Elucidating Hepatic Metabolism in Non-alcoholic Fatty Liver Disease (ECHO)

The goal of this observational, cross-sectional, case-control clinical study is to investigate the metabolic adaptations underlying the progression of nonalcoholic fatty liver disease (NAFLD), and to test the hypothesis that hepatic mitochondrial reductive stress contributes to progression of NAFLD.

The main question it aims to answer is:

Do patients with advanced NAFLD compared to patients with mild NAFLD and healthy controls have increased hepatic mitochondrial reductive stress as determined by the ketoisocaproate breath test and by plasma beta-hydroxybutyrate to acetoacetate ratio (b-OHB/AcAc)?

Study Overview

• Status: Enrolling by invitation
• Conditions: Non-alcoholic Fatty Liver Disease (NAFLD)
• Intervention / Treatment: Diagnostic test: Ketoisocaproic acid breath test; Diagnostic test: Ammonium Chloride; Diagnostic test: Bicarbonate de sodium; Diagnostic test: Deuterated Water; Diagnostic test: Oral Glucose Tolerance Test

Detailed Description

In this study the investigators study the metabolic adaptations underlying the progression of nonalcoholic fatty liver disease (NAFLD), namely hepatic mitochondrial reductive stress, ureagenesis, de novo lipogenesis and gluconeogenesis in patients with advanced and mild NAFLD and in healthy controls.

At the first visit, an informed consent will be obtained, followed by assessment of inclusion/exclusion criteria and medical history. Participants fulfilling the criteria will be enrolled in the study. A physical examination will be performed and laboratory test will be taken. Body composition will be determined with bioelectrical impedance and dual-energy x-ray absorptiometry (DEXA).

At the second visit, hepatic lipid content will be measured with magnetic resonance spectroscopy.

At the third visit, participants will pick up containers for overnight urine collection and doses of deuterated water and a standardized meal replacement bar. The fourth visit is a clinical study visit. In a specified order, participants will drink tracer doses of 15NH4Cl, 13C-bicarbonate and 13C-alpha-ketoisocaproate. An oral glucose tolerance test is performed in the end of the study day. Breath samples are collected at different time points for determination of 13C enrichment of CO2. Furthermore, "arterialized" blood samples are taken to obtain beta-hydroxybutyrate to acetoacetate ratios (b-OHB/AcAc) at different timepoints.

Whole-body oxidation of lipids, carbohydrates and protein will be determined using indirect calorimetry.

• Study Type: Observational
• Enrollment (Estimated): 30

Contacts and Locations

Study Locations

• Finland
  • Uusimaa
    • Helsinki, Uusimaa, Finland
      • Helsinki Central University Hospital

Participation Criteria

Eligibility Criteria

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

• Sampling Method: Non-Probability Sample

Study Population

General population and hospital outpatients

Description

The following inclusion/ exclusion criteria will be employed:

1. Participants must be able to communicate meaningfully with the investigator and must be legally competent to provide written informed consent.
2. Subject must be likely to be available to complete all protocol-required study visits or procedures, to the best of the subject's and investigator's knowledge.
3. Participants must be aged between 18-75 years.
4. Participants are not allowed to have alcohol consumption of 350 g/week or more in women and 420 g/week or more in men.
5. Participants are not allowed to have history of liver disease other than NAFLD as judged by history and physical examination and standard laboratory tests.
6. Participants are not allowed to have claustrophobia or metal implants to allow magnetic resonance studies.
7. Participants are not allowed to be pregnant or lactating.
8. No known or anticipated difficulties in cannulation of peripheral veins.
9. No history or evidence of any other clinically significant disorder, condition or disease other than those outlined above that, in the opinion of the investigator may compromise the ability of the subject to give written informed consent, would pose a risk to subject safety, or interfere with the study evaluation, procedures or completion.

Study Plan

How is the study designed?

Number of groups / cohorts

3

Cohorts and Interventions

Group / Cohort	Intervention / Treatment
Advanced NAFLD; Intrahepatic triglyceride content ≥ 5.56 % as determined by magnetic resonance specroscopy and liver stiffness measurement ≥ 8 kPa using transient elastography (Fibroscan)	Diagnostic test: Ketoisocaproic acid breath test; 13C-alpha-ketoisocaproic acid breath test to estimate hepatic mitochondrial reductive stress (1 mg/kg body weight; oral dose; study duration 390 min); Diagnostic test: Ammonium Chloride; 15N-ammonium chloride stable isotope test to estimate ureagenesis (20 mg/kg body weight; oral dose; study duration 390 min); Diagnostic test: Bicarbonate de sodium; 13C-bicarbonate breath test to estimate body bicarbonate pool size and gastric emptying (0.5 mg/kg body weight; oral dose; study duration 390 min); Diagnostic test: Deuterated Water; Deuterated water stable isotope test to estimate hepatic de novo lipogenesis and gluconeogenesis (3 g/kg body water; oral dose; duration: overnight); Diagnostic test: Oral Glucose Tolerance Test; A standard 2-hour 75-gram oral glucose tolerance test to assess glucose tolerance and whole body metabolism
Mild NAFLD; Intrahepatic triglyceride content ≥ 5.56 % as determined by magnetic resonance specroscopy and liver stiffness measurement < 8 kPa using transient elastography (Fibroscan)	Diagnostic test: Ketoisocaproic acid breath test; 13C-alpha-ketoisocaproic acid breath test to estimate hepatic mitochondrial reductive stress (1 mg/kg body weight; oral dose; study duration 390 min); Diagnostic test: Ammonium Chloride; 15N-ammonium chloride stable isotope test to estimate ureagenesis (20 mg/kg body weight; oral dose; study duration 390 min); Diagnostic test: Bicarbonate de sodium; 13C-bicarbonate breath test to estimate body bicarbonate pool size and gastric emptying (0.5 mg/kg body weight; oral dose; study duration 390 min); Diagnostic test: Deuterated Water; Deuterated water stable isotope test to estimate hepatic de novo lipogenesis and gluconeogenesis (3 g/kg body water; oral dose; duration: overnight); Diagnostic test: Oral Glucose Tolerance Test; A standard 2-hour 75-gram oral glucose tolerance test to assess glucose tolerance and whole body metabolism
Healthy control; Intrahepatic triglyceride content < 5.56 % as determined by magnetic resonance specroscopy and liver stiffness measurement < 8 kPa using transient elastography (Fibroscan)	Diagnostic test: Ketoisocaproic acid breath test; 13C-alpha-ketoisocaproic acid breath test to estimate hepatic mitochondrial reductive stress (1 mg/kg body weight; oral dose; study duration 390 min); Diagnostic test: Ammonium Chloride; 15N-ammonium chloride stable isotope test to estimate ureagenesis (20 mg/kg body weight; oral dose; study duration 390 min); Diagnostic test: Bicarbonate de sodium; 13C-bicarbonate breath test to estimate body bicarbonate pool size and gastric emptying (0.5 mg/kg body weight; oral dose; study duration 390 min); Diagnostic test: Deuterated Water; Deuterated water stable isotope test to estimate hepatic de novo lipogenesis and gluconeogenesis (3 g/kg body water; oral dose; duration: overnight); Diagnostic test: Oral Glucose Tolerance Test; A standard 2-hour 75-gram oral glucose tolerance test to assess glucose tolerance and whole body metabolism

What is the study measuring?

Primary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Hepatic mitochondrial reductive stress as determined by plasma beta-hydroxybutyrate-to-acetoacetate ratio	Ratio of beta-hydroxybutyrate and acetoacetate concentrations in arterialized plasma	Fourth study visit (2 months)
Hepatic mitochondrial reductive stress as determined by the ketoisocaproic acid breath test	Breath 13CO2 enrichment after ingesting 13C-alpha-ketoisocaproate measured as area under the curve	Fourth study visit (2 months)

Secondary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Plasma metabolomics	Arterialized plasma analyzed by nuclear magnetic resonance	Fourth study visit (2 months)
Plasma metabolomics	Arterialized plasma analyzed by mass spectrometry	Fourth study visit (2 months)

Other Outcome Measures

Outcome Measure	Measure Description	Time Frame
Fractional Rate of Ureagenesis	Hepatic incorporation of ammonia to urea (i.e. ureagenesis), as determined by plasma [15N1]urea enrichment after ingestion of a tracer dose of 15NH4Cl	Fourth study visit (2 months)
Fractional Rate of Hepatic de novo lipogenesis	Hepatic de novo lipogenesis, as determined by deuterium enrichment in plasma triglyceride-palmitate after ingestion of tracer doses of deuterated water	Fourth study visit (2 months)
Fractional Rate of Gluconeogenesis	Fractional gluconeogenesis, as determined by deuterium enrichment in plasma glucose after ingestion of tracer doses of deuterated water	Fourth study visit (2 months)
Ammonia incorporation to glutamine	Hepatic incorporation of ammonia to glutamine (i.e. glutamine synthesis), as determined by plasma [15N1]glutamine enrichment after ingestion of a tracer dose of 15NH4Cl	Fourth study visit (2 months)
Bicarbonate pool size	Breath 13CO2 enrichment after ingesting 13C-bicarbonate measured as area under the curve	Fourth study visit (2 months)

Collaborators and Investigators

• Sponsor: Helsinki University Central Hospital
• Investigators: Principal Investigator: Panu K. Luukkonen, MD, PhD, University of Helsinki

Study record dates

Study Major Dates

• Study Start (Estimated): May 1, 2025
• Primary Completion (Estimated): December 31, 2029
• Study Completion (Estimated): December 31, 2030

Study Registration Dates

• First Submitted: March 14, 2025
• First Submitted That Met QC Criteria: April 16, 2025
• First Posted (Actual): April 24, 2025

Study Record Updates

• Last Update Posted (Actual): April 24, 2025
• Last Update Submitted That Met QC Criteria: April 16, 2025
• Last Verified: April 1, 2025

More Information

Terms related to this study

• Keywords: liver stiffness; Hepatic mitochondrial dysfunction; Non-alcoholic Fatty Liver Disease NAFLD; 13C-α-Ketoisocaproic Acid Breath Test
• Additional Relevant MeSH Terms: Digestive System Diseases; Liver Diseases; Fatty Liver; Non-alcoholic Fatty Liver Disease
• Other Study ID Numbers: HUS/176/2025

Plan for Individual participant data (IPD)

• Plan to Share Individual Participant Data (IPD)?: NO
• IPD Plan Description: Due to European Union GDPR regulations.

Drug and device information, study documents

• Studies a U.S. FDA-regulated drug product: No
• Studies a U.S. FDA-regulated device product: No
• product manufactured in and exported from the U.S.: No