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Riedijk et al. 10.1073/pnas.0607965104. |
SI Appendix
Calculations of GIT and Whole-Body Methionine Kinetics. Whole-body methionine kinetics. Whole-body kinetics were calculated by using methionine isotopic enrichments during both the IV and ID tracer infusion protocols, and the results are presented in Table 1. Additionally, methionine flux is calculated by using the [13C]methionine carbon isotopomer (Qc) and the [2H]methionine methyl isotopomer (Qm), respectively, as described by Storch et al. 1989.
Whole-body methionine flux = (IEinfusate/IEplasma - 1) × tracer infusion rate, [1]
where whole body flux is expressed as mmol·kg-1·h-1, IEinfusate is isotopic enrichment (MPE) of methionine infusate during IV or ID infusion, and IEplasma is isotopic enrichment (MPE) of methionine isotopomer in plasma during IV or ID infusion. Tracer infusion rate is expressed in (mmol·kg-1·h-1).
Body CO2 production = (IEinfusate/IEplasma - 1) × tracer infusion rate, [2]
where IEinfusate is the isotopic enrichment of the infused [13C]bicarbonate (MPE), and IEplasma is the enrichment of [13C]bicarbonate in plasma (MPE); body CO2 production is expressed in mmol·kg-1·h-1.
Transsulfuration of methionine to cysteine is reflected by [1-13C]methionine oxidation (mmol×kg-1×h-1) to 13CO2 and is calculated as follows:
Whole-body transsulfuration (TS) = (IE 13CO2/IEplasma) × (Eq. 2), [3]
where IE 13CO2 is enrichment of CO2 (APE) during IV or ID infusion of [1-13C]methionine, and IEplasma is plateau isotopic enrichment of methionine in plasma during IV or ID infusion.
Whole-body remethylation, transmethylation, and protein synthesis are expressed in mmol·kg-1·h-1 calculated as follows:
Whole-body remethylation (RM) = Qm (Eq. 1) - Qc (Eq. 1). [4]
Whole-body transmethylation (TM) = RM (Eq. 4) + TS (Eq. 3). [5]
Whole-body protein synthesis (PS) = Qc (Eq. 1) - TS (Eq. 3). [6]
Net GIT Methionine, Homocysteine, and CO2 Balance: The following equations were used to calculate the rates of net balance of methionine, homocysteine, and CO2 (Table 3); amino acid (AA) can be substituted with concentrations of methionine, homocysteine, or CO2. Arterial AA input was calculated as follows:
Arterial AA input = arterial (AA)C × PBF, [7]
where arterial AA input is expressed in mmol·kg-1·h-1, arterial amino acid concentration (AA)C is expressed in mmol·liter-1, and PBF is portal blood flow (liters·kg-1·h-1); this rate is noted as in Fig. 2B. Note that PBF reflects plasma flow after correction for hematocrit. Portal AA output noted as in Fig. 2C is:
Portal AA output = portal (AA)C × PBF. [8]
Net portal AA balance is then calculated by:
Net portal AA balance = Eq. 8 - Eq. 7, [9]
where net portal AA balance is expressed in mmol·kg-1·h-1. In the case of homocysteine and CO2, the net balance reflects their release into the portal blood. The net utilization of methionine by the GIT was:
Net GIT Met utilization = dietary Met intake - Eq. 9, [10]
where dietary Met intake is expressed is as mmol·kg-1, and net GIT Met utilization is in mmol·kg-1·h-1.
Fractional GIT Met utilization = Eq. 10/dietary Met intake × 100, [11]
where fractional GIT Met utilization is expressed as percentage (%).
Kinetics of [13C]Methionine, [13C]Homocysteine, and 13CO2 in the GIT. The following equations were used to calculate the unidirectional kinetics of [13C]methionine, [13C]homocysteine, and 13CO2 flux in the GIT as shown in Fig. 2. Tracer kinetics for these metabolites are derived from the product of metabolite concentration and isotopic enrichment. The values for methionine, homocysteine, or CO2 can be substituted in these equations where (AA) is shown. Arterial tracer input is as follows:
Arterial tracer AA input = arterial (AA)C × arterial (AA)IE × PBF, [12]
where arterial tracer AA input is expressed in mmol·kg-1·h-1, and arterial amino acid isotopic enrichment (AA)IE expressed in mole percent excess (MPE).
Portal tracer AA output = portal (AA)C × portal (AA)IE × PBF. [13]
Unidirectional GIT tracer utilization by the GIT during IV tracer infusion protocol is noted as in Fig. 2F and is calculated by:
Unidirectional arterial tracer utilization by GIT = Eq. 12 - Eq. 13. [14]
This flux (mmol·kg-1·h-1) represents the utilization or uptake of methionine extracted by the GIT from the arterial circulation. The fractional utilization of arterial tracer by the GIT during the IV tracer infusion protocol is calculated by:
Fractional GIT utilization of arterial tracer = Eq. 14/Eq. 12. [15]
The amount of arterial methionine used by the GIT is calculated by:
Total arterial methionine used by GIT = Eq. 7 × Eq. 15. [16]
The dietary methionine tracer not metabolized the GIT in first pass during ID tracer infusion protocol represents absorption and is calculated by:
Dietary tracer absorption = Eq. 13 - Eq. 12. [17]
The rate of dietary methionine utilization by the GIT during the ID methionine tracer infusion is calculated using the following equation:
Dietary tracer utilization by GIT = dietary tracer intake - Eq. 17. [18]
The true amount of dietary methionine used on first pass by the GIT must be corrected by the amount of dietary methionine that is absorbed into the portal vein (and thus is not metabolized by GIT) and then reenters the GIT from the arterial circulation. This methionine will be used by the GIT in the same proportion as the intravenously administered methionine tracer (see Eq. 15). The true amount of dietary methionine used on first pass by the GIT must to be corrected by the amount of dietary AA that renters the portal circulation via the arterial circulation; this rate is noted as in Fig. 2D. Thus, during ID infusion the equation is:
Fractional dietary first pass AA utilization =
(dietary tracer intake - Eq. 13 - (((1 - Eq. 15)) × Eq. 12))/dietary tracer intake. [19]
The true amount of dietary AA that is used by the GIT is calculated by:
First-pass AA use by GIT = (Eq. 11) × dietary intake AA, [20]
where first-pass AA use by the GIT is expressed in mmol·kg-1·h-1. Some of the AA appearing in the portal vein is derived neither from the diet nor from the systemic circulation. This is due to recycling of AA in the GIT derived from proteolysis, either intracellular or luminal, or from microbial production; this rate is noted as in Fig. 2H. Recycling (mmol·kg-1·h-1) of methionine was calculated with the results from both the IV and ID tracer studies with the following equation:
Recycled methionine GIT release = (Eq. 8) - (dietary intake - Eq. 20) -
(Eq. 7 - Eq. 16). [21]
The unidirectional rates of [13C]homocysteine and 13CO2 that are derived from GIT [13C]methionine metabolism and released into the portal circulation are calculated by substituting their respective concentrations and enrichments into Eq. 14; these calculations represent the rates of transmethylation and transsulfuration, respectively, derived from [13C]methionine metabolized by the GIT; these rates are noted as, in Fig. 2 I and J, respectively.
GIT transmethylation = unidirectional [13C]homocysteine + 13CO2 flux. [22]
GIT transsulfuration = unidirectional 13CO2 flux. [23]
Protein synthesis by the GIT was calculated by using the equation; this rate is noted as in Fig. 2K:
Protein synthesis by GIT = (Eq. 10) - (Eq. 22) - (Eq. 23). [24]
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