Application of an automated enzymatic technique for the determination of non-esterified fatty acids in bovine blood

Brookes, P; Dew, AM; Pike, BV; Roberts, CJ

doi:10.1136/vr.114.17.421

Cited by 7 publications

(5 citation statements)

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“…Higher values in serum have been attributed to activation of de-esterifying enzymes (e.g., lecithinases) or lipolytic enzymes during clotting or possible EDTA inhibition of the reaction (McGann and Hodson, 1991). In contrast, NEFA concentrations were higher (albeit not significantly) in EDTA plasma compared with serum in sheep (Morris et al, 2002), and in a previous study in 10 dairy cows (Brookes et al, 1984). A reason for the discrepancies between these results and our data is not readily apparent, but could be related to differences in sample handling and processing, because the same analytical technique was used for these 3 studies.…”

Section: Discussionmentioning

confidence: 81%

“…The current protocol for blood sampling for NEFA testing is to collect blood into gel and clot activator or serum separator tubes (SST), allow the blood to clot, centrifuge the sample, transfer the serum into another tube, then ship the serum chilled or frozen overnight for analysis (NEFA and Metabolic Profile Sample Handling Guidelines). A previous study in dairy cows showed that NEFA concentrations were slightly higher in EDTA plasma than in serum in 8 of 10 cows (Brookes et al, 1984). However, to our knowledge, there have been no published reports on the effect of other anticoagulants (e.g., heparin), use of SST, or other preanalytical variables, including delayed separation of serum or plasma from cells, storage temperature, and duration of storage, on NEFA and BHBA concentrations in dairy cows.…”

Section: Introductionmentioning

confidence: 73%

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Effect of Anticoagulant and Storage Conditions on Bovine Nonesterified Fatty Acid and β-Hydroxybutyrate Concentrations in Blood

Stokol

Nydam

2005

Journal of Dairy Science

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The objective of this study was to determine if nonesterified fatty acid (NEFA) and beta-hydroxybutyrate (BHBA) concentrations were affected by anticoagulants or gel and clot activator tubes (serum separator tubes, SST), storage of samples as whole blood, separated plasma or serum at 24 degrees C or 4 degrees C for 24 to 72 h, or storage as serum at -40 degrees C for 1 mo. Blood was collected from dairy cows into EDTA, heparin, nonanticoagulant tubes, and SST, and analyzed immediately to obtain baseline NEFA and BHBA concentrations. Portions were stored as whole blood or separated plasma or serum at 4 or 24 degrees C and assayed daily for 24 (whole blood) and 72 (separated samples) h. Serum samples were frozen at -40 degrees C and assayed at 24 h or weekly for 1 mo. Baseline NEFA concentrations were unaffected by anticoagulants; however, they were significantly higher in SST compared with nonanticoagulant tubes. Concentrations of NEFA were stable in all samples at 4 degrees C, whereas they sequentially increased from 24 to 48 h at 24 degrees C. Changes were more dramatic in heparinized samples. Serum could be stored frozen for up to 1 mo with minimal changes in NEFA concentrations. Concentrations of BHBA were stable under all conditions evaluated. Our results indicated that blood for NEFA testing should be collected into EDTA or nonanticoagulant tubes (but not SST), separated promptly from cells, and maintained at 4 degrees C until analysis.

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Section: Discussionmentioning

confidence: 81%

Section: Introductionmentioning

confidence: 73%

Effect of Anticoagulant and Storage Conditions on Bovine Nonesterified Fatty Acid and β-Hydroxybutyrate Concentrations in Blood

Stokol

Nydam

2005

Journal of Dairy Science

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“…4 ) method (Boehringer Mannheim GmbH). Serum was stored at -20" before determination of urea (Boehringer Mannheim GmbH) and nonesterifed fatty acids (NEFA, Brookes et al 1984). Rumen pH, rumen VFA and rumen NH,-N were determined as described previously.…”

Section: Exptmentioning

confidence: 99%

Nutrient supply and growth of cattle offered straw-based diets

Ortigues¹,

Smith²,

Oldham³

et al. 1989

Br J Nutr

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An experiment was conducted using steers cannulated at the rumen, duodenum and ileum to study the effects of increasing the levels of barley and fishmeal in straw-based diets. Diets A, B, C and D contained ammonia-treated straw, barley and fishmeal in the ratios, 67:33:0, 66:23:11, 53:47:0 and 52:36:12 (by weight) and were offered in daily amounts of 3.9,3.9,4.8 and 4.8 kg dry matter. The effects of barley were attributable to increased intakes of digestible organic matter and consequently to increased flows of microbial matter to the duodenum. There were no modifications in the balance of energy to nitrogenyielding nutrients available for absorption. Introducing fishmeal into diets improved digestibility of cellulose and xylose by up to 6 7 and 4.7 % respectively, and shifted digestion towards the large intestine. Second, it increased amino acid N supply to the small intestine which averaged 52.2, 63.2, 68.8 and 84.0 g/d with diets A, B, C and D. Some changes were also noted in the balance of amino acids absorbed. Consequently, the contribution of amino acids to metabolizable energy intake increased with the proportion of fishmeal in diets (0.17, 0.20, 0.18 and 0.21 for diets A, B, C and D).Growth rates measured in heifers amounted to 259,431,522

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“…1 Other analytical techniques for the separation and quantitation of various saturated and unsaturated free fatty acids have been described. 24,36,40,41 In addition to the above techniques, a number of enzymatic methods 4,8,16,17,22 for the colorimetric detection of NEFA in serum/plasma are now available including a commercially available kit a (''NEFA C'' Kit). This latter technique involves conversion of NEFA to their corresponding enoyl-Coenzyme A (CoA) products with generation of hydrogen peroxide (H 2 O 2 ; Fig.…”

mentioning

confidence: 99%

Detection of Nonesterified (Free) Fatty Acids in Bovine Serum: Comparative Evaluation of Two Methods

2004

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Abstract. A method comparison study for the determination and quantitation of nonesterified fatty acids (NEFAs) in serum, using the commercial ''NEFA C'' enzymatic test kit, was performed using the spectrophotometric method recommended by the manufacturer and a modified procedure optimized for the use of a microplate reader, a 96-well microtiter plate, and small sample volumes (10 l). Linearity, sensitivity, and precision using the test kit were determined for each method of detection. The assay was linear from 0 to 1.97 mEq/liter for both procedures, and the limits of detection were determined to be 0.22 (Ϯ0.074) and 0.05 (Ϯ0.002) mEq/liter for the spectrophotometer and microplate reader, respectively. Pairs of measurements for bovine serum samples were compared and evaluated by a mean difference plot method and not regression analysis, a method that has been shown to be inappropriate for method comparison studies. The difference plot was used to evaluate the systematic bias between the 2 methods. Random error is reported on the basis of SD differences, and ''limits of agreement'' are used to describe the maximum differences likely to occur between the 2 methods. Results suggest that the microplate reader method can be used reliably in place of the recommended spectrophotometric method. The microplate reader method is preferred because of its high throughput capabilities, simultaneous analysis of all the standards and samples, use of small sample and reagent volumes, and reduction in labor requirements and costs.The measurement of nonesterified fatty acids (NEFAs) in dairy cows can be used to assess dry cow nutritional management and, in conjunction with cholesterol determination, serves as an indictor of postpartum disease risk. 6,11,13,20 Increased energy demand during late gestation and early lactation, combined with a decline in dry matter intake before parturition, make dairy cows uniquely susceptible to negative energy balance (NEB). Plasma NEFA concentrations can be used as a measure of adequate energy intake, with increased concentrations associated with a NEB. Several studies have shown a positive association between increased plasma NEFA concentrations during the last 2-3 weeks of gestation and the occurrence of postpartum diseases such as ketosis, fatty liver, displaced abomasums, retained placentas, and metritis. 6,11,13,20 Feeding strategies designed to increase energy intake during this period are desirable for optimal postpartum health and performance. Thus, monitoring of plasma NEFA is a useful tool for dry cow management.A large number of methods for detection and isolation of NEFA have been previously reported. These include thin-layer, 12 gas-liquid, 37 and high-performance liquid chromatography (HPLC) 23,29,36,38,40 applied to

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Application of an automated enzymatic technique for the determination of non-esterified fatty acids in bovine blood

Cited by 7 publications

References 0 publications

Effect of Anticoagulant and Storage Conditions on Bovine Nonesterified Fatty Acid and β-Hydroxybutyrate Concentrations in Blood

Effect of Anticoagulant and Storage Conditions on Bovine Nonesterified Fatty Acid and β-Hydroxybutyrate Concentrations in Blood

Nutrient supply and growth of cattle offered straw-based diets

Detection of Nonesterified (Free) Fatty Acids in Bovine Serum: Comparative Evaluation of Two Methods

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