Arginine-vasopressin marker copeptin is a sensitive plasma surrogate of hypoxic exposure

Abstract

Background: A reduced oxygen supply puts patients at risk of tissue hypoxia, organ damage, and even death. In response, several changes are activated that allow for at least partial adaptation, thereby increasing the chances of survival. We aimed to investigate whether the arginine vasopressin marker, copeptin, can be used as a marker of the degree of acclimatization/adaptation in rats exposed to hypoxia.

Methods: Sprague-Dawley rats were exposed to 10% oxygen for up to 48 hours. Arterial and right ventricular pressures were measured, and blood gas analysis was performed at set time points. Pulmonary changes were investigated by bronchoalveolar lavage, wet and dry weight measurements, and lung histology. Using a newly developed specific rat copeptin luminescence immunoassay, the regulation of vasopressin in response to hypoxia was studied, as was atrial natriuretic peptide (ANP) by detecting mid-regional proANP.

Results: With a decreasing oxygen supply, the rats rapidly became cyanotic and inactive. Despite continued exposure to 10% oxygen, all animals recuperated within 16 hours and ultimately survived. Their systemic blood pressure fell with acute (5 minutes) hypoxia but was partially recovered over time. In contrast, right ventricular pressures increased with acute (5 minutes) hypoxia and normalized after 16 hours. No signs of pulmonary inflammation or edema were found despite prolonged hypoxia. Whereas copeptin levels increased significantly after acute (5 minutes) hypoxia and then returned to near baseline after 16 hours, mid-regional proANP levels were even further increased after 16 hours of exposure to hypoxia.

Conclusion: Plasma copeptin is a sensitive marker of acute (5 minutes) exposure to severe hypoxia, and subsequent regulation can indicate recovery. Copeptin levels can therefore reflect clinical and physiological changes in response to hypoxia and indicate recovery from ongoing hypoxic exposure.

Keywords: acclimatization; adaptation; atrial natriuretic peptide; copeptin; critical illness; hypoxia; vasoactive peptides.

Figure 1

Experimental timeline. After 16 hours of either normoxia or hypoxia, the animals were anesthetized and instrumented, and the systolic right ventricular pressure was measured at the following points: 1, normoxia at baseline; 2, acute hypoxia; 3, 16 hours of hypoxia; 4, recovery (normoxia, hyperoxia and hypoxia, with measurements as indicated in minutes). While the animals were under anesthesia, the inspired gas mixture was adjusted as illustrated, and blood was collected at the indicated time points. Abbreviations: Nx, normoxia (21% O₂); Hx, hypoxia (10% O₂); Hyp, hyperoxia (100% O₂); hr, hours.

Figure 2

Body weight gradually decreased with 8–24 hours of hypoxia, after which no further changes were observed. Weight loss is reported as a percentage by comparing the weight of the animal at the beginning of the experiment with that at the end. *P<0.05 versus normoxia at 24 hours; ^#P<0.05 versus hypoxia at 8 hours. Abbreviations: Nx, normoxia (21% O₂); Hx, hypoxia (10% O₂); hr, hours.

Figure 3

Stabilization of HIF-1α in liver homogenates. Representative immunoblot of a normoxic control (first lane) compared with three hypoxic (16 hours) samples (lanes 2–4). All hypoxic liver samples (n=6) showed stabilization of HIF-1α. Abbreviations: Nx, normoxia (21% O₂); Hx, hypoxia (10% O₂); HIF-1α, hypoxia-inducible factor-1α; h, hours.

Figure 4

Systolic right ventricular pressure (SRVP) measured by right heart catheterization. Two separate experiments were performed, in which rats were exposed to 16 hours of ambient room air (baseline) followed by an acute hypoxic phase (5 minutes) or 16 hours of hypoxia followed by a normoxic recovery phase. *P<0.05, n=7. Abbreviations: Nx, normoxia (21% O₂); Hx, hypoxia (10% O₂); RV, right ventricular; min, minutes; hr, hours.

Figure 5

Fluid accumulation in the lungs was assessed by two approaches. (A) The wet lung to body weight ratio was calculated after 8, 16, 24, and 48 hours of hypoxia (*P<0.05). (#P<0.05 for difference between 8 hours and 16 hours of hypoxia). (B) The wet to dry lung ratio was measured at baseline and after 16 hours of hypoxia, n=6. Abbreviations: Nx, normoxia (21% O₂); Hx, hypoxia (10% O₂); hr, hours.

Figure 6

Constituents of bronchoalveolar lung fluid, with quantification of albumin and cells in BALF isolated from control animals and animals exposed to 16 hours of hypoxia. (A) Albumin content (µg/µL). (B) Total cell number (per mL). (*P<0.05). Abbreviations: Nx, normoxia (21% O₂); Hx, hypoxia (10% O₂); BALF, bronchoalveolar lung fluid.

Figure 7

Regulation of plasma copeptin and mid-regional proANP levels in response to hypoxia. Two separate experiments were performed, in which rats were exposed to 16 hours of ambient room air (baseline) followed by an acute hypoxic phase (5 minutes) or 16 hours of hypoxia followed by a normoxic recovery phase (5 minutes), 10 minutes of hyperoxia (100% oxygen), or repeated exposure to hypoxia for 5 and 10 minutes. (A) Plasma copeptin (pmol/L). (B) Plasma sodium (mM). (C) Plasma mid-regional proANP (pmol/L). *P<0.05 versus normoxia (baseline); ^#P<0.05 versus repeated acute hypoxia (5 minutes), n=5. Abbreviations: Nx, normoxia (21% O₂); Hx, hypoxia (10% O₂); Hyp, hyperoxia (100% O₂); hr, hours; m, minutes; proANP, proatrial natriuretic peptide; MR, mid-regional.