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.

Methods
Steller sea lion pups were captured live at the Chiswell Island rookery in southcentral Alaska in June 2016. These pups were handled as part of collaborative ongoing research by the Alaska SeaLife Center and the Alaska Department of Fish and Game under Marine Mammal Protection Act permit numbers 18537-00 and 18438-00. Blood samples were drawn from anesthetized pups at the vein of the caudal gluteal plexus directly into blood collection tubes treated with anticoagulants (ethylenediaminetetraacetic acid or sodium heparin). 

The calibration of the ketometer was tested at the beginning of each day of use using low (0.4–0.8 mmol/L) and high (3.1–5.1 mmol/L) control solutions provided by the manufacturer. Within ten minutes of collecting a blood sample, the whole blood [β–HBA] was measured by the Precision Xtra™ ketometer in triplicate technical replicates following manufacturer instructions for both types of anticoagulant blood collection tubes.

Samples in blood collection tubes were kept chilled in the field until centrifuging at the end of the day. Plasma aliquots were frozen and stored at -80°C. 

Weeks following the field collections, the [β–HBA] of thawed (Thaw 1) plasma aliquoted from NaHep treated blood collection tubes were measured using both the Precision Xtra™ ketometer and the Reference Assay, the established method utilized in our research laboratory, via commercially-available assay kit (Sigma Aldrich, now Millipore Sigma, St. Louis, MO, Kit #MAK041). Both plasma Reference Assay and ketometer tests were conducted on the same day in triplicate technical replicates at Thaw 1. The remaining plasma samples were refrozen to -80°C. Four months later, these same plasma samples (Thaw 2) were again tested using only the Precision Xtra™ to evaluate the effect of an additional freeze-thaw cycle on the accuracy of the results. 

Data were reported as the mean (mmol/L) of the technical replicates, and the percent coefficient of variation (%CV) was calculated for each set of technical replicates; the desired %CV was <10% to meet laboratory standards.

Calculations of assay metabolite concentrations were made using Softmax Pro (v. 4.8) software or the open-access interface MyAssays.com, applying a four-parameter logistic curve.