Activity
of Lipoprotein
L i p a s e in A d i p o s e T i s s u e s f r o m S t e e r s D. R. RAO 1 and G. E. HAWKINS Department of Animal and Dairy Sciences Auburn University Auburn, AL 36830
ABSTRACT
Mean lipoprotein lipase activities (/Jeq fatty acids released/h per g tissue) and lipid content (%) in adipose tissue of steers from brisket, perirectal, subcutaneous abdominal, and mesenteric areas were 32, 32, 30, 50, and 73, 77, 64, and 85. The enzyme activity was higher in mesenteric tissue than in the other sites which did not differ from each other. Mesenteric adipose tissue had higher and subcutaneous abdominal adipose tissue had lower lipid content than did brisket and perirectal adipose tissue. Lipid content and enzyme activity were correlated. 38. INTRODUCTION
Lipoprotein lipase (LPL) catalyzed the hydrolysis of circulating triglyceride moieties of lipoproteins into glycerol and free fatty acids before the uptake of fatty acids by tissue cells (1, 7, 14). Thus, lipoprotein lipase plays a key role in deposition of fat in tissues and milk fat synthesis by the mammary cells. The activity of this enzyme has been reported in the tissues of steers and many species (9). However, the relative activity of lipoprotein lipase in various sites of adipose tissue from steers was not known. We have studied activities of this enzyme and lipid content in four sites of adipose tissue from steers. A preliminary report was in (12). MATERIALS AND METHODS
Samples of adipose tissue were taken from brisket, perirectal, subcutaneous abdominal, and mesenteric areas immediately after slaughter of eight steers at the meats laboratory at Auburn University. The three Angus, two Hereford, and three crossbreds were 436 to 602
Received May 19, 1975. 1Department of Food Science and Technology, Alabama A&M University, Normal 35762.
days of age and weighed 373 to 457 kg at slaughter. The samples were rinsed in ice-cold .15 M KCL, frozen immediately on dry ice, and subsequently stored at - 1 4 C for 3 to 20 days before assay. The enzyme activity did not change when the tissue was frozen for at least 3 wk. Preparation of Tissue for Lipoprotein Lipase Assay
The method of McBride and Korn (11) as modified by Askew et al. (1) was used to prepare the adipose tissue for LPL assay. A weighed amount of frozen tissue was cut into small pieces, placed in 10 volumes of cold .15 M KCL, and homogenized (VirTis, Model 45) at 35,000 rpm for 1 min. After centrifugation at 800 x g for 10 rain, the supernatant fluid was decanted through glass wool to remove fat and cellular debris. The resulting filtrate formed the source of lipoprotein lipase. Initial velocity studies indicated that .1 to 1.0 ml of this filtrate h y d r o l y z e d the substrate linearly up to 60 min at 38 C. All extraction operations were at 0 C . Preparation of Substrate
The substrate was prepared from a commercial coconut oil emulsion, Ediol (Lipostrate-CB, stable 50% emulsion of coconut oil; Cal Biochem, Los Angeles, CA). To one part of pure Ediol, six parts of distilled water were added. This diluted Ediol was "activated" by incubating with an equal volume of fresh cow serum in a screw capped vial for 30 min at 38 C in a water bath equipped with a shaker. Activated Ediol served as the substrate for the assay of lipoprotein lipase. Lipoprotein Lipase Assay
The incubation mixture consisted of 1.0 ml of 10% bovine serum albumin (Fraction V; Sigma Chemical Co., St. Louis, MO) adjusted to pH 8.5 with concentrated NH4OH, .3 ml of substrate, and .5 ml of enzyme preparation. 161
162
RAO AND HAWKINS
The mixture was incubated in screw capped vials at 38 C for 30 rain in a shaking water bath (80 oscillations/min). Since there was no difference in titration value between boiled enzyme preparation and .15 M KCL, .5 ml of the latter was added to all control vials. Free fatty acids were extracted and titrated according to the method of Dole and Meinertz (4). Total Iipids and protein in adipose tissue were determined according to Folch (6) and Lowry et al. (10), respectively. Statistical analyses were as outlined by Steele and Torrie (15).
e~
¢4,Z
o6r~
÷1
+1
+~
+1
+1
+1
+1
+L
RESULTS AND DISCUSSION
The mean lipid and protein content and the lipoprotein lipase activity in brisket, perirectal, subcutaneous abdominal, and mesenteric adipose tissues are in Table 1. The percentage of lipids in the mesentery was higher (P.05). The LPL activity per gram of wet tissue in the mesentery was higher (P
of Lipoprotein
L i p a s e in A d i p o s e T i s s u e s f r o m S t e e r s D. R. RAO 1 and G. E. HAWKINS Department of Animal and Dairy Sciences Auburn University Auburn, AL 36830
ABSTRACT
Mean lipoprotein lipase activities (/Jeq fatty acids released/h per g tissue) and lipid content (%) in adipose tissue of steers from brisket, perirectal, subcutaneous abdominal, and mesenteric areas were 32, 32, 30, 50, and 73, 77, 64, and 85. The enzyme activity was higher in mesenteric tissue than in the other sites which did not differ from each other. Mesenteric adipose tissue had higher and subcutaneous abdominal adipose tissue had lower lipid content than did brisket and perirectal adipose tissue. Lipid content and enzyme activity were correlated. 38. INTRODUCTION
Lipoprotein lipase (LPL) catalyzed the hydrolysis of circulating triglyceride moieties of lipoproteins into glycerol and free fatty acids before the uptake of fatty acids by tissue cells (1, 7, 14). Thus, lipoprotein lipase plays a key role in deposition of fat in tissues and milk fat synthesis by the mammary cells. The activity of this enzyme has been reported in the tissues of steers and many species (9). However, the relative activity of lipoprotein lipase in various sites of adipose tissue from steers was not known. We have studied activities of this enzyme and lipid content in four sites of adipose tissue from steers. A preliminary report was in (12). MATERIALS AND METHODS
Samples of adipose tissue were taken from brisket, perirectal, subcutaneous abdominal, and mesenteric areas immediately after slaughter of eight steers at the meats laboratory at Auburn University. The three Angus, two Hereford, and three crossbreds were 436 to 602
Received May 19, 1975. 1Department of Food Science and Technology, Alabama A&M University, Normal 35762.
days of age and weighed 373 to 457 kg at slaughter. The samples were rinsed in ice-cold .15 M KCL, frozen immediately on dry ice, and subsequently stored at - 1 4 C for 3 to 20 days before assay. The enzyme activity did not change when the tissue was frozen for at least 3 wk. Preparation of Tissue for Lipoprotein Lipase Assay
The method of McBride and Korn (11) as modified by Askew et al. (1) was used to prepare the adipose tissue for LPL assay. A weighed amount of frozen tissue was cut into small pieces, placed in 10 volumes of cold .15 M KCL, and homogenized (VirTis, Model 45) at 35,000 rpm for 1 min. After centrifugation at 800 x g for 10 rain, the supernatant fluid was decanted through glass wool to remove fat and cellular debris. The resulting filtrate formed the source of lipoprotein lipase. Initial velocity studies indicated that .1 to 1.0 ml of this filtrate h y d r o l y z e d the substrate linearly up to 60 min at 38 C. All extraction operations were at 0 C . Preparation of Substrate
The substrate was prepared from a commercial coconut oil emulsion, Ediol (Lipostrate-CB, stable 50% emulsion of coconut oil; Cal Biochem, Los Angeles, CA). To one part of pure Ediol, six parts of distilled water were added. This diluted Ediol was "activated" by incubating with an equal volume of fresh cow serum in a screw capped vial for 30 min at 38 C in a water bath equipped with a shaker. Activated Ediol served as the substrate for the assay of lipoprotein lipase. Lipoprotein Lipase Assay
The incubation mixture consisted of 1.0 ml of 10% bovine serum albumin (Fraction V; Sigma Chemical Co., St. Louis, MO) adjusted to pH 8.5 with concentrated NH4OH, .3 ml of substrate, and .5 ml of enzyme preparation. 161
162
RAO AND HAWKINS
The mixture was incubated in screw capped vials at 38 C for 30 rain in a shaking water bath (80 oscillations/min). Since there was no difference in titration value between boiled enzyme preparation and .15 M KCL, .5 ml of the latter was added to all control vials. Free fatty acids were extracted and titrated according to the method of Dole and Meinertz (4). Total Iipids and protein in adipose tissue were determined according to Folch (6) and Lowry et al. (10), respectively. Statistical analyses were as outlined by Steele and Torrie (15).
e~
¢4,Z
o6r~
÷1
+1
+~
+1
+1
+1
+1
+L
RESULTS AND DISCUSSION
The mean lipid and protein content and the lipoprotein lipase activity in brisket, perirectal, subcutaneous abdominal, and mesenteric adipose tissues are in Table 1. The percentage of lipids in the mesentery was higher (P.05). The LPL activity per gram of wet tissue in the mesentery was higher (P
