University of AIberta The Effects of Diet and Tracer Administration Route on Tryptophan Requirements of Neonatd Pislets Suzan Stephanie Ck-itkovic @ A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfiilrnent of the requirements for the degree of Master of Science Xutrition and Metabolism Department of Agricultural, Food, and Nutritional Science Edmonton, Alberta Fall 2000
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University of AIberta
The Effects of Diet and Tracer Administration Route on
Tryptophan Requirements of Neonatd Pislets
Suzan Stephanie Ck-itkovic @
A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfiilrnent of
the requirements for the degree of Master of Science
Xutrition and Metabolism
Department of Agricultural, Food, and Nutritional Science
Edmonton, Alberta
Fall 2000
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ABSTRACT
Previous research has demonstrated that enterally fed piglets require -30% more
methionine and -55% more threonine than parenterally fed piglets. To determine whether
tryptophan requirements of the pislet is affected by the route of diet infusion, we used the
indicator amino acid oxidation (IAAO) technique in newborn piglets receivins an
elemental diet either enterally or parenterally. Using breakpoint analysis, the mean
tryptophan requirement \vas detemined to be O, 13 (10.02 SE) and 0.15 (i0.02 SE) g k g ' d
for enterally and parenterarly fed piglets, respectively. Thus, it is unlikely that tqptophan
is preferentially utilized by the gut. However, it has been demonstrated that a substantial
ponion of phenyIalanine (commonly used as an indicator amino acid) is utilized on first
pass by the splanchnic bed. To determine whether rnetabolism of tracer during IQLAO
afXects phenylalanine kinetics and requirernent estimates, enterally fed pislets received L-
[ l -"C-phenylalanine either intragastrically (IG) or intravenously (IV) dunt-tg oxidation
studies. dthough est iniates of phenylalanine kinetics were significantly affected bj- tracer
administration route, p henylalanine osidation, when espressed as a percentage of dose
osidized, \vas similar for piglets in both groups. The mean tryptoptian requirements were
O. i 1 (i0.03 SE) ~JksJd and 0.13 (=O.OZ SE) g/kg/d for pislets given IG and IV tracers.
Therefore. the use of an IG administered isotopic tracer with breath sampling provides a
similar estimate of rryptophan requirements in piglets.
DEDICATION
To Mom and Dad, who have always encouraged me to pursue my goals and strive to be my very best. Love you both.
1 would like to acknowledge the exceptional guidance and support of my supervisors, Dr.
Ronald Bail and Dr. Paul Pencharz. 1. am very grateful for the opportunity to expand my
knowledge and abilities, and to be able to share this experience with so many capable and
caring people. SpeciaI thanks to al1 of them: Dr. Rob Bertolo, Dr. Janet Brunton, Dr-
Sonke Mohn, and fellow graduate students Kate ShovelIer. Anastasia Nimchuk. Laurie
Drozdowski, Robyn Harte, Garson Law and Raja Elango. FinalIy, 1 would like to thank
the staff at the Metabolic Unit as well as Gary Sedgwick for their technical assistance.
h i n o Acid Profile of Commercial Parenteral Amino Acid Solutions .................................................................................... (Bal1 et al.. 1996) -5
Tryptophan Requirement Estimates in Humans ...................................... -36
........................ .h ino Acid Requirements of Humans According to A_oe 3 s
Tryptophan Requirernents of Pigs (1-5 k_o) .............................................. 39
- h i n o Acid Content of Cornpiete ElementaI Diet ................................... -4s
Vitamin Content of Compfete Elemental Diet ......................................... -49
Minerai Content of Complete ElementaI Diet ......................................... -50
Amino Acid Composition of Test Diets ................................................... 5 1
Plasma h i n o Acid Concentrations of Enterally Fed Pislets (IV Tracer) ......................................................... .. . 58
.............. Plasma Amino Acid Concentrations of Parenterally Fed Piglets 60
...................... PhenyIalanine Kinetics in Enterally Fed Piglets (IV Tracer) 63
Phenylalanine Kinetics in Parenterally Fed Piglets .................................... 6s
Tryptop han Requirement by Breakpoint Analysis in Enterally Fed ........................ .................................................... Pislets (IV Tracer) .. 73
Tryptophan Requirement by Breakpoint Analysis in Parenterally .............. ......................................................................... Fed Piglets .. 73
Phenylalanine Kinetics in Enterally Fed Pielets (IV Tracer) .................. 103
Table 4.5 Tryptophan Requirement by Breakpoint Analysis in Enterally Fed ........................................................ ............ Pigiets (IG Tracer) .... 10s
Table 4.6 Tryptophan Requirement by Breakpoint Analysis in Enterally Fed ................................................................................ Piglets (IV Tracer) 10s
Table 4.7 Estimates of Phenylalanine Kinetics: Cornparison of Enterally Fed Piglets Given TG vs IV Tracer ............................................................... 2 10
LIST OF FIGURES
Figure 2.1
Figure 2.2
Fisure 2-3
Fisure 3.1
Figure 3 -2
Figure 3 -3
Figure 3 -4
Figure 3.5
Fisure 3 -6
Figure 3.7
Figure 3.9
Figure 4.1
Fisure 4.2
The Kynurenine Pathway o f Tryptophan MetaboIism ...-. .. . - - .. . . . ...... .. . . . . . . -26
Phenylalanine Osidation in Enteraily Fed Piglets (IV Tracer) Based on 14 C-Phenylalanine Radioactic-ity in Collected Plasma. .. ....... .. .--- .-. - -.. . . . ..... 66
"COL Radioactivity in Collected Breath of Parenterally Fed Piplers. .... .. ..69
Phenylalanine Osidation as a ?/a of Dose in Parenterally Fed Piglets.. . . . . . .. 70
Phenylalanine Osidation in Parenterally Fed Pislets Based on "C- P henylalanine Radioactivity in Collected Plasma.. . . ...... . . . . . . . . . . . . . .... .. . . . . . . . . .7 1
T y t o p h a n Concentrations in Plasma of Piglets- Comparing -Ail Esperiments .._.-. ..... . ..... .... .......... . ... ... . . . . . . . . - - - - . . -p. ... .. -......-..-. ---. -. .....-..-..-.. 76
Phenylalanine Concentrations in Plasma of PigIets- Comparing A l Espenments ...... ...-----.. .------.. ..-...---. ......-..-....----.--.- .-. . ........-.- ... .....-.. .......- 77
Figure 4.3 Phenyialanine Oxidation as a % of Dose in Enterally Fed Pigiets ........................................................................................... (TG Tracer). -99
Figure 4.4 Phenylalanine Oxidation in Enterally Fed Piglets (IG Tracer) Based on ........................... "C-Phenylalanine Radioactivity in Collected Plasma. -100
Figure 4.5 14CQ2 Radioactivity in Collected Breath of Enterally Fed PigIets (IV Tracer). .......................................................................................... i 04
Figure 4.6 Phenylalanine Oxidation as a % of Dose in Enterally Fed PigIets (IV Tracer) ......................................................................................... 1 0 5
Figure 4.7 Phenylalanine Oxidation in Enterally Fed Piglets (IG Tracer) Based on ............................. "C-Phenylalanine Radioactivity in Coilected Plasma 1 06
F i s r e 4.8 Plasma Phenylalanine Specific Radioactivity: Comparing AI Esperiments .......................................................................................... 1 12
during TPN feeding may be greatly diferent than when entera1 nutrients are provided
(Bertolo et al., 1999). There is evidence to sujgest that first pass metabolism of the sut
may infiuence whole body amino acid requirements. Bertolo et al. (1998) esamined
threonine requirements in neonatal piglets fed identical diets either parenterally o r
enterally. Usin3 the 1-440 method, they determined that the threonine requirement for
TPN fed piglets was approximately 40 % of the requirement estimated for their enterally
fed counterparts. This is supported by Stoll et al. (1998), who measured the appearance
of labeiled amino acids in piglet portal blood folloivin; an intragastric infusion of [u-'~c]
algal protein in combination nith enteral feedinss. Tt was determined that uptake of the
gut accounted for 61% of the threonine provided in the oral tracer. Similarly, 35 % of
both lysine and plienylalanine were absorbed by the sut on first pass (Stoll et al., 199s).
The lysine (House et al.. 1998) and phenylaIanine (House et al., 1997) requirements of
TPK fed piglets has also been detemined using the 1-440 method. When compared to
NRC recomrnendations for enteraIly fed pigIets, the parenterally fed animals had
approsirnately 30 % lower requirements for both lysine and phenylaIanine. FinaIly, the
methionine requirement in parenterally fed piglets was estimated ro be approximately 70 $4
of the entera1 requirement based on indicator amino acid oxidation (Shoveller et al., 2000).
Overall, it has been shown that rouçhly one third of d ie ta l essential amino acids is
taken up during intestinal first pass metabolism (Stoll et al., 1998). However, the extent to
which each of these indispensable amino acids is absorbed and utilized by the gut differs.
17
Currently, the proportion of tryptophan initially taken and rnetabolized by the =ut is
unknown. Unlike threonine, which gea t ly contnbutes to intestinal mucin production (G.
Law, MSc. thesis, 3000). tryptophan is not currentIy believed to be preferentially utilized
by the g t . Although dietary tryptophan requirements have been defined for the enterafly
fed young pig (Firth and Johnson, 1956; Bal1 and BayIey, 1954) and human (Lazarus-
Brunner et al., 1998; Leverron et al., 1956; Young et al., 1972), the parenteral
requirement for tryptophan has yet to b e elucidated-
2.4 TRACER STUDIES: ORAL VS INTRAVENOUS ISOTOPE
Severai isotopicalIy labelled amino acids have been used as tracers to examine
amino acid kinetics. Typically, leucine, phenylalanine and lysine are used in osidation
studies. due to the fact that they have reasonably few rnetabolic tàtes, and that they have a
carboxyl carbon that is irreversibly osidized, and thus appears in breath CO,. Amino acid
requirernent studies, using osidation techniques. can be conducted with two possible
routes of isotope administration: oral o r intravenous. IV tracers have been commonly
used in pigs (Bert010 et al., 19%; House et al., 1997,1998) as well as in aduIt humans
(Zello et al., 1990,1993; Duncan et al., 1995; Lazams-Brunner et al., 1998). As stated
previously, IV inhsion techniques are invasive, and are considered inappropriate for use in
special populations, Iike premature infants. Oral administration of isotope is potentially
less invasive. Oral tracers were used in the early development of the I.UO method in pigs
(Ba11 and Bayley, 1954; Kim et al., 1983)- Oxidation studies usins oral tracers have been
15
done in adults as well (Basile-Filho et al., 1997; Sanchez et al., 1995; Bross et al., 1998).
There are several issues surrounding the use of oral versus IV îracers. There is
substantial first pass metabolism of labelled amino acids by the splanchnic tissues
(Matthews et al., 1993; Biolo et al., 1993; Hoerr et ai., I 991; Krempf et al., 1990; Stoll et
al., 1998; van Goudoever et al., 2000). The fraction of gastncally fed [phenyl-%,]-
phenylalanine utilized by the splanchnic bed on first pass was estimated to be
approGmately 30% in the adult human (Matthews et al., 1999). This measure was denved
by esamining plasma isotopic enrichment of phenylalanine followin_o the oral
administration of tracer-
The sastrointestinal tract is likely the predominant organ involved in splanchnic
extraction of amino acids. L.sin_o a combination of mass balance and tracer inhsion
tecliniques, Stoll et al ( 1998) determined that approsimately 3 5% of enteral "c-
phenylalanine provided in the diet as [U-"Cl-algal protein was taken up on first pass by
the p t . n-ith the rernainder appearïng in the portal blood. This is very similar to previous
estirnates of entire splanchnic phenylalanine extraction or first-pass removal of amino acids
by both the -mt and liver (Biolo et al., 1993; Matthews et al., 1999). Indeed, it has been
estimated that 75% of total splanchnic metabolism of intragastric phenylalanine tracer is
due to gut metabolisrn in young pigs (Stoll et al., 1997). SimiIarly, 8% of splanchnic
extraction of gastrically administered, labelled Ieucine is due to y t metabolisrn, and the
remainins 15% due to the Iiver, in dojs (Yu et al., 1990).
Small intestinal utilization of nutrients, both quantitatively and qualitatively, is
affected by the route of nutrient provision. In the fastins and even the fed state, the portal
drained viscera (PDV) use arterial amino acids (Rerat et al-, 1992; Yu et al., 1990, I992).
Approsimately I 1, 1 1, and 5% of the respective intake of labelled lysine. ieucine and
phenylalanine was taken up by PDV via the arterial circulation in enterally fed piglets
(Stoll et al., 1998). What is the metabolic fate of these arnino acids absorbed by the gut?
Arterially presented labelled amino acid utilization may be different from the use of
Iurninally derived amino acid tracers (Aipers, 1972). As for gastric tracers, only 1 8, 2 1 ,
and 18% of 13C-lysine, "C -1eucine and "C -phenylalanine metabolized by the gut in the
first pass was incorporated into mucosal protein (Stoll et al., 199s). For phenylalanine,
approsimately 50% of the rernaining absorbed tracer \vas likely convened to tyrosine in
the mucosa, and the rest may have been osidized (Stoll et al., 199s). Although the
proportion of tracers used for mucosal protein synthesis appears to be reIathpely similar for
different amino acids. the fate of the majority of the tracer amino acid çrsatly depends
upon the metabolic pathways unique to each individual amino acid.
The impact of the *t OR tracer metabolism has substantial implications for
measurements of amino acid kinetics. The route of isotope infusion may affect the tracer
dilution in the plasma pool- There are three possible scenarios which can be considered:
the delivery of both diet and tracer intravenousIy, enteral feedins and intravenous isotope
administration, or the use of both diet and tracer intragastrically. Parenteral feedins in
combination with IV tracer infiision is considered to be an appropriate means of
determining accurate measures of plasma tracer enrichment, flux and oxidation. In this
20
case, the infusion and sarnpling of labelled amino acid is in the central or plasma pool.
When diet is given enterafly and tracer intravenously, the ,sut preferentialIy utilizes the
unlabelled enteral amino acids on first pass rather than the Iabelled tracer in the arterial
circu1ation (Stol1 et ai., 1998). Consequently, plasma e ~ c h m e n t , which can be
generalized as the arnount of label incorporated into the plasma amino acid pool, may be
greater than that of the g ~ t . Considering that the gut may account for over one quarter of
whole body protein turnover (Reeds et al., 1999). the jack of tracer utilization by these
tissues would likely lead to an underestimation of whole body protein synthesis.
Phenylalanine fius is caiculated as the dose of labelled phenylalanine divided by the plasma
enrichment of the tracer, and therefoie disproportionately high plasma enrichments would
result in lower rates of phenylalanine flus. Phenylalanine osidation when calculated using
plasma plienylalanine flus, may potentially be lower in enterally fed individuals receiving
IV versus IG tracer. Therefore, caiculated estimates of whole body plienylalanine
o'cidation. as well as protein synthesis, rnay be underestimated when IV instead of IG
tracers are used for the enterally fed individual-
Alon_o similar lines, the gastric infusion of diet and tracer may alter amino acid
tracer kinetics. As stated previously, approximately 30% of orally administered tracer is
taken up on first pass by the splanchnic tissues. As a result, the isotopic enrichment of
plasma would be markedly reduced in those receiving IG versus IV tracers. As stated
previously, estimations of flux are determined as the dose of tracer divided by plasma
enrichrnent. Consequently, the use of oral tracer cornpared to IV tracer rnay result in
2 1
lotver plasma enrichments and higher plasma fluxes. Several studies have demonstrated
significantly lower plasma amino acid fluxes in enteralIy fed adults receiving IG versus IV
tracers (Sanchez et al., 1995; Hoerr et ai., 199 1 ; Krernpf et al., 1990).
Whether flux is an accurate measure of amino acid kinetics in enterally fed
individuals receiving oral tracers is currently unresohed. 1s the resulting plasma isotopic
enrichment a representation of just the plasma pool or of the whole body pool? Some
researchers have corrected flux estimations based on the amount of tracer lost in first pass
uptake (Sanchez et al., 1995). In contrast, it may be argued that when both tracer and diet
are given enterally, the loss of tracer is a reflection ofthe amino acids being utilized, and
that the subsequent plasma enrichment is more representative of the whole body pool.
Clearly, a greater understanding of this area is needed.
The collection of labelled breath CO, is a cornmon component of amino acid
osidation studies. \Jrhen appearance of label in breath is espressed as a percent of dose
infused ,aastrically, the issue of label dilution may be avoided. Perhaps unlike plasma,
breath may more accurately reflect intraceliular amino acid enrichment. Whether this
notion holds true for IV infùsed tracers given concurrently with enteral diet is unknown.
However; recent studies have found no significant differences between whole body
osidation rates of tracers given IV versus IG (El-Khoury et al., 199s; Bross et al., 1998).
The issue of plasma tracer ennchment is critical in direct oxidation studies (J.D.
House, PhD. thesis, 1995). However, when the indicator arnino acid osidation method is
used. it is the siope of the line and breakpoint in breath CO, that is most important in the
3 3 --
determination o f the test amino acid (Zello et al., 1995). Consequently, the route of tracer
infusion may not be an important issue when indicator oxidation techniques are used,
however, no data presently exists to support this point of view.
In conclusion. the route o f tracer administration rnay play a major role in the
estimation o f amino acid kinetics. First pass metabolism of tracers by splanchnic tissues.
primady the intestine, alter the appearance of labelled amino acids in plasma. Subsequent
determination of plasma flux is also affected. The interpretation of whether plasma
constitutes the whole body amino acid pool is under question. Although the effect of
tracer route on amino acid kinetics has been esarnined. no one to date has actually
cornpared amino acid requirement estimates directly usin- either IV or IG tracers.
2.5 TRYPTOPMAN UTILIZATION AND 3IETABOLISM
Tryptoplian is a neutral arnino acid conraining an indole and aromatic side group.
Tryptophan is the least abundant amino acid in most proreins (Block and Weiss. 1956),
and has been estimated to constitute 1 and 1 .j % o f total amino acids in typical plant and
animal proteins. respectively (Peters, 199 1). Tq-ptophan is an indispensable amino acid
for rats (Rose, 1918), infants and children (Holt and Snyderman. 1965), and adults (Rose,
1957). Tryptophan plays an unique role in protein synthesis, and is a precursor for severa
important molecules in the body, including niacin, NAD-, NADP- , and serotonin.
Oxidation of this amino acid occurs via one of two main pathways: the iqnurenine
pathway o r throuçh the serotonin pathway. Current tryptophan requirement estimates are
based on several studies conducted in human and pigs. These aspects o f tryptophan
nutrition and metabolism will b e discussed in the following sections.
2-51 PROTEEU SYNTHESIS
Tryptophan is one of 20 amino acids required for tissue protein synthesis. Young
et ai. (1983) estimated the average adult male synthesizes approximately 3 g o f protein per
kg o f body weight (or -2755-250 g protein) daily when at nitrogen equilibrium. This is
equivaknt to 3.5 g o f tryptophan per day that is used for protein synthesis, and three times
the average daily tryptophan intake of such individuals. Therefore. a tremendous flux of
amino acids course through this pathway, making it quantitatively the most significant
utilization of tryptophan (Peters. 199 1 ).
Tryptophan has been su~ges ted to play a unique role in the regulation of protein
synthesis. The feeding of a solution containing tr).ptophari alone stimulated ribosome
aggregation and protein synthesis in the liver of rats and mice, whereas solutions of other
indikidual aniino acids. includins isoleucine, methionine, or threonine did not have such an
effect (Sidransky et al, 197 1). Others have confirmed that dietary tryptophan may induce
hepatic protein synthesis (Park et al., 1973; Jorgenson and Majumdar, 1975; Majumdar,
1982; Ponter et al., 1994), as well as increase the nbosomal fraction of porcine muscle
(Lin et al., 1958). The proposed mechanism by which tryptophan stimulates hepatic
protein synthesis may be by either increasing mRNA synthesis, or by increasing
nucleocytoplasmic translocation of mRNA, thereby increasing the supply of message to
24
areas in the ce11 where translocation occurs (Sidransicy et ai., 1984). These effects may be
mediated by tryptophan binding sites on cell nuclei (Cosgrove et al., 1992; Sidranshy and
Vemey, 19%; 1997; SidransLy et al., 1992). The amino acids alanine, phenylalanine,
tyrosine, and histidine, as well as the hormone 3,5,3'-trïiodothyronine (T,), have been
shown to compete with 'H-tryptophan binding to hepatic nuclei in vitro (Sidransky et al..
1992; Sidranslcy and Verney, 1999). Unlike tryptophan, these molecules did not stimulate
hepatic protein synthesis, but the binding of L-alanine and T, to t-ptophan receptors on
hepatic nucIei prevented the s t i r n d a t o ~ effect of tryptophan on hepatic protein synthesis
(Sidransky et al., 1990, 1992). Therefore, tryptophan appears to be involved in the
regulation of protein synthesis in a manner that is separate fiom its roIe as a component of
protein.
2.5.2 OXIDATIVE PATHLVAY VLA KYNURENINE
Quantitatively, the second most important route of tryptophan metabolism. afier
protein synrhesis, is its osidation via the kynurenine pathway (Fisure 2.2). This pathway
accounts for approsimately 95% or more of daily tryptophan cataboIism (Peters, 199 1 ).
The first and rate-limitinç step is the irreversible conversion of tryptophan to
formylkynurenine. This reaction is catalyzed by trvptophan-3,3-dioxygenase, which is a
haem-dependent, Iiver cytosolic enzyme (Badarvy, 1951). Both the amounr and actib-ity of
tryptophan-2,3-dioxysenase is increased by the presence of twtophan (Peters, 199 f ), and
this enzyme is aIso induced by corticosteroids, Lyurenine, and glucocorticoids (Knox,
25
1966; Sainio and Sainio, 1990). Conversely, this reaction is subject to feedback inhibition
by NADH and NADPH (Cho-Chung and Pitot, 1967).
A second enzyme is also capable of catalyzing the first step of the Iqwrenine
pat hway. Indoleamine-2,3 -dioxygenase (DO) esists in several tissues, includin~ the
intestine, stomach, lungs and brain, as well as in macrophages and monocytes (Sainio et
al., 1996). This enzyme differs fiom tryptophan-2,3 -dioxygenase because it utilizes
superoxide anion instead of moIecular oxygen as the oxidizing agent (Hayakhi et al.,
1984). ID0 has a much broader substrate specificity, and so will act upon not only
tryptophan, but also serotonin, tsptarnine, and 5-hydroxytryptophan (Hayaishi et al.,
1984). ID0 is not stimulated by either tryptophan or glucocorticoid (Hayaishi, 1996). but
is l~ighly inducible by interferon gamma (EN-G) (Yoshida et al.. 1979, 19s 1; Ozaki et al.,
1987; Takikawa et al., 1990; Werner et al.. 1989; Taylor and Feng, 199 1).
The estent to which tryptophan is catabolized by I D 0 varies between species. but
in Iiumans, it is believed that tryptophan-2,3-dioxygenase is normaI1ÿ the primary enqrne
catabolizing trvptophan (Leklem, 1971; Knowles et al., 1989). However, when the
immune system is stimulated, induction of ID0 by IFN-G may cause this enzyme to be
quantitatively more important in the pathway of tryptophan metabolism. Intraperitoneal
administration of bacterial endotosin induced approsimateIy a 100-fold increase in ID0
activity in the mouse lung, and decreased hepatic tryptophan-2,3-dioxygenase to Iess than
50% of its normal activity (Hayaishi et al., 1981). Induction of ID0 in cancer patients
receiving EN-G has been shown to reduce plasma tryptophan concentrations by 50-80%,
27
although other plasma amino acids remained unchanged (Brown et al., 1989; Bqrne et al.,
1986; Carlin et aI., 1989). Correspondingly, the concentrations of hynurenine and other
metabolites of this pathway increase substantially in plasma and urine (5-500-fold increase)
Prown, 1996; Brown et al., 1991)- It appears that although the provision of escess
dietary tryptophan to the immune chaIlenged may improve plasma tryptophan levels and
prevent tryptophan deficiency, supplementation may also fùrther increase the accumulation
of kynurenine pathway metabolites. Build-up of these metabolites rnay result in
subsequent complications. such as dementia seen in HIV patients (Brown, 1996).
The ne\? step in the kynurenine pathway is the conversion of formylkynurenine to
arninocarboxymuconate-semialdehyde (or acroleylaminofùmarate). At this point. the
pathway branches. The carbon skeleton of aminocarbo~ymuconate-semialdehyde can be
converted ro acetyl-CoA and osidized conlpletely to CO, via the Citric Acid Cycle.
Alternarively, aminocarbosymuconate-semialdehyde can be converted to quinolate by way
of non-enqmatic cyclization (Peters, 199 1). Normally, the majority of
aminocarbos~muconate-semialdehyde is osidized to CO,, and the significant conversion of
tryptophan through quinoIate to niacin onIy occurs when the capacity of one of the
enzymes in the former pathway (namely, aminocarbos)muconate-semialdehyde
decarboxylase) becomes limiting (Bender, 1982).
There are several important compounds that are produced via the kynurenine
pathway, each of which have specific fùnctions in the body. Picolinate is produced by the
non-enzymatic cyclization of aminomuconic sernialdehyde, and it plays a role in intestinal
28
zinc absorption (Evans and Johnson, 1980). Quinolinate is an iron chelator, and is
involved in the replation of gluconeogenesis (Veneziale et al., 2967). Niacin (vitamin
B,), othemise known as nicotinate, is produced by the further metabolisrn of quinolinate
(Figure 22). If dietary niacin intake is insufficient, 60 mg of tryptophan is required for
each 1 mg of niacin formed (Homitt et al., 1956).
Niacin's coenzyme derivatives, Nicotinic Adenine Dinucleotide @AD') and
NADP-, are other products of tryptophan metabolism. Both of these coenzymes are
ubiquitous, yet they have different fùnctions. NADH transfers electrons fiom intemediate
moIecules into the electron transport chain, and NAD- parricipates in several osidative
reductions, including: gIycolysis. osidative carbosylation of pyruvate. osidation of
acetate through the Citric Acid Cycle. and the beta-osidation of fatty acids. NADPH is
involved in the reductive biosynthesis of fatty acids, cholesterol, steroid hormones,
glutamate and deosyribonucleotides (Hunt and Groff, 1990). A fùrther rnetabolite of - NAD- is poly(ADP-ribose). Poly(ADP-ribose) is a homopotymer of ADP-nbose which is
necessary for the repair of damased DNA (Hayaishi et al., 1984). Therefore, several
metabolites produced from the cataboIism of tryptophan via the kynurenine pathway,
particularly niacin, NAD-, and N-ADP-, are of great importance for several daily metabolic
processes.
2.5.3 SEROTONIN (5-HT) PATHWAY
The biosynthesis of serotonin occurs in several tissues, including enterochromafin
a.b.cd denots siLgndicancs by Tukcy-s multiple cornparison test (~4.05): NS = not signitlcant (p>O.O5): ND = not detectabk: Trp dztection 1imit:-10 pmoVL
1
Amino Acid (pmofi)
Aspartate
Glutamate - Hydroqproline
Serine
Asparagine
Glycine
Glutamine
Taurine
Histidine
CituIline
TIirconine
Alanine
Arsinine
Prolinc
T>~osinc
Valine
Mcdiionine
C'stine
Isoleucine
Leucine
P henylal aninc
Trxptoph,m
Omi thine
Lysine
0.20
15
166
9Sab
193
17
745
100'
42
57'
856
800"'
135
474
21b
32 - - 12
IO3
236
2Sd
2Sk
86
442
0.15
18
154
1
223
18
1159
177b
13jk
30
89"&
530
75ybC
149
537
-- 7 7b
197"9Gb
27
14
109
228
ANOVA p value
NS
NS
-00 1 1
NS
NS
NS
.O177
-0123
NS
-0131
NS
-0012
NS
NS
-0006
-0408
KS
NS
NS
NS
.O00 1
-000 1
NS
NS
0.10
19
208
91b
263
20
1098
213b
204""4Sk
43
104""85*
S 17
655"
158
546
4sb
27Sb
0.30
28
288
9 1""
277
19
985
1 6 1 " ~ ~
136&
62
6-Ck
0.025
25
207
65'-'
0.05
17
140
118"
ND
132
551
0.40
20
12
174
277
SE
306
5 1
979
549"
277"
87
119"
395
5 s p
149
739
1 5
392"
8Zb
ND
93
444
62.3
44.8
9.1
35.0
9.7
14.8
1.8
0.9
6.4
11.4
4.1
5.3
20.3
235
4 1
746
44Tb
5 1
439
43 lC
107
480
122"
276b
572
1022"
35
717
24"
267b
27
1 O
125
277
44cd
45"-7"
115
462
24
13
120
209
128
99"b
201
16
762
17Zb
136"
39
71k
883
78AJb
141
583
i S b
21Sb
26
10
111
244
1 5 9 - 1
101
513
32
15
141
269
62&
ND
13 1
573
16.5
4-3
17.7
3.6
84.0
35.2
12.5
5.0
6.1
- 75d 21'
100
487
decreased from 1 18 to 82 pmol/L as tryptophan intake increased from 0.025 to 0.05
dkg,/d (p<O.OS), and dropped again to 25 yrnol/L at 0.15 g tryptophankgld (p<0.05). - Plasma phenylalanine concentrations were similar for pislets fed Q.lSO.40 g
tryptophadkgld. Tyrosine concentrations in plasma significantly declined (p<0.05)
between tryptophan levels of 0.025-0.10 g k d d , and remained stable among tryptophan
intakes of 0.10-0.40 g k g d . Hydrosyproline concentrations increased significantly from
65 tu 93 p m o K for tryptophan intakes of O . O Z to 0.05 ,o/kg/d, respectively, and
increased again to 119 pmoVL at tq-ptophan level of 0.15 @g/d (p<O.Oj). Plasma
hydrosyproline levels were not siyificantly different for piglets given 0.1 5-0.40 g
tryptophan/k_o/d. Both glutamine and taurine concentrations in plasma significantly
decreased as tryptophan intakes increased from 0.025 to 0.10 gikgid, and remained similar
for tryptophan levels of O. 10-0.40 cJk=/d. Plasma valine concentrations dropped as
tryptophan intakes increased from 0.025 to 0.05 =/kg/d, and valine levels were constant in
piglets receiving 0.05-0.40 g tsptophanBrg,/d. Lastiy, plasma tryptophan concentrations
were below detection for dietary tryptophan levels 0.025-0.10 =/kg/d. Plasma tqptophan
concentrations increased (p<0.05) as the intake of tryptophan rose from 0.15-0.30 dk=/d.
3 -4.2.2 ParenteralIy Fed Piglets
Plasma phenylalanine concentrations were not significantly affected by graded
tryptophan intakes in parenterally fed piglets (Table 3.6). As dietary tryptophan intake
increased ftom 0.035 to 0.05 3/kg/d, plasma tyrosine concentrations decreased from 328
59
Table 3.6 Plasma Arnino Acid Concentrations of ParenteralIy Fed Piglets
Tryptophan Intake (c&/d)
a.b.c.d denote si_gnificance by Tukq-s multiple cornparison test ( ~ 4 . 0 5 ) ; NS = not si_mificant (p=-0.05): ND = not dstectabie; Trp detection 1imit:- 1 O.fnoi/L
1
.4NOV,4 p d u e
NS
0.0017
NS
NS
NS
.O0 13
-0003
NS
-0368
NS
SS
NS
SS
.O0 1 1
XS
NS
NS
KS
NS
NS
.O042
NS
NS
Arnino Acid (pmon)
Aspanate
Glutamate
Hydrossproline
Serine
Gl:-cule
Glutmine
Taurine
Histidine
C i t n i h e
Thrconinc
SE
3- 1
16.0
4.4
36.0
107.0
27.1
15-3
1 .O
11.2
25.7
19.1
7.7
- - - 22.3
20.6
11.7
L -2
1.3
6.3
9.9
8.2
2.9
8.0
38.9 1
0.025
6 1
325"
44
535
1527
418"
317"
8
15 3"
194
0.20
39
137'
57
232
459
109'
1
33'
420
431
94
529
58'
1
15
12
93
184
67
17k
77
517
0.05
47
22Sb
50
575
0.30
34
167bc
58
290
569
83b
137'
O
4 ldb
329
397
7 2
452
72'
136
26
12
85
167
78
- 78ab 71
431
Alanine
-4r~inine
ProIine
T>-rosine
Valine
Methionine
C>-s tine
kolcucine
Leucine
Phen>-lalanine
Tqptophan
Omithine
Lysine
0.40
26
1 3
80
305
751
I15b
132'
- 3
GOab
412
495
1 OS
546
67'
180
15
23
94
220
75
39"
94
521
512
117
772
195'
2-44
12
20
149
23 1
127
ND
123
452
142
854
328"
265
12
18
137
229
153
ND
125
806
0-10
39
23.Cb
60
337
0.15
30
149'
63
277
467
95
620
1 lGk
214
13
17
118
236
52
ND
120
669
9jb
129'
1467
347"
220b
418
62
561
64'
136
I6
143
95
182
65
4'
112
652
185'
151' I
465 726
4
77"'
283
583
3
62"'
323
3
40"'
330
to 195 ,umol/L (p<0-05) and continued to decrease to 64 pmoi/L at a dietary tryptophan
IeveI of 0.1 5 g k d d (p<O.OS). Further graded increases in tryptophan resulted in sirniiar
plasma tyrosine concentrations. Plasma glutamate concentrations followed a similar
pattern: as tryptophan intake increased from 0.025 t o 0.05 p/kg/d, and fiom 0.05 to 0.15
&g/d, glutamate concentrations significant ly declined (p<0.05) and then remained - constant between dietary tryptophan levels of O.2O-O.i1O ~y/kg/d. Glutamine concentrations
dropped significantIy between tryptophan levels of 0.025 and 0.10 @g/d and were sirnilar
benveen tryptophan intakes of 0.15-0.40 g/k&i. Plasma taurine concentrations were
reduced in a stepwise fashion from tryptophan intakes of 0.025 to 0.05 Ck-d (p<0.05).
and €rom tryptophan levels of 0.05-0.10 o/kg/d (p<0.05). Taurine concentrations were
not significantly different from piglets receiving 0.10-0.40 J tvptophankg/d. FinaIly.
pIasma tryptophan concentrations fer pigIets given 0.025-0.15 d k d d of dietary
tryptophan were below the detectable ranse, approsimately 10 pmol/L, usinç HPLC-
Tryptophan concentrations in plasma significantly increased as tqprophan intake increased
above 0.15 @g/d.
3.43 Phenyialanine Kinetics
3 .K. 1 EnteralIy Fed Pislets
Dunnj the oxidation period, plateau values for breath "CO, production,
phenylalanine SR4 and tyrosine SR4 were reached tvithin 2 hours afier the initiation of a
primed, constant infision of tracer in al1 piglets. Data on phenylalanine kinetics for
enteraIly fed piglets are summarized in Table 3 -7. Phenylalanine intake was not
significantly different arnong diet ary tryptophan Ievels (p>O .OS). P henylalanine SRA
significantly increased from 28.2 xlo3 to 50.6 x103 dpdpmol as dietary tryptophan
increased from 0.035 to 0.1 5 @gd, but then dropped at tryptophan Ievels of 0.20 and
0.30 zAg/d. Phenylalanine S M was not sicgificantly different arnong the 0.15 to 0.30 g
tsptophankg./d intake groups. The ratio of tyrosine SPA to phenylalanine SRA in plasma
significantly declined as dietat-y tryptophan increased from 0.025-0.10 =/'kg/d- The
tyrosine to phenylalanine ratio was similar for piglets receiving tryptophan intakes of O. 10-
and release from protein breakdown differed between dietary treatment groups, there is
not a clear trend present.
Phenylalanine oxidation, espressed as "CO, significantly decreased from 523 s 1 O3
to 307 s103 d p d k g h as tryptophan intake rose €rom 0.025 to 0.50 @kg/d (Figure 3 2).
Values for this parameter continued to decline to 34 XIO' dpmlkzh as dietary tryptophan
increased to 0.15 @@d (p<0.05). Further increases in dietary ts-ptophan did not
significantly affect ''CO, values. As tryptophan treatment levels increased from 0.025 to
0.10 gkg/d, percentage of dose oxidized (Figure 3 -3 ) and plasma phenylalanine oxidation
(Figure 3.4) significantly decreased. Vatues for these two parameters remained consistent
in concentration for tryptophan levels above 0.15 gkg/d . Phenylalanine balance increased
significantly when dietary tryptophan rose from 0.025-0.10 z&=/d (p<0.05). Further
graded increases in tryptophan intake had no sigificant effect on phenylalanine balance.
62
Table 3.7 Phenylalanine Kinetics in Enterally Fed Piglets (IV Tracer)
o. 10
1 GO"
Plasma T\T SRA (s 10'
Phe Flux i.Q) (Llm01lk~11)
Plie Osidation (€1 ! umol ,k~ ' f~ j
Phe Balance 1 ( 1 4 )
U/o Dose Osidized
a. h.c.d denots si_gnificanct. by Tuksy's multiple cornparison test (pCO.05): NS = not si_miiticant (p>O.O5) * n = l
L-[l"C]-Phenylalanine Osidation in Enteraliy Fed Pijlets (IV Tracer)
Figure 3.2 Tryptophan Requirernent of EnteraILy Fed Piglets Receiving IV Tracer b). 1-1 2-Phase Linear Regression. CO1 radioactivity in collected breath of individual pislets
receiving different levels of tryptophan.
L-["Cl-PhenyIalanine Osidation as a % of Dose in Enterally Fed Piglets (IV Tracer)
Figure 3.3 Tryptophan Requirement of Enterally Fed Piglets Receiving IV Tracer bu ?-Phase Linear Regession. Phenylalanine osidation as a O./. of dose in individual piglers receiving different levels of tryptophan.
L-["Cl-Phenylalanine Oxidation in Enterally Fed Piglets (IV Tracer)
Figure 3.4 Tryptophan Requirement of Enterally Fed Piglets Receiving IV Tracer by ?-Phase Linear Regession. "C-Phenylalanine radioactivity in collected plasma of indiridual piglets receiving different levels of tryptophan
3.4.3 -2 Parenterally Fed Pisjets
During the osidation period, plateau values for breath "CO, production,
phenylalanine SRA and tyrosine S M were reached within 2 hours after the initiation of a
primed, constant infusion of tracer in al1 piglets. Data on phenylalanine kinetics for
parenterally fed piglets are summarized in Table 3 -8- Plasma phenylalanine intake, flux.
SRA, non-osidative disposal, and release from protein breakdown was not significantly
dieerent among diet treatments. Plasma tyrosine S U decreased significantly from 9.7
XIO' to 3.1 x103 dpdpmol as dietary tryptophan increased from 0.075-0.15 @g/d.
Similarly, the ratio of tyrosine to phenylalanine S M in plasma significantly declined from
34.2% to 9.8 % as tryptophan levels rose from 0.025 to 0.10 ~ A d d , respectively. The
ratio of tyrosine to phenylalanine was simiIar among diet levels of O. 10-0.40 g
tryptophan/kg/d.
Plienylalanine osidation. espressed as breath ''CO2, as calculated from plasma
SR4 data. or as a percentage of dose osidized, \vas significantly affected by dietav intake
oftryptophan. As tryptophan increased from 0.025 to 0.15 c$kg/d. breath "CO, (Figure
3.5) and percentage of dose oxidized (Figure 3 -6) significant ly decreased (p<O.05). Wit h
subsequent increases in tryptophan intake from 0.1 5-0.40 gkg/d, there was no change in
these measures. Plasma phenylalanine oxidation also significantly decreased from 5.8 to
3.3 ,umoVk=/h when dietary tryptophan rose fiom 0.025 to 0.10 ç/lig/d, and phenylalanine
osidation was not significantly diEerent among diet levels of 0.10-0.40 g tryptophadlidd
(Fisures 3.7). Phenylalanine balance was similar for al1 dietary treatments (p>0.05).
67
Table 3.8 Phenylalanine Kinetics in Parenterally Fed Piglets
L-["Cl-Phenylalanine Oxidation in Parenterally Fed Piglets -100000-
Figure 3.5 Tryptophan Requirement of Parenrerally Fed Pislets by ?-Phase Linear Regressioii. 14C0, radioactivity in collected breath of individual piglets recei\-in_o different levels of tryptophan.
L-[l'C]-Phenylalanine Osidation as a % of Dose in Parenterally Fed Piglets
Figure 3.6 Tryptophan Requirement ofParenteraIly Fed Piglets by ?-Phase Linear Regression. PhenylaIanine osidation as a % of dose in individual piglets receiving different levels of tryptophan.
L-["Cl-Phenylalanine Oxidation in Parenterally Fed Piglets
Figure 3.7 Tryptophan Requirement of Parenterally Fed Pijlets by ?-Phase Linear Regession. "C-Phenylalanine radioactib-ity in collected plasma of individual piçlets receiving different Ievels of tq-ptophan.
3.4.4 Breakpoint Analysis
To determine tryptophan requirements, a two phase linear regression crossover
model was used, in which data points were partitioned between two regression lines. The
data partitioning seIected w2s based on the model that produced the highest regression
coefficients and the lowest residual error. The breakpoint, or estimation of the mean
tryptophan requirement, and the corresponding confidence intervaIs are surnmarized in
Table 3.9 for the enterally fed anirnals. The breakpoint estimated by breath ''CO2 lias
O. 122 + 2 tryptophanlkg/d (CI: 0.085-0.159), by percentage of dose osidized was 0.127
d k d d (CI: O.OS9-0.164), by phenylalanine osidation was 0.10 1 ~ A d d (CI: O.OS 1-0.12 1) - and by phenylalanine balance was 0.10; @g/d (CI: 0.083-0.124). Al1 of these measures
yielded similar estimates due to the fact that the standard error of the estimate (SEE) for
each of these measures overlapped.
The breakpoint and the corresponding confidence intemals for parenterally fed
pislets are summarized in Table 3.10. The breakpoint estirnated by breath l'CO2 and
phenylalanine oxidation rate was determined to be 0.142 g tryptophan/kg/d (95% CI:
0.102-0.1 S3 and O.lO7-0.17S- respectively). The breakpoint determined by plienylalanine
osidation as a percentage of dose was 0.145 g tryptophan.k=/d (95% CI: O. 104-0.1 S 5 ) .
PhenyIalanine balance data did not e-xhibit a suitable pattern for breakpoint analysis, and so
was not included in this analysis. NI three parameters used produced similar tryptophan
requirement estimates for parenterally fed piglets.
Table 3.9 Tryptophan Requirement by Breakpoint Analysis in Enterally Fed Piglets (IV Tracer)
Parameter CL, i ~ o o t SE of ( g k d d ) MSE Estimate
P heny lalanine Oxidation
P henylalanine Balance
% Dose Osidized
Table 3.10 Tqptophan Requirement by Breakpoint h a l y s i s in Parenterally Fed Piglets
Parameter Breakpoint Root 1 SE of
P henylalanine Osidation
5% Dose Osidized
3.5 DISCtJSSION
Tryptophan metabolism is comprised of a complex pathway which precludes the
use o f direct oxidation methodology. The indicator amino acid oxidation technique has
been demonstrated prevïously to be an appropriate method of determining amino acid
requirements in enterally fed (Bertolo et al., 1998) and parenterally fed (House et al., 1997:
1995) piglets- Therefore, the IAAO method was applied to determine both entera1 and
parenterai t q ~ t o p h a n requirernents for neonatal pigiets.
Dietary tryptophan intake significantiy influenced the plasma concentrations of
several amino acids. When tqptophan intake was most limiting for protein synthesis in
enterally fed piglets, the concentrations o f phenylalanine, tyrosine, glritamine, taurine, and
vaIine were significantly larger than when tryptophan was supplied at levels of 0.10 g k d d
or greater. Similarly, parenterally fed piglets dernonstrated marked elevations in
glutamate, glutamine, taurine and tyrosine concentrations at dietary trlptophan intakes of - less tlian 0.10 @g/d (p<O.OS). A pre\-ious esperiment detailin2 the lysine requirernent of
piglets using the IA.40 method reported comparable elevations in the plasma
concentrations of @utamine, valine, and phenylalanine (House et al., 19%). When
tryptophan was supplied at the lowest dietary treatment levels, the body's ability to
catabolize the escesses of the phenylalanine and tyrosine was likely exceeded, resu1ting in
the observed accumulation of these amino acids in plasma. These results also suggest that
the use of these indispensable arnino acids were masimized when tryptophan was supplied
at its requirement level. The observed nse in plasma glutamine concentrations in the
74
lowest tryptophan treatment g o u p s may possibly be due to its role in the transfer o f
nitrogen. Ammonia (NH,') g o u p s arising from the catabolism of excess amino acids
react with glutamate to form gIutamine. Finally, the reason for the increase in plasma
taurine concentration ir, the the lowest tryptophan diet groups remains uncIear.
Plasma tryptophan concentrations increased from 2 1-57 prnollL and from 3-39
pmoVL, respectively, when enterally and parenteralIy fed piglets were given 0.15-0.40 g
fed pislets demonstrated greater (although not significant. p>0.03) plasma tryptophan
concentrations, which increased linearly and in parallel with the parenterally fed animals
(Figure 3. S).
Concentrations of plasma phenylalanine and tyrosine significantly differed between
the enterally and parenterally fed aninials. The mean plasma phenylalanine concentration
for enterally fed animals was 55.9 gmol/L compared to 92.3 prnol/L for piglets receivin~
parenteral diets (Figure 3.9). Tyrosine concentrations eshibited a similar pattern (Fisure
3.10). Average tyrosine concentrations in plasma for the enterally and parenterally fèd was
60.3 and 128 -3 ;c moVL, respectively. These are substantial differences, and suggest that
the g t is likely ritilizing phenylalanine on first pass. StoIl et aI. (199s) measured the
appearance of labelled amino acids in piglet portal blood followin,o an intragastric infusion
of [U-'3C] algal protein in combination with enteral feedings. They determined rhat
intestinal first pass metabolism accounred for approximately 35% of phenylalanine intake.
This resulr supports Our findings, however, Stol1 et al. (1998) also detennined that 6 1% of
75
Figure 3.8 Tryptophan Concentrations in Plasma of Piglets Receiving Parenteral Diet and IV Tracer (IV (IV)). Entera1 Diet and IV Tracer (IG (IV)) or Enteral Diet and Tracer (IG (TG)). Data are means * pooled SE.
Figure 3.9 Phenylalanine Concentrations in Plasma of Piglers Receiving Parenteral Diet and Tracer (IV (IV)), Enteral Diet and IV Tracer (IG (IV)), or Entera1 Diet and Tracer (IG (IG)). Data are means % pooled SE.
Figure 3.10 Tyrosine Concentrations in Plasma of PigJets Receiving Parenteral Diet and Tracer (IV (IV)), Enteral Dier and IV Tracer (IG (IV) j, or Enteral Diet and Tracer (IG (IG)). Data are means * pooled SE.
dietary threonine were utilized on first pass. We found that plasma threonine
concentrations in enterally fed piglets were approsimately twice as large as those in the
parenterarly fed group- Zt is unclear why threonine concentrations in plasma do not
support the pattern seen with phenylalanine and tyrosine, however, Bertolo et al. (1 999)
found sirnilar chanses in arnino acid concentrations in enteraIly compared to parenterally
fed piglets. Enterally fed anirnals demonstrated threonine piasma concentrations that
wliere approsimately 2-fold larger than parenterally fed pigIets, but this diftèrence was not
statistically sisnificant due to large variability. It is possible that animals given parenterîl
diets had greater threonine osidation than the enterally fed _moup.
The safe intake of phenylalanine for parenterally fed pigiets has been estimated to
be 0.48 =/k/d, which is the amount provided in the elemental diets. Gut utilization of
phenylalanine on first pass was witnessed in tliis esperiment (Fisure 3.9). and has been
estimated to account for 3576 of enterally fed labelled phenylalanine (Stol1 et al., 199s). as
mentioned previously. It is likely that phenylalanine \vas the nest limiting amino acid, due
to the fact thar percentase of dose osidized for animals receiving test diets contaixiinl
tqptoplian levels above the estimated requirement was approsimateiy 0.5-0.8%. Thus,
phenyIalanine rnay have become Iimiting in enterally fed animals.
Plasma phenylalanine SRA in piglets fed enterally was significantly lower at the
lowest dietary tryptophan level than at tryptophan requirement. Due to the fact that
plasma phenylalanine concentrations were significantly higher in the lowest tryptophan
treatment sroups, it may be that the labelled phenylalanine was diluted by the additional
79
non-labelled phenylalanine in the plasma. Aithough phenylalanine S M was not
sigificantly different among tsp tophan treatment groups for parenterally fed piglets, a
similar pattern existed for these animals. In contrast, tyrosine S R 4 and plasma tyrosine
concentrations for par enter al!^ fed piglets was substantially greater at the lowest
tryptophan intake, and then decreased and remained constant at tryptophan levels at and
above requirement. It appears that there was a greater accumuIation of label in the
tyrosine pool, in proportion to total tyrosine plasma concentrations, compared to
phenylalanine. Phenylalanine is converted to tyrosine and then is further osidized to CO?.
The compIete osidation of tyrosine may be the rate Iimiting step in the catabotism o f
phenylalanine, more so than the conversion o f phenylalanine to tyrosine. Indeed, the ratio
of plasma tyrosine S M to phenylalanine S M was significantly greater for the lowest
dietary tryptophan Ievels, indicatins an accumulation of tracer was primanly in the tyrosine
pool. The change in the combined phenyIalanine and tyrosine SR4 fioni the lowest diet
level to that at requirement, however. accounted for less than 156 of the infused tracer.
Consequently, it is untikely that breakpoint estimates were affected by the observed
changes in plasma amino acid concentrations and SR4.
Calculated rneasures o f phenyIa1anine intake, flus, non-osidative disposal and
breakdown from protein were not significantly different among die taq tryptophan intakes
for parenterally fed piglets. Previous work which used the I M O method to determine
Iysine and threonine requirements in parenterally fed neonatal pislets also found no
differences in these parameters over dietary treatment levels (House et al., 199s; C. Chen
80
MSc thesis, 1997). Although enterally fed piglets receiving different levels of dietary
tryptophan demonstrated significant differences in flux, non-osidative disposa1 and
breakdown of phenylalanine, values for these rneasures did not appear to follow a clear
trend when analyzed with Tukey's multiple comparison test. Phenylalanine intake did not
differ among tryptophan treatment levels in enterally fed piglets. and were not different
between animals receiving either route of feeding
Phenylalanine osidation, espressed as breath 14C02, calculated €rom plasma
phenylalanine S R 4 or expressed as a percentage of dose osidized, decreased significantly
when dietary tryptophan increased from insufficient Ievels to requirement, for both
enterally and parenterally fed animals. The decline in phenylalanine osidation
demonstrated that amino acid osidation decreased as the limiting amino acid increased,
reflecting an increase in protein synthesis until the requirement for tryptoplian was met.
Phenylalanine balance increased along with the increase in tryptophan intake for enterally
fed piglets. as espected, hokvever, there \vas no change in apparent phenylalanine balance
in parenterally fed animals. Phenylalanine balance was determined by the subtraction of
plasma phenylalanine osidation from dietary phenylalanine intake. Plasma phenylalanine
osidation appeared to be greater in enterally fed compared to parenterally fed piglets ai
the lowest dietary tryptophan level (20.0 vs 8.8 ,umol/kg/h), as espected for piglets with
healthy, functioninç gastrointestinal tracts. It is likely that our inability to see significance
in balance data for parenterally fed piglets is due to the lower level of phenylalanine
osidation rates determined for animals receiving deficient dietary tryptophan levels.
s 1
The breakpoint, or tryptophan requirement estimate, is cornrnonly based on the
model which produces the highest regression coefficient and the lowest amount of
variability. Using this criteria for enterally fed piglets, plasma phenylalanine osidation
appeared to be the most appropnate model. Due to the fact that plasma phenylalanine
osidation values were caicuIated from phenylalanine SRA, and that phenylalanine SEL4
was significantly affected by dietary tryptophan intakes, it is possible that a greater amount
of error would be associated with breakpoint estirnates derived from plasma 'phenylalanine
osidation and balance data. Therefore, percentage of dose oxidized was used to
determine the breakpoint for both enterally and parenterally fed piglets. The mean
tryptophan requirement for enterally fed animals was 0.127 ~ A - g d with a safe upper lirnit
of 0.164 g/kg/d. The average tryptophan requirement estimate for parenterally fed piglets
\vas 0.145 @g/d with a safe upper limit of O. 1 S5 zA=/d. The breakpoint estimates for
enterally fed piglets are well within the confidence intervals and standard error of the
estimate (SEE) for parenterally fed pislets (Tables 3 -9, 3.10). Therefore, the enteral and
parenteral tryptophan requirernent in neonatal piglets appears to be sirniIar. This finding
suggests that the gut is not preferentially utilizing tryptophan for any processes other tlian
protein qnthesis. Unlike tryptophan, the parenteral threonine requirernent has been found
to be only - 45% of the mean enteral threonine requirement (Bert010 et al., 1 998), and the
parenterat requirement of methionine in the absence of cysteine was - 69% of the mean
requirement in enterally fed piglets (Shoveller et al., 2000). Thus, the amino acid needs of
the parenterally fed individual must be determined empirically, and cannot be estimated
82
based on assumptions made concerning splanchnic first pass metabolism.
The tryptophan requirements determined in the enterally and parenterally fed
animals are similar to the hXC (1998) current recomrnendations. For 3 kg pigglets, the
rnean tryptophan requirement was estimated at 0.153 @g/d. This estimate falls within the
upper confidence intervals of both the enterally and parenterally fed anirnals in this study.
The NRC ( 1998) recomrnendations for 3 kg piglets are based heavily on the work of Bal1
and Bayley (1984). Bal1 and Bayley (1984) estimated the trptophan requirement of 2.5
E;s pislets fed an enteral, semi-punfied diet usin2 the IAAO rnethod. They determined that
the young piglet requires 2 gkç of a 140 g proteinks diet. which is the equivalent of
approximately 0.83 ç tryptophad 1005 protein. Our requirement estimates for enrerally
and parenterally fed piglets was 0.117 and O. 145 J tryptophan/kg/d. or approsimately 0.80
and 0.92 3 tryptophad 1 00 protein fed, respectively. Tlierefore. these current tryptophan
requirement estimates are similar to tliose preMously reported. and do not diKer fiom the
hRC 's current recommendations.
4.0 EFFECT OF ROUTE OF ISOTOPE D\I'mSION ON TEIE TRYPTOPHAN
REQUIREMENT DETERMIXED BY tNDICATOR AMTNO ACID
0,XIDATION
4.1 iDrTRODUCTION
Arnino acid oxidation is increasingly being used to determine amino acid
requirements (Bert010 et al., 1998; Brunton et al., 1998). CIassically, the tracer has been
given IV in order to avoid splanchnic metabolism. Controversy exists, however. with
regard to the comparability of requirernent estimates from IV versus IG tracer
administration. The issues include not only splanchnic extraction of tracer. but aIso
isotopic dilution of plasma, and subsequent estimates of tracer kinetics which are
determined using plasma enrichment estimates.
When labe1led amino acids are given orally. a significant proportion of tracer is
estracted by the splanchnic tissues on first pass (hlatthews et al., 1993; Biolo et al., 1992,
Hoerr et al., 199 1; Krempf et al., 1990; Stoll et aI., 19%; van Goudoever et al., 2000).
For esample, approsimately 30-35% of gastrically fed phenylalanine was estimated to be
taken up by the spIanchnic bed on first pass in humans (Matthews et al., 1999) and piglets
(Stoll et al., 1998). Such splanchnic extraction has implications for the apppearance of
label in plasma and estimates of tracer kinetics.
The route of tracer administration aIso affects isotope dilution in the plasma pool.
Consequently, estimates of arnino acid kinetic parameters, such as plasma specific
84
radioactivity (SRA) and plasma tracer flux, may be different when tracers are given IG
versus IV. lsotopic enrichment (or SRA) of IabeIled amino acids have been shown to be
significantly lorver and flux significantly higher in plasma of subjects receivinz oral versus
IV tracers (Sanchez et al., 1995; Hoerr et al., 199 1; Krempf et al., 1990). These
rneasurements are often used to calculate amino acid osidation and other kinetic
parameters and thus may potentially alter subsequent amino acid requirement estimates,
which are dependent upon amino acid or Iabei osidation as an end point.
\VhiIe several investigators have studied the effect of the route of tracer
administration on arnino acid kinetics (Sanchez et al., 1995; El-Khoury et ai., 1998; Hoerr
et al., 199 1 ; 1993; Yu et aI., 1990; 1992), no one has yet studied the efect of the route of
isotope administration on an amino acid requirement estimate. En the present experiment
ive studied whether the route of isotope (1-"C-phenylalanine) administration (IG \.S. Il,.)
\i-ouId result in any change in the estinlate of tqptophan requirements in enterally fed
piglets. We chose t o do so with rryptophan as the test amino acid, because in the prel-ious
experiment we showed that by passing the splanchnic bed did not alter the tryptophan
NS = not sigiticant (p>O.O5); ND = not detsctabler Trp detection 1imit:-10 prnol/L
0.20
15
166
9Yb
193
17
7
lGlb
100"
42
5
856
SOOAb
135
474
21b
1 5 ~ 6 ~
35
12
IO3
236
23d
2~~
56
442 tsa (p<0.05):
Amino Acid (umoVL)
Aspartate
Glutamate
Hwoqprol ine
Serinc
Asparagine
Glycine
Glutamine
Taurine
Histidine
Citrulline
Tlmonine
Alanine
Ar~inine
Proline
T~rosine
VaIine
~Msthionine
Cystine
[soIeucine
Lericine
Phen!-f alanine
Tqptophan
Ornithine
L>-sine ~ b . c . d drnote sipiticance
0-05
17
140
64d
235
41
746
44Tb
5 1
104"~
439
43lc
107
480
122"
276"77Sb
24
13
120
209
81b
ND
93
444 Tukq ' s
0.025
25
207
G j C d
306
5 1
979
549"
277"
87
1 19"
9
_78jbc
149
739
135"
392"
20
12
174
277
118"
ND
132
551
by
0.30
28
288
91ak
277
19
985
223"
13Gk
62
6
572
1022"
95
717
2-Ib
267b
27
10
125
277
4
45"b
115
462
0.10
19
208
9926
0.15
18
154
119"
0.40
12
128
9gab
201
16
762
172"
13Gk
39
71k
883
78Aab
141
583
18"
21gb
26
10
1 1 1
244
l_Fd
57"
101
1
SE
1.9
16.5
4.3
17.7
3.6
54.0
35.2
12.5
5.0
6.1
62.3
44.8
9.1
35.0
9.7 -
14.8
1.3
0.9
6.4
11.4
7.1
4.1
5.3
20.3
263
20
1098
213b
204a"48k
43
S5"&
817
655&
158
546
48b -
32
15
141
269
G l t "
ND
13 1
573 multiple
ANOVA p \-alue
NS
NS
.O0 1 1
NS
NS
WS
-0177
-0123
NS
.O13 1
NS
.O012
NS
NS
-0006
-0403
NS
NS
NS
NS
.O00 1
.O00 1
NS
NS
223
18
1159
1 7 7 ~
1 3 5 ~
30
8gJk
830
75SJK
149
537
-- 7 7b
1 9 7 ~
27
14
109
228
75" +
21"
100
487 cornpanson
similar for piglets fed 0.15-0.40 g tryptophannig/d. Tyrosine concentrations in plasma
sigificantly declined (p<O.OS) between tryptophan levels of O.O?S-O.10 3/kg/d, and
rernained stable among tryptophan intakes of 0.10-0.40 *g/d- Hydrosyproline
concentrations increased significantly fiom 65 to 92 p m o K for tryptophan intakes of
0.025 to 0.05 f l g d , respectiveIy, and increased again to 1 19 p m o K at tryptophan level
of 0.1 5 slidd (p<0-05). Plasma hydrosyproline levels were not significantly different for
piglets given 0.15-0.40 g tryptophankg/d. Both glutamine and taurine concentrations in
plasma significantly decreased as tryptophan intakes increased from 0.025 to 0.10 d k g d .
and remained similar for tryptophan levels of 0.10-0.40 dkyd. Plasma valine
concentrations dropped as tryptophan intakes increased from 0.025 to 0.05 z&o/d. and
valine levels were constant in pigIets receiving 0.05-0.40 g tryptophan/kg/d. Lastly,
plasma tryptophan concentrations were below detection for dietary tryptophari leve1s
0.025-0.10 =/kg/d. Plasma tryptophan concentrations increased (p<0.05) as the intake of
tryptophan rose from 0.15-0.40 g/kcJd.
4-43 Phenylalanine Kinetics
4.4.3.7 EnteraIly Fed Piglets (with TG Tracer)
Durinj the oxidation period, plateau values for breath ''CO2 production,
phenylalanine SRA and tyrosine SRA were reached within 2 hours afier the initiation of
tracer infusion in al1 piglets. Phenylalanine intake and tyrosine SR4 was not significantly
different among diet treatments (Table 4.3). PhenylaIanine SRA, however, significantly
96
Table 4.3 Phenylalanine Kinetics in Enteraily Fed Pislets (IG Tracer)
Tryptophan Intake ( ~ A g l d )
Parameter r--
96 Dose Osidizsd
denote significance by Tukey's multiple cornparison test (p<0.05); NS = not significant (p>0.05)
L-[l'C]-~henylalanine Oxidation in Enterally Fed Piglets (IG Tracer)
Figure 4.2 Tryptophan Requirement of Enterally Fed Piglets Receiving 1G Tracer bu '-Phase Linear Regression. '* COz radioactivity in collected breath of individual piglers receiving different levels of tryptophan.
L-[l-?C]-Phenylalanine Oxidation in Enterally Fed Piglets (IG Tracer) as a 5% of Dose
Figure 4.3 Tryptophan Requirenient of Enterally Fed Piglets Receiving IG Tracer by '-Phase Linear Regression. Phenylalanine osidation as a ?6 o f dose in individual piglets receiuing different levels o f tryptophan.
L-["Cl-PhenyIalznine Oxidation in Enterally Fed Piglets (IG Tracer)
Figure 4.4 Tryptophan Requirement of Enterally Fed Piglets Receivins IG Tracer by 14 2-Phase Linear Regression. C-Phenylalanine radioactivity in collected plasma of
individual piglets receiving different IeveIs of tqpophan.
increased from 22.5 x103 to 42.4 x10' dpdpmol as dietary tqptophan increased fiom
0-025 to O. 15 ç/kg/d. Additional intake of tryptophan above 0.15 ç/kg/d did not result in
any significant changes in plasma phenylalanine SRA (p>0.05). Although the ratio of
plasma tyrosine S RA to phenyldanine Sm p henylalanine flux, non-osidative disposa1 and
release from protein breakdown differed between diet treatment groups, there is not a
clear trend present.
Phenyialanine osidation, espressed as 14C02, significantly decreased from 42 1 s 10;
to 11 1 s10"~m/kg/h as dietary tryptophan rose from 0.05 to 0.10 g k g d (Fijure 4.2).
Further gaded increases in tryptophan resulted in similar l'CO2 values (p>0.05).
Percentaçe of dose osidized (Figure 4 3 ) , plasma phenyIalanine osidation (Figure 4.4), and
phenylalanine balance significantly decreased as dietary tryptophan increased from 0.05 to
0.10 g/kg/d, and was not significantly different among diet levels of 0.10-0.40 g
tryptophadkrd.
4-43 -2 Enterally Fed Pislets (with IV Tracer)
Plasma phenylalanine kinetics for enterally fed piglets receiving IV tracer lias been
discussed previously in section 3 -43.1. During the osidation penod, plateau values for
breath "COl production, phenylalanine SRA and tyrosine SRA were reached within 2
hours afier the initiation of a primed, constant infusion of tracer in al1 piglets. Data on
phenylalanine kinetics for enterally fed piglets are summarized in Table 4.4. Phenylalanine
intake was not siçnificantly different amonç dietary tryptophan levels (pO.05).
101
Phenylalanine S M sigificantIy increased from 28.2 xlo3 to 50.6 x10' dpm/pmol as
dietary tryptophan increased h m 0.025 to 0.15 gkg/d, but then dropped at tryptophan
Ievels of 0.20 and 0.30 S/kYd. Phenylalanine SRA was not significantly diffèrent among
the 0.15 to 0.30 g tryptophankdd intake groups. The ratio of tyrosine SR4 to
phenyIalanine SRA in plasma significantly declined as dietary tryptophan increased from
0.025-0.10 e g d . The tyrosine to phenylalanine ratio was similar for pigiets receivinç
tryptophan intakes of 0.10-0.40 s&~Jd. Although plasma tyrosine SEM, phenylalanine
flux, non-oxidative disposa1 and release fiom protein breakdown differed between diet
treatment groups, there is not a ctear trend present.
Phenylalanine osidation, espressed as CO^, sigdïcantly decreased from 523 s10'
to 307 x1o3 dpmkgh as tryptophan intake rose from 0.025 to 0.50 =/kg/d (Figure 4.5).
Values for this parameter continued to decline to 34 ':IO' d p d k g h as dietary tryptophan
increased to 0.15 ~ A d d (p<0.05). Further increases in dietary tryptophan did not
signi ficantly affect "CO, values. As tryptophan treatment levels increased from 0.025 to
0. I O ~ A d d , percentage of dose osidized (Figure 3.6) and plasma phenyIaIanine osidation
(Figure 3.7) significantly decreased. Values for these two parameters rernained consistent
in concentration for tryptophan levels above O. 15 @@d. Phenylalanine balance increased
significantly when dietary tryptophan rose from 0.025-0.10 gkg/d (p<0.05). Further
gradecl increases in tryptophan intake had no significant effect on phenylalanine balance.
Table 4.4 PhenyIalanine Kinetics in Enterally Fed Piglets (IV Tracer)
Tryptophan Intake (gkg/d)
ANOVA p \ .due Parameter 1
Corrected V1-'CO2 ( S 1 o3 DPbükgh)
Plasma P heS RA (S 10' DPhUpmol)
Plasma T?T SRA (s10' DPWumol )
-
r ~ ~ : P h e (%)
Phe Flux (QI ( prnoLk:k)
P l x Osidation (E) ( p r n o ÿ k g ~ )
Phe Intnke (1) (pmol/kg/li)
Non-osid. Losscs (S=Q-E) (prnoükfli)
Brenkdonn (B=Q-1) (,umoi/kfli)
Phe Balance (1-E) (~lm0l/k=/h)
% Dose Osidized
~ b . c . d dtrnotc
-
-
-
7
siLmiIicance h>- Tukq's mulriplc 1 cornparison test ( p d . 0 5 ) : N S = not si_rniîicant (p>1).05)
--
L-["Cl-Phenylalanine Osidation in Enterally Fed Piglets (IV Tracer)
Figure 4.5 Tryptophan Requirement of Enterally Fed Piglets Receiving IV Tracer by '-Phase Linear Regression. ''CO, radioactivity in collected breath of individual pisiets receivins different Ievels of tryptophan.
L-["Cl-Phenylalanine Oxidation as a % of Dose in Enterally Fed Piglets (IV Tracer)
Figure 4.6 Tryptophan Requirement of Enterally Fed Piglets Receivino, IV Tracer by 2-Phase Linear Regression. Phsnylalanine osidation as a ?4 of dose in individual piglers receiving different Ievels of tryptophan.
L-["Cl-Phen~lalanine Oxïdation in Enterally Fed Piglets (IV Tracer)
Figure 4.7 Tryptoptian Requirement of Enterally Fed Piglets Receivinj IV Tracer by ?-Phase Linear Regression. l'C-Plienylalanine radioactivity in collected plasma of individual piglets receivinz different leveIs of tryptophan
4.4.4 Breakpoint Analysis
To detemine tryptophan requirements, a two phase linear regression crossover
rnodel was used, in which data points were partitioned between two regression lines. The
data partitionhg selected was based on the rnodel that produced the hishest regression
coefficients and the Iowest residual error. The breakpoints and corresponding confidence
intervals for enterally fed piglets receiving IG tracer are surnmarized in Table 4.5. The
breakpoint estimated by breath "CO2 was determined to be 0.11 1 g tryptophadkdd (95%
CI: 0.068-0.154), by percentage of dose oxidized was 0. 1 13 tryptophan/k5/d (95% CI:
0.072-0.154), and by plasma phenylalanine oxidation \vas 0.1 17 g tryptophadkg'd (CI:
0.07 1-0.163). Al1 of these measures yielded similar requirement estimates. Phenylalanine
balance data did not eshibit a suitable pattern for breakpoint analysis, and so was not
included in this analysis.
The breakpoints and respective confidence intervals for enterally fed piglets given
IV tracer have been discussed previously in section 3.4.4, and are shown in Table 4.6.
The breakpoint estimated by breath ''CO2 was 0.122 2 tryptophan/kg/d (CI: 0.085-0.159).
by percentage of dose osidized was 0.127 &Jld (CI: 0.089-0.161), by phenylalanine
oxidation was 0.10 1 ~ g / d (CI: 0.08 1-0.12 1) and by phenylalanine balance was 0.103
d k y d (CI: 0.083-0.134). Ail of these measures yielded similar estimates due to the fact - that the standard error of the estimate (SEE) for each of these measures were seen to
overIap.
Table 4.5 Tryptophan Requirement by Breakpoint Analysis in Enterally Fed Pislets (IG Tracer)
Table 4.6 Tryptophan Requirement by Breakpoint .haIysis in Enterally Fed Pislets (IV Tracer)
Parameter
IJC02
P henyIalanine Osidation
% Dose Osidized
Breakpoint
(@g/d)
0.111
0.1 17
0.113
Parameter
"CO,
Phenylalanine Osidation
P henylalanine Balance
% Dose Osidized
CI,,,, (+dg/d)
0.068
0.07 1
0.073.
Breakpoint A d )
O- 125
0.1 13
0.1 14
O. 137
c~,, (gkgld)
0.154
0.163
0.154
CI ,,,, (g/k@d)
0.098
0.085
0.077
0.089
i
0.53
0.57
0.56
C T ~ ~ (g/kg/d)
0.152
0-141
O. 150
0.164
6
0.68
0.72
0.72
0.71
Root MSE
135.3 slo3
6.04
1 .S6
Root hl1 SE
121.7 s103
4.15
5 -42
1.66
SE of
Estirnate
0.025
0.027
0.024
SE of Estimate
0.0 16
0.0 16
0.073
0.022
4.5 DISCUSSION
Plasma concentrations of amino acids appeared to be similar for enterally fed
pislets receiving either IG or IV tracer. As seen in the previous study, however, plasma
phenylalanine, tyrosine, glutamine and taurine concentrations were ~i~pificantly greater at
tryptophan intakes below O. I O c,/k3/d compared to concentrations of these amino acids
when tryptophan was provided at or above the estimated requirement (Tables 4.1. 4.3).
This observation suggests that when tryptophan is supplied at the lowest treatment levels,
protein synthesis is dramatically limited, resulting in saturation of amino acid catabolic
pathways, and subsequent accumulation of phenylalanine and tyrosine in plasma. The rise
in observed glutamine concentrations in plasma in the lowest tqptophan diet groups is
likely due to its roIe in the transfer of nitrogen in the body. FinalIy, it is unclear as to why
taurine concentrations were affected by dietary tryptophan intakes.
Estimates of phenylalanine kinetics were affected by the route of isotope infusion
(Table 4.7). Athough phenylalanine intake was not significantly different between
enterally fed piglets receiving either IG or IV tracer, phenylalanine S R 4 (or plasma
enrichment of labelled phenylalanine) was significantly higher for piglets given enteral diet
and IV tracer (p<0.05) (Fipre 4.8). Previous esperiments in adults have aIso reported
higher plasma enrichrnents in both fasted and fed subjects receiving IV compared to IG
tracers (Hoerr et aI., 199 1 ; Matthews et al., 1993, 1999). Splanchnic utilization of
phenyIalanine is likely the reason for this observed difference. When labelled
phenylalanine was given IG, a substantial proportion may have been taken up by the gut
1 O9
Table 4.7 Estimates of Phenylalanine Kinetics: Cornparison of Enterally Fed Pislets (IG Tracer) with Enterally Fed PieJets (IV Tracer) $
* NS = not significant, p > 0.05; Means and SEE calcuiated for piglets receiving adequate tryptophan intakes (diets containhg 0.15- 0.40 g Trpkgld)
Parameter
Corrected V1'CO, (X 1 o3 D P L ~ E J ~ )
PIasrna Phe SRA (x t O' DPhUgrnol)
Plasma Tyr S M (x 1 O3 DPWpmoI)
Tyr:Phe
P he Flux ( Q ) (,m~ol/kg/h)
Phe Osidation (E) (pniol/kg/h)
Phe Tntake (1) (gmoVkg/Ii)
Phe Balance (LE) (y moL/kg/h)
?,6 Dose Osidized
P Value*
NS
0-023
N S
hrS
0.005
0.03 7
h7S
NS
N S
OraI Diet / IG Tracer
Means
58.9
39.0
2-6
O. I
199.3
1.5
103.4
101.0
0.8
Oral Diet / IG Tracer
SEE
10.2
1.7
O. 7
0.0
14.3
0.2
0.9
0.9
0.2
Oral Diet / IV Tracer
Means
36.5
46.9
3.4
O. 1
115.S
0.9
1 03 -4
102.6
0.6
Oral Diet / IV Tracer
SEE
4-8
2.9
0.3
0.0
12.0
0.2
1.4
1.3
0.1
on first pas , resulting in less label appearin~ in plasma. Indeed, Stol1 et al. (1998)
determined that -35% of labelled phenylaIanine was taken up by the splanchnic bed
following the enteral infusion of diel wirh L3C-algal protein. h o t her kinetic parameter,
phenylalanine flux, was also afTected by p t utilization of IG tracer. Flux was calculated
as the amount of label infùsed divided by plasma phenylaIanine SRA. As espected, piglets
receiving enteral diet and IG tracer had significantly higher plasma phenylalanine fluses
compared to enterally fed piglets @ven IV tracers (pcO.05) (Figure 4.9). This result is
simiIar to observations in fasted and fed adults siven IG or IV tracers (Krernpf et al.,
1990; Hoerr et al., 199 1; Sanchez et al,, 1995; 1996; El-Khoury et al., 1998).
The subsequent estirnate of phenylalanine oxidation calculated from piasma S U
was affected by the differences in plasma SEL. between the piglets given IG or IV tracer
(p<O.O5). Calculated phenylalanine osidation was deterrnined as the rate of "CO2
production divided by plasma phenylalanine SRA. Previous osidation studies, using either
13C-lysine or l'C-leucine tracers, did not demonstrate differences in labelled amino acid
osidation. El-Khoury et al (1998) cornpared lysine osidation rates in adult men receiving
"C-lysine either iG or IV. They found significantly higher plasma lysine fluxes in those
given ZG tracer, but no differences in plasma lysine osidation calculated fkom plasma
enrichment. When 13C-leucine was administered to adults (Hoerr et al., 1991; 1993) or
dogs (Yu et al., 1990; 1992), calculated leucine oxidation was not siçnificantly different
between those subjects or animals receiving the tracer either IV or IG.
Bross et al (1998) compared phenylalanine kinetics from several independent
111
(- )
0 .03 0.05 O. 1 O. 15 U.2 ci. 3 Ci.4
Trp Iniak (gkp'dj
Figure 4.8 Plasma Phenylalanine Specific Radioactivity ( S M ) of Piglets Receiving Parenteral Diet and Tracer (IV (IV)). Entera1 Diet and IV Tracer (IG (IV)), or Enterai Diet and Tracer (IG (IG)). Data espressed as means = pooled SE.
Figure 4.9 Plasrna Phenylalanine Flux of Piglets Receiving Parenteral Diet and Tracer (IV (IV)), Enteral Diet and 11' Tracer (IG (Iij)). or Enteral Diet and Tracer (IG (IG)). Data espressed as means = pooled SE.
studies in which adults were given "C-phenylalanine either IV or IG, in combination with
enteral feedings. Calculated phenylalanine osidation rates were similar between al1
studies, in which phenylalanine intake was constant in relation to total protein intake,
regardless of the route of isotope infusion press et al., 1998).
Others, however, have reponed significant differences in phenylalanine osidation in
adults receiving IG vs IV infusion of "C-phenylalanine (Sanchez et al., 1995; 1996).
Sanchez et al (1995) calculated phenylalanine osidation rates to be 3.32 k 0.76 and 1.16 * 0.32 prnoVkzg/h for enterally fed adults receiving 13C-phenylalanine IG or IV, respectively.
Thus, the IG infiised tracer resulted in a phenylafanine osidation estimate which was
approsimately 64 % greater than that of the IV tracer. and was reported to be statistically
different (Sanchez et al., 1995). Our mean calculated phenylalanine osidation rates for
piglets recei~~ing adequate dietary tryptophan (0.15-0.40 g/ko/d) was found to be 1.45 =
0.59 and 0.85 = 0.57 pmoVkgh for anirnals given IG and IV tracers. respecrively. We
found tliat piglets infused IG with the phenylalanine tracer had - 43 06 Iiigher calculated
phenylalanine osidation rate than piglets receiving IV tracer. These differences in
calculated phenylalanine osidation rates are Iikely due to phenylalanine flux rates. As
mentioned previously, phenylalanine oxidation was calculated as the rate of breath "CO2
production divided by plasma S M . Altematively, this calculation can be espressed as:
Due to the fact that the only diRerence in treatments between piglets was that of isotope
infùsion route, it is clear that actual phenylalanine oxidation rares do not di#er between
groups given IG or IV tracers, only the calcuIated estimate of phenylalanine oxidation
changed. When phenylalanine oxidation is espressed as a proportion of flux (which is
anaIosous to % dose oxidized), osidation was seen to account for 0.8 * 0.57 and 0.6 % * 0.28 of IG and IV infused tracer (p>0.05). Indeed, Sanchez et al (1995) reponed the ratio
of phenylalanine osidation to phenylalanine flux for subjects given IG and IV tracers to be
0.04 i 0.0 1 (or 4%) and 0.06 * 0.03 (or 6%), respectively. Clearly, although this study
and Sanchez et al (1995) found significant differences in plasma phenylalanine oxidation
rates when IG and IV tracers were used, there were no significant differences in tracer
osidation when espressed as a % of dose osidized.
Changes in plasma enrichment ( S M ) and flux between IG and IV tracer in adults
and piglets identically treated suj;est that plasma is not an appropriate precursor pool
from which to sarnple during amino acid osidation studies. Plienylalanine osidation. when
espressed as ?/o of dose, is likely the most accurate measure. This is due to the fact that
botb the dose of tracer infüsed and the subsequent collection of label in breath can be
confidently determined and involve low levels of esperimental error. As mentioned
previously, ?4 dose osidized was not significantly different between enterally fed piglets
given IG or IV tracers. In addition, the rate of "CO2 production was similar between the
14 two alternative tracer goups (p>0.05). Tlierefore, C-phenylalanine ofidarion rates
determined by the appearance of "CO, in breath provides more accurate estirnates of
whoie body phenylalanine oxidation*
Ultimately, the most important question to be addressed is whether the route of
115
tracer administration significantly affects the estimate of tryptophan requirement.
Tryptophan requirernents, espressed as plasma phenylalanine osidation, ''CO2 production
rates, o r percentage of dose osidized, were similar for piglets infused with isoropically
labelled phenylalanine IG or IV during enteral feeding (Tables 4.5, 4.6). The mean
tryptophan requirement for enterally fed piglets determined by a 2-phase linear regression
crossover mode1 and based on % dose oxidized was found to be 0.1 13 i= 0.024 and 0. f 27
= 0.022 g/kg/d for animals given IG and IV tracers, respectively. Therefore, although the
route of tracer administration alters estimates of phenylalanine SR4 and flux, ultimately
the tryptophan requirement. as determined by the IA40 technique. remains unafected.
5.0 GENERAL SUNIRIARY AND FUTURE DIRECTIONS
Low birth weizht infants require unique nutritional management eariy in life due to
the rnetabolic immatunty of the gastrointestinal tract and biochemicd pathways. The use
o f the piglet mode1 is a crucial step in deterrnining amino acid requirements for both the
parenterally and enterally fed premature infant. Although the requirement for other amino
acids, such as threonine and methionine. are substantially greater (approsimately 2 fold) in
enteral versus TPN fed pigiets, this appears not t o be true for tryptophan. The mean
tr'fptophan requirement was determined to be 0.137 k 0.022 and 0.145 = 0.023 Ykg/d
(based on ?/o dose osidized) for enterally and parenterally fed piglets, a difference of abour
12%. This is fiirttier evidence that al1 indispensable amino acids are not equally utilized by
the gut on first pass, and thus the entera1 and parenteral requirernent for each indispensable
amino acid must be enipirically detennined in the neonaial piglet.
Commercial amino acid solutions currentIy used to make TPN contain a larse
range of tryptophan concentrations. Mrhen adjusted to the arnino acid intake o f the piglets
in this study, the solutions provide 0.20-0.32 g tryptophadkg/d. This study susgests that
these solutions provide 160-250% of the requirement. However, before it can be
recommended that tryptophan levels be lowered in TPhT formulas, neonatal needs for
branched chain arnino acids (isoleucine, leucine and valine) must be defined. Tryptophan
cornpetes with both branched chain and arornatic (phenylalanine and tyrosine) amino acids
for transport in the zut and brain. Due to the fact that uptake of these amino acids into
117
brain depend on their relative concentration in plasma, the appropriate ratio of tryptophan
to branched chain and aromatic amino acids must be determined. Untii t k s is done,
lowering tryptophan concentrations in TPN solutions cannot be recommended.
Ultimately, the goal is to determine the ideaI arnino acid profile for the low birth
weight infant, so that protein synthesis is maximized and metabolic imbalances are reduced
to a minimum- Once al1 of the indispensable amino acids are defined in the neonatal pislet
and the ideal profiIe is ascertained, the nest step wiII be to determine if this profile
achieved al1 ofthe needs of the low birth weight infant. Osidation studies are not
cominonly conducted using premature infants due to the invasive procedures involved (ie:
IV tracer infusion, frequent blood sampling, and prolonged adaptation tc diets containing
escess or deficient amounts o f amino acids). Bross et al (1998) has arsued that several of
these dificulties can be overcome. making osidation studies more feasible. They
demonstrated that osidation studies using oral '3C-lysine or '3C-plienylaianine, and
measurement of breath and urine insread of blood could be used to estimate tracer
kinetics, within an S h o u penod. In addition, no pnor adaptation to an esperimental
amino acid diet was required (Bross et al., 1998).
The issue of whether orally administered isotopes affect tracer kinetics and
subsequent requirement estimates was addressed in the current study. As espected, oral
compared to IV infùsed "C-phenylalanine resulted in Lower plasma phenylalanine SR4 and
higher plasma fluses, likely due to first pass utilization of phenylalanine by the gut. The
route of isotope infusion significantly alters estimates of phenylalanirie kinetics in
I l 8
identically treated animais firther suggesting that plasma should not be considered an
appropriate precursor pool. Phenylalanine oxidation rates based o n breath "CO,
collection, and ultimately tryptophan requirement estimates, were not dtered by the route
of infusion of tracer. This study has provided further evidence that the IAAO technique is
an excellent rnethod for the determination of amino acid requirements. Along with the
work of Bross et a1.(1998), it is likely that this technique may be applied to the low birth
weight infant once the ideal amino acid profiie has been established in the neonatal piglet.
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Two Phase Linear Regression Crossover Mode1
The mean requirement for tryptophan was estimated by breakpoint analysis using a
hvo phase linear regression crossover model, using the program of J.D. House (Ph-D.
thesis, 1995). With this model, the data points are partitioned between two separate Iinear
regression lines and the intersection of the two Iines, or the breakpoint. is determined.
The SAS program used is s h o m below. PhenyIalanine oxidation rates (pheos)
were used as the dependent variable, and dietary tryptophan intake (trpin) was used as the
independent variable. The values included on the first line were the rates of osidation
associated with tryptophan intakes of less than 0.05 @&d, mhereaî the remaining data
points were used for the second regression line.
Proram Statement :
* - data entered as piglet number, tryptophan intake, and the oxidative response (% dose osidized) **
croc p r i n t ; proc çim; moatl ph2o:-:=trpin dvalu? trpincdvalue; proc rsg outest=outl23 covout outsscp=outsums; nodel pheox=t r p i n dvalue erpdval; proc p r i n t data= out123; FECC ~ r i z z Uata= cüts'üiis;
Output Statement :
General
Variable: PZEOX
D r
L i n e a r Modêls
S m of Squa re s
Mean Square F Source V a l u e
13-50 Model 3
22
Total 25
3-Square
0 , 0 4 7 5 8 3
23.03050177
15 .55350433
4 4 - 18OOÇ615
C.V.
55.14369
C û r r e c t e d
Meàn Squzre F Value
S o u r c t Typs III ss Mean Square F V a l u e
Model: MODEL1 Depenaent Vzriable: PEEOX
--alysis of Variance
Mean Square Source
Model .-. 1r1or C Total
R-square Adj R-sq
R o c t MSE Dcp M e a n C.V.
Pazameter Standard T for H O : Es~imats E r r o r Paraneter=û Prob > I T i Var iable
CGV INTERCEP PHEOK 0.8400 ù.142û -0.8037 -C.152 9.80 . CCV TnPIx n--- = a = O X O . e 4 0 9 - 0 . 5 0 3 7 4.1234 0.603 -4.12 . CCYJ DVALUE PEEOX O . U 4 O S -0.1520 G.5'037 1.324 -35.07 . COV TRPDVAL PZCOV O.S4Oe 0.8037 -4.1235 -35.1170 1056.24 -
SSCP INTEXCEP 23.005 4 .1490 5.000 O. 2510 00.82ü SSCO T R P I N 0.149 O. 4399 O. 251 O. 0088 4.757 SSCP DVALUk 9.000 O. 2510 8.000 O. 2510 41.650 SSCP T R P D V X 9.251 0.0088 O. 251 O. 0098 1.351 SSCP PEEOX 50.820 4.7569 41.650 1- 2507 350.171 N 28.000 28.0000 2 8 . 0 0 0 28.0000 28.000
Linear Regression Eauation:
The equation used for the linear regression model was as follows:
Y= Al + BIx + (A2-A1)D + (B2-Bl)(Ds) + E
Uliere Y represents the individual observations for the dependent variable (amino acid
oxidation), A l and A2 represent the intercepts of the first and second lines, respectively.
and B 1 and B I represent the dopes of the first and second lines, respectively. The first
line. the line with slope, was &en a D value of 1, and the second line, wïth minimal slope
was given a D value of O. E represents the residual error of the model.
The equations of the two lines were:
Line 1: Y = ( A l + A S - A l ) + ( B l + B 2 - B l ) s
Line 2: Y= A I + B l s
Equating the two functions and solving for s or the crossover point yields:
X= - ( X - & A I)/(B2-B 1 )
The parameters in the Output statement allow for the rapid determination of the
breakpoint or crossover point as folIows:
.A1 = INTERCEP = 1.33736
B = TRPW = - 1.925273
( M - A l ) = DVALLE = 2.655433
(B2-Bl) = TRPDL'AL = -16.406360
Therefore, the crossover point was calculated as:
Crossover or Breakpoint = -DVALUE/TR.PD\I'AL = -3.65843 3/- 16.406360
= O. 162 g/kg/d
95% Confidence Interval Prooram:
The safe Ievel of intake of an amino acid was estimated to be one that would meet
the needs of the upper 95% of the population. Therefore 95% confidence limits
corresponding to the rate of amino acid oxidation, as affected by the amino acid intake,
were determined usin3 Fieller's theorem (as done by J.D. House, Ph.D. thesis, 1995).
SAS Prooram:
This program used the parameters €rom the Output staternent corresponding to the
crossover or breakpoint analysis.
BETA2 = DV.4LC.E = 2.658
BETA i 2 = TRPDVAL = - 16.406
VTBETM = DVALLE * DVALUE = 1.395
\'BETA13 = TRPDVAL " TRPDVAL = 1036.240
COVBETA = DVALUE * TRPDVAL = -35.071
Progam Statement:
options 1s=75 nodate pageno=l forrttdlim='-'; c i t l e l 'Tryptophan Requirement During TPN1; title2 '95% Confidsnce Interval- Oxidation Rate'; data one;
beta2= 2.658; 5etz12= -16.406; vbeta2= 1.395; vbetal2= 1086.240; covbeta= -35.071; t=l.725; ratio=-(betaZ/bttalZ); za=vbsta2/ (beta2**2) ; Db=-~bet212/ (betal2-"2) ; ab=covbeta/(beta2'betal2); varratlo=(rati0**2)*(aa+bb-2~a5); s3ratFo=sqrt(varratio); cloh-er=ratro- t*seratio; cu~~er=ratio+ t * s e r a t i o ; pïoc print; run;
Output Statement:
T r y ~ t o ~ h a n Requ i re rnen t Durlng TPN
4 5 : Confidence I ~ C e r v a l - Oerdatron Xace
OES SZT.32 DETA12 VEZTP.2 VSST'P.12 COVSETA T ?AT10