Assessment of BHB Concentration Agreement Among Sampling Locations and the Impact of Ketosis on Erythropoietin Levels: A Two-Part Study
Phase: NA
Conditions: Ketosis
Sponsor: Central Jutland Regional Hospital

BHB agreement and EPO 
Version: Final  
04-12-2025 
 Assessment of BHB Concentration 
Agreement Among Sampling Locations 
and the Impact of Ketosis on 
Erythropoietin Levels: A Two-Part 
Study  
Aim and perspective 
This study's main goal (part 1) is to determine 
i. 
agreement in beta-hydroxybutarate (BHB) of capillary and venous blood 
estimates and subsequently whether agreement is dependent on BHB level, and 
ii. 
agreement in BHB of finger and earlobe capillary samples, and 
iii. 
agreement in BHB of venous estimates (estimated by point-of-care device) and 
full blood (estimated by use of hydrophilic interaction liquid chromatography 
tandem mass spectrometry (HLCMS)) 
The main goal (part 2) is to determine 
i. 
effects of ketosis on erythropoietin(EPO) short-term and long-term 
ii. 
derived effects on erythropoiesis and iron metabolism from two-week intermittent 
ketosis 
 
This study will establish (part 1) whether it is appropriate to select a specific sampling site for 
measuring BHB in patients and performing research, and whether there is a difference 
between measurements capillary and venous blood. The agreement between the KetoSure, 
a point-of-care-test (POCT) device, and the gold standard will also be examined, offering 
insight into any discrepancies with point-of-care estimates obtained by electrochemical 
estimations and HLCMS. High BHB measurement validity is required in both clinical and 
experimental contexts. First, BHB quantification may aid in clinical decision-making for 
suspected hyperinsulinemia, hypoglycemia of uncertain etiology, diabetic ketoacidosis, and 
other diseases. Second, a substantial amount of research has been conducted on BHB 
inference and ketones in general in recent years, and the validity of the interpretation of 
these studies is highly dependent on measurement precision in regards to which sample 
type and sampling site is applied. 
In addition, the effects of ketosis on EPO concentrations will be investigated (part 2) from 
blood samples obtained during part 1 (acute effects) and at day 7 and day 14 where 
participants are intermittently ketotic allowing also for investigations on EPO-derived effects 
on erythropoiesis and iron metabolism.  

BHB agreement and EPO 
Version: Final  
04-12-2025 
Background 
During an experiment in which samples were obtained from two distinct places, namely the 
finger pulp and the earlobe, the KetoSure ketonometer disclosed a discrepancy in BHB 
estimates, prompting us to review the relevant literature on discrepancy between sampling 
sites. 
 
In glucose determination, such discrepancy between capillary and venous blood 
concentration is well-known[1]. In contrast to our incidental findings, capillary glucose 
estimations are higher than venous plasma estimates. Several hypotheses, with variances in 
hematocrit being the most prevalent, have been suggested as explanations for these 
discrepancies; however, conclusions change depending on the analyte of interest[2]. In 
addition to the comparator, the POCT detection method, kind of enzymatic reaction, and pH 
value are of utmost importance[3]. 
As reported in a 2016 study, the accuracy of capillary BHB measurement has been tested in 
various contexts and with various ketonometers[4]. Primarily with the intent of improving its 
ability to identify diabetic ketoacidosis. Similarly, the accuracy of glucose estimates from 
different capillary sample sites has varied in response to both glucose concentration and 
capillary sampling site[5]. 
 
To our knowledge, neither the agreement between venous blood and full blood+HLCMS 
values nor the disparity between different sampling sites for the extraction of capillary 
samples have been investigated. 
 
Two investigations have indicated a link between ketosis and EPO[6,7]. Both, however, had 
other primary endpoints, and thus were not meant to answer this particular research 
question. In addition, only the correlation between ketosis and EPO levels has been studied. 
Thus, erythropoietic effects of EPO in the bone marrow (and elsewhere) have not been 
studied. The incorporation of ambulatory blood testing, and the extension of the ketosis, 
such effects can be investigated over an appropriate time frame and provide the opportunity 
to investigate not only the relationship between exogenous ketosis in general, but also any 
temporal aspects thereof, as the study design includes both an "acute" (part 1) and a 
"chronic" (part 2) factor. 
References 
1.  
Boyd R, Leigh B, Stuart P. Capillary versus venous bedside blood glucose 
estimations. Emerg Med J. 2005;22(3):177-179. doi:10.1136/emj.2003.011619 
2.  
Osteresch B, Cramer B, Humpf HU. Analysis of ochratoxin A in dried blood spots  
Correlation between venous and finger prick blood, the influence of hematocrit and 
spotted volume. Journal of Chromatography B. 2016;1020:158-164. 
doi:10.1016/j.jchromb.2016.03.026 
3.  
Kotwal N, Pandit A. Variability of capillary blood glucose monitoring measured on 
home glucose monitoring devices. Indian J Endocrinol Metab. 2012;16(8):248. 
doi:10.4103/2230-8210.104052 
4.  
Brooke J, Stiell M, Ojo O. Evaluation of the Accuracy of Capillary Hydroxybutyrate 
Measurement Compared with Other Measurements in the Diagnosis of Diabetic 

BHB agreement and EPO 
Version: Final  
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Ketoacidosis: A Systematic Review. Int J Environ Res Public Health. 2016;13(9):837. 
doi:10.3390/ijerph13090837 
5.  
Lightbody TO, Nightingale P, Reddy Kolanu V, Sapey E. The Benefits and Limitations 
of Point of Care Testing for Patients with Diabetes Who are Acutely Unwell. J 
Diabetes Metab. 2017;08(09). doi:10.4172/2155-6156.1000761 
6.  
Evans E, Walhin JP, Hengist A, Betts JA, Dearlove DJ, Gonzalez JT. Ketone 
monoester ingestion increases post-exercise serum erythropoietin concentrations in 
healthy men. American Journal of Physiology-Endocrinology and Metabolism. 
Published online November 30, 2022. doi:10.1152/ajpendo.00264.2022 
7.  
Lauritsen KM, Søndergaard E, Svart M, Møller N, Gormsen LC. Ketone Body Infusion 
Increases Circulating Erythropoietin and Bone Marrow Glucose Uptake. Diabetes 
Care. 2018;41(12):e152-e154. doi:10.2337/dc18-1421 
8.  
Topping J, Reardon M, Coleman J, et al. A Comparison of Venous versus Capillary 
Blood Samples when Measuring Blood Glucose Using a Point-of-Care, Capillary-
Based Glucometer. Prehosp Disaster Med. 2019;34(5):506-509. 
doi:10.1017/S1049023X19004850 
  
  
Methods 
Design 
The study is performed with an analytical cross-sectional design, i.e., participants are 
sampled during three different exposures (different degrees of ketosis) on a single study 
day(part 1) and on two out-patient occasions on day 7 and day 14 (part 2). 
Part 2 is a participant-blinded randomized controlled design where participants receive in a 
1:1 fashion either the intervention (ketone monoester drink) daily or a taste- and volume-
matched placebo drink 
Study day (part 1) 
The study day duration will be approximately four hours with participants reporting in at Klinik 
for Diabetes & Hormonsygdomme, Viborg Regional Hospital at 8 A.M. in the overnight fasted 
state. A standard 18G venous catheter is inserted in a median cubital, basilic, or cephalic 
vein for blood sampling. Baseline and following, blood samples are drawn on the hour for 3 
hours. Subsequently, participation is over, the catheter is removed with observation and 
compression 5-10 min. upon which the study participation is over for the part of the 
participants. 
Ambulatory investigations (part 2) 
Following the study day (part 1) participants are provided the full amount of either the ketone 
monoester drink or the comparator (placebo-drink). The drinks are provided in small plastic 
bottles  

BHB agreement and EPO 
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Methods 
In order to obtain ketosis, participants ingest a common sports drink, KE4, which contains a 
ketone monoester. Upon this, ketosis is established with a substantial increase in BHB for 
estimation in the venous and whole blood, along with capillary measurements from the 
earlobe and finger pulp. The dosage will be 500 mg/kg for the study day (part 1) and daily, 
divided in two or three dosages as preferred, for the following 14 days (part 2) 
Feasibility 
No substantial practical challenges are expected  all equipment and competences are 
present in the Department of Internal Medicine and Department of Clinical Biochemistry.  
No recruitment issues are expected  this is a small-scale study with minimal impact in 
relation to participation. 
The study is fully funded by independent research funds within departments involved 
although external funding will be sought for. 
Analytic methods 
BHB is estimated by the Ketosure
 for capillary measurements (finger pulp and earlobe), 
for venous blood samples, and HLCMS for full blood estimation of BHB as the reference 
standard. 
Statistical considerations 
Analytical approach 
Following a visual examination of the graphical representation for linearity, differences are 
calculated using the paired t-test, agreement is determined using the Bland-Altman plot, and 
Pearson's r is used to determine correlations. Supporting calculations are performed using 
When comparisons over 
observations from part 1 and 2 are performed an repeated measurements anova equivalent 
analysis is applied. 
Sample size and power calculation 
No previous studies with data on BHB agreement have been performed. Based on our 
incidental findings, only very few subjects would be needed. The data is, however, far too 
small to base sample size calculations on.  
 
Based on Boyd et al.[1] with highly correlated estimates of glucose and the acceptance of a 
type 1 error (alpha) of 0.05, type 2 error of 20% (beta), a sample size of 13 participants is 
required to provide adequate statistical power to detect a difference of 0.58 mM in glucose 
estimates between capillary and venous blood samples with the standard deviation of 0.68 
mM. Others have similarly found a clinically meaningful difference of 1 mM between 
sampling sites[8]. With alpha and beta set as mentioned, a sample size of 20 participants is 

BHB agreement and EPO 
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required to provide adequate statistical power to detect a difference of 1 mM (SD = 1.5), 
should such a difference exist. 
 
As such, we find it reasonable to include 15 patients in our study resulting in a power of 91% 
if expecting a meaningful difference of 1 mM and an average standard deviation of the two 
studies mentioned of 1.1 mM. Similar number of participants have been used elsewhere with 
regards to part 2 of this project and sample size as suggested is fully justified to test 
hypotheses of part 2. 
Participants 
Recruitment 
Subjects will be recruited through notices on specific websites (sundhed.dk, 
forsøgsperson.dk) and social media (Facebook) along with board notice on selected local 
educational institutions, hospitals, and in newspapers. 
First contact will be by telephone with the primary investigator(s) followed by the formal 
information on study participation (se section on recruitment and informed consent) 
Inclusion criteria 
Age 18-60 years 
BMI 19-30 kg/m2 
Expected ease of catheter insertion 
Considered of sound health 
Oral and written informed consent 
Exclusion criteria 
Inability to fully understand the consent including consent forms 
Inability to cooperate to the study 
Any electrolyte disorders, kidney disease or otherwise compromised renal function including 
excess risk hereof, e.g. hypertension, albuminuria, autoimmune disease, family history of 
kidney disease 
Any liver or bile disease, or an increased risk of getting one, such as from hepatotoxic drugs, 
alcoholism, gallstones, pancreatitis, an autoimmune disease, or a history of liver disease in 
the family. 
Diabetes mellitus or any metabolic and/or hormonal disease including 
diagnosed/undiagnosed reactive hypoglycemia or similar disorders. This includes treatment 
with drugs, and dietary supplements with inference on key metabolic or hormonal markers, 
e.g. insulin, glucagon, lipids, and GLP-1 
Any use of illegal or otherwise use of medicinal products without prescription 
Anemia or other know disease of the hematopoietic system 
Previous bariatric surgery 
Previous myocardial infarction or uncontrolled myocardial ischemia 
Recent intended/unintended weight loss 

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Allergies to catheters or adhesives 
Safeguards to minimize pain, discomfort, fear and 
other risks 
Short-term and long-term risks and side effects 
There are neither short-term nor long-term serious hazards or adverse effects to concern. 
KE4 is a bitter sports drink that can cause moderate, temporary nausea and bloating. 
Appetite may be slightly reduced over the full study period (part 2). Compared to sweet, 
salty, or umami-flavored beverages, the digestive adverse effects are equivalent to those of 
bitter-flavored beverages in general. This is a well-known side effect of bitter-tasting 
beverages in certain individuals. 
As with the pricking of the fingers and earlobes, there is a small risk of infection following 
catheter placement, as well as a slight degree of discomfort. 
Risk of radioactive radiation 
There is no radiation involved in this study 
Collection of new biological material 
What and how much material 
A total of 150 mL of blood is drawn in this study. This includes unavoidable spill from 
repeated sampling from indwelling catheter in part 1. No spillage is associated with part 2 
since blood sampling is performed only twice in an outpatient setting (at the hospital 
laboratory for blood sampling) 
Purpose 
For analysis in bulk. 
How long and what is the purpose of storage 
All biological samples are stored for the full period of data collection and 18 months after in 
order to analyse all samples in bulk. Samples are stored at -80 ºC. 
Biobank 
A research biobank is developed for the bulk analysis of all samples not analyzed on the day 
of the study, i.e. measurements using the Ketosure
 POCT device. The analyses are 
conducted within a few months at the earliest opportunity and surplus material is preserved 
as a biobank for future yet undetermined research. 

BHB agreement and EPO 
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Information from patient records 
Access to participants' electronic patient records (exclusively and limited to solely laboratory 
results section) is included in study participation consent and necessary for practical 
reasons, i.e., information on medication, blood pressure readings. Only information 
otherwise routinely obtained as part of participants' treatment and are registered with the 
purpose of analysis within the frame of the study. Any information for use in the study given 
prior to oral and written consent are forwarded to the investigator. 
Consent gives the study investigators as well as any control authority direct access to obtain 
information in the patient's health records, etc., including electronic health records, in order 
essary for the implementation 
of the research project and for the purposes of any and all controls, including self-monitoring, 
quality control, and monitoring, which these are required to perform. 
Handling of personal data in the study 
The Data Protection Act is upheld in accordance with The General Data Protection 
Regulation in all aspects of the study. 
All data are pseudonymized by de-identification when entered in data analysis sheet. A copy 
of the de-identification code kept only by primary investigator. 
Access to data is equally open only to investigators until final analysis. Hereafter other 
investigators have access by which time all data are completely anonymized. After 
publication all data are open to public scrutiny. 
Financial information 
The study is initiated by primary investigator Henrik Holm Thomsen 
The study is financed from independent research funds within the Department of Internal 
Medicine, Regional Hospital Viborg. Additional external funding will be sought. All funding 
will be related to study expenses only, excluding any investigator salary which is cared for 
according to individual agreements with respective employers. All financial transactions are 
administered according to the research practices of the Central Denmark Region and 
Regional Hospital Viborg. 
 
Research investigators and collaborators have no financial interests in the study. 
Remuneration and/or other benefits to participants 
Participants will be offered an inconvenience allowance of DKK 750 for completed study day 
participation (part 1) and DKK 750 for participation in part 2 (outpatient), thus DKK 1500 in 
total, with proportionate remuneration in the event of premature withdrawal. Requirements of 
the Committee Law §20, stk. 1, nr. 3 are upheld.  

BHB agreement and EPO 
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Recruitment of participants and informed consent 
Potential participants are recruited through post on social media (Facebook and Twitter). 
 
The first formal contact will be by telephone with the primary investigators (Henrik Holm 
Thomsen or Birgitte Sandfeld Paulsen) who are contacted by potential participant through 
notice of the study. Following this, the mandatory information about study participation, the 
interview, and the examination will be conducted as either a face-to-face meeting conducted 
with regard to privacy or as a video call,  per request of the potential study participant. 
Potential study participants are informed that they are allowed to bring a companion to the 
interview. 
 
Potential participants are always approached in a friendly and professional manner with a 
focus on minimizing any coercion. 
 
 
After receiving information about study background and participation, potential participants 
are given reasonable time (one to two weeks or as deemed necessary by potential 
participant) to consider participation and allowing for further questions. Hereafter, they 
submit the signed consent form and give their oral consent. 
It is emphasized to the potential participants that participation can be halted or stopped at 
any time upon request. Participants are not obliged to give the reason(s) for withdrawing 
consent and/or participation. 
Special consideration will also be on ensuring that potential participants fully understand the 
study procedures. 
Dissemination of results 
The aim is to publish results, be them 'negative', 'positive' or inconclusive, in an 
internationally recognised peer-reviewed medical journal. If not accepted for publication, 
results are disseminated through the registration at clinicaltrials.gov where the study will be 
registered.  
Ethics 
Participation is associated with minute risks and discomfort and obtaining blood samples are 
done on routine visits to outpatient clinic as already planned. Hence, the study is believed to 
be justified with obtaining novel knowledge to better treatment options for future patients. 
Information about the compensation scheme 
The study is covered by the Danish Compensation Association