Effects of supplemental fat on growth performance and quality of beef from steers fed corn finishing diets.

To measure the effects of dietary fat on feedlot performance and carcass characteristics, and on beef appearance, moisture binding, shelf life, palatability, and fatty acid content, 126 crossbred beef steers (321.1 +/- 0.57 kg of BW) were allotted to a randomized complete block (3) design with a 3 x 2 + 1 factorial arrangement of dietary treatments. The main effects were level of yellow grease (0, 3, or 6%) and alfalfa hay (3.5 or 7%) in corn-based diets containing 15% potato by-product (PB). The added treatment was 6% tallow and 7% alfalfa in a barley-based diet containing 15% PB. Dry matter intake and ADG were not affected by diet; however, G:F and diet NE content increased linearly (P < 0.10) with yellow grease. Kidney, pelvic, and heart fat (2.0 to 2.3 +/- 0.07) and yield grade (2.8 to 3.1 +/- 0.09) increased linearly (P < or = 0.05) with yellow grease. Steers fed corn plus 6% yellow grease had lower (P < 0.05) beef firmness and beef texture scores but greater (P < 0.01) fat color score than those fed barley plus 6% tallow. Moisture retention of beef was not affected by dietary treatment, except purge score during retail storage, which was decreased linearly (P < 0.01) from 2.1 to 1.6 +/- 0.06 by level of yellow grease. Steaks from steers fed barley plus 6% tallow had greater (P < 0.05) shear force than those from steers fed corn plus 6% yellow grease, and beef flavor increased linearly (P < 0.05) from 6.2 to 6.7 +/- 0.11 as the level of yellow grease increased. Level of yellow grease linearly increased (P < 0.01) transvaccenic acid (TVA) by 61% and CLA content of beef by 48%. Beef from steers fed corn plus yellow grease had lower (P < 0.05) palmitoleic and oleic acids and greater (P < 0.05) linoleic, TVA, and CLA than beef from steers fed the barley-tallow diet. Feeding yellow grease increased diet energy content, which increased carcass fatness, and altered beef fatty acid content, which increased beef flavor without affecting moisture retention, shelf life, or cooking properties of the beef. Additionally, beef from steers fed corn plus 6% yellow grease was more tender and had more polyunsaturated fatty acid content and CLA than beef from steers fed barley plus 6% tallow.

A direct method for fatty acid methyl ester synthesis: application to wet meat tissues, oils, and feedstuffs.

A simplified protocol to obtain fatty acid methyl esters (FAME) directly from fresh tissue, oils, or feedstuffs, without prior organic solvent extraction, is presented. With this protocol, FAME synthesis is conducted in the presence of up to 33% water. Wet tissues, or other samples, are permeabilized and hydrolyzed for 1.5 h at 55 degrees C in 1 N KOH in MeOH containing C13:0 as the internal standard. The KOH is neutralized, and the FFA are methylated by H(2)SO(4) catalysis for 1.5 h at 55 degrees C. Hexane is then added to the reaction tube, which is vortex-mixed and centrifuged. The hexane is pipetted into a gas chromatography vial for subsequent gas chromatography. All reactions are conducted in a single screw-cap Pyrex tube for convenience. The method meets many criteria for fatty acid analysis, including not isomerizing CLA or introducing fatty acid artifacts. It is applicable to fresh, frozen, or lyophilized tissue samples, in addition to oils, waxes, and feedstuffs. The method saves time and effort and is economical when compared with other methods. Its unique performance, including easy sample preparation, is achieved because water is included rather than eliminated in the FAME reaction mixtures.

Enzymatic method for measuring subnanomolar concentrations of oxygen in gaseous or aqueous solutions.

A method that combines glucose oxidase with [U-14C]glucose to measure subnanomolar to decamillimolar concentrations of oxygen is presented. The method is inexpensive and easy to use. It is independent of sample pH and enzyme inhibitors, including detergents and metal ions. This method can be directly applied to gaseous or aqueous samples and multiple assays can be run simultaneously.

Methyl viologen as a preferred electron acceptor in metabolic experiments.

Widely used artificial electron acceptors, including dichlorophenol-indophenol, methylene blue, and phenazine ethosulfate, were shown to induce the nonenzymatic decarboxylation of pyruvate. This characteristic rendered these electron acceptors unsuitable for use in metabolic experiments. In contrast, methyl viologen stimulated the pentose phosphate pathway in mouse preimplantation embryos without decarboxylating pyruvate in the process. Furthermore, methyl viologen had no effect on the Embden-Myerhoff pathway and was not overtly toxic to mouse embryos. These features made methyl viologen a preferred artificial electron acceptor for metabolic studies.

Glucose metabolic pathways in the anaerobic rumen fungus Neocallimastix frontalis EB188.

Primary pathways for glucose metabolism were established in the anaerobic rumen fungus Neocallimastix frontalis EB188. This highly capable cellulolytic organism demonstrated a strict anaerobic integration of metabolic pathways. Glycolysis in N. frontalis EB188 was coupled to malate dehydrogenase, 'malic' enzyme and specified hydrogenosome reactions. Pyruvate, as in most life forms, was a pivotal compound. The major fermentation products of N. frontalis EB188 were acetate, ethanol and lactate, with the concomitant generation of H2. On the basis of its unique characteristics and streamlined fermentation pathways, it was concluded that N. frontalis EB188 should be an important contributor to programs generating energy and selected chemicals from currently intractable biomass.

Calculation of the pentose phosphate and Embden-Myerhoff pathways from a single incubation with [U-14C]- and [5-3H]glucose.

A method that simultaneously determines Embden-Myerhoff pathway and pentose phosphate pathway (PPP) activities from an incubation with [U-14C]- and [5-3H]glucose is presented. The method relies on the use of unlabeled pyruvate and lactate to dilute out radiolabel entering the tricarboxylic acid cycle. Gluconeogenesis from pyruvate is prevented by the use of an incubation chamber that maintains a CO2 (and bicarbonate) free environment. The method, which includes the contribution by the recycling steps of the PPP, is especially useful when biological material is limited or developmental timing is critical.

Characterization of a retinylmonophosphatase in the plasma membrane of mouse brain.

Retinylmonophosphatase (RMPase) activity in mouse brain paralleled the subcellular distribution of the plasma-membrane marker Na+ + K+-dependent ATPase. The enzyme had a pH optimum between 5.5 and 7.0. The enzyme demonstrated linear kinetics with respect to time and both protein and substrate concentrations. RMPase was saturated by low retinyl monophosphate (RMP) concentrations and exhibited an apparent Km of 4.6 microM. The enzyme did not require MgCl2 for activity, and in fact assays were routinely run in the presence of 10 mM-Na2EDTA. In general, detergents inhibited the enzyme, with 0.05% Triton X-100 causing a 30% loss of activity. Phosphatidic acid was also inhibitory, but phosphatidylcholine and sphingomyelin stimulated phosphatase activity. RMPase was inhibited 35% by 5 mM concentrations of fluoride, phosphate or pyrophosphate. A series of other phosphorylated compounds, including glucose 6-phosphate, alpha-glycerophosphate, ATP, AMP, p-nitrophenyl phosphate and thiamin pyrophosphate, showed little or no inhibition. RMPase activity differed in several characteristics from that previously reported for dolichylmonophosphatase. It is concluded that RMP could play a distinct role in the plasma membrane.

Quantitative determination of the pentose phosphate pathway in preimplantation mouse embryos.

A quantitative calculation was made of the pentose phosphate pathway (PPP) activity in preimplantation mouse embryos from the 2-cell through the late blastocyst stage. This activity varied with development and showed repeated high and low values. Peak activities occurred at both the 2-cell (15.8%) and compacted morula (13.6%) stages, with lowest activity at the development of the late blastocyst (3.2%). The metabolic effectors dimitrophenol (DNP) and phenazine ethosulfate (PES) had opposite effects on PPP activity. Dinitrophenol, although stimulating total CO2 production, virtually eliminated PPP activity while PES stimulated the pentose cycle activity 6-fold. These results indicated that the PPP was under metabolic control and that the embryos had a potential for much larger PPP activities. There was no correlation between the C-1/C-6 ratio obtained from the metabolism of [1-14C] and [6-14C] glucose and calculated PPP activities. A metabolic incubation chamber was devised for these experiments that exhibited certain unique features, including continuous collection of 14CO2 and 3H2O. Single embryos were placed in the chamber and sampled momentarily for metabolic activity. Subsequently, such embryos were successfully transferred to pseudopregnant recipients.

The subcellular distribution of beta-carotene in bovine corpus luteum.

The distribution of beta-carotene was determined in various subcellular fractions of bovine corpus luteum. It was found in significant amounts in all subcellular fractions examined including nuclear, mitochondrial, microsomal, cytosolic, and floating lipid. Much of the beta-carotene found in the crude nuclear and mitochondrial fractions was loosely bound and could be removed with repeated washings. In contrast, the microsomal beta-carotene could only be removed by detergent extraction suggesting that it is an integral component of this membrane preparation. In the cytosol fraction beta-carotene was bound to high-molecular-weight protein(s), quite possibly a plasma-derived lipoprotein. The subcellular distribution of beta-carotene in corpus luteum is quite similar to the distribution of its metabolite, retinol, in liver. This finding coupled with other recently published data suggests that beta-carotene could play a distinct role in corpora lutea function.

Vitamin A and beta-carotene in bovine and porcine plasma, liver, corpora lutea, and follicular fluid.

Bovine and porcine blood plasma, liver, corpora lutea, and follicular fluid were obtained from local abattoirs for study of distribution of vitamin A and beta-carotene. Retinol, retinyl esters, and beta-carotene were separated on alumina columns and subjected also to thin-layer chromatography. Retinol and retinyl esters were in corpora lutea and follicular fluid of both species. Concentrations of beta-carotene were high in bovine plasma, corpus luteum, and follicular fluid. In contrast, beta-carotene was lower in porcine tissues. Retinol, retinyl esters, and beta-carotene were closely correlated in bovine follicular fluid and blood plasma; however, correlations between bovine plasma and corpora lutea were not significant except for retinol. Only porcine retinol was closely correlated with plasma and follicular fluid, whereas correlations were nonsignificant between plasma and corpora lutea retinol, retinyl esters, and beta-carotene. Further studies, therefore, are needed to elucidate the physiological role of vitamin A and beta-carotene in regulating ovarian functions.

The Na+,K+-ATPase: a plausible trigger for voltage-independent release of cytoplasmic neurotransmitters.

A comparison was made between the releasability of eight neurotransmitters from eight regions of mouse brain in response to either 60 mM-K+ or 20 microM-ouabain, a specific inhibitor of the Na+,K+-ATPase. With few exceptions, all transmitters were released by either or both agents from each brain region examined. Potassium was superior in releasing the biogenic amines and acetylcholine, while the putative amino acid transmitters were generally releasable by both agents. Measurements of tissue depolarization using [3H]-tetraphenylphosphonium uptake indicated that 60 mM-K+ is capable of depolarizing brain tissue above the threshold necessary for initiating an action potential, but 20 microM-ouabain is not. The pattern of release by ouabain coupled with its failure to depolarize brain tissue at 20 microM suggests that inhibition of the Na+,K+-ATPase is capable of releasing cytoplasmic neurotransmitters in a voltage-independent manner.

The subcellular distribution of carnosine, carnosine synthetase, and carnosinase in mouse olfactory tissues.

The dipeptide, carnosine, its synthetic enzyme, carnosine synthetase, and its degradative enzyme, carnosinase, appear to be localized in the cytosol of mouse olfactory bulb and epithelium. Mouse olfactory bulbs and epithelium were prelabeled in vivo with [3H]carnosine following intranasal irrigation with [3H]beta-alanine. [3H]carnosine co-distributed in olfactory bulb with lactate dehydrogenase with only 10% in the crude mitochondrial fraction. Similar results were also seen with endogenous carnosine distribution. Over 70% of the carnosine present in the crude mitochondrial fraction was localized in synaptosomes following sucrose gradient centrifugation. However, further fractionation of vesicle containing fractions from osmotically lysed crude mitochondrial fractions indicated that [3H]carnosine was not associated with vesicles. Nearly 70% of all the [3H]carnosine present in olfactory epithelium was soluble with most of the remainder in the crude nuclear fraction. The enzymes carnosine synthetase and carnosinase were clearly soluble in olfactory epithelium with 98% and 85% of the activity in the cytosol. Less than 2% was found in the crude mitochondrial fraction. In olfactory bulb both enzymes also appeared soluble.

Cellular localization of alpha-ketoglutarate: glyoxylate carboligase in rat tissues.

alpha-Ketoglutarate: glyoxylate carboligase activity has been reported by other laboratories to be present in mitochondria and in the cytosol of mammalian tissues; the mitochondrial activity is associated with the alpha-ketoglutarate decarboxylase moiety of the alpha-ketoglutarate dehydrogenase complex. The cellular distribution of the carboligase has been re-examined here using marker enzymes of known localization in order to monitor the composition of subcellular fractions prepared by differential centrifugation. Carboligase activity paralleled the activity of the mitochondrial matrix enzyme citrate synthase in subcellular fractions prepared from rat liver, heart and brain as well as from rabbit liver. Whole rat liver mitochondria upon lysis released both carboligase and citrate synthase. The activity patterns of several other extramitochondrial marker enzymes differed significantly from that of carboligase in rat liver. In addition, the distribution pattern of carboligase was similar to that of alpha-ketoglutarate decarboxylase and of alpha-ketoglutarate dehydrogenase complex. The data indicate that alpha-ketoglutarate: glyoxylate carboligase activity is located exclusively within the mitochondria of the rat and rabbit tissues investigated. There is no evidence for a cytosolic form of the enzyme. Thus the report from other laboratory that the molecular etiology of the human genetic disorder hyperoxaluria type I is a deficiency of cytosolic carboligase must be questioned.