ELSEVIER Biochimica et Biophysica Acta 1300 (1996) 42-48 BB Biochi~ic~a et Biophysica A~ta Identification of an alkaline sphingomyelinase activity in human bile O Lena Nyberg a, Rui-Dong Duan b,, , Jan Axelson c, Ake Nilsson d a Swedish Dairies' Association, S-223 70 Lund. Sweden b Center of Experimental Research. Cell Biology Deparmwnt 1. {h2ilersity Hospital, S-221 85 Lund. Sweden Department of Surgetlv, Unit,ersity Ho~j~ital, S-221 85 Lund. Sweden d Gasttwenlerology Diz'ision, Department ~['Medicine, Unit'ersiO' Hospital. S-221 85 Lund, Sweden Received 19 June 1995; revised 10 October 1995; accepted 7 December 1995 Abstract The hydrolysis of sphingomyelin has been found to generate important signals regulating cell proliferation, differentiation and apoptosis. However, the enzymes responsible for digestion of dietary sphingomyelin have not been well documented. This study demonstrates the occurrence of a sphingomyelinase (SMase) in both human hepatic bile and gallbladder bile. The enzyme was equally found in both bacteria negative and positive bile samples and in samples obtained from patients with or without gallbladder diseases. A bacteria-free gallbladder bile was used for characterization. It was found that bile SMase hydrolyzed sphingomyelin to phosphorylcholine and ceramide with negligible activity against either phosphatidylcholine or p-nitrophenyl phosphate. The enzyme preferred an alkaline condition and the optimal pH was 9. The activity of this alkaline SMase was bile salt dependent and was fully activated by 4 6 mM bile salts. Triton X-100, the non-ionic detergent did not activate bile SMase. Ca 2+ and Mg 2+ ions had no significant effect at optimal bile salt concentration. The molecular mass of this enzyme was about 85 kDa as measured by Sephadex G200 gel chromatography. In conclusion, we demonstrated a SMase in bile which differs markedly from the known acid and neutral SMase. Its potential important roles in sphingomyelin digestion and gallbladder diseases require further investigation. Keywords: Sphingomyelinase; Bile; (Human) 1. Introduction The metabolism of sphingomyelin (SM) has received considerable attention in recent years, mainly because SM hydrolysis products have important signalling effects on multiple cellular functions, particularly on cell prolifera- tion and differentiation [1,2]. Furthermore, the metabolism of SM is closely related to the mobilization and esterifica- tion of cholesterol [3,4], a key component for cardio- vascular diseases and gastrointestinal diseases such as cholelithiasis [5]. SM is hydrolyzed by sphingomyelinase (SMase), which cleaves SM to ceramide and phospboryl- choline. Two types of SMases have been identified. One is the lysosomal acid SMase with an optimal pH around 5 [6], and the other is neutral SMase, which is Mg 2+ depen- dent and present mainly in plasma membranes with opti- mal pH 7.5 [7]. SM is also a component of the normal diet. In bovine milk SM comprises 0.2% of the milk fat, occurring mainly * Corresponding author. Fax: +46 46 137277. 0005-2760/96/$15.00 ~ 1996 Elsevier Science B.V. All rights reserved SSDI 0005-2760(95)00245-6 in the fat globule membrane [8,9]. It is also found in meat and sea food [10]. In contrast to those well studied cellular SMases, little is known about the SMase responsible for digestion of SM in gastrointestinal tract. Our knowledge on gastrointestinal SMase is largely based on studies per- formed more than two decades ago by Nilsson [1 1,12], who demonstrated SMase and ceramidase activity in hu- mall intestinal content and rat intestinal mucosa and an active interconversion of the sphingosine portion to palmitic acid. Recently, Schmelz et al. extended these observations and found that digestion of SM occurred at most levels of the intestinal tract [13]. Several lines of evidences have pointed to the importance of the digestion and absorption of SM in the gastrointestinal tract. For example, dietary SM was found to inhibit the uptake of cholesterol by enterocytes [14] and administration of SM suppressed the promotion of chemical carcinogen-induced colon cancer in experimental mice [ 15]. In this study we demonstrated the presence of a SMase activity in human bile, which is different from neutral and acid SMases and may potentially be important for both SM digestion and gallbladder diseases.