Whole-genome association study of fatty acid composition in a diverse range of beef cattle breeds.

Fatty acid composition of adipose tissue associated with meat is an important factor for the beef industry because of its implications for human health, processing, meat quality, and palatability. Individual fatty acid composition is a trait under genetic control, so improvement via selective breeding of cattle is possible. The objective of this study was to investigate the genetic architecture of fatty acid composition and identify genes associated with this trait in 3 breed types: Bos indicus (Brahman), Bos taurus (4 breeds), and tropically adapted composites (2 breeds). Using high-density data, regions on chromosomes 1, 9, 14, 16, 19, 23, 26, 29, and X were associated with fat composition and quantity traits. Known candidate genes, such as fatty acid synthase (FASN; chromosome 19) and stearoyl-CoA desaturase (SCD; chromosome 26), were confirmed in our results. Other candidate genes and regions represent novel association results, requiring further validation.

Enriching M. sternomandibularis with alpha-tocopherol by dietary means does not protect against the lipid oxidation caused by high-pressure processing.

We hypothesized that elevating the concentration of alpha-tocopherol in beef muscle tissue by dietary means would increase lipid stability following high-pressure processing. Beef M. sternomandibularis was obtained from cattle that had medium (4.92 microg/g) and high (7.30 microg/g) concentrations of alpha-tocopherol. Post-rigor, paired muscles samples were subjected to pressures of 0.1 (atmospheric), 200 or 800 MPa for 20 min at approximately 60 degrees C. Following high-pressure processing, measurements were made immediately (d 0) or on samples stored in the dark for 6 d at 4 degrees C (d 6). Intramuscular lipid was similar for each group (4.02% vs. 4.26%, respectively; P=0.78), but lipid from the medium alpha-tocopherol muscle was more saturated and less monounsaturated than muscle from the high alpha-tocopherol group. High-pressure processing at 800 MPa and 60 degrees C did not reduce the amount of alpha-tocopherol but significantly reduced the concentration of linoleic acid (18:2n-6) in muscle from both production groups of cattle. Thiobarbituric acid reactive substances increased linearly with treatment pressure only in d 6 samples (day x pressure interaction P=0.0001) and were higher overall (P=0.02) in the high alpha-tocopherol muscle than in the medium alpha-tocopherol muscle. At d 6, lipid peroxides were decreased (P=0.007) by high-pressure treatment and were higher (P<0.0001) in the high alpha-tocopherol group than in the medium alpha-tocopherol group. Therefore, muscle from the high alpha-tocopherol cattle in this study had a greater accumulation of lipid peroxides by d 6, making the muscle from those cattle more susceptible to oxidation.

National sampling and gross composition of Australian retail pork cuts 2005/2006.

After consideration of five potential sampling designs, 13 retail pork cuts were purchased from randomly selected supermarkets and butchers' stores in urban areas across the socioeconomic scale in three States of Australia in late 2005 and early 2006. They were analysed, raw and cooked, for gross composition (fat, lean, bone and gristle). Gross composition varied considerably within cut associated with large divergences in interpretation of standard pork cuts by butchers. There were no notable differences in gross composition across States, across the socioeconomic range of suburbs of purchase or between outlet types (butcher vs supermarket). Cuts tended to be larger and leaner than those in similar studies in the 80s and 90s. Due to increasing uniformity in breeding and feeding of pigs in Australia, sampling designs in future surveys could be simplified.

Effect of glycogen concentration and form on the response to electrical stimulation and rate of post-mortem glycolysis in ovine muscle.

The associations between the muscle glycogen concentration and form and the rate of post-mortem glycolysis in ovine muscle were investigated. Twenty-two merino wethers (18-24 months) were allocated to either roughage or concentrate pelleted diets for 34 days prior to slaughter. An exercise depletion/repletion model was applied four days prior to slaughter to generate differences in muscle glycogen levels at slaughter. Muscle biopsies were taken from the m. semimembranosus (SM) and m. semitendinosus (ST) prior to and immediately after exercise for muscle glycogen determination. At slaughter, one side was electrically stimulated and both sides were conventionally chilled for 24h. The pH response to electrical stimulation (ΔpH) and the rate of pH decline adjusted to a constant temperature of 38°C over the initial 6h post-mortem period was determined in three muscles (m. longissimus thoracis et lumborum LTL, SM and ST). In addition, the concentrations of glycogen, proglycogen (PG), macroglycogen (MG) and lactate in the three muscles immediately after slaughter were determined. The glycogen loss due to exercise was influenced by diet (P<0.01; concentrate 63% and roughage 73%) but did not differ between muscles. The rates of repletion significantly varied between muscles (SM>ST) and diet (concentrate>roughage). The available glycogen (glycogen(A)) and MG concentrations at slaughter varied significantly depending on the diet (P<0.01) and muscle (P<0.001). The percentage of MG relative to MG+PG varied between muscles (46%, 50% and 57% for the ST, LTL and SM). The concentration and form of available glycogen at slaughter did not influence the response to electrical stimulation after adjusting for pre-stimulation pH (P<0.01). The ΔpH varied significantly between muscles (0.39±0.03, 0.26±0.02 and 0.20±0.03 for the ST, LTL and SM) after adjusting for pre-stimulation pH. Differences in the temperature adjusted rate of pH decline were observed between the muscles (LTL>SM>ST). Importantly, a positive linear association (P=0.05) was found between muscle glycogen(A) concentration at slaughter and the rate of pH decline (temperature adjusted).

Fatty acid indices of stearoyl-CoA desaturase do not reflect actual stearoyl-CoA desaturase enzyme activities in adipose tissues of beef steers finished with corn-, flaxseed-, or sorghum-based diets.

We hypothesized that stearoyl-CoA desaturase (SCD) enzyme activity would not correlate with fatty acid indices of SCD activity in steers fed different grains. Forty-five Angus steers (358 +/- 26 kg BW) were individually fed for 107 d diets differing in whole cottonseed (WCS) supplementation (0, 5, or 15% of DM) and grain source (rolled corn, flaxseed plus rolled corn, or ground sorghum grain) in a 3 x 3 factorial arrangement. Flaxseed- and corn-fed steers had greater (P < 0.01) G:F (0.119 and 0.108, respectively) than sorghum-fed steers (0.093). Marbling score was decreased by WCS (P = 0.04), and LM area was decreased (P < 0.01) by sorghum. Plasma 14:0, 16:0, 16:1n-7, and 18:2n-6 were greatest in corn-fed steers, whereas plasma 18:3n-3 and 20:5n-3 were greatest in the flax-seed-fed steers (P < 0.01). Plasma 18:1trans-11 was least in sorghum-fed steers, and plasma cis-9,trans-11 CLA was barely detectable, in spite of high intestinal mucosal SCD enzyme activity (118 to 141 nmol*g tissue(-1).7 min(-1)). Interfascicular (i.f.) and s.c. cis-9,trans-11 CLA remained unchanged (P > or = 0.25) by treatment, although 18:1trans-11 was increased (P < or = 0.02) in steers fed corn or flaxseed. Steers fed flaxseed also had greater (P < 0.01) i.f. and s.c. concentrations of 18:3n-3 than steers fed the other grain sources. Oleic acid (18:1n-9) was least and total SFA were greatest (P < 0.01) in i.f. adipose tissue of steers fed 15% WCS. Lipogenesis from acetate in s.c. adipose tissue was greater (P < 0.01) in flaxseed-fed steers than in the corn- or sorghum-fed steers. Steers fed flaxseed or corn had larger i.f. mean adipocyte volumes (P < 0.01) than those fed sorghum and tended (P = 0.07) to have larger s.c. adipocyte volumes. Several fatty acid indices of SCD enzyme activity were decreased (P < or = 0.03) by WCS in i.f. adipose tissue, including the 18:2cis-9,trans-11/ 18:1trans-11 ratio. The 18:2cis-9,trans-11/18:1trans-11 ratio also tended to be decreased (P = 0.09) in s.c. adipose tissue by flaxseed; however, SCD enzyme activities in i.f. and s.c. adipose tissue were not affected by dietary WCS (P > or = 0.47) or grain source (P > or = 0.37). Differences in SFA seemed to be independent of SCD enzyme activity in both adipose tissues, suggesting that duodenal concentrations of fatty acids were more important in determining tissue fatty acid concentrations than endogenous desaturation by SCD.

Effect of vitamin E supplementation on α-tocopherol and ß-carotene concentrations in tissues from pasture- and grain-fed cattle.

The effects of dietary vitamin E supplementation of grain-fed cattle on lipid oxidation and meat colour have been extensively investigated, but little attention has been given to pasture-fed cattle where meat is likely to contain naturally high amounts of α-tocopherol and carotenoids. In the work described, we evaluated the effects of pasture-feeding alone and with vitamin E supplementation on tissue levels of anti-oxidants and compared the findings with those obtained for grain-fed cattle with and without supplementation. Sorghum was the major component of the grained-based ration. α-Tocopherol concentrations in plasma, muscle and fat tissues of pasture-fed cattle were not affected by vitamin E supplementation (2500 IU/head/day for 132 days prior to slaughter) while those of grain-fed cattle increased significantly. The α-tocopherol concentrations in the supplemented grain-fed cattle were similar in muscle and liver to pasture-fed animals but were lower in their fat (P<0.05). The major carotenoid present in all tissues studied from pasture-fed was ß-carotene and its contents in plasma, liver, fat and muscles were decreased (P<0.05) by supplementation with vitamin E. Carotenoids were essentially absent in grain-fed cattle except for small amounts in liver. The implication of this study for the meat industry is that cattle grazed on good pasture can achieve concentrations of α-tocopherol in muscles and other tissues at least as high as those obtained by supra-nutritional supplementation of grain-fed cattle with vitamin E. However, α-tocopherol supplementation of pasture-fed cattle reduced tissue concentrations of ß-carotene, which would reduce carcase fat yellowness and make pasture-fed cattle more acceptable to some Asian markets.

Lipid stability and meat colour of beef from pasture- and grain-fed cattle with or without vitamin E supplement.

Meat from pasture-fed cattle can have high contents of α-tocopherol and other anti-oxidants originating from naturally occurring compounds present in grasses. However, meat from pasture-fed cattle may have an increased demand for endogenous anti-oxidants because of its high content of polyunsaturated fatty acids, which in turn, may affect its colour and lipid stability. In the work described, we evaluated the effects of pasture-feeding alone and with vitamin E supplementation and compared the findings with those obtained for grain-fed cattle (predominantly sorghum) with and without supplementation. Within each nutritional background, vitamin E supplementation did not alter meat colour or colour stability of fresh or 47-day aged muscle during 7-day aerobic storage. However, both control and supplemented grain-fed product had better meat colour (more redness) compared with meat from grass-fed cattle. These differences in redness between pasture- and grain-fed fresh beef were not apparent after ageing. The treatments did not affect the lipid stability of fresh meat during aerobic storage; however, supplementation reduced (P<0.01) lipid oxidation in grain-fed aged beef compared with pasture-fed aged beef, despite both having similar α-tocopherol contents. Pasture-fed beef had more linolenic acid, less linoleic acid and, overall, was more polyunsaturated than grain-fed beef (P<0.05). In summary, vitamin E supplementation of pasture-fed cattle did not alter muscle tocopherol contents but pasture-fed beef (both control and supplemented) was more susceptible to lipid oxidation following ageing than vitamin E supplemented grain-fed beef.

Delta9 desaturase activity in bovine subcutaneous adipose tissue of different fatty acid composition.

Two experiments were conducted to investigate the relationship between delta9 desaturase (stearoyl-coenzyme A desaturase) activity and fatty acid composition in subcutaneous adipose tissue from cattle of different backgrounds. In Experiment 1, subcutaneous adipose tissue samples were taken from carcasses of pasture-fed cattle and feedlot cattle fed for 100, 200, or 300 d. Adipose tissue from pasture-fed cattle had significantly lower total saturated fatty acids and higher total unsaturated fatty acids than feedlot cattle. Desaturase activity correspondingly was 60-85% higher in pasture-fed cattle than in feedlot cattle. There was no difference in the fatty acid composition or desaturase activity among samples from the 100-, 200-, and 300-d feedlot cattle. In Experiment 2, adipose tissue samples were collected from carcasses of feedlot cattle fed for 180 d with either a standard feedlot ration (control group), or a ration containing rumen-protected cottonseed oil (CSO) for the last 70-80 d. Adipose tissue from the CSO-fed cattle was more saturated than that from the control group, having significantly more 18:0 and less 16:1 and 18:1. Correspondingly, adipose tissue from the CSO group had significantly lower desaturase activity. The elevated 18:2 in adipose tissue from the CSO group confirmed that unsaturated fatty acids (including cyclopropenoid fatty acids) were protected from biohydrogenation. Further studies are needed to determine whether the repression of desaturase activity results from direct inhibition by cyclopropenoic acids or by higher dietary contents of 18:2.

A comparison of fat composition of Japanese and long-term grain-fed Australian steers.

Subcutaneous and intermuscular fat samples were collected from carcases of four major breeds of steers in Japan: Wagyu, Wagyu × Angus, Dairy and Murray Grey. For comparison, we also collected subcutaneous fat samples from carcases of long-term grain-fed (350-455 days) Angus, Jersey and Angus × Hereford steers, and short-term grain-fed (70-100 days) Murray Grey steers in Australia. Fatty acid profiles were determined on all samples and triacylglycerol composition, thermal properties, fat cell size and lipid and connective tissue contents were determined on representative samples. Compared with the Japanese samples which were soft to very soft when assessed subjectively, samples of Australian fat were generally hard and somewhat fibrous in appearance. These tactile and visual differences in the hardness of the subcutaneous fat between the Japanese and Australian beef were confirmed by the physical and chemical properties determined. Markedly different melting patterns were observed for the Australian and Japanese fat samples. The Japanese fat had considerably less saturated and more unsaturated fatty acids resulting in much higher unsaturated/saturated ratios (1.9) compared with the Australian samples (1.0). This resulted primarily from the high contents of oleic and palmitoleic acids and the low content of stearic acid of the Japanese samples. The triacylglycerols from the Japanese fat had considerably less tri-saturated and di-saturated fatty acids and more di-monounsaturated and tri-monounsaturated fatty acids in their structure. Differences were observed when the Japanese subcutaneous fat samples were grouped by their meat quality grades. From Grade 5 to Grade 2, there was a significant decrease in marbling score (9.3 to 2.5) and in the ratio of palmitoleic to stearic acid (1.7 to 1.2) and an increase in the connective tissue content (1.5 to 2.1%). Compared with subcutaneous fat, intermuscular fat had a higher content of saturated and a lower content of unsaturated fatty acids resulting in a lower ratio of unsaturated to saturated fatty acids and of palmitoleic to stearic acid. It was concluded that the fatty acid composition and the triacylglycerol structure of fat plays the predominant role in determining the lustre, texture and properties of fat desired by the Japanese market: the soft character of fat from Japanese cattle results primarily from its low content of stearic acid and consequent lower melting temperatures. Fat cell size and the lipid and connective tissue contents of fat appear to be less important.

Positional analysis of triacylglycerols from bovine adipose tissue lipids varying in degree of unsaturation.

The objective of this study was to demonstrate that changing the fatty acid composition of bovine adipose tissue concurrently changed (i) proportions of triacylglycerol species, (ii) fatty acid composition of triacylglycerol species, and (iii) positional distribution of the component fatty acids of the triacylglycerol species. To achieve this, we took advantage of adipose tissue lipids, from cattle fed in Australia and Japan, that varied widely in fatty acid composition and melting points. Treatment groups produced in Australia were cattle fed: a cornbased diet (MUFA1); a grain-based diet containing whole cottonseed (SFA); a grain-based diet containing protected cottonseed oil (PUFA); and a grain-based diet that resulted in high contents of trans fatty acids (TFA). Treatment groups produced in Japan (MUFA2 and MUFA3) were diets of unknown composition fed for over 300 d. The MUFA1, MUFA2, and MUFA3 samples all were rich in monounsaturated fatty acids, varying only in the proportions of the individual monounsaturates. The SFA, PUFA, and TFA samples had relatively high concentrations of stearic acid (18:0), PUFA, and TFA, respectively. Slip points (indicative of melting points) were 45.1, 41.5, 38.5, 30.7, 28.4, and 22.8 degrees C, for the SFA, TFA, PUFA, MUFA1, MUFA2, and MUFA3 groups, respectively (P < 0.05). Triacylglycerols were separated by high-performance liquid chromatography on a silver nitrate-impregnated column into sn-1,2,3-saturated fatty acid triacylglycerol (SSS); [triacylglycerols containing two saturated acids and one trans-monounsaturated fatty acid (SMMt sn-positions unknown)]; sn-1-saturated, 2-monounsaturated, 3-saturated triacylglycerol (SMS); sn-1-saturated, 2-monounsaturated 3-trans-monounsaturated triacylglycerol (SMMt); sn-1-saturated, 2,3-monounsaturated fatty acid triacylglycerol (SMM); sn-1-saturated, 2-polyunsaturated, 3-trans-monounsaturated triacylglycerol; sn-1,2,3-monounsaturated fatty acid triacylglycerol (MMM); and sn-1-saturated, 2-polyunsaturated, 3-monounsaturated triacylglycerol. Fatty acid methyl esters of each triacylglycerol species also were determined, and further analysis indicated sn-2, and sn-1/3 positions. As the percentage oleic acid increased in the total lipid extract, the proportions of SMM and MMM increased (e.g., from 31.4 and 2.4% in the SFA group to 55.4 and 17.8% in the MUFA3 group). The elevated 18:0 in the SFA group (26%) was reflected in increased percentages of SSS and SSM, and caused an increase in the proportion of 18:0 in all triacylglycerol species relative to the other treatment groups. The percentage of 18:0 in the sn-1/3 positions was elevated markedly in the SMS fraction of the SFA group (to 44%); this would account for the high melting point of the fat of these animals. We conclude that long-term feeding of cattle is sufficient to produce significant alterations in fatty acid composition in bovine adipose tissue. Alterations in the fatty acid composition of bovine adipose tissue changed both the distribution and the composition of the triacylglycerol species, which, in turn, accounted for marked differences in melting points among treatment groups.

The in vitro destruction of rumen fluid carotenoids by plant lipoxygenases.

An ability to destroy carotenoids in the rumen of cattle may be an effective method of limiting their absorption from the small intestine which, in turn, is likely to result in reduced adipose tissue colour. Plant lipoxygenases are well known for their ability to bleach beta-carotene. As lutein is the major carotenoid present in most forage grasses, we have investigated the bleaching of lutein as well as beta-carotene by lipoxygenase isolated from soybeans. In vitro studies, using micellar preparations of carotenoids, indicated that lutein was rapidly oxidised and that the progress of the reaction was similar to that observed for beta-carotene. The polyunsaturated fatty acid linoleate was essential. When bovine rumen fluid was used as a source of carotenoid for in vitro studies with preparations of lipoxygenase, a rapid decrease in carotenoid and chlorophyll concentrations was observed, again requiring the addition of linoleic acid. The direct addition of soya flour to bovine rumen fluid resulted in the effective bleaching of the pigments without the inclusion of linoleate. When compared with flours from a variety of other plant sources, soya flour was most effective. The inclusion of dietary sources of lipoxygenase may be an effective method for controlling carotenoid uptake in certain ruminant species.

A comparison of beta-carotene-splitting activity isolated from intestinal mucosa of pasture-grazed sheep, goats and cattle.

The in vitro enzymatic conversion of beta-carotene to retinal by partially purified preparations from the intestinal mucosa of sheep, goats and cattle was demonstrated. At pH 7.8 and 37 degrees C, this enzyme from sheep, goats and cattle displayed Michaelis-Menten type kinetics with a Vmax (nmol substrate split/mg protein/h) of 0.21, 0.27, and 0.04 and an apparent Km of 4.66 x 10(-6) M, 13.4 x 10(-6) M and 1.27 x 10(-6) M respectively. Maximal reaction was obtained by addition of an appropriate combination of detergent and bile salt, sodium dodecyl sulphate and egg lecithin. The cattle enzyme was inhibited by sodium glycocholate. The activity of sheep enzyme was higher (P < 0.05) than that of goats and cattle. The measured activity of the preparations from sheep, goats and cattle grazed on the same pasture reflected their contents of the reaction product, retinol, in the liver.

A relationship between bovine fat colour and fatty acid composition.

Subcutaneous adipose tissue was obtained from pasture-grazed (n = 13) and short-term (70 days) grain-fed (n = 13) cattle. The yellow colour of the adipose tissue was assessed by Minolta b(∗) value readings and by determination of total carotenoids and the two measurements gave a correlation coefficient of 0·79 (P < 0·01). The fatty acid composition of the samples varied with fat colour. As the b(∗) value and the carotenoid content of the fat increased, there was a significant increase in the total percentage of cis mono-unsaturated fatty acids and a decrease in saturated fatty acids (P < 0·01). Consequently, the ratio of cis mono-unsaturated to saturated fatty acids was found to be higher in those samples exhibiting a greater yellow colour.

Beta-endorphin and cortisol concentrations in plasma of blood samples collected during exsanguination of cattle.

Blood samples were collected during exsanguination from a group of 36 cattle slaughtered at a research abattoir and from a group of 36 cattle slaughtered at a commercial abattoir. Beta-endorphin and cortisol values were measured in plasma from all blood samples. The mean beta-endorphin values for the two groups of animals (19·2 and 20·9 pmol/litre) did not differ significantly. The mean cortisol values for the two groups of cattle did differ significantly (P < 0·001), with the commercial abattoir group having the greater mean value (123 nmol/litre versus 41 nmol/litre). Although the commercial abattoir group had an elevated mean cortisol value there were no dark cutting carcasses in the group.

The assessment of pre-slaughter and slaughter treatments of livestock by measurement of plasma constituents-A review of recent work.

The measurement of plasma constituents in a blood sample can provide information on the stress status of the animal. The interpretation of results obtained for constituents of blood samples collected at exsanguination must consider the effect of the slaughter process on the constituent. Both electrical and mechanical stunning methods can cause dramatic increases in catecholamine levels and minor increases in glucose levels. Thus, there are difficulties in the interpretation of catecholamine and, to a lesser extent, glucose, values in blood samples collected post-stunning. Cortisol levels appear to be unaffected by stunning methods and measurement of this constituent in post-slaughter blood samples has been used to assist in the evaluation of transport and abattoir treatments. Beta-hydroxybutyrate concentrations may assist in evaluating nutritional stress prior to slaughter while the limited evidence available suggests that beta-endorphin measurements will be of value in assessing pain and other stressors prior to slaughter. Adreno-corticotrophic hormone, calcium and magnesium, free fatty acids, glucose, lactate and thyroid hormones have all been used on occasions to assist in the evaluation of stress status. In some cases it was not possible to demonstrate a clear relationship between plasma constituents that indicate stress, and stress-related meat quality defects.

Primary structure of ovine apolipoprotein C-II and the production of antibodies directed towards a synthetic fragment (residues 46-59).

Two distinct activator proteins for lipoprotein lipase were isolated from ovine plasma and purified to homogeneity by reverse phase HPLC. The two proteins were partially sequenced (up to residue 59) and the results show that they are identical except that 6 residues were missing from the N-terminal of the smaller protein. The complete sequence of the proteins has been deduced from amino acid composition studies and by comparison with the sequence information available from other species. Antibodies were produced in BALB/c mice to a synthetic peptide corresponding to a highly hydrophilic region (residues 46-59) of the activator protein. The antibodies cross-reacted with the two forms of activator and with ovine lipoproteins. This work with a synthetic fragment of ovine activator protein confirms that the technique is useful for investigating antibody production and specificity directed against native lipoproteins.

Characterization of polyclonal antibodies to cell-surface antigens from ovine adipose tissue.

High titres of polyclonal antibodies to specific proteins of ovine adipose tissue plasma membranes were raised in horses and chickens following repeated injections of purified plasma membranes. Horse antiserum was highly species specific, reacting only weakly with rat adipose tissue plasma membranes. A protein of molecular weight 68,000 was most antigenic in that it was readily precipitated; however proteins of 25,000, 82,000 and 94,000 were also precipitated when the reaction was performed for longer with a higher antiserum concentration. Chicken egg yolk IgY reacted strongly with ovine adipose tissue plasma membranes as did those preparations from horse, but IgY was ineffective in immunoprecipitating solubilized membrane proteins and exhibited no cytotoxic reaction when incubated with intact ovine adipocytes. However, horse antiserum produced a strong complement-dependent cytotoxic reaction with ovine adipocytes, as measured by leakage of lactate dehydrogenase. This work suggests that the membrane protein of molecular weight 68,000 is likely to be an important antigenic marker for ovine adipocytes.

Post-heparin triacylglycerol lipases in ovine plasma.

Lipoprotein lipase and hepatic lipase have been shown to be present in the post-heparin plasma of sheep. Intravenous injection of heparin into sheep produced a rapid increase in the free fatty acid concentration and lipolytic enzyme activity of the plasma, both peaking within 5-15 min and then falling to pre-heparin levels within 30-60 min. Lipolytic activity was not detected in plasma before heparin treatment. Two distinct lipolytic activities were separated from the plasma by chromatography on heparin-Sepharose 6B. Lipoprotein lipase was identified on the basis that the lipolytic activity was dependent upon the addition of plasma, inhibited by 1M NaCl, and inhibited by a specific antiserum against lipoprotein lipase. The second lipolytic activity of plasma was identified as hepatic lipase, as it was not dependent upon plasma for activity, nor was it inhibited by 1M NaCl or antiserum against lipoprotein lipase. Its properties were identical to the lipase extracted from the liver of sheep. Lipoprotein-lipase activity, but not hepatic-lipase activity, was dependent upon the nutritional state of the sheep at the time of heparin injection. However, hepatic lipase comprised a significant proportion of the total lipolytic activity.

Isolation of two distinct activator proteins for lipoprotein lipase from ovine plasma.

Two distinct activator proteins for lipoprotein lipase (LPL) have been isolated in approximately equal amounts from ovine plasma. These low molecular weight proteins were readily separated from each other on the basis of size and charge. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated proteins of Mr about 8000 and 5000, with pI in urea-containing gels of about 5.1 and 4.8 respectively. Each of the ovine activator proteins was as effective as human apolipoprotein C-II (apo C-II) in activating LPL, with 1 microgram/ml giving near to maximum activation, and in lowering the apparent Km of LPL for triolein substrate. As the ratio of activator to triolein increased from 0.16 to 5.2 (micrograms/mg) the apparent Km fell from about 0.5 to 0.18 mM. Whereas human apo C-II and the two ovine activators were equally effective in stimulating the hydrolysis of triolein, differences were observed between the human and ovine activators when p-nitrophenylbutyrate was used as substrate. Unlike human apo C-II, which produced significant inhibition of p-nitrophenylbutyrate hydrolysis, the ovine activators were without effect. This suggests that the interaction between the ovine activators and LPL is different from that of human apo C-II.