Beta-hydroxybutyrate ketone concentrations via ketometer and colorimetric assay in Steller sea lion whole blood and plasma

We evaluated the Precision Xtra™ ketometer during a larger study categorizing free-ranging Steller sea lion (Eumetopias jubatus; SSL) pup fasting status, necessitating identification of plasma β-hydroxybutyrate concentrations ([β-HBA]) around a < and ≥0.3 mmol/L threshold. Whole blood samples mixed with sodium heparin (NaHep) or ethylenediaminetetraacetic acid liquid anticoagulants were tested <10 minutes after collection (n=14; triplicate technical replicates). Plasma (stored at -80°C, NaHep, Thaw1) measured via our laboratory's Reference Assay (Sigma Aldrich, St. Louis, MO, Kit #MAK041) served as the standard [β-HBA] for ketometer comparisons. Our observed β-HBA range (0.0–1.6 mmol/L), consistent with published [β-HBA] of free-ranging Otariid pups, represented the lower 20% of the ketometer's range (0.0–8.0 mmol/L). The maximal coefficient of variation (%CV) of ketometer technical replicates was 9.1% (NaHep, whole blood). The majority of ketometer technical replicate sets (84%, including all matrices, anticoagulants, and thawings) were identical (CV=0%). We found linear relationships and agreement of ketometer [β-HBA] between whole blood preserved with different anticoagulants and between whole blood and plasma (Thaw1) measurements. The ketometer produced results with linearity to the Reference Assay for both whole blood and plasma (Thaw1). We identified a non-linear relationship between plasma at Thaw1 and Thaw2 (tested four months apart, NaHep), as only samples with higher SSL [B-HBA] decreased in concentration, and all others remained the same. With respect to categorizing SSL pup fasting, the ketometer % Accuracy, %Sensitivity, and %Specificity for samples with Reference Assay β-HBA <0.2 and >0.4 mmol/L were 100%. We adopted a modified procedure: plasma samples with mean ketometer concentrations ±0.1 mmol/L of 0.3 mmol/L β-HBA were re-evaluated using the Reference Assay, improving measurement precision from tenths (ketometer) to thousandths (assay) mmol/L. The Precision Xtra™ ketometer was valuable to our application, over the range of [β-HBA] observed in SSL pup plasma and whole blood samples.

本研究在一项针对野生北海狮（Eumetopias jubatus，SSL）幼崽禁食状态进行分类的大型研究中，对Precision Xtra™酮体检测仪（ketometer）开展性能评估，本次评估需围绕<0.3 mmol/L与≥0.3 mmol/L的阈值，对血浆β-羟丁酸（β-hydroxybutyrate, β-HBA）浓度([β-HBA])进行分类判定。采集的全血样本经肝素钠（NaHep）或液体乙二胺四乙酸（EDTA）抗凝后，于采集后10分钟内完成检测，共纳入14份样本，每份样本设置3次技术重复。以本实验室采用的参考检测方法（Reference Assay，使用西格玛奥德里奇Sigma Aldrich公司美国密苏里州圣路易斯分部生产的试剂盒（货号MAK041））检测的血浆样本（肝素钠抗凝，于-80℃冻存，首次解冻（Thaw1））的β-HBA浓度作为标准值，用于与酮体检测仪的检测结果进行比对。本研究测得的β-HBA浓度范围为0.0~1.6 mmol/L，与已发表的野生海狮科（Otariid）幼崽的β-HBA浓度范围一致，该范围处于该酮体检测仪检测范围（0.0~8.0 mmol/L）的下20%区间内。酮体检测仪的技术重复最大变异系数（%CV）为9.1%（肝素钠抗凝全血样本）；84%的技术重复组（涵盖所有样本基质、抗凝剂及解冻方式）的变异系数为0%，即检测结果完全一致。研究发现，不同抗凝剂保存的全血样本之间，以及全血与首次解冻的血浆样本之间，酮体检测仪测得的β-HBA浓度呈线性相关且一致性良好；无论全血还是首次解冻的血浆样本，酮体检测仪的检测结果均与参考检测方法的结果呈线性相关。此外，我们观察到首次解冻与二次解冻（Thaw2）的血浆样本（肝素钠抗凝，两次检测间隔4个月）之间的β-HBA浓度呈非线性相关：仅野生北海狮幼崽β-HBA浓度较高的样本出现浓度下降，其余样本浓度无变化。在针对野生北海狮幼崽禁食状态的分类任务中，当参考检测方法测得的β-HBA浓度<0.2 mmol/L与>0.4 mmol/L时，酮体检测仪的准确率（%Accuracy）、灵敏度（%Sensitivity）及特异度（%Specificity）均为100%。我们优化了检测流程：对于酮体检测仪测得的平均浓度与0.3 mmol/L的β-HBA差值在±0.1 mmol/L范围内的血浆样本，采用参考检测方法进行复检，将检测精度从酮体检测仪的十分位（mmol/L）提升至参考检测方法的千分位（mmol/L）。综上，在野生北海狮幼崽血浆及全血样本的实测β-HBA浓度范围内，Precision Xtra™酮体检测仪可有效应用于本研究场景。