Reduction of glucose and insulin concentrations during in vitro incubation of whole blood

Introduction

Incubation of whole blood has been used in numerous in vitro investigations. The purpose of the present study was to test the hypothesis that glucose and insulin concentrations declined during incubation.

Methods

Six young, healthy and fasting males donated blood. Aliquots containing heparin-anticoagulated whole blood were added to with different quantities of insulin (Actrapid; Novo Nordisk, Bagsvaerd, Denmark) and bacterial endotoxin (Escherichia coli lipopolysaccharide serotype 026:B6; Difco Laboratories, Detroit, MI, USA). Aliquots were incubated for 6 hours at 37°C in an atmosphere of humidified 5% CO₂ and 95% air. Concentrations of blood glucose were measured every hour, whereas insulin was measured at baseline and 6 hours. The Wilcoxon signed-rank test was used to compare medians.

Results

The glucose concentration in aliquots to which insulin 30 nmol/l and lipopolysaccharide 1,000 ng/ml were added decreased linearly and approached zero after 6 hours (median 0.6 vs. 4.75 mmol/l, P = 0.027; presented as a line chart of medians with range in Figure 1). The insulin content in blood samples without any additions was reduced by more than 50% during incubation (median 22.5 vs. 48.0 pmol/l, P = 0.028; depicted as box plots with median lines, 25th to 75th percentile boxes and 10th to 90th percentile error bars in Figure 1). Similar glucose and insulin measurements were performed during incubation of other selected aliquots. The reductions of glucose and insulin content were analogous to the results above; the magnitude of reduction seemed to be independent of the addition of insulin and/or lipopolysaccharide.

Figure 1

Glucose and insulin concentrations during incubation.

Conclusion

The concentration of glucose was reduced to almost zero during 6 hours of incubation; the decline was probably due to glycolysis in blood cells rather than the effects of insulin. The resulting hypoglycaemia may affect cellular functions, and addition of glucose should therefore be performed during in vitro incubations. The decline in insulin content was unexpected, as insulin is mostly degraded in the liver and kidney, but to some extent also in blood cells and extracellularly.