The goal of this observational study is to learn if some components of blood or exhaled breath can diagnose people having more fat in their livers than is normal, because of their poorer metabolic health (for example, because of obesity and diabetes). The main questions it aims to answer are:

1. Can a method find participants with higher liver fat than healthy participants?
2. Can a method find participants in whom higher liver fat was a cause of liver inflammation or stiffness?

Participants will:

• fast overnight
• have a routine blood draw
• easily exhale a few times into a special device or a plastic bag and fill in a short dietary questionnaire (if participating in a breath test)
• optionally swallow capsules with an orange peel extract and fish oil before exhaling, which can help get better results from breath (capsules will be medically safe and approved)

Patients with MASLD and healthy volunteers will be offered an observational study evaluating the diagnostic role of new non-invasive experimental methods (serum bile acids, breath VOC, and plasmatic spectroscopic patterns) assessing the presence and severity of liver fibrosis and steatosis.

First, this study aims to differentiate patients with metabolic dysfunction-associated steatotic liver disease (MASLD) and metabolic dysfunction-associated steatohepatitis (MASH) from healthy controls (or simple steatosis) using three experimental methods.

Second, the investigators aim to stratify patients with MASLD according to the presence/grade of steatosis and fibrosis (any, significant F2-3, advanced F3, cirrhosis F4) using the same methods.

Experimental methods tested in this study include:

1. analyzing the fasting spectrum of serum bile acids using liquid chromatography-mass spectrometry
2. analyzing the disease-specific spectroscopy patterns given by vibrational and chiroptical spectroscopy of blood plasma
3. analyzing trace concentrations of volatile organic compounds (VOC) in exhaled human breath using the Selected ion flow tube mass spectrometry. This contains the so-called stress test to monitor breath VOC (d-Limonene and triethylamine) after their regulated ingestion in the form of capsules

These parameters may eventually be combined with anthropometric and laboratory parameters (such as age or BMI).

Clinical examinations, blood sampling, ultrasound examinations of the liver, and liver elastography will be performed as part of routine care.

The results of the blood parameters can be retrospectively evaluated.

Approximately 60-80 participants in total were anticipated for each method. For patients who participate in breath tests, adequate insurance must be guaranteed.

Statistical processing: individual parameters will be evaluated in an exploratory and a validation group or by the PLS-DA algorithm with repeated cross-validation. A difference of p <0.05 will be considered a statistically significant change.

Inclusion Criteria for Participants with MASLD:

• Presence of liver steatosis (detected either by hepatic ultrasonography/CAP/histologically) or MASH/simple steatosis confirmed by liver biopsy
• Absence of secondary causes of fat accumulation in the liver and other liver disease: ruled out other etiologies of liver disease, such as viral hepatitis, drug-induced liver disease, autoimmune liver disease, biliary tract disease, and hereditary metabolic diseases

Inclusion Criteria for Negative Controls:

• Absence of liver disease
• For breath analysis: absence of liver steatosis (normal liver ultrasound image and CAP less than 248 dB/m)
• For plasma spectroscopy and bile acid analysis: Body Mass Index ≤ 25 and waist-hip ratio ≤ 0.95 plus normal liver ultrasound
• Absence of diabetes mellitus and metabolic syndrome
• Normal liver function tests, lipid spectrum, fasting glycemia
• Alcohol intake less than 20 g/ day (women) or 30 g/ day (men)

Exclusion Criteria:

• Non-compliance with the investigation program
• Failure to sign the informed consent form
• Liver biopsy/clinic discrepancy
• For LMN a TMA "stress test": fish, see fruit and citrus fruit allergy
• Pregnancy
• For bile acid analysis: Treatment with BA or BA sequestrants
• For bile acid analysis: Portal hypertension (does not apply for MASH patients)
• For bile acid analysis: Cirrhosis (does not apply for MASH patients)
• For plasma spectroscopy: Cirrhosis
• Anamnesis of alcohol abuse (based on GGT, carbohydrate-deficient transferrin, urinary ethyl-glucuronide, and patient's history)

Patients with MASLD of primary care clinic, healthy volunteers