A simple CO₂ equilibration method for measuring blood oxygen isotope compositions

Blood water oxygen isotope compositions can provide valuable insights into physiological processes and ecological patterns. While blood samples are commonly drawn for medical or scientific purposes, blood fractions are infrequently measured for oxygen isotopic compositions (δ¹⁸ O) because such measurements are time consuming and expensive.

We sampled blood from sheep, goats, and iguanas raised in field and animal laboratories into serum, EDTA, heparin, and uncoated plastic vials commonly used in medical and scientific research, then separated red blood cell (RBC) and plasma or serum blood fractions. These were injected into helium-flushed Exetainer tubes where they naturally outgassed endogenous CO₂ (goat blood), or into He- and CO₂ -flushed tubes (iguana blood). The CO₂ gas was sampled on a GasBench II system, and δ¹⁸ O was measured by an isotope ratio mass spectrometer (IRMS).

Repeated δ¹⁸ O measurements were stable over multiple days. The addition of desiccated blood solids to water standards had little impact on their δ¹⁸ O measurements, suggesting that organic molecular constituents within blood serum and plasma do not interfere with blood water δ¹⁸ O values. We observed slight but statistically significant δ¹⁸ O offsets between plasma, serum and RBC fractions. Mass-dependent body water turnover times for iguanas were derived from the data.

We demonstrate that a simple blood-CO₂ equilibration method using the GasBench can quickly, reliably and accurately characterize water δ¹⁸ O in the plasma, RBC, and whole blood fractions of mammalian and reptilian blood samples (precision ≤ 0.1‰). This method will expand the application of blood stable isotope analysis in physiological and medical research.