CHAPTER 2 METHODS
2.3 The doubly-labelled water method
2.3.6 Step 5: Calculation of outcome variables
From the elimination spreadsheets, variables were copied into one separate spreadsheet for all the infants with a successful measurement. Table 2.1 is a list of the variables transferred into this spreadsheet.
Table 2.1 List of variables from original data sets as they were transferred to the spreadsheet for
calculation of outcome variables.
Column Variable name Variable label or formula for calculationa A Study ID Date of birth and initials
B Sex girl = 1, boy = 0
C Age1_day0 Age visit 2 (day 0) (d)
D Age1_d02 Age1_day0 * Age1_day0a
E Age1_day7 Age visit 3 (day 7) (d)
F Age1_d72 Age1_day7 * Age1_day7a
G Age2_day0 Age visit 6 (day 0) (d)
H Age2_day02 Age2_day0 * Age2_day0a
I Age2_day7 Age visit 7 (day 7) (d)
J Age2_day72 Age2_day7 * Age2_day7a ANTHROPOMETRY
K Weight_Age1_d0 Weight Age 1 day 0 (g)
L Weight_Age2_d0 Weight Age 2 day 0 (g)
M Weight_Age1_d7 Weight Age 1 day 7 (g)
N Weight_Age2_d7 Weight Age 2 day 7 (g)
O dWeight1 Change in Weight d7 - d0, Age 1 (g)a
P dWeight2 Change in Weight d7 - d0, Age 2 (g)a MASS SPEC DATA
Q Error1 mass spec error Age 1
R Error2 mass spec error Age 2
S Space_ratio1 Space ratio Age 1
T Space_ratio2 Space ratio Age 2
U TEE1 TEE Age 1 (kJ/d)
V TEE2 TEE Age 2 (kJ/d)
W FM1 Fat mass Age 1 (g)
X FM2 Fat mass Age 2 (g)
Y Fat_percent1 Fat % Age 1
Z Fat_percent2 Fat % Age 2
AA O18Space1 O18 dilution space Age 1 (mol)
AB O18space2 O18 dilution space Age 2 (mol)
AC D2Space1 D2 dilution space Age 1 (mol)
AD D2Space2 D2 dilution space Age 2 (mol)
AE kO1 elimination constant O18 Age 1 (/d)
AF kD1 elimination constant D2 Age 1 (/d)
AG kO2 elimination constant O18 Age 2 (/d)
AH kD2 elimination constant D2 Age 2 (/d)
AI TBW1 TBW Age 1 (g)
AJ TBW2 TBW Age 2 (g)
AK FFM1 Fat free mass Age 1 (g)
AL FFM2 Fat free mass Age 2 (g)
a Variables that were calculated.
Age1: 1st time-point, Age2: 2nd time-point, TEE: Total Energy Expenditure,
Subsequently, outcome variables were calculated using the approach developed by Lucas and colleagues279 and refined by Davies and Wells250,255. Table 2.2 is a list of all the calculations in the sequence they were performed.
Energy deposited in growth
Egrowth (kJ/d) was calculated from weight gain over the 7 days of the TEE
measurement, as previously described elsewhere255. FFM on day 0 was calculated using an age- and sex-specific factor of hydration of FFM, which was derived from regression equations on data of the reference child249. FM on day 0 was
then calculated as body weight minus FFM. Weight gain over the 7 days was assumed to have a linear increase in the proportions of FM and FFM. Gain in FM (g) was calculated directly as the difference in FM between day 0 and day 7. Gain in FFM (g) was corrected for age- and sex- specific changes in hydration and in the proportion of FFM that is protein, using regression equations derived from data on the reference child249, resulting in a value of protein gain. Finally, the energy stored as fat and protein was calculated, assuming a standard caloric value of 38·7 kJ/g for fat and 23·6 kJ/g for protein335 and summarised to produce a value for Egrowth (kJ/d). The standard calorific values were used rather
than specific calorie conversion factors obtained for breast milk (fat: 36.87 kJ/g, protein: 19.99 kJ/g)336 in order to facilitate comparison with other studies.
Total metabolisable energy intake
Daily TEI was calculated as the daily average TEE (kJ/d) plus the daily average
Egrowth (kJ/d), as described by Lucas and colleagues279.
Metabolisable breast milk intake
The elimination rate of deuterium was used to calculate amount of daily milk intake (g/d) 273 and correcting for environmental water influx244:
Milk intake (g/d) = 0.88
Metabolisable milk energy content
Milk energy content (kJ/g) was calculated as TEI (kJ/d) divided by milk intake (g/d)279.
Table 2.2 List of calculated variables for metabolisable milk intake, Egrowth, metabolisable energy intake and milk energy content.
Column Variable name Formula for calculation
AM Water_efflux_1 D2Space1 * kD1 * 18.02/0.99 (g/d)
AN Water_efflux_2 D2Space2 * kD2 * 18.02/0.99 (g/d)
AO Water_stored_1 dWeight1 (g) * TBW1 (g) / Weight_Age1_d0 (g) /7d (g/d)
AP Water_stored_2 dWeight2 (g) * TBW2 (g) / Weight_Age2_d0 (g) /7d (g/d)
AQ Water_influx_1 Water_efflux_1 + water stored_1 (g/d)
AR Water_influx_2 Water_efflux_2 + water stored_2 (g/d)
AS Milk_water_1 0.937 * Water influx_1 (g/d)
AT Milk_water_2 0.937 * Water influx_2 (g/d)
AU Milk_intake_1 Milk water_1 / 0.96 (g/d)
AV Milk_intake_2 Milk water_2 / 0.96 (g/d)
BODY COMPOSITION DATA
AW TBW_Age1_d0 O18space1 * 18.02 / 1.01 (g)
AX TBW_Age2_d0 O18space2 * 18.02 / 1.01 (g)
AY Age_sex_TBW_frac_FFM_Age1_d0 Age-sex-specific regression of TBW fraction of FFM Age1_d0
AZ Age_sex_TBW_frac_FFM_Age2_d0 Age-sex-specific regression of TBW fraction of FFM Age2_d0
BA FFM_Age1_d0 TBWAge1_d0 / Age-sex TBW fraction FFM from regression (g)
BB FFM_Age2_d0 TBWAge2_d0 / Age-sex TBW fraction FFM from regression (g)
BC FM_Age1_d0 Weight_Age1_d0 - FFM_Age1_d0 (g)
BD FM_Age2_d0 Weight_Age2_d0 - FFM_Age2_d0 (g)
BE Age_sex_protein_frac_FFM_Age1_d0 Age-sex-specific regression of protein fraction of FFM_Age1_d0
BF Age_sex_protein_frac_FFM_Age2_d0 Age-sex-specific regression of protein fraction of FFM_Age2_d0
BG protein_mass_Age1_d0 FFM_Age1_d0 * Age_sex_protein_frac_FFM_Age1_d0 (g)
BH protein_mass_Age2_d0 FFM_Age2_d0 * Age_sex_protein_frac_FFM_Age2_d0 (g) Age1: 1st time-point
Age2: 2nd time-point
TEE: Total Energy Expenditure FM: Fat Mass
k: Elimination rate constant TBW: Total Body Water FFM: Fat Free Mass
Table 2.2 continued List of calculated variables for metabolisable milk intake, Egrowth, metabolisable energy intake and milk energy content.
Column Variable name Formula for calculation
BI Age_sex_protein_frac_FFM_Age1_d0 Age-sex-specific regression of protein fraction of FFM_Age1_d0
BJ Age_sex_protein_frac_FFM_Age2_d0 Age-sex-specific regression of protein fraction of FFM_Age2_d0
BK protein_mass_Age1_d0 FFM_Age1_d0 * Age_sex_protein_frac_FFM_Age1_d0 (g)
BL protein_mass_Age2_d0 FFM_Age2_d0 * Age_sex_protein_frac_FFM_Age2_d0 (g)
BM TBW_frac_BW_Age1_d0 TBW_Age1_d0 (g) / Weight_Age1_d0 (g)
BN TBW_frac_BW_Age2_d0 TBW_Age2_d0 (g) / Weight_Age2_d0 (g)
BO Age_sex_TBW_frac_BW_Age1_d0 Age-Sex-specific regression TBW fraction of Weight_Age1_d0
BP Age_sex_TBW_frac_BW_Age1_d7 Age-Sex-specific regression TBW fraction of Weight_Age1_d7
BQ Age_sex_TBW_frac_BW_Age2_d0 Age-Sex-specific regression TBW fraction of Weight_Age2_d0
BR Age_sex_TBW_frac_BW_Age2_d7 Age-Sex-specific regression TBW fraction of Weight_Age2_d7
BS regression_proportion_Age1 proportion TWB_frac_Weight_Age1 d7 / d0
BT regression_proportion_Age2 proportion TWB_frac_Weight_Age2 d7 / d0
BU TBW_Age1_d7 Weight_Age1_d7 * TBW_frac_Weight_Age1_d0 * regression proportion_Age1 (g)
BV TBW_Age2_d7 Weight_Age2_d7 * TBW_frac_Weight_Age2_d0 * regression proportion_Age2 (g)
BW Age_sex_TBW_frac_FFM_Age1_d7 Age-sex-specific regression of TBW fraction of FFM Age1_d7
BX Age_sex_TBW_frac_FFM_Age2_d7 Age-sex-specific regression of TBW fraction of FFM Age2_d7
BY FFM_Age1_d7 TBW_Age1_d7 / Age-sex-TBW_frac_FFM_Age1_d7 (g)
BZ FFM_Age2_d7 TBW_Age2_d7 / Age-sex-TBW_frac_FFM_Age2_d7 (g)
CA FM_Age1_d7 Weight_Age1_d7 - FFM_Age1_d7 (g)
CB FM_Age2_d7 Weight_Age2_d7 - FFM_Age2_d7 (g)
CC Age_sex_protein_frac_FFM_Age1_d7 Age-sex-specific regression of protein fraction of FFM_Age1_d7
CD Age_sex_protein_frac_FFM_Age2_d7 Age-sex-specific regression of protein fraction of FFM_Age2_d7
CE protein_mass_Age1_d7 FFM_Age1_d7 * Age-sex-specific protein fraction of FFM_Age1_d7 (g)
CF protein_mass_Age2_d7 FFM_Age2_d7 * Age-sex-specific protein fraction of FFM_Age2_d7 (g) Age1: 1st time-point
Age2: 2nd time-point
TEE: Total Energy Expenditure FM: Fat Mass
k: Elimination rate constant
Table 2.2 continued List of calculated variables for metabolisable milk intake, Egrowth, metabolisable energy intake and milk energy content.
Column Variable name Formula for calculation
CG dFM_Age1 FM_Age1_d7 - FM_Age1_d0 (g)
CH dFM_Age2 FM_Age2_d7 - FM_Age2_d0 (g)
CI dprotein_mass_Age1 Protein_mass_Age1_d7 - Protein_mass_Age1_d0 (g)
CJ dprotein_mass_Age2 Protein_mass_Age2_d7 - Protein_mass_Age2_d0 (g)
ENERGY DATA
CK Estored_Age1 dFM_Age1 (g) * 38.7 (kJ/g) + dprotein_Mass_Age1 (g) * 23.6 (kJ/g) (kJ)
CL Estored_Age2 dFM_Age2 (g) * 38.7 8kJ/g) + dprotein_Mass_Age2 (g) * 23.6 (kJ/g) (kJ)
CM Estored_Age1_kJ_d Estored_Age1 / 7 days (kJ/d)
CN Estored_Age2_kJ_d Estored_Age2 / 7 days (kJ/d)
CO TEI_Age1 Estored_Age1_kJ_d + TEE1 (kJ/d)
CP TEI_Age2 Estored_Age2_kJ_d + TEE2 (kJ/d)
CQ Emilk_Age1 TEI_Age1 (kJ/d) / milk_intake_Age1 (g/d) (kJ/g)
CP Emilk_Age2 TEI_Age2 (kJ/d) / milk_intake_Age2 (g/d) (kJ/g) Age1: 1st time-point
Age2: 2nd time-point
TEE: Total Energy Expenditure FM: Fat Mass
k: Elimination rate constant TBW: Total Body Water FFM: Fat Free Mass