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  Ketosis is a common metabolic disorder frequently observed in dairy cows during the early lactation period.It is a metabolic condition characterized by increased levels of ketone bodies in blood,urine and milk.The major ketone body is beta-hydroxybutyrate (BHBA).Determination of BHBA in milk samples is an important tool in the diagnosis of subclinical/clinical ketosis in dairy cows.In this report, we describe a novel method based on the traditional spectrophotometric approach for measuring BHBA levels in milk of dairy cows.From two commercial dairy farms,milk samples were taken from dairy cows within 2 months after lactation.The spectrophotometric approach involve d two reactions.BHBA,in the prese nce of NAD,was oxidized by BHBA dehydrogenase to AcAc and NADH. Then NADH and nitroblue tetrazoli um were reduced by diaphorase to fo rmazan and NAD,respectively.The amount of formazan was measured photometrically.Samples were prepa red as follows:milk was deproteiniz ed with perchloric acid,centrifuged, the supernatant was neutralized with KOH solution.The standard curve was made by a series of dilutions the BHBA standard solution.A reaction buffer was prepared,and comprised Tris buffer,Triton x-100,NBT,BHB A dehydrogenase,diaphorase and oxamic acid.Tris-buffer(pH 8.5), the reaction solution and prepared milk sample were added to centrifug e tubes,in order,and shaken vigorou sly.The resulting mixture was kept at 30 min at room temperature in the dark and assayed using a UV spectro photometer.The following paramete rs were established:linearity,recove ry,repeatability,decision limit and specificity.The fluorometric detectio n procedure as follows.The sample buffer comprised Tris-buffer pH 8.5, oxamic acid,NAD,rezasurin,Triton X-100,BHBA dehydrogenase and diaphorase.The end-point process was read after 20 min.The results showed that the standard-curve equa tion of the UV spectrophotometer method was y=0.2582x+0.0269 (R~2=0.9967).The reaction buffer did not react with AcAc,L-succinic acid, L-fumaric acid and acetone.The assa y was highly specific with a minimu m detection limit of 0.01 mmol/L and measuring range of up to 5 mmo I/L.The recovery was between 99.35%and 100.22%and repeatabili ty was 99.8%.The comparison betw een the spectrophotometric method and the fluorometric method reveale d a close correlation(r=0.9939).The BHBA level detected by the spectrop hotometric method in milk samples ranged from 10 to 437μmol/L;the average concentration was 57μmol /L.The spectrophotometric assay is easily realized because the materials are relatively cheap compared with other methods and the instrument is conventional.The technique shows a broader limit of quantitation with high accuracy.The method is ideal for measuring a large number of samples and can be used as an alternative assay method for the analysis of BHBA in milk.……