IAAO method — Protocol24 hours befo

IAAO method — Protocol
24 hours before the study day, the infants consumed their test สูตร (ที่รวมถึง the specific level of the test amino acid being given for each particular infant) for
adaptation, with the feeding regime conforming to that of the hospital. During the study day, the feeding regime was changed to hourly bolus feeding of the infant's test

สูตรto ensure a metabolic steady state in feed condition. In order to quantify

individual CO2 production, the infants received a primed (14 umol/kg) continuous (9
umol/kg/h) infusion of [13C]bicarbonate (sterile pyrogen free, 99%13C APE;
Cambridge Isotopes, Woburn, MA) for 3 hours (Riedijk, 2005). Directly following the

3 hour bicarbonate infusion, a primed (34 umol/kg) continuous (27 pinol/kg/h) enteral

infusion of L-[1-13C]ฟีนิลอะลานีน (99% 13C APE; Cambridge Isotopes) was given for
4 hours. To minimise invasiveness the tracers were given enterally by means of a gastric tube.

Breath samples were collected by means of the direct sampling method described

by van der Schoor (2004). At baseline, สอง duplicate breath samples were obtained
before tracer infusion. During the last 45 minutes of [13C]bicarbonate infusion, and
during the last hour of L[1-13C]ฟีนิลอะลานีน infusion, duplicated samples were

collected every ten and fifteen minutes, respectively. Samples were stored at room
temperature until monthly shipment to the Netherlands for analysis at the mass
spectrometry laboratory of the Erasmus Medical Center, Rotterdam. Expired 13CO2
enrichment was measured by isotope mass spectrometry (ABCA; Europe Scientific,

van Loenen Instruments, Leiden, the Netherlands), and reported in units of atom

percent excess (APE).
For each participant, the estimated body CO2 production was calculated, as described previously (Riedijk, 2005). The rate of fractional [1-13C]ฟีนิลอะลานีน oxidation was calculated using this equation:
Fractional ฟีนิลอะลานีน oxidation (%) = [IEPHE x iB x IEB] x 100%
Where IEPHE is the excess 13C isotopic enrichment in expired air during [1-
13C]ฟีนิลอะลานีน infusion (APE), iB is the infusion rate of [13C]bicarbonate
(gmol/kg/h), iPHE is the infusion rate of [1-13C]ฟีนิลอะลานีน (umol/kg/h), and IEB is
the excess 13C isotopic enrichment in expired air during [13C]bicarbonate infusion (van Goudoever, 1993).








53
The results of the IAA() studies for อะมิโนแอซิด: ไลซีน, เมไธโอนีน, ธรีโอนีน,
ทริปโตฟาน, วาลีน, ไอโซลิวซีน and ลิวซีน, are described in Examples la-lg, and with
the results also illustrated graphically in Figures 2-8. As ฟีนิลอะลานีน was used as the
indicator amino acid, each of Figures 2-8 plots the oxidation rates of the ฟีนิลอะลานีน 5 (F13CO2) against dietary intake of the test amion aci.d Each point represents an
individual infant who was fed an amino acid based สูตรwhere each essential amino
acid was present in excess except the essential amino acid to be tested, where the test
amino acid was present in an amount ranging from deficient to excess.


10 ecommended dietary intake ranges
The recommended dietary intake range for the อะมิโนแอซิดจำเป็น in each of
Examples la-lg was then determined using the following strategy.
First, the dietary requirement (breakpoint) of the IAA() results was estimated using a biphasic linear regression crossover model and was taken as the primary parameter.
15 To ensure that nearly all individuals meet the amino acid requirements, a safe level
of intake was defined. The safe level of protein intake proposed by the WHO
(WHO/FAO/UNU, 2007) was used, defining the safe level of protein intake as 125% of
the average protein requirement. In this instance, the safe level of amino acid intake
was therefore calculated to be >125% of the primary parameter (breakpoint value)
20 obtained as described above, to reach the population safe requirement value.
An assumption was made that this primary parameter (breakpoint value) is higher
than the requirement of protein bound อะมิโนแอซิด based on the study by Metges
(2000) which showed > 20% higher first pass oxidation rate when free อะมิโนแอซิด are
ingested compared to protein bound อะมิโนแอซิด. Therefore 20% of the estimated
25 requirement was subtracted from the population safe requirement value, which is
believed to be conservative (i.e. , an overestimate rather than underestimate).
The net effect of the above สอง calculations is therefore that the breakpoint equals the recommended intake (measured in mg° kg-1. d-1).
The recommended intake measured in mg. kg-I d' was then converted to units of 30 mg (of amino acid) / 100 mL (ของสูตรทารก). This calculation was made by
dividing the recommended intake (mg. kg-l-d-1) by the volume intake by an infant of
150 mL.kg-i.d-1 (Shaw, 2001) and multiplying by 100. This is described below as the






54
recommended dietary intake for each amino acid, expressed in mg (amino acid) / 100 mL (infant formula).
This recommended dietary intake range was also converted into units of g (amino
acid) / 100 g โปรตีน as follows. The calculations were based onระดับปริมาณโปรตีนที่รับเข้าไป 5 of 0.9 —1.4 g (protein) / 100 mL (infant formula), หรือ 900 — 1,400 mg (protein) / 100 mL
(infant formula) (Koletzko, 2009). This ระดับปริมาณโปรตีนที่รับเข้าไป correlates ถึง 1.4 ถึง 2.1 g
protein / 100 kcal infant สูตรbased on an energy level of 66 kcal ต่อ 100 mL The
recommended dietary intake for the amino acid expressed in mg / 100 mL was
therefore divided by the upper limit of ระดับปริมาณโปรตีนที่รับเข้าไป (1,400 mg / 100 mL) and 10 multiplied by 100 to reach the lower limit of the proposed interval. The recommended
dietary intake for the amino acid expressed in mg / 100 mL was also divided by the lower limit of ระดับปริมาณโปรตีนที่รับเข้าไป (900 mg / 100 mL) and multiplied by 100 to reach the upper limit of the proposed interval.


15 Example la: ไลซีน
Using the method described above, the breakpoint for ไลซีน by a group of 21
infants was estimated to be 130 mg. kg-1 d-' (this is also shown in Figure 2), and the
recommended dietary intake for ไลซีน was then determined to be 87 mg / 100 mL (of
infant formula). Converted into g / 100 g โปรตีน as described above, the result for 20 ไลซีน is 6.2 — 9.7 g / 100 g โปรตีน.


Example lb: เมไธโอนีน
Using the method described above, the mean breakpoint for เมไธโอนีน by a group
of 33 infants was estimated to be 38 mg.kg-i.d-1 (this is also shown in Figure 3). It is 25 noted that this estimation was made based on a diet that contained an excess of ซิสเทอีน
(91 mg.kg-1. d.-1). The recommended dietary intake for เมไธโอนีน was then determined
to be 25 mg / 100 mL (ของสูตรทารก). Converted into g / 100 g โปรตีน as described
above, the result for เมไธโอนีน is 1.8 — 2.8 g / 100 g โปรตีน.


30 Example ic: ธรีโอนีน
Using the method described above, the breakpoint for ธรีโอนีน by a group of 32 infants was estimated to be 68 mg• kg-1. d-1 (this is also shown in Figure 4) and the recommended dietary intake for ธรีโอนีน was then determined to be 45 mg / 100 mL






55
(ของสูตรทารก). Converted into g / 100 g โปรตีน as described above, the result for ธรีโอนีน is 3.2 — 5.0 g / 100 g โปรตีน.


Example ld: ทริปโตฟาน
5 Using the method described above, the breakpoint for ทริปโตฟาน by a group of 30
infants was estimated to be 15 mg.kg-1.d-1 (this is also shown in Figure 5) and the
recommended dietary intake for ทริปโตฟาน was then determined to be 10 mg / 100 mL (ของสูตรทารก). Converted into g / 100 g โปรตีน as described above, the result for ทริปโตฟาน is 0.7 —1.1 g / 100 g โปรตีน.
10
Example le: วาลีน
Using the method described above, the breakpoint for วาลีน by a group of 28
infants was estimated to be 110 mg.kg-I.d-1 (this is also shown in Figure 6) and the
recommended dietary intake for วาลีน was then determined to be 73 mg / 100 mL (of 15 infant formula). Converted into g / 100 g โปรตีน as described above, the result for
วาลีน is 5.2 — 8.1 g / 100 g โปรตีน.


Example lf: ไอโซลิวซีน
Using the method described above, the breakpoint for ไอโซลิวซีน by a group of 22 20 infants was estimated to be 105 mg.kg-1- d-1 (this is also shown in Figure 7) and the
recommended dietary intake for ไอโซลิวซีน was then determined to be 70 mg / 100 mL
(ของสูตรทารก). Converted into g / 100 g โปรตีน as described above, the result for
ไอโซลิวซีน is 5.0 — 7.8 g / 100 g โปรตีน.


25 Example lg: Letnine
Using the method described above, the breakpoint for ลิวซีน by a group of 33
infants was estimated to be 140 mg.kg-l.d-1 (this is also shown in Figure 8) and the
recommended dietary intake for ลิวซีน was then determined to be 93 mg / 100 mL (of
infant formula). Converted into g / 100 g โปรตีน as described above, the result for
30 ลิวซีน is 6.6 —10.3 g / 100 g โปรตีน.







56
Example 2: ฮิสทิดีน, ฟีนิลอะลานีน, ซิสเทอีน and ไทโรซีน requirements in infants
The IAAO methodology was not performed for the อะมิโนแอซิดจำเป็น: ฮิสทิดีน
and ฟีนิลอะลานีน. Moreover, the IAAO methodology was (cannot) be used for the 5 conditionally อะมิโนแอซิดจำเป็น: ซิสเทอีน and ไทโรซีน. Thus, the levels of these
อะมิโนแอซิด were determined as explained below.

ผลลัพธ์ (อังกฤษ) 1:

IAAO method — Protocol24 hours day before the study, the infants consumed their test (including the specific amino acid level of the test being given for each particular infant) for. adaptation, with the feeding regime conforming to that of the hospital. During the study day, the feeding regime was changed to hourly bolus feeding of the infant's testTo ensure a steady state metabolic formula in order to quantify In feed condition.individual CO2 production, the infants received a primed (14 umol/kg) continuous (9umol/kg/h) infusion of [13C]bicarbonate (sterile pyrogen free, 99%13C APE;Cambridge Isotopes, Woburn, MA) for 3 hours (Riedijk, 2005). Directly following the3 hour bicarbonate infusion, a primed (34 umol/kg) continuous (27 pinol/kg/h) enteralInfusion of L-[1-13C] phenylalanine (99% APE 13C; Cambridge Isotopes) was given for.4 hours. To minimise invasiveness the tracers were given enterally by means of a gastric tube.Breath samples were collected by means of the direct sampling method describedBy van der Schoor (2004). At baseline, two duplicate samples were obtained breath.before tracer infusion. During the last 45 minutes of [13C]bicarbonate infusion, andDuring the last hour of L [1-13C] phenylalanine infusion, duplicated samples were.collected every ten and fifteen minutes, respectively. Samples were stored at roomtemperature until monthly shipment to the Netherlands for analysis at the massspectrometry laboratory of the Erasmus Medical Center, Rotterdam. Expired 13CO2enrichment was measured by isotope mass spectrometry (ABCA; Europe Scientific,van Loenen Instruments, Leiden, the Netherlands), and reported in units of atompercent excess (APE).For each participant, the CO2 production was calculated, estimated body as described previously (Riedijk, 2005) The rate of fractional [1-13C] phenylalanine oxidation was calculated using this equation:Phenylalanine Fractional oxidation (%) = [IEPHE x iB x IEB] x 100% Where IEPHE is the excess 13C isotopic enrichment in expired air during [1-13C] phenylalanine infusion (APE), iB is the bicarbonate infusion rate of [13C](Kg/h/gmol), the infusion rate of iPHE is [1-13C] phenylalanine (umol/kg/h), and IEB is.the excess 13C isotopic enrichment in expired air during [13C]bicarbonate infusion (van Goudoever, 1993). 53The results of the IAA () aesit: studies for amino lysine, nin, thari thai mail-o-o, nin. Tre pato Phan, valine, and Leu Leu scene scene solutions, Examples are described in la-lg, and with. The results also illustrated graphically in Figures 2-8. As phenylalanine was used as the. Indicator amino acid, each of the Figures 2-8 plots of the oxidation rates phenylalanine dietary intake (F13CO2) against the test of Each point represents an amion aci.d. An infant who was fed individual amino acid based formula where each essential amino. acid was present in excess except the essential amino acid to be tested, where the test amino acid was present in an amount ranging from deficient to excess.10 ecommended dietary intake rangesThe recommended dietary intake range for the amino dacham as in each of the SI.Examples la-lg was then determined using the following strategy.First, the dietary requirement (breakpoint) of the IAA() results was estimated using a biphasic linear regression crossover model and was taken as the primary parameter.15 To ensure that nearly all individuals meet the amino acid requirements, a safe levelof intake was defined. The safe level of protein intake proposed by the WHO(WHO/FAO/UNU, 2007) was used, defining the safe level of protein intake as 125% of the average protein requirement. In this instance, the safe level of amino acid intake was therefore calculated to be >125% of the primary parameter (breakpoint value) 20 obtained as described above, to reach the population safe requirement value. An assumption was made that this primary parameter (breakpoint value) is higherThan the requirement of protein bound amino aesit based on the study by Metges.(2000) which showed a 20% higher first pass > oxidation rate when free amino aesit are. Compared to ingested protein bound amino aesit. 20% of the estimated Therefore. 25 requirement was subtracted from the population safe requirement value, which is believed to be conservative (i.e. , an overestimate rather than underestimate).The net effect of the two calculations is therefore above the breakpoint that equals the recommended intake (measured in mg° kg-1. d-1).The recommended intake measured in mg. d ' kg-I was then converted to units of 30 mg (of amino acid)/100 mL (baby formula). This calculation was made by. dividing the recommended intake (mg. kg-l-d-1) by the volume intake by an infant of 150 mL.kg-i.d-1 (Shaw, 2001) and multiplying by 100. This is described below as the 54recommended dietary intake for each amino acid, expressed in mg (amino acid) / 100 mL (infant formula).This recommended dietary intake range was also converted into units of g (amino Acid/100 g of protein) as The calculations were based on follows. level 5 of the input protein to 0.9 1.4 g (protein) —/100 mL (infant formula), or 900 1400 mg (protein) —/100 mL. (Infant formula) (Koletzko, 2009). This level of protein that are input to. Correlates to 1.4 to 2.1 g Protein/100 kcal infant formula based on an energy level of 66 kcal. Per 100 mL The recommended dietary intake for the amino acid expressed in mg / 100 mL was Therefore the upper limit of the level divided by protein that is input to. (1,400 mg / 100 mL) and 10 multiplied by 100 to reach the lower limit of the proposed interval. The recommendedDietary intake for the amino acid expressed in mg/100 mL was also divided by the lower limit of the input to protein levels. (900 mg / 100 mL) and multiplied by 100 to reach the upper limit of the proposed interval.Example 15: la lysineUsing the method described above, the breakpoint for lysine by a group of 21. infants was estimated to be 130 mg. kg-1 d-' (this is also shown in Figure 2), and the Recommended dietary intake for lysine was then determined to be 100 mg/mL (87 of. Infant formula), g/100 g Converted into 3270 proteins as described above, the result is 20 for lysine 6.2 9.7 g/100 g of protein —.Example: thai o nin lbUsing the method described above, mean the breakpoint for the Memphis group by a thai o-nin. Of infants was estimated to be 33 38 mg.kg-i.d-1 (this is also shown in Figure 3) It is noted that 25. this estimation was made based on a diet that contained an excess of Xin hostel e Green. (Mg.kg-1. d. 91-1), The recommended dietary intake for mail. thai o nin was then determined. To be 25 mg/100 mL (baby formula) Converted into g/100 g. protein as described. Above, the result is thai for mail o nin 1.8 2.8 g/100 g of protein —.Example: 30 o' nin thari icUsing the method described above, the breakpoint for a group of nin by thari o infants was estimated to be 68 32 mg • kg-1. d-1 (this is also shown in Figure 4) and the recommended dietary intake for Obihiro thari was then determined to be nin 45 mg/100 mL. 55(Baby formula) Converted into g/100 g. protein as described above, the result is nin video for thari 3.2 g/100 g of protein — 5.0.Example: ld Tre pato Phan5 Using the method described above, the breakpoint for the trip by a group of 30 pato Phan.infants was estimated to be 15 mg.kg-1.d-1 (this is also shown in Figure 5) and theRecommended dietary intake for a day trip was then determined to be pato Phan 10 mg/100 mL (baby formula) Converted into g/100 g. protein as described above, the result is 0.7 for Tre pato Phan — 1.1 g/100 g. Protein.10.Example: le valineUsing the method described above, the breakpoint for a group of valine by 28.infants was estimated to be 110 mg.kg-I.d-1 (this is also shown in Figure 6) and theRecommended dietary intake for valine was then determined to be 73 mg/100 mL (15 of infant formula) Converted into g/100 g. protein as described above, the result for. Valine is 5.2 g/100 g of protein — 8.1.Example: lf I solo scene LeuUsing the method described above, the breakpoint for an SEO by a group of 22 Leu-scene 20 infants was estimated to be 105 mg.kg-1-d-1 (this is also shown in Figure 7) and the. Recommended dietary intake for an SEO was then determined to be scene Leu 70 mg/100 mL. (Baby formula) Converted into g/100 g. protein as described above, the result for. I solo scene is 5.0 g Leu/100 g protein--7.8.25 Example lg: LetnineUsing the method described above, the breakpoint for a group of 33 scene by Leu.infants was estimated to be 140 mg.kg-l.d-1 (this is also shown in Figure 8) and theRecommended dietary intake for Leu was then determined to be scene mg/100 mL (93 of. Infant formula), g/100 g Converted into 3270 proteins as described above, the result for. 30 g scene is 6.6 Leu — 10.3/100 g. Protein. 56.Example 2: Hiroshima sathi Dean, phenylalanine, and Rosa e mountain hostel Taitung-scene requirements in infants.The methodology was not performed for the IAAO amino silicone dacham: hip distresses sathi Dean. And phenylalanine. Moreover, the IAAO methodology was used for the (cannot) be conditionally 5. Amino silicone silicone dacham: @ hostel e green and Thus, the Tai Chi roll scene of these levels. Amino aesit were determined as explained below.

ผลลัพธ์ (อังกฤษ) 2:

IAAO method - Protocol
24 hours before the Study Day, the infants Consumed their Test formula (which includes the specific level of the amino acid Test being given for each particular infant) for
Adaptation, with the feeding regime conforming to that of the Hospital. During the study day, the feeding regime was changed to hourly bolus feeding of the infant's test formulas to ensure a metabolic steady State in feed condition. In Order to quantify individual CO2 Production, the infants received a primed (14 UMOL / kg) continuous (. 9 UMOL / kg / H) infusion of [13C] bicarbonate (sterile pyrogen free, 99% 13C APE; Cambridge Isotopes, Woburn, MA) for 3 hours (Riedijk, 2005). Directly following the 3 hour bicarbonate infusion, a primed (. 34 UMOL / kg) continuous (27 Pinol / kg / H) enteral infusion of L-[1-13C] phenylalanine (99% APE 13C; Cambridge Isotopes) was given for 4 hours. To Minimise the invasiveness tracers. were given Enterally by means of a gastric tube. Breath samples were Collected by means of the Direct sampling method described by Van der Schoor (2004th). At baseline, two Duplicate breath samples were obtained before Tracer infusion. During the last 45 minutes of [. 13C] bicarbonate infusion, and during the last hour of L [1-13C] phenylalanine infusion, samples were duplicated and Collected Every Ten Fifteen minutes, respectively. Samples were stored at Room Temperature Monthly until shipment to the Netherlands for. Analysis at the mass spectrometry Laboratory of the Erasmus Medical Center, Rotterdam. Expired 13CO2 enrichment was measured by Isotope mass spectrometry (ABCA; Europe Scientific, Van Loenen Instruments, Leiden, the Netherlands), and reported in Units of Atom percent excess (APE). . For each participant, the estimated CO2 Production was calculated Body, As described previously (Riedijk, 2005th). The rate of fractional [1-13C] phenylalanine oxidation was calculated using Equation this: Fractional phenyl alanine. The oxidation (%) = [x iB x IEPHE IEB] x 100% Where is the excess IEPHE 13C isotopic enrichment in Air expired during [1- 13C] phenylalanine infusion (APE), iB is the infusion rate of. [13C] bicarbonate (Gmol / kg / H), IPHE is the infusion rate of [1-13C] phenylalanine (UMOL / kg / H), and IEB is the 13C isotopic enrichment in excess expired during Air [. 13C] bicarbonate infusion (Van Goudoever, in 1993). 53 The results of the IAA () Studies for amino acids: lysine, methionine, a threonine, a trip in the van, Wa. Lean, isoleucine and leucine, are described in Examples la-lg, and with the results also illustrated graphically in Figures 2-8. As phenylalanine was used As the amino acid Indicator, each of Figures. 2-8 plots the oxidation rates of the phenylalanine 5 (F13CO2) Intake of dietary against the Test Point Amion Aci.d Each represents an individual infant was fed an amino acid based formula Who where each Essential amino acid was present. in excess except the Essential amino acid to be tested, where the Test amino acid was present in an amount ranging from deficient to excess. 10 Ecommended dietary Intake Ranges The Recommended dietary Intake Range for the amino acid required in each of Examples. la-lg was then determined using the following Strategy. First, the dietary Requirement (breakpoint) of the IAA () results was estimated using a biphasic linear regression crossover Model and was taken As the primary parameter. 15 To ensure that nearly all individuals Meet. the amino acid requirements, a Safe level of Intake was defined. The Safe level of protein Intake Proposed by the WHO (WHO / FAO / UNU, 2007th) was used, Defining the Safe level of protein Intake As 125% of the average protein Requirement. . In this instance, the Safe level of amino acid Intake was therefore calculated to be> 125% of the primary parameter (breakpoint value) 20 obtained As described above, to reach the population Safe Requirement value. An assumption was Made that this primary parameter. (breakpoint value) is Higher than the Requirement of protein bound amino acid based on the Study by Metges (in 2000) which Showed> 20% Higher First Pass oxidation rate when free amino acids are ingested compared to protein. bound amino acid. Therefore 20% of the estimated 25 Requirement was subtracted from the population Safe Requirement value, which is believed to be Conservative (IE, an overestimate rather than underestimate). The net Effect of the above two calculations is. therefore that the breakpoint equals the Recommended Intake (measured in mg ° kg-1. D-1). The Recommended Intake measured in mg. kg-I D 'was then converted to Units of 30 mg (of amino acid) / 100 mL. (infant formula). This was Calculation Made by Dividing the Recommended Intake (mg. kg-LD-1) by the volume Intake by an infant of 150 ML.kg-ID-1 (Shaw, in 2001) and Multiplying by 100. This is described Below the As. 54 Recommended dietary Intake for each amino acid, expressed in mg (amino acid) / 100 mL (infant Formula). This Recommended dietary Intake Range was also converted into Units of G (amino acid) / 100 G protein as follows. The calculations were. based on the amount of protein you get 5 of 0.9 -1.4 g (protein) / 100 mL (infant formula), or 900 - 1,400 mg (protein) / 100 mL (infant Formula) (Koletzko, 2,009th). This level of protein content. Get the latest correlates to 1.4 to 2.1 G protein / 100 kcal infant formula based on an energy level of 66 kcal per 100 mL The Recommended dietary Intake for the amino acid expressed in mg / 100 mL was therefore divided by the Upper Limit of volume. The proteins get into (1,400 mg / 100 mL) and 10 multiplied by 100 to reach the Lower Limit of the Proposed interval. The Recommended dietary Intake for the amino acid expressed in mg / 100 mL was also divided by the Lower Limit of volume. The proteins get into (900 mg / 100 mL) and multiplied by 100 to reach the Upper Limit of the Proposed interval. 15 Example la: Lysine Using the method described above, the breakpoint for Lysine by a Group of 21 infants was estimated. to be 130 mg. kg-D-1 '(this is also shown in Figure 2), and the Recommended Intake for dietary lysine was then determined to be 87 mg / 100 mL (of infant Formula). Converted into G / 100. g protein, as described above, the result for 20 lysine is 6.2 - 9.7 g / 100 g protein. Example LB: methionine pm Using the method described above, the mean breakpoint for methionine at by a Group of 33. infants was estimated to be 38 mg.kg-id-1 (this is also shown in Figure 3). It is 25 noted that this estimation was made ​​based on a diet that contained an excess of acid poured caffeine (91 mg. kg-1. d.-1). The recommended dietary intake for methionine and then was determined to be 25 mg / 100 mL (baby formula). Converted into g / 100 g protein As described above, the Result for methionine and is 1.8 - 2.8 g / 100 g protein. 30 Example IC: threonine pm Using the method described above, the breakpoint for Argyle Rio. This was by a group of 32 infants was estimated to be 68 mg • kg-1. d-1 (this is also shown in Figure 4) and the recommended dietary intake for threonine and was then determined to be 45 mg /. 100 mL 55 (of infant formula). Converted into g / 100 g protein, as described above, the result for threonine and is 3.2 - 5.0 g / 100 g protein. Example LD: Trips van 5 Using the method described above, the breakpoint for trips. van by a Group of 30 infants was estimated to be 15 Mg.kg-1.d-1 (this is also shown in Figure 5) and the Recommended dietary Intake for trips van was then determined to be 10 mg /. 100 mL (baby formula). Converted into g / 100 g protein, as described above, the result for trips van is 0.7 -1.1 g / 100 g protein. 10 le Example: valine Using the method described above, the breakpoint for valine by a. Group of 28 infants was estimated to be 110 Mg.kg-Id-1 (this is also shown in Figure 6) and the Recommended dietary Intake for valine was then determined to be 73 mg / 100 mL (of 15 infant formula). . Converted into g / 100 g protein, as described above, the result for valine is 5.2 - 8.1 g / 100 g protein. Example lf: isoleucine Using the method described above, the breakpoint for isoleucine by a. Group of 22 infants was estimated to be 20 105 Mg.kg-1- D-1 (this is also shown in Figure 7) and the Recommended dietary Intake for isoleucine was then determined to be 70 mg / 100 mL (of. infant formula). Converted into g / 100 g protein, as described above, the result for isoleucine is 5.0 - 7.8 g / 100 g protein. 25 Example lg: Letnine Using the method described above, the breakpoint for leucine by a Group of 33 infants. was estimated to be 140 Mg.kg-LD-1 (this is also shown in Figure 8) and the Recommended Intake for dietary leucine was then determined to be 93 mg / 100 mL (of infant Formula). Converted into G / 100. As described above g protein, the Result for 30 leucine is 6.6 -10.3 g / 100 g protein. 56 Example 2: The histidine, phenylalanine, CSIS only caffeine and Tyrosine requirements in. infants The IAAO methodology was not performed for the amino acid needed: a histidine and phenylalanine. Moreover, the IAAO methodology was (can not) be used for the 5 conditionally. Amino acid required: Cisco pour Tatooine and tyrosine. Thus, the levels of these amino acids were determined as explained below.

ผลลัพธ์ (อังกฤษ) 3:

IAAO method - Protocol
24 hours before the, study day the infants consumed their test formula (including the specific level. Of the test amino acid being given for each particular infant), for
adaptation with the feeding regime conforming to that. Of the hospital. During the study day the feeding, regime was changed to hourly bolus feeding of the infant 's test

.The formula to ensure a metabolic steady state in feed condition. In order to quantify

individual CO2 production the infants,, Received a primed (14 umol / kg) continuous (9
umol / kg / h) infusion of [13C] bicarbonate (sterile, pyrogen free 99%13C APE;
Cambridge. ,, Isotopes Woburn MA) for 3, hours (Riedijk 2005). Directly following the

3 hour, bicarbonate infusionA primed (34 umol / kg) continuous (27 pinol / kg / h) enteral

infusion of L - [1-13C] phenylalanine (99% 13C APE; Cambridge Isotopes). Was given for
4 hours. To minimise invasiveness the tracers were given enterally by means of a gastric tube.

Breath samples. Were collected by means of the direct sampling method described

by van der Schoor (2004, At baseline).Two duplicate breath samples were obtained
before tracer infusion. During the last 45 minutes of [] bicarbonate, 13C infusion. And
during the last hour of L [1-13C] phenylalanine, infusion duplicated samples were

collected every ten and, fifteen minutes. Respectively. Samples were stored at room
temperature until monthly shipment to the Netherlands for analysis at the mass
.Spectrometry Laboratory of the Erasmus, Medical Center Rotterdam. Expired 13CO2
enrichment was measured by isotope mass. Spectrometry (ABCA; Europe, Scientific

van Loenen, the Leiden Instruments, Netherlands), and reported in units of atom

percent. Excess (APE).
For, each participant the estimated body CO2 production was calculated as described, previously (Riedijk,, 2005).The rate of Fractional [1-13C] phenylalanine oxidation was calculated using this equation:
Fractional phenylalanine oxidation. (%) = [IEPHE x iB x IEB] x 100%
Where IEPHE is the excess 13C isotopic enrichment in expired air during [1 -
13C] phenylalanine. Infusion (APE), iB is the infusion rate of [13C] bicarbonate
(gmol / kg / h),IPHE is the infusion rate of [1-13C] phenylalanine (umol / kg / h), and IEB is
the excess 13C isotopic enrichment in expired. Air during [] bicarbonate 13C infusion (van, Goudoever 1993).









The 53 results of the IAA () studies for amino acid:? Lysine, methionine, methionine, Mei is three
tryptophan valine, isoleucine, leucine, and andAre described in, Examples la-lg and with
the results also illustrated graphically in Figures 2-8. As phenylalanine was. Used as the
indicator, amino acid each of Figures 2-8 plots the oxidation rates of the phenylalanine 5 (F13CO2 against.) Dietary intake of the test amion aci.d Each point represents an
.Individual infant who was fed an amino acid based formula where each essential amino
acid was present in excess except the. Essential amino acid to, be tested where the test
amino acid was present in an amount ranging from deficient to excess.


10. Ecommended dietary intake ranges
The recommended dietary intake range for the amino acid necessary in each of
.Examples la-lg was then determined using the following strategy.
First the dietary, requirement (breakpoint) of the IAA (). Results was estimated using a biphasic linear regression crossover model and was taken as the primary parameter.
15 To. Ensure that nearly all individuals meet the amino acid requirements a safe, level
of intake was defined.The safe level of protein intake proposed by the WHO
(WHO / FAO / UNU 2007), was used defining the, safe level of protein intake. As 125% of
the average protein requirement. In this instance the safe, level of amino acid intake
was therefore calculated. To be > 125% of the primary parameter (breakpoint value)
20 obtained as, described above to reach the population safe requirement. Value.
.An assumption was made that this primary parameter (breakpoint value) is higher
than the requirement of protein bound amino acid Based on the study by Metges
(2000) which showed > 20% higher first pass oxidation rate when free amino acid are
ingested. Compared to protein bound amino acid. Therefore 20% of the estimated
.25 requirement was subtracted from the population safe, requirement value which is
believed to be conservative (i.e, an. Overestimate rather than underestimate).
The net effect of the above two calculations is therefore that the breakpoint equals. The recommended intake (measured in mg ° kg-1. D-1).
The recommended intake measured in mg.Kg-I d 'was then converted to units of 30 mg (of amino acid) / 100 mL (infant formula). This calculation was made by
dividing. The recommended intake (mg. Kg-l-d-1) by the volume intake by an infant of
150 mL.kg-i.d-1 (Shaw 2001), and multiplying. By 100. This is described below as the







recommended 54 dietary intake for each, amino acidExpressed in Mg (amino acid) / 100 mL (infant formula).
This recommended dietary intake range was also converted into units. Of G (amino
acid) / 100 g protein as follows. The calculations were based on levels of protein admitted to 5 of 0.9 - 1.4. G (protein) / 100 mL (infant formula), or 900 1 400 -, Mg (protein) / 100 mL
(infant formula), (Koletzko2009). This levels of protein admitted to correlates to 1.4 to 2.1 G
protein / 100 kcal infant formula based on an energy. Level of 66 kcal per 100 mL The
recommended dietary intake for the amino acid expressed in mg / 100 mL was
therefore. Divided by the upper limit of level, protein content, take (1400 mg / 100 mL) and 10 multiplied by 100 to reach the lower limit of the proposed interval. The recommended
dietary intake. For the amino acid expressed in mg / 100 mL was also divided by the lower limit of level protein input (900 mg. / 100 mL) and multiplied by 100 to reach the upper limit of the proposed interval.


15 Example La: lysine
.Using the method described above the breakpoint, for lysine by a group of 21
infants was estimated to be 130 mg. Kg-1. D - '(this is also shown in Figure 2), and the
recommended dietary intake for lysine was then determined to be 87 mg / 100 mL. (of
infant formula). Converted into g / 100 g protein as described above the result, for 20 lysine is 6.2 - 9.7 g / 100 g protein.


Example LB: Mei don
Using methionine the method, described above the mean breakpoint for Mei parathion methionine by a group.
of 33 infants was estimated to be 38 mg.kg-i.d-1 (this is also shown in Figure 3). It is 25 noted that this estimation. Was made based on a diet that contained an excess of cysteine
(91 mg.kg-1. D.-1).The recommended dietary intake for Mei parathion methionine was then determined
to be 25 mg / 100 mL (infant formula). Converted into G. / 100 g protein as described
above the result, for Mei parathion methionine is 1.8 - 2.8 g / 100 g protein.


30 Example IC: three methionine
Using. The method, described aboveThe breakpoint for three methionine by a group of 32 infants was estimated to be 68 Mg - kg-1. D-1 (this is also shown in Figure. 4) and the recommended dietary intake for three methionine was then determined to be 45 mg / 100 mL







55 (infant formula). Converted. Into g / 100 g protein as, described above the result for three methionine is 3.2 - 5.0 g / 100 g protein.


.Example LD: tryptophan
5 Using the method, described above the breakpoint for tryptophan by a group of 30
infants was estimated. To be 15 mg.kg-1.d-1 (this is also shown in Figure 5) and the
recommended dietary intake for tryptophan was then determined. To be 10 mg / 100 mL (infant formula). Converted into g / 100 g protein as, described aboveThe result for tryptophan is 0.7 - 1.1 g / 100 g protein.
10
Example Le: valine
Using the method described above the breakpoint,, For valine by a group of 28
infants was estimated to be 110 mg.kg-I.d-1 (this is also shown in Figure 6) and the
recommended. Dietary intake for valine was then determined to be 73 mg / 100 mL (of 15 infant formula).Converted into g / 100 g protein as described above the result, for
valine is 5.2 - 8.1 g / 100 g protein.


Example LF:? Isoleucine
Using the method, described above the breakpoint for isoleucine by a group of 22 20 infants was estimated to. Be 105 mg.kg-1 - D-1 (this is also shown in Figure 7) and the
.Recommended dietary intake for isoleucine was then determined to be 70 mg / 100 mL
(infant formula). Converted into g / 100 g. Protein as, described above the result for
isoleucine is 5.0 - 7.8 g / 100 g protein.


25 Example LG: Letnine
Using the. Method described above the breakpoint, for leucine by a group of 33
infants was estimated to be 140 mg.Kg-l.d-1 (this is also shown in Figure 8) and the
recommended dietary intake for leucine was then determined to be 93 mg. / 100 mL (of
infant formula). Converted into g / 100 g protein as described above the result, for
30 leucine is 6.6 - 10.3. G / 100 g protein.








Example 56 2: phenylalanine, histidine,Cysteine and tyrosine requirements in infants
The IAAO methodology was not performed for the necessary: amino acid histidine.
and phenylalanine. Moreover the IAAO, methodology was (cannot) be used for the 5 conditionally necessary: amino acid cysteine. And tyrosine. Thus the levels, of these
.Amino acid were determined as explained below.
.