Assessment of BHB Concentration Agreement Among Sampling Locations and the Impact of Ketosis on EPO, and More

Assessment of BHB Concentration Agreement Among Sampling Locations and the Impact of Ketosis on Erythropoietin Levels: A Two-Part Study

This study aims to address two key aspects. First, the suitability of selecting a specific sampling site for BHB measurement in patients and research, as well as potential differences between capillary and venous blood measurements. Additionally, the study will investigate the effects of ketosis on EPO concentrations, sex hormone levels, hemodynamic markers, and blood pressure.

This investigation will utilize blood samples collected from baseline and at Day 15, between which participants are exposed to intermittent ketosis or placebo in a randomized parallel design.

Study Overview

• Status: Completed
• Conditions: Ketosis
• Intervention / Treatment: Dietary supplement: Placebo; Dietary supplement: Ketone monoester

Detailed Description

Aim and Perspective The primary objective of this study is to ascertain The agreement between estimates of beta-hydroxybutyrate (BHB) in capillary and venous blood and whether this agreement is influenced by the level of BHB.

The agreement in BHB measurements between finger and earlobe capillary samples. The agreement in BHB measurements between venous estimates obtained through a point-of-care device (KetoSure) and full blood estimates obtained through hydrophilic interaction liquid chromatography tandem mass spectrometry (HLCMS).

A central aim of the study is also to investigate The impact of ketosis on short-term and longer-term erythropoietin (EPO) levels.

The resulting effects of EPO on erythropoiesis and iron metabolism during a two-week period of intermittent ketosis.

The effects of ketosis on sex hormones including derived factors. The effects of ketosis on hemodynamic markers and blood pressure.

The study aims to determine the appropriateness of selecting a specific sampling site for BHB measurement in both patient care and research. Additionally, it seeks to identify any differences between BHB measurements from capillary and venous blood samples. The study will also examine the concordance between the KetoSure point-of-care-test (POCT) device and the established gold standard, offering insight into any discrepancies arising from electrochemical estimations and HLCMS.

Accurate BHB measurement is crucial in clinical and experimental settings. Firstly, precise BHB quantification can inform clinical decision-making for conditions such as suspected hyperinsulinemia, uncertain etiology hypoglycemia, and diabetic ketoacidosis. Secondly, given the extensive research on BHB inference and ketones in recent years, the credibility of these studies heavily hinges on the precision of measurements concerning sample type and sampling site selection.

During the randomized study period, the effects of ketosis on EPO concentrations, sex hormone levels, hemodynamic markers, and blood pressure measurements will be explored. These analyses will be conducted using blood samples collected from baseline through the acute study visit as well as on Day 15, when participants receive intermittent BHB ketone ester supplementation or matched placebo according to randomization.

Analytical approach Following a visual assessment of graphical linearity representation, differences will be calculated using the paired t-test, agreement determined using the Bland-Altman plot, and correlations assessed using Pearson's r. Further calculations will employ Lin's concordance correlation coefficient of absolute agreement. An analysis equivalent to repeated measurements ANOVA will be applied. No imputation of missing data will be conducted, and steps will be taken to ensure data completeness before participants leave the research facilities.

Sample Size and Power Calculation Given the absence of prior studies on BHB agreement data, our study's sample size calculations are based on relevant literature. Citing Boyd et al., and considering correlated glucose estimates, a sample size of 13 participants will provide sufficient statistical power (alpha = 0.05, beta = 20%) to detect a difference of 0.58 mM in glucose estimates between capillary and venous blood samples (SD = 0.68 mM). A sample size of 20 participants is justifiably required to detect a clinically significant difference of 1 mM (SD = 1.5) under the same parameters. Consequently, we will include 16 patients in our study.

Up to 150 mL of blood will be collected during the study, including repeated sampling through an indwelling catheter during acute testing and follow-up outpatient sampling at Day 15.

Purpose of Storage Biological samples will be stored at -80 °C for the duration of data collection and for 18 months thereafter to allow for batch analyses. A research biobank will be established for samples not analyzed immediately, with surplus material preserved for potential future research.

Handling of Patient Information Access to electronic health records is limited to laboratory results necessary for study analyses and is included in informed consent. Data will be pseudonymized during analysis. De-identification codes will be retained by the primary investigator.

Data Privacy and Sharing Data access is restricted to investigators until completion of final analyses. After anonymization, data may be shared upon reasonable request. Public data sharing is under consideration following publication.

Financial Information The study is investigator-initiated and funded through independent research funds within the Department of Internal Medicine, Regional Hospital Viborg. No investigators have financial interests in the intervention.

• Study Type: Interventional
• Enrollment (Actual): 16
• Phase: Not Applicable

Contacts and Locations

Study Locations

• Denmark
  • Viborg, Denmark, 8800
    • Department of Internal Medicine, Viborg Regional Hospital

Participation Criteria

Eligibility Criteria

• Ages Eligible for Study: Adult
• Accepts Healthy Volunteers: Yes

Description

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
• electrolyte disorders
• acute or chronic kidney disease or ompromised renal function including excess risk
• servere hypertension
• autoimmune disease
• liver or bile disease
• diabetes mellitus
• reactive hypoglycemia or similar disorders
• treatment with drugs, and dietary supplements with inference on key metabolic or hormonal markers, e.g. insulin, glucagon, DDP-IV inhibitors, GLP-1 RA, sulfunylurea
• 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
• allergies to catheters or adhesives

Study Plan

How is the study designed?

Design Details

• Primary Purpose: Basic Science
• Allocation: Randomized
• Interventional Model: Parallel Assignment
• Masking: Triple

Number of Arms

2

Arms and Interventions

Participant Group / Arm	Intervention / Treatment
Experimental: Ketosis; Ketosis (the condition being investigated) is obtained by ingestion of a ketone monoester	Dietary supplement: Ketone monoester; Supraphysiological ketosis induced by ingestion of a ketone monoester dietary supplement administered intermittently during the randomized two-week intervention period.; Other Names: KetoneAid Ke4 Pro Ketone Ester
Placebo Comparator: Control; The control arm is a drink matched in taste, volume, appearence, and viscosity to that of the active/experimental arm	Dietary supplement: Placebo; The placebo vehicle is matched to the ketosis intervention in the experimental arm with regards to taste, volume, viscosity, appearence, and packaging

What is the study measuring?

Primary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Change in Venous Plasma Beta-hydroxybutyrate (BHB) From Baseline to Peak During Acute Ketosis Exposure	Venous plasma beta-hydroxybutyrate (BHB) concentration measured in mmol/L. The outcome represents the change from baseline (0 minutes) to the maximum observed BHB concentration during the acute ketosis exposure.	Baseline (0 minutes) to peak concentration during acute ketosis exposure (0-180 minutes)
Serum Erythropoietin Concentration at Day 15	Serum erythropoietin (EPO) concentration measured in IU/L. Values represent the mean concentration at the end of randomized treatment.	Day 15 (end of randomized treatment)
Estradiol Concentration at Day 15	Plasma estradiol measured in pmol/L at Day 15. Values represent the mean concentration at the end of randomized treatment.	Day 15 (end of randomized treatment)

Secondary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Serum Ferritin Concentration at Day 15	Serum ferritin concentration measured in µg/L at Day 15. Values represent the mean concentration at the end of randomized treatment.	Day 15 (end of randomized treatment)

Other Outcome Measures

Outcome Measure	Measure Description	Time Frame
Plasma Testosterone Concentration at Day 15	Testosterone concentration measured in nmol/L at Day 15. Values represent the mean concentration at the end of randomized treatment.	Day 15 (end of randomized treatment)
Hematocrit at Day 15	Hematocrit, expressed as erythrocyte volume fraction (EVF), measured percent (%). Values represent the mean concentration at the end of randomized treatment.	Day 15 (end of randomized treatment)
Hemoglobin Concentration at Day 15	Hemoglobin concentration measured in mmol/L at Day 15. Values represent the mean concentration at the end of randomized treatment.	Day 15 (end of randomized treatment)

Collaborators and Investigators

• Sponsor: Central Jutland Regional Hospital
• Investigators: Principal Investigator: Henrik H Thomsen, Ph.D., Department of Internal Medicine, Viborg Regional Hospital, Denmark

Study record dates

Study Major Dates

• Study Start (Actual): August 24, 2023
• Primary Completion (Actual): January 30, 2024
• Study Completion (Actual): August 30, 2024

Study Registration Dates

• First Submitted: August 23, 2023
• First Submitted That Met QC Criteria: September 19, 2023
• First Posted (Actual): September 25, 2023

Study Record Updates

• Last Update Posted (Actual): April 28, 2026
• Last Update Submitted That Met QC Criteria: April 7, 2026
• Last Verified: April 1, 2026

More Information

Terms related to this study

• Keywords: ketosis; sex hormones; ketone monoester; erythropoitin
• Additional Relevant MeSH Terms: Metabolic Diseases; Acid-Base Imbalance; Acidosis; Nutritional and Metabolic Diseases; Ketosis
• Other Study ID Numbers: 1-16-02-279-23

Plan for Individual participant data (IPD)

• Plan to Share Individual Participant Data (IPD)?: NO

Drug and device information, study documents

• Studies a U.S. FDA-regulated drug product: No
• Studies a U.S. FDA-regulated device product: No