The human heart produces hormones that help regulate blood pressure and fluid balance in the body. One of these hormones is atrial natriuretic peptide (ANP). ANP lowers blood pressure by relaxing blood vessels and increasing urinary excretion by the kidneys.

Previous research has demonstrated that ANP naturally carries a small sugar molecule attached. This sugar moiety is produced endogenously and can modify the biological activity of ANP. When this sugar is present, ANP may affect blood vessels and renal function differently compared with the non-glycosylated form.

The present study examines how this sugar modification alters the physiological effects of ANP. This is achieved by administering ANP, either with or without the attached sugar molecule, via intravenous infusion. The study aims to determine whether glycosylated ANP differs from the native form in its effects on blood pressure and fluid balance.

Introduction Atrial natriuretic peptide (ANP) is a critical component of the natriuretic peptide family and plays a central role in cardiovascular homeostasis. ANP is primarily synthesized and secreted by atrial myocytes in response to atrial stretch and related stimuli. The peptide exerts biological effects through binding to specific receptors, leading to activation of A-type guanylyl cyclase (GC) (also known as NPR-A) and subsequent production of cyclic guanosine monophosphate (cGMP). This signaling cascade induces vasodilation, natriuresis, and diuresis, thereby contributing to regulation of blood pressure and fluid balance.

The concept of proteoforms refers to the various molecular forms that a protein can assume, arising from genetic variation, alternative splicing, and post-translational modification. ANP proteoforms therefore comprise molecular variants of ANP with potentially distinct biological activities and clinical implications.

ANP proteoforms have diagnostic and prognostic value in cardiovascular disease. Elevated ANP and related peptides indicate increased cardiac stress and are observed in conditions such as heart failure, hypertension, and acute coronary syndromes. Measurement of ANP, together with B-type natriuretic peptide (BNP) and N-terminal proBNP (NT-proBNP), is widely used in clinical practice to assist diagnosis and guide management (Volpe 2021).

Beyond diagnostic utility, ANP possesses therapeutic potential. Vasodilatory and natriuretic properties make ANP an attractive candidate for treatment of conditions associated with fluid overload and hypertension. Recombinant ANP analogues have been used clinically in acute heart failure.

Recent advances highlight strategies to enhance natriuretic peptide activity. Inhibition of neprilysin prolongs peptide half-life and biological effect. Combined neprilysin inhibition and angiotensin receptor blockade has demonstrated clinical benefit in heart failure (McMurray 2013).

Post-translational modification, including glycosylation, further diversifies ANP function. Glycosylated ANP refers to peptide forms containing covalently attached carbohydrate groups, typically linked to serine or threonine residues. This modification alters receptor binding, biological activity, and resistance to proteolytic degradation. Glycosylation may therefore influence the physiological and therapeutic profile of ANP. Recent observations suggest that glycosylation modulates interaction with natriuretic peptide receptors and downstream signaling pathways (Hansen 2019).

Objectives, Hypothesis, and Endpoints The primary objective of the study is to clarify the physiological significance of glycosylated ANP in humans.

The primary hypothesis is that glycosylated ANP regulates blood pressure without inducing diuresis.

Primary endpoints include diuresis and natriuresis during intervention. Secondary endpoints include changes in blood pressure and plasma and urinary concentrations of natriuretic peptides, renin-angiotensin-aldosterone parameters, electrolytes, cyclic nucleotides, metabolic substrates, stress hormones, and amino acids.

Methods and Study Design The study is designed as a randomized, double-blind, placebo-controlled crossover investigation. Each participant acts as an individual control. Randomization assigns infusion with ANP, glycosylated ANP (gANP), or saline on separate study days.

Following written informed consent, screening is performed after overnight fasting and includes clinical assessment, laboratory evaluation, and urine testing.

On study days, participants are admitted after a 10-hour fast. Procedures include bladder emptying, venous catheter placement, baseline sampling, and continuous blood pressure monitoring. Infusions of ANP, gANP, or saline are administered according to protocol.

Blood samples are obtained at predefined intervals, and urine is collected during and after infusion. Ultrasonographic assessment of vascular parameters and bladder volumes is performed where applicable.

Study Population Inclusion criteria comprise healthy male individuals aged 18-30 years with normal body mass index and hemoglobin levels.

Exclusion criteria include recent illness, chronic disease, hypotension, smoking, substance abuse, recent blood donation, and medication use incompatible with study requirements.

Safety and Ethics Both ANP and gANP are associated with blood pressure reduction. Continuous monitoring is implemented, and infusion is discontinued if symptomatic hypotension occurs.

The study is conducted in accordance with ethical guidelines, with approval from the relevant ethics committee.

Biobank and Data Handling Biological samples are coded and stored according to regulatory requirements. Analyses are performed at designated institutions, and remaining material is handled in accordance with approved protocols.

Funding and Registration The study is supported by Innovation Fund Denmark. A patent application related to glycosylated ANP has been filed.

Trial registration was completed following recognition of registry requirements, with all study parameters finalized prior to participant enrollment and approved by the ethics committee.

Inclusion Criteria:

• Healthy male volunteers
• Age: 18-30 years
• BMI: 20-25 kg/m²
• Body weight ≤90 kg (amended protocol)
• Normal hemoglobin
• Ability to provide informed consent

Exclusion Criteria:

• Acute illness within two weeks
• Chronic cardiovascular, renal, hepatic, pulmonary, metabolic or malignant disease
• Hypotension or history of syncope
• Smoking
• Substance or alcohol abuse
• Recent blood donation (<3 months)
• Medication that cannot be safely interrupted