5 (analytical) and 1.0 mm (prepara- tive). Up to 50 mg of lipids per TLC plate were used for frac- tionation; every fraction was scraped off, extracted with var- ious mixtures of chloroform and methanol, and centrifuged, and the liquid phase was evaporated under a nitrogen atmo- sphere. Sample Characteristics. A total of four milk samples were examined, each one in triplicate. The raw cow’s milk samples used were collected within the first 48 h after milking * Author to whom correspondence should be ad- dressed (telephone +301-8648963; fax +301-32183040. † University of Athens. ‡ School of Food Technology and Nutrition. 3047 J. Agric. Food Chem. 1996, 44, 3047-3051 S0021-8561(95)00619-4 CCC: $12.00 © 1996 American Chemical Society