DOI: 10.5061/dryad.qfttdz0ps
title: Beta-hydroxybutyrate ketone concentrations via ketometer and colorimetric assay in Steller sea lion whole blood and plasma

description: 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 &lt; and ≥0.3 mmol/L threshold.
 Whole blood samples mixed with sodium heparin (NaHep) or
 ethylenediaminetetraacetic acid liquid anticoagulants were tested
 &lt;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 &lt;0.2 and
 &gt;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.
description: 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 (<em>Thaw
 1</em>) plasma aliquoted from NaHep treated blood collection tubes
 were measured using both the Precision Xtra™ ketometer and the
 <em>Reference Assay</em>, 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
 <em>Reference Assay</em> and ketometer tests were conducted on
 the same day in triplicate technical replicates at<em> Thaw
 1.</em> The remaining plasma samples were refrozen to -80°C. Four
 months later, these same plasma samples (<em>Thaw 2</em>) 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 &lt;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.
description: # Beta-hydroxybutyrate ketone concentrations via ketometer and
 colorimetric assay in Steller sea lion whole blood and plasma
 [https://doi.org/10.5061/dryad.qfttdz0ps](https://doi.org/10.5061/dryad.qfttdz0ps) The data consist of five separate measurements of beta hydroxybutyrate (mmol/L) on blood or plasma samples collected from each of 14 Steller sea lion pups.  The five measurements vary by method, blood compartment, anticoagulant, and/or storage history.   **The data are organized into 13 columns:**   SSL Pup                      range 1–14; corresponds to ID in Table 3 and Figure 1 in linked publication   PupID                         unique identifier for each Steller sea lion pup   Method                      technique used to obtain B-HBA concentration:  A=Assay; K=Ketometer   Matrix                         blood compartment tested:  WB=whole blood; PL= plasma   Anticoagulant           chemical that blood collection tube was treated with to prevent coagulation: NaHep=sodium heparin; EDTA= ethylenediaminetetraacetic acid   StorageHistory         condition of the blood compartment being tested:  A=freshly collected (&lt;10 minutes); B=frozen @ -80°C for weeks prior to *Thaw 1*; C=refrozen @ -80°C after *Thaw1* and thawed again four months later (*Thaw2*)   BHBA\_Rep1              technical replicate #1, concentration of beta hydroxybutyrate concentration (mmol/L)   BHBA\_Rep2              technical replicate #2, concentration of beta hydroxybutyrate concentration (mmol/L)   BHBA\_Rep3              technical replicate #3, concentration of beta hydroxybutyrate concentration (mmol/L)   TestDate                    date test conducted:  YYYY-MM-DD   BHBA\_Mean             mean of the technical replicates (mmol/L)   BHBA\_SD                  standard deviation of the technical replicates    BHBA\_CV                  percent coefficient of variation of the technical replicates:  CV= (SD/Mean)\*100