Knox D P, Jones D G
Vet Res Commun. 1984 May;8(2):103-15. doi: 10.1007/BF02214702.
An enzymatic 'reaction rate' micro-method for the rapid routine estimation of D-B-hydroxybutyrate (D-B- HOB ) in ruminant plasma, using an I.L. Multistat III centrifugal analyzer, is described. Reaction conditions were optimized to give a linear response for plasma D-B- HOB concentrations between 100 and 2500 mumoles per litre, at 30 degrees C and pH 9.0. For the standardized method the within-run and between-run coefficients of variation for deproteinised ovine plasma were consistently less than 3.5%. There was good agreement between plasma concentrations obtained by the present method and both original U.V. end-point technique (r = 0. 927b = 0.950) and a colorimetric end-point procedure (r = 0.937. b = 0.879). Untreated ovine and bovine plasma consistently exhibited high 'blank' activity and this was directly correlated with plasma lactate dehydrogenase (LDH) activity in both species (r = 0.971; p less than 0.001 and r = 0.949; p less than 0.001 respectively). The distribution of LDH activity in man was similar to sheep but, contrastingly , non-specific interference was extremely low in human plasma and unrelated to LDH. Horse, chicken and rat had negligible 'blank' activity and comparatively low LDH levels. In both cattle and sheep non-specific interference was abolished by perchloric acid precipitation. In the sheep subtraction of 'blank' activity gave D-B- HOB concentrations for untreated plasma comparable to those in deproteinised samples. However, in the bovine, D-B- HOB levels remained significantly (t = 6.44; p less than 0.001) higher even after 'blank' correction. In contrast to man and other non-ruminants, perchloric acid precipitation is essential in ruminants to avoid false overestimation of plasma D-B- HOB levels. Plasma with EDTA as anticoagulant and serum gave concentrations of D-B- HOB approximately 60% lower, than samples containing heparin or oxalate/fluoride. However, heparin was associated with much higher (up to 50%) non-specific NAD reduction than oxalate/fluoride. High levels of acetoacetate (400-1000 mumoles per litre) reduced the recovery of D-B- HOB from ovine plasma by less than 10%. This effect was negated by the inclusion of hydrazine hydrate in the reaction mixture. Perchlorate ion concentrations above 25 mumoles per litre per test dramatically inhibited the assay in ovine plasma, and therefore precipitation conditions must be carefully controlled. Plasma with oxalate/fluoride as anticoagulant showed the greatest stability in storage; 24 hours at room temperature, one week at +4 degrees C and at least one month at -20 degrees C.
本文描述了一种使用I.L. Multistat III离心分析仪，用于快速常规测定反刍动物血浆中D-β-羟基丁酸（D-β-HOB）的酶促“反应速率”微量方法。反应条件经过优化，在30℃和pH 9.0时，血浆D-β-HOB浓度在100至2500微摩尔/升之间可给出线性响应。对于标准化方法，脱蛋白羊血浆的批内和批间变异系数始终小于3.5%。本方法获得的血浆浓度与原始紫外终点技术（r = 0.927，b = 0.950）和比色终点法（r = 0.937，b = 0.879）均具有良好的一致性。未经处理的羊和牛血浆始终表现出高“空白”活性，且这与两个物种的血浆乳酸脱氢酶（LDH）活性直接相关（分别为r = 0.971；p < 0.001和r = 0.949；p < 0.001）。人类LDH活性分布与绵羊相似，但与之形成对比的是，人血浆中的非特异性干扰极低且与LDH无关。马、鸡和大鼠的“空白”活性可忽略不计且LDH水平相对较低。在牛和羊中，高氯酸沉淀可消除非特异性干扰。在绵羊中，扣除“空白”活性后，未经处理血浆的D-β-HOB浓度与脱蛋白样品相当。然而，在牛中，即使经过“空白”校正，D-β-HOB水平仍显著更高（t = 6.44；p < 0.001）。与人类和其他非反刍动物不同，在反刍动物中高氯酸沉淀对于避免血浆D-β-HOB水平的错误高估至关重要。以EDTA为抗凝剂的血浆和血清中D-β-HOB浓度比含有肝素或草酸盐/氟化物的样品低约60%。然而，肝素导致的非特异性NAD还原比草酸盐/氟化物高得多（高达50%）。高浓度的乙酰乙酸（400 - 1000微摩尔/升）使羊血浆中D-β-HOB的回收率降低不到10%。反应混合物中加入水合肼可消除此效应。每次测试中高氯酸根离子浓度高于25微摩尔/升会显著抑制羊血浆中的测定，因此沉淀条件必须仔细控制。以草酸盐/氟化物为抗凝剂的血浆在储存时稳定性最佳；室温下可保存24小时，4℃下可保存一周，-20℃下至少可保存一个月。
