Appendix
Calculations
Whole body kinetics of protein and lipids were calculated by conventional isotope dilution
equations using a stochastic model during steady state enrichment (1) and glucose kinetics
were estimated using the Steele equation (2). The rate of appearance (Ra) of unlabeled
substrate can be derived from the plasma isotope enrichment calculated by:
Ra = i x (Einf/Epl - 1)
(1)
where i is the infusion rate of the labeled tracer, Einf is the tracer enrichment of the infusate
and Epl the tracer enrichment in plasma (Mole fraction per cent excess), respectively.
Glucose metabolism
Estimates of whole-body glucose kinetics were made at isotopic steady-state,
effectively attained during the last 40 min of the basal period and the last 30 min of the HEC.
Mean values of plasma D-[6,6-2H2]glucose enrichment (Mole fraction percent excess) and of
exogenous glucose infusion rate were used for data calculation. Under steady state
conditions total glucose rate of appearance (Ra) is equal to the rate of disappearance (Rd)
(2). The rates of glucose disappearance reflect glucose utilization.
Total endogenous glucose production (EGP) rate was calculated during the last 40 min of the
basal period and the last 30 min of the HEC by subtracting the exogenous glucose infusion
rate from the glucose rates of appearance (Ra) (3).
Total endogenous glucose production (EGP; mg.kg-1.min-1) =
Glucose Ra – Glucose infusion rate
(2)
Insulin sensitivity (mg.kg-1.min-1/µU.mL-1) as measured with the HEC and pointed out as Mvalue, is the rate of glucose disappearance (Rd) divided by plasma insulin concentration
during steady state (4).
Insulin sensitivity (mg.kg-1.min-1/µU.mL-1) = Rd/Plasma insulin
(3)
Insulin stimulated glucose disposal (mg.kg-1.min-1) was considered equal to the sum of EGP
and GIR during steady state glucose rate of disappearance (5).
Insulin stimulated glucose disposal (mg.kg-1.min-1 ) = EGP + GIR
(4)
Protein Metabolism
Whole body plasma leucine flux, an index of protein metabolism, was calculated
using plasma α-ketoisocaproate (α –KIC) enrichment expressed as mole fraction per cent
excess during the last 40 min of the baseline period and the last 30 min of the HEC period,
as described previously (6). Leucine Oxidation was obtained as follows:
VCO2 x (E13CO2/69.18)/13C α-KIC
(5)
where VCO2 is the rate of carbon dioxide elimination measured in milliliters per minute and
converted to millimoles per hour by multiplying by 60 min and dividing by 22.4, which is the
number of 1 in 1 mole of an ideal gas at standard temperature and pressure. We have
previously shown that 69.18 is the correction factor for
13
CO2 under recovery obtained from
parenterally fed critically ill children (7).
Under the assumption that 1 gram of mixed muscle protein contains approximately
621 µmol of leucine (8), whole body protein turnover was calculated from the model
described by Golden and Waterlow (9). Briefly, the model is described in the following
equation;
Ra = S + O = B + I.
(6)
Where Ra is the rate of appearance into the plasma pool, S corresponds to protein synthesis
and O to oxidation, and therefore losses from the plasma amino acid pool, while B
represents protein breakdown and I dietary intake, and therefore entry of amino acids into
the plasma pool (10).
Protein synthesis (S) was calculated by subtracting the rates of leucine oxidation (O) from
the plasma leucine flux (Ra).
Prot Synthesis = Leucine Ra – Leu ox
(7)
Protein breakdown (B) was calculated by subtracting the leucine intake (I; tracer infusion +
leucine content of parenteral nutrition) from the leucine flux (Ra).
Prot Breakdown = Leu flux – Leu intake
(8)
Protein balance was then calculated subtracting whole body protein breakdown from whole
body protein synthesis.
Prot bal = Prot Synthesis – Prot Breakdown
(9)
Lipid Metabolism
Glycerol flux was calculated during the last 40 min of the baseline period, and during
the last 30 min of the hyperinsulinemic euglycemic clamp according to the steady state tracer
dilution equations reported previously (11). As part of their parenteral nutrition the patients
were provided with lipids intravenously which contained 2.25 mg.mL-1 glycerol. Lipolysis was
calculated by subtracting the glycerol intake (tracer infusion + glycerol intake through
parenteral nutrition) from the glycerol flux (Ra).
Lipolysis = Glycerol Ra – Total glycerol intake
(10)
References of the calculations
1.
Altszuler, N., R. C. De Bodo, R. Steele, et al. Carbohydrate metabolism of
hypophysectomized dogs as studied with radioactive glucose. Am J Physiol 1956;
187(1):25-31.
2.
Steele, R., J. S. Wall, R. C. De Bodo, et al. Measurement of size and turnover rate of
body glucose pool by the isotope dilution method. Am J Physiol 1956; 187(1):15-24.
3.
Bier, D. M., R. D. Leake, M. W. Haymond, et al. Measurement of "true" glucose
production rates in infancy and childhood with 6,6-dideuteroglucose. Diabetes 1977;
26(11):1016-23.
4.
Matsuda, M., and R. A. DeFronzo. Insulin sensitivity indices obtained from oral
glucose tolerance testing: comparison with the euglycemic insulin clamp. Diabetes Care
1999; 22(9):1462-70.
5.
Jani, R., M. Molina, M. Matsuda, et al. Decreased non-insulin-dependent glucose
clearance contributes to the rise in fasting plasma glucose in the nondiabetic range. Diabetes
Care 2008; 31(2):311-5.
6.
Matthews, D. E., K. J. Motil, D. K. Rohrbaugh, et al. Measurement of leucine
metabolism in man from a primed, continuous infusion of L-[1-3C]leucine. Am J Physiol
1980; 238(5):E473-9.
7.
Sy, J., A. Gourishankar, W. E. Gordon, et al. Bicarbonate kinetics and predicted
energy expenditure in critically ill children. Am J Clin Nutr 2008; 88(2):340-7.
8.
Argaman, Z., V. R. Young, N. Noviski, et al. Arginine and nitric oxide metabolism in
critically ill septic pediatric patients. Crit Care Med 2003; 31(2):591-7.
9.
Golden, M. H., and J. C. Waterlow. Total protein synthesis in elderly people: a
comparison of results with [15N]glycine and [14C]leucine. Clin Sci Mol Med 1977; 53(3):27788.
10.
el-Khoury, A. E., M. Sanchez, N. K. Fukagawa, et al. Whole body protein synthesis in
healthy adult humans: 13CO2 technique vs. plasma precursor approach. Am J Physiol
1995; 268(1 Pt 1):E174-84.
11.
Beylot, M., C. Martin, B. Beaufrere, et al. Determination of steady state and
nonsteady-state glycerol kinetics in humans using deuterium-labeled tracer. J Lipid Res
1987; 28(4):414-22.