A hyperinsulinaemic euglycaemic clamp has been used together with indirect calorim etry for simultaneous measurement of substrate utilization and glucose metabolism. Correct urine collection is an important parame- ter influencing the results of substrate utilization estimated by indirect calorimetry. The aims of our study were a) to improve the timing of urine collection for measuring substrate oxidation during hyperinsulinaemic euglycaemic clamp and b) to evaluate the circadian variation in urinary urea nitrogen excretion in healthy subjects. The study was performed on 11 healthy subjects during 3 days with a standard dietary intake. Urinary urea excretion (UE) was measured by standard technique in urine samples collected a) during 24 hours (periods I-VII) and b) during hyperinsulinaemic (1 and 10 mU/kg/min) euglycaemic (5 mmol/l) clamp combined with indirect calorimetry (basal period: -120-0 min, period I: 0-120 min, period II: 120-240 min). The circadian variation in UE during 24 hours was not statistically significant. During hyperinsulinaemic euglycaemic clamp UE did not change (period I: 462 ± 157 vs period II: 436 ± 138 mmol/24 hours, NS), but the serum urea level decreased significantly (period I: 4.29 ± 0.61 vs period II: 3.8 ± 0.63 mmol/l; p > 0.001). There was a significant relationship between UE corrected for changes in urea pool size (period Ic and IIc) compared to uncorrected UE (period I and II) (period I: 462 ± 157 vs period 1c: 240 ± 170 mmol/24 hours; period 2c: 219 ± 122 vs period II: 436 ± 138 mmol/24 hours, p > 0.001). We conclude that urine collections made during the clamp and corrected for changes in urea pool size are most suitable for measurement of substrate utilization during hyperinsulinaemic euglycaemic clamp. Urine collections during clamp cannot be replaced by any urine collection from tested periods (periods I-VII) nor one 24-hour urine sample. Finally, we found no significant variation in UE during 24 hours in healthy subjects.

        Key words: substrate utilization, urinary urea excretion, insulin, glycaemic clamp, indirect calorimetry.