Volume 120, Issue 5, Pages 1193-1202 (April 2001) Compensatory phospholipid digestion is required for cholesterol absorption in pancreatic phospholipase A2–Deficient mice  Bonnie L. Richmond, Amy C. Boileau, Shuqin Zheng, Kevin W. Huggins, Norman A. Granholm, Patrick Tso, David Y. Hui  Gastroenterology  Volume 120, Issue 5, Pages 1193-1202 (April 2001) DOI: 10.1053/gast.2001.23254 Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 1 Cholesterol transport from intestinal lumen to lymphatics in rats. Lymph fistula rats were infused with an emulsion that consisted of 40 μmol triolein, 7.8 μmol of egg phosphatidylcholine, and 7.8 μmol [14C]cholesterol in 3.0 mL of phosphate-buffered saline in the absence (●) or presence of the PLA2 inhibitor FPL 67047XX (■). Lymph was collected for 1 hour before lipid infusion and served as the fasting lymph. After the onset of lipid infusion, lymph was collected every 2 hours during the first 4 hours and then hourly for the remaining time period. Aliquots of the lymph were taken for radioactivity determination by scintillation counting to determine the amount of radiolabel absorbed. Gastroenterology 2001 120, 1193-1202DOI: (10.1053/gast.2001.23254) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 2 Triglyceride transport from intestinal lumen to lymphatics in rats. Lymph fistula rats were infused with an emulsion that consisted of 40 μmol tri-[3H]olein, 7.8 μmol of egg phosphatidylcholine, and 7.8 μmol cholesterol in 3 mol of phosphate-buffered saline in the absence (●) or presence of the PLA2 inhibitor FPL 67047XX (■). A separate group of rats were infused with emulsion consisting of 177 μmol tri-[3H]olein, 15.6 μmol cholesterol, and 9.9 μmol of egg phosphatidylcholine (▴). Lymph was collected for 1 hour before lipid infusion and served as the fasting lymph. After the onset of lipid infusion, lymph was collected every 2 hours during the first 4 hours and then hourly for the remaining time period. Aliquots of the lymph were taken for radioactivity determination by scintillation counting to determine the amount of radiolabel absorbed. The data point represents the mean with standard deviation from 3 animals in each group. Gastroenterology 2001 120, 1193-1202DOI: (10.1053/gast.2001.23254) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 3 Polymerase chain termination reaction amplification of PLA2 gene from wild-type, and PLA2 gene–targeted mice. Genomic DNA was isolated from tail or ear punches of wild-type (lane 1), PLA2 heterozygous (lane 2), and PLA2 homozygous gene–targeted mice (lane 3), and then amplified by PCR with PLA2 oligonucleotide primers described in Table 1. The products were electrophoresed in polyacrylamide gels. The sizes of the PCR products were determined by comparison with molecular weight markers (Mr). The high molecular weight bands in lanes 2 and 3 represent the disrupted PLA2 gene products, whereas the lower molecular weight bands in lanes 1 and 2 represent the native undisrupted PLA2 amplification products. Gastroenterology 2001 120, 1193-1202DOI: (10.1053/gast.2001.23254) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 4 Immunoblot of pancreatic extract from wild-type and PLA2 gene–targeted mice. One hundred micrograms of total pancreatic protein from control PLA2(+/+), heterozygous PLA2(+/−), and homozygous PLA2(−/−) mice was electrophoresed in duplicates on a 10% sodium dodecyl sulfate–polyacrylamide gel and either stained with Coomassie Blue (A) or transferred to nitrocellulose and blotted with rabbit antirat PLA2 (B). The molecular weight standards (Mr) and the expected position of PLA2 are indicated. Gastroenterology 2001 120, 1193-1202DOI: (10.1053/gast.2001.23254) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 5 Phospholipid hydrolytic activity in pancreatic extract of wild-type and PLA2 gene–targeted mice. Micellar substrate was prepared by sonicating egg phosphatidylcholine mixed with cholate in a molar ratio of 0.75 in buffer containing 500 μmol/L Tris-HCl, pH 8.0, 135 mmol/L NaCl, and 1 mmol/L CaCl2. Fifty microliters of the substrate was incubated at 37°C with 50 μg of pancreatic protein from PLA2(+/+) (●) or PLA2(−/−) (○) mice and 15 μL of trypsin. The reaction was stopped by the addition of 15 μL of buffer containing 10 mmol/L sodium carbonate, 10 mmol/L sodium borate, pH 10. Hydrolytic activity was monitored based on the release of free fatty acids measured at OD of 550 nm. Gastroenterology 2001 120, 1193-1202DOI: (10.1053/gast.2001.23254) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 6 Cholesterol absorption efficiency in wild-type and PLA2 gene–targeted mice. The control wild-type PLA2(+/+) mice and their heterozygous PLA2(+/−) and homozygous PLA2(−/−) knockout littermates were each fed by stomach tube a 50-μL bolus lipid test meal consisting of 10 μCi/mL of [14C]cholesterol, 1 μCi/mL of [3H]sitostanol, 5 μg/mL of cholesterol, and 35 μg/mL of either phosphatidylcholine (2) or dioleyl ether phosphatidylcholine (▨). Feces were collected over a 24-hour period and extracted with chloroform/methanol to recover excreted sterols. The percentage of cholesterol absorbed was determined based on the amount of nonabsorbed [14C]cholesterol excreted in the feces after normalizing for recovery with the nonabsorbable sterol [3H]sitostanol. The data represent the mean ± SEM from 6 mice in each group for the phosphatidylcholine study and 10 mice in each group for the dioleyl ether phospholipid study. The bars with different letters are different significantly at P < 0.05. Gastroenterology 2001 120, 1193-1202DOI: (10.1053/gast.2001.23254) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 7 Phospholipid absorption from intestinal lumen to lymphatics in mice. Lymph fistula of control (●) and PLA2 knockout (○) mice were infused with a test meal containing 35 μg/mL of palmitoyl-2-[14C]oleoyl-phosphatidylcholine and 5 μg/mL of cholesterol. Lymph was collected for 1 hour before lipid infusion and served as the fasting lymph. After the onset of lipid infusion, lymph was collected every 2 hours during the first 4 hours and then hourly for the remaining time period. Aliquots of the lymph were taken for radioactivity determination by scintillation counting to determine the amount of radiolabel absorbed. The data represent mean with standard deviation from 4 wild-type and 3 PLA2(−/−) mice. Gastroenterology 2001 120, 1193-1202DOI: (10.1053/gast.2001.23254) Copyright © 2001 American Gastroenterological Association Terms and Conditions