The Journal of Nutrition Vol. 128 No. 11 November 1998, pp. 1913-1919

Development of a Minimally Invasive Protocol for the Determination of Phenylalanine and Lysine Kinetics in Humans during the Fed State^1,2,3

Rachelle Bross^{*, **}, Ronald O. Ball^{*, **,} , and Paul B. Pencharz^{*, , **, , 4}

^* Departments of Nutritional Sciences and  Paediatrics, University of Toronto, Toronto, ON, Canada M5S 3E2; ^** The Research Institute, The Hospital for Sick Children, Toronto, ON, Canada M5G 1X8; and  Department of Agricultural, Food & Nutritional Sciences, University of Alberta, Edmonton, AB, Canada T6G 2P5

	ABSTRACT

Abstract Introduction Methods Results Discussion References

The primed, continuous intravenous infusion of amino acids labeled with ¹³C together with measurement of isotopic enrichment in plasma is commonly used to study amino acid metabolism. However, a less invasive, oral infusion that also produces an isotopic steady state in CO₂ and urine would be useful, particularly for pediatric studies. We measured the ¹³C enrichments of expired CO₂, plasma and urine free phenylalanine and lysine and estimated flux and oxidation rates in adult humans (n = 12) who received a 4-h oral, primed, equal dose infusion of either L-[1-¹³C]phenylalanine, L-[1-¹³C]lysine (D-lysine = 1.6%) or L-[1-¹³C]lysine (D-lysine  0.2%). Steady fed state conditions were established by feeding subjects eight hourly meals beginning 4 h before the start of the oral infusion protocol. Isotopic plateau in CO₂, plasma and urine was achieved within 120 min of phenylalanine or lysine infusion. At isotopic plateau, the mean ratio of plasma to urine enrichment was 1.0 ± 0.04 (SEM), 0.39 ± 0.03 and 0.97 ± 0.02 for L-[1-¹³C]phenylalanine, L-[1-¹³C]lysine (D-lysine = 1.6%) and L-[1-¹³C]lysine (D-lysine 0.2%), respectively. There was good agreement between isotopic enrichment in plasma and urine for L-[1-¹³C]phenylalanine and L-[1-¹³C]lysine (D-lysine  0.2%). However, use of L-[1-¹³C]lysine (D-lysine = 1.6%) resulted in significantly higher enrichment in urine, probably due to renal tubular discrimination of D-lysine. Mean flux rates for phenylalanine and lysine were consistent with the literature. Thus, the oral, primed, equal dose infusion produces the isotopic steady-state condition required for amino acid flux and oxidation determination within an 8-h period.

	INTRODUCTION

Abstract Introduction Methods Results Discussion References

Advances in the use of stable isotopes have increased the availability of suitable methods for the study of human amino acid and protein metabolism. Direct and indicator amino acid oxidation (IAAO)⁵ are currently two tracer techniques that have been used to reassess the human adult requirements for several indispensable amino acids (Basile-Filho et al. 1997, Lazaris-Brunner et al. 1998, Sanchez et al. 1995, Zello et al. 1993).

No reports were found in the literature of amino acid requirement estimates derived by oxidation techniques for infants or children. The experimental designs used in both oxidation techniques involve invasive and impractical interventions such as intravenous infusion of the stable isotope labeled amino acid, measurement of precursor isotopic abundance in blood and adaptation to an experimental, metabolic diet for 2-7 d, all of which preclude their ethical use in infants and children and other vulnerable groups.

Noninvasive methods have been incorporated into the stochastic model for the study of protein and amino acid metabolism. Oral or intragastric infusion of isotope, for 12-60 h has been used to study amino acid and protein turnover in both infants and adults (Basile-Filho et al. 1997, de Benoist et al. 1984, Sanchez et al. 1995, Waterlow et al. 1978b, Wykes et al. 1992). Furthermore, urine has been used to sample plasma amino acid enrichment. De Benoist et al. (1984), Wykes et al. (1990) and Zello et al. (1994) showed good correlation between plasma and urinary amino acid enrichments using the isotopes L-[1-¹³C] leucine, L-[1-¹⁵N] glycine and L-[1-¹³C]phenylalanine. Despite the development and application of noninvasive methods, no one study has combined these methods into a 1-d protocol that could be followed to study amino acid kinetics.

Consumption of a diet deficient in or containing excessive amounts of an amino acid for an extended period (days) is contraindicated in healthy infants and in specific clinical pediatric or adult populations. For example, lengthy exposure to an imbalanced amino acid intake could compromise metabolic control in individuals with an inborn error of amino acid metabolism. Therefore, current protocols that require adaptation for 3-5 d before the infusion study cannot be used. A short-term protocol (<1 d) needs to be developed.

The objective of this study was therefore to develop a minimally invasive model that would allow the study of indispensable amino acid kinetics in vulnerable groups. The goals of the model were as follows: 1) to limit the need for the experimental diet to only the day of study in combination with 2) oral administration of the isotope and 3) the use of urine to sample plasma amino acid enrichment. Successful development of a 1-d protocol would allow methods for the determination of indispensable amino acid requirements by oxidation techniques to be expanded to include groups previously excluded from study because of age or clinical condition.

	MATERIALS AND METHODS

Abstract Introduction Methods Results Discussion References

Subjects.  Thirteen healthy adult female and one healthy adult male volunteer (age 28.4 ± 1.1 y; weight 60.9 ± 3.4 kg; height 163.9 ± 2.2 cm; body mass index 22.7 ± 0.9 kg/m²) participated on an outpatient basis in the Clinical Investigation Unit at The Hospital for Sick Children (HSC), Toronto, Canada. All subjects were screened by questionnaire for chronic diseases and atypical exercise or dietary habits. Subjects were nonsmokers who had maintained a stable body weight for several months and had taken no medication in recent weeks. Written informed consent was obtained for the study, which was approved by the University of Toronto Human Experimentation Committee and the Human Subject Review Committee of the HSC.

Experimental design.  Four experiments were conducted. The objective of Experiment 1 was to determine whether the experimental diet could be limited to only the day of the study. This was evaluated by determining the pattern and stability of background ¹³C enrichment in expired CO₂, while subjects consumed the experimental diet without prior adaptation. Six subjects participated in Experiment 1. The objectives of Experiments 2 and 3 were to determine whether L-[1-¹³C]phenylalanine and L-[1-¹³C]lysine, respectively, administered orally, could produce an isotopic steady state in plasma, urine and expired CO₂ within 4 h. Five subjects each participated in Experiments 2 and 3. Experiment 4 was conducted to evaluate and account for the effect of D-lysine in the tracer in Experiment 3. Two subjects participated in Experiment 4.

Diet and energy intakes.  The experimental diet was developed for amino acid kinetic studies; its composition has been reported in detail previously (Zello et al. 1990a). Briefly, a flavored liquid formula (protein-free powder, Product 80056, Mead Johnson, Evansville, IN; Tang, Don Mills, Canada; Koolaid, Don Mills, Canada) and protein-free cookies (HSC Research Kitchen) supplied the main source of energy in the diet. A crystalline amino acid mixture, based on the amino acid composition of egg protein, was consumed at 1.0 g/(kg · d) and provided the only source of amino nitrogen in the diet. The macronutrient composition of the experimental diet, expressed as a percentage of dietary energy, was 53% carbohydrate, 38% fat and 9% protein. The diets were prepared and weighed (Mettler Scale, model PE 2000, Nanikon, Switzerland) in the research kitchen of the HSC and were portioned into isoenergetic, isonitrogenous meals. The diet was consumed as hourly meals, and each meal represented one twelfth of the subject's total daily requirement. Total energy intakes were based on each subject's calculated resting metabolic rate (FAO 1985), multiplied by a factor of 1.7. This method for determining energy requirements has been shown to result in maintenance of subjects' weight during short-term amino acid oxidation studies (Bell et al. 1985, Duncan et al. 1996, Zello et al. 1993). Subjects were instructed to maintain their usual level of physical activity and to fast for 10-12 h overnight before the study. Standing height was measured without shoes (nearest 0.1 cm) with a wall-mounted stadiometer and subjects were weighed (Toledo Scale, model 2020, Windsor, Canada) within 1 wk of the study day and again on the morning of the study.

Study protocols.  On each study day, subjects were maintained in a temperature-controlled metabolic facility at the HSC. The first step (Experiment 1) was to establish whether an isotopic steady state could be reached in breath ¹³CO₂ enrichment without prior adaptation to the experimental diet. Breath ¹³CO₂ production (F ¹³CO₂) was measured in six subjects while they consumed hourly meals of the experimental diet for a period of 6 h. Simultaneous breath collection and measurement of CO₂ production rate (V_CO2) occurred at min 30, 75, 105, 135, 165, 195, 225, 255, 285, 315 and 345 while the subjects lay in a semirecumbant position on a hospital bed. The subjects breathed in a normal fashion while wearing a ventilated face mask (Scott 80216730, Sensormedics, Anaheim, CA). Once the subject's air flow had stabilized (CO₂ concentration = 0.5-0.8%), the expired breath was collected using a vacuum extraction system (Pump VB0025, Vortex Blower, Spencer Turbine Company, Windsor, CT) combined with a gas flow meter. To trap the respiratory CO₂ , the expired breath was bubbled at a rate of 500 mL/min through 10 mL of a 1 mol/L NaOH solution in a modified reflux condenser for 7 min. The resulting NaH¹³CO₃ solution was then injected (Monojet, Sherwood Medical, St. Louis, MO) into vacutainer glass tubes (Vacutainer Brand 6441, 100 × 16 mm, Becton Dickinson, Mississauga, Canada). The vacutainers were evacuated with a syringe to remove air introduced during the injection and frozen at 20°C until analysis. To determine CO₂ volume, the CO₂ analyzer (Beckman Medical Gas Analyzer LB-2, Fullerton, CA) was calibrated using standardized gases (nitrogen, CO₂ and O₂, Linde Medical Gas, Union Carbide, Toronto, Canada), and barometric pressure and temperature were accounted for.

Having established how long it took for background ¹³CO₂ enrichment to reach a steady state, we proceeded to Experiments 2 and 3, in which the following stable isotopes were used: L-[1-¹³C]phenylalanine with an enrichment of 99% (Tracer Technologies, Somerville MA) and L-[1-¹³C]lysine · HCl · H₂O with an enrichment of 99% (Cambridge Isotopes Laboratories, Woburn, MA). The chemical and isotopic purity of the labeled amino acids was confirmed by gas chromatography-mass spectrometry (GC-MS). Isometric purity (<2% D-isomer) was assessed by GC using a chiral column for [¹³C]phenylalanine and by chiral HPLC for [¹³C]lysine. The stock solutions of [¹³C]phenylalanine (20 g/L) and [¹³C]lysine (10 g/L) were prepared with sterile water by passage through a 0.22-µm Millipore filter (Millipore, Bedford, MA) under a laminar flow hood and then dispensed into multiple dose vials. Each batch was demonstrated to be sterile and free from bacterial growth over 7 d in culture. On the basis of the results of preliminary pilot studies, each subject received an oral priming dose of L-[1-¹³C]phenylalanine (19.37 µmol/kg) or L-[1-¹³C]lysine (21.89 µmol/kg) at time 0. Equal oral doses of L-[1-¹³C]phenylalanine (4.24 µmol/kg) or L-1-[¹³C]lysine (4.79 µmol/kg) were administered every 30 min beginning 15 min after the prime. Isotope administration involved swallowing the prime or equal infusion dose, followed by water, to rinse the tube that contained the isotope.

On each study day, hourly meals were consumed beginning at time 240 min (0800 h) and were continued for a total of eight hourly meals (4 before isotope administration and 4 after; the fifth meal was given at time 0, immediately after the isotope prime). The level of dietary phenylalanine or lysine in the meals was reduced by an amount that corresponded to the amount of [¹³C]phenylalanine or [¹³C]lysine administered during the oral tracer infusion period. Water (150 mL)was consumed with each meal to ensure hourly production of urine. Subjects voided hourly, but urine was not collected before time 60 min. Three baseline samples of breath (45, 30 and 15 min) and two urine samples (60 and 0 min) were taken during the hour before the administration of the isotopes began. Once the isotope infusion was started, hourly urine collections continued, breath collections occurred at min 45, 75, 105, 135, 165, 195, 210 and 225 and blood samples were taken at min 160, 200 and 240. The total length of the experiments was 8 h.

Hourly urine samples were mixed with 1 mL of 3.4 mol/L HCl as preservative and stored at 20°C until analysis. Breath collections and V_CO2 measurements were performed as described above. Blood was collected via a 21-gauge needle inserted into a superficial dorsal vein in the left hand. The line was kept patent by administering heparin (10,000 USP units/L) between blood sampling. Arterialized venous blood was obtained by heating the hand inside a thermostatic chamber maintained at 60°C for 15 min before the blood was sampled (Zello et al. 1990c). Arterialized venous blood (3 mL) was drawn into heparinized syringes (Aspirator, Marquest Medical Products, Englewood, CO). The blood samples were kept on ice until centrifugation at 1,500 × g at 4°C. The plasma was then frozen at 20°C until analysis.

Due to the presence of 1.6% D-[1-¹³C]lysine in the lysine, an additional experiment (Experiment 4) was conducted in two parts. In part 1, 30 urine samples and 42 blood samples, taken from two adult subjects participating in another amino acid kinetics study carried out in this laboratory, were analyzed to determine background [¹³C]lysine enrichment. Samples were taken during the baseline period of the protocol, after the subjects had consumed four hourly meals of the same experimental diet as described above, immediately before isotope administration. In part 2, two infusion studies were conducted with L-[1-¹³C]lysine (0.2% D-[1-¹³C]lysine, Mass Trace, Woburn, MA) to determine whether urine and plasma enrichments of [¹³C]lysine were equal when D-lysine was not present in the infusion. The first of the two infusion studies was conducted in the fed state and involved both a 4-h baseline period in which hourly meals were consumed followed by a 4-h oral, primed, equal dose L-[1-¹³C]lysine infusion. The second study was conducted in fasting subjects and involved a 4-h oral, primed, equal dose L-[1-¹³C]lysine infusion. Three paired urine and blood samples were collected at 30-min intervals in the hour preceding the start of the isotope infusion (60,30 and 0 min). Water was consumed with each dose of isotope to ensure adequate production of urine. The subjects voided, but urine was not collected during the first 120 min of the infusion. Four paired urine and blood samples were collected during the final 120 min of the infusion protocol, at 140, 180, 220 and 240 min, which corresponded to the time at which isotopic plateau was achieved in Experiment 3. The procedures followed for collecting and storing urine and blood samples and for the administration of isotope were identical to those for the lysine infusion study described in Experiment 3.

Analytical methods.  The percentage enrichment of the expired ¹³CO₂ was measured on a dual-inlet magnetic sector isotope ratio mass spectrometer (VG Micromass 602D, Cheshire, England) using techniques described in earlier work (Jones et al. 1986). Breath ¹³CO₂ enrichments were expressed as atoms percent excess (APE) over a reference standard of compressed CO₂ gas.

The isotope enrichment values for urine and plasma free [1-¹³C]phenylalanine or [1-¹³C]lysine were measured on a gas chromatograph (Hewlett-Packard model 5890 Series II, Mississauga, Canada) attached to a quadrupole mass spectrometer (VG Trio-2). Plasma (200 µL) and urine samples (500 µL) were deproteinized and acidified with an equal volume of 200 g/L trichloroacetic acid and centrifuged at 9,000 × g. Amino acids were separated from the supernatant by a cation exchange resin (Dowex 50W-X8, 100-200 mesh H⁺ form, Bio Rad Laboratories, Richmond, CA) and were derivatized by the method described by Patterson et al. (1991) to their N-heptafluorobutyryl n-propyl esters. Separation of the amino acid derivatives was performed with helium as the carrier gas on a 30 m × 0.32 mm (i.d.) × 1.0 µm (film thickness) fused silica capillary column (HP5, Hewlett-Packard) coupled directly to the ion source, which was operated under conditions of negative chemical ionization with ammonia as the reactant gas. Each amino acid was analyzed by splitless injection, on an automatic sampler (HP 7673 injector). Selected ion chromatographs were obtained by monitoring mass-to-charge ratios of 383 and 384 for [¹³C]phenylalanine, and 560 and 561 for [¹³C]lysine, corresponding to the unenriched (m) and enriched (m+1) peaks, respectively. The areas under the peaks were integrated by a Digital DECp 450D₂LP computer, using a Lab-Base program (VG Biotech, Altringham, UK).

Estimation of isotope kinetics.  A stochastic model was used to evaluate phenylalanine and lysine kinetics (Waterlow et al. 1978a). Isotopic steady state in the metabolic pool was represented by plateaux in ¹³CO₂ enrichment in breath, and in [¹³C]phenylalanine and [¹³C]lysine enrichment in plasma and urine. This state was achieved in breath, urine and plasma by 120 min from the start of both the [¹³C]phenylalanine and [¹³C]lysine isotope infusions and was maintained to the end of the study at 240 min. The mean breath isotope enrichment values of the three baseline samples and the five plateau samples were used to determine APE above baseline at isotopic steady state. The mean ratio of the enriched peak (m+1) to the unenriched peak (m) in urine for both baseline and plateau samples was used to calculate molecules percent excess (MPE) for [¹³C]phenylalanine and [¹³C]lysine (<0.2% D-isomer). The mean ratio of the enriched peak (m+1) to the unenriched peak (m) in urine for baseline samples and the mean ratio of the enriched peak (m+1) to the unenriched peak (m) in plasma for plateau samples were used to calculate MPE for [¹³C]lysine (1.6% D-isomer). Plasma enrichments at plateau were used because urinary enrichment of [¹³C]lysine was affected by the presence of 1.6% D-[1-¹³C]lysine in the infused [¹³C]lysine.

The isotopic enrichment in urine/plasma and breath was calculated using standard equations (Matthews et al. 1980, Rosenblatt et al. 1992).

Flux (Q) was measured from the dilution of the infused tracer L-[1-¹³C]phenylalanine and L-[1-¹³C]lysine in the plasma and urine metabolic pools at isotopic steady state (Matthews et al. 1980).

The rate of phenylalanine or lysine oxidation [µmol/(kg · h)] was calculated from ¹³CO₂ exhaled in breath (F ¹³CO₂), corrected for retention in the body bicarbonate pools (Hoerr et al. 1989) in the fed state (Matthews et al. 1980).

Statistical analysis.  Results are expressed as means ± SEM. In Experiment 1, the significance of the change in background APE of ¹³C over the 6-h study period was tested by regression analysis (SAS Institute 1985) and by one-way ANOVA on the data from individual subjects. In Experiments 2 and 3, isotopic steady state in the metabolic pool was represented by plateaux in urine and plasma for [¹³C]phenylalanine, in plasma for [¹³C]lysine and in breath ¹³CO₂, for both labeled amino acids. In Experiment 4, for [¹³C]lysine (<0.2% D-isomer), isotopic steady state was represented by plateaux in urine and plasma. Attainment of isotopic steady state (plateau) was evaluated in each subject, in breath, plasma and urine data. Plateau was evaluated by visual inspection followed by repeated linear regression analysis in which data points, beginning at time 0 min, were removed until a regression line with a slope not different from zero was achieved. In Experiments 2, 3 and 4, the difference in amino acid enrichment of plasma and urine was evaluated by paired t test. Further, the difference of the mean ratio from 1 was evaluated by t test. The relationship between enrichment in plasma and urine of each amino acid was also assessed by linear regression analysis. Agreement between plasma and urine [¹³C]phenylalanine and [¹³C]lysine enrichment was also assessed by the method of Bland and Altman (1986). Results were considered statistically significant at P-values  0.05.

	RESULTS

Abstract Introduction Methods Results Discussion References

In Experiment 1, complete data sets of 11 breath samples were obtained in four subjects. The final CO₂ sample in one subject was unsuccessfully trapped in NaOH and another subject became ill for reasons unrelated to the experiment and did not provide the final two samples. The effect of small hourly meals on background ¹³C enrichment in breath CO₂ and V_CO2 in the six subjects is shown in Figure 1. The mean change in background ¹³CO₂ enrichment for the six subjects after four meals was 0.003471 ± 0.00041 APE and after six meals for five subjects was 0.003505 ± 0.00033 APE. For each subject, the slope of the line for breath ¹³CO₂ enrichment data collected after 225 min was not significantly different from zero. For data collected between 225 and 360 min, the CV among points within a slope was 1.52-3.31%.

View larger version (14K): [in this window] [in a new window]   Fig 1. Experiment 1: effect of experimental diet on 13CO2 enrichment expressed as atoms percent excess (APE; ) and on rate of CO2 production (VCO2; ). A plateau in 13CO2 enrichment and VCO2 was achieved for all subjects beginning at 225 min. Values are means ± SEM, n = 12. Slopes of the 13CO2 enrichment or VCO2 vs. time regression lines were not significantly different from zero after 225 min, P > 0.05.

Meal consumption had a significant effect on V_CO2 such that it increased from 230 ± 15 mL/min to 282 ± 24 mL/min (P = 0.002) between the first and last breath samples. V_CO2 increased with each meal until 225 min, after which V_CO2 did not change significantly until the end of the study.

During the [¹³C]phenylalanine and [¹³C]lysine infusion studies, isotopic steady state was achieved in breath ¹³CO₂ by 120 min from the start of the [¹³C]phenylalanine and [¹³C]lysine infusions and was maintained to the end of the study at 240 min. The oral infusion protocol for [¹³C]phenylalanine and [¹³C]lysine produced mean enrichment curves for data collected between 120 and 240 min, with slopes not significantly different from zero (P = 0.73 for [¹³C]phenylalanine and P = 0.16 for [¹³C]lysine). The CV for breath ¹³CO₂ enrichment determinations for baseline and plateau enrichments were 2.90 ± 0.53 and 2.61 ± 0.23% for the [¹³C]phenylalanine infusion and 2.67 ± 0.76 and 2.40 ± 0.30% for the [¹³C]lysine infusion, respectively.

The mean isotopic enrichment of [¹³C]phenylalanine and [¹³C]lysine in urine before isotope infusion and at isotopic steady state in plasma (P) and urine (U) is shown in Table 1. Plasma samples were not available for one subject in Experiment 2 during the expected plateau period. At isotopic steady state, plasma [¹³C]phenylalanine was similar to that of urine (P = 0.31) and the plasma to urine enrichment ratio (P:U ratio) was not different from 1 (P = 0.36). From the Bland and Altman (1986) analysis, the mean bias in [¹³C]phenylalanine enrichment between urine and plasma was 0.015 MPE, where bias is the mean of the mathematical differences between the plasma and urine enrichments. The limits of agreement (mean bias ± 2 SD) ranged from 0.054 to 0.085. The mean bias represents 2.5-4.8% of the range of average enrichments observed in subjects at plateau in plasma. Conversely, the mean plasma [¹³C]lysine enrichment in subjects who received the [¹³C]lysine that contained 1.6% D-[¹³C]lysine was approximately one third that of urine (P = 0.002), and the resulting P:U ratio was significantly different from 1 (P < 0.0001). Independent qualitative analysis of the [¹³C]lysine isotope, a plateau urine sample and a plateau plasma sample, by electron impact GC-MS using a chiral column (Mass Trace), revealed a significant m+1 peak of D-[1-¹³C]lysine in the urine sample. Quantification of the D- and L-lysine peaks in the urine sample was not possible. The ability to resolve and quantitate amino acid enantiomers is dependent on their retention times and required column temperatures. Because of lysine's long retention time and high column temperature (near the maximum temperature for the chiral column), resolution of D- and L-lysine is less than for other amino acids (Abdalla et al. 1987). The plasma sample contained only negligible amounts of the D-isomer. At isotopic steady state, mean plasma [¹³C]lysine enrichment in the two subjects who received the [¹³C]lysine with <0.2% D-isomer was similar to that of urine (P = 0.24) and the P:U ratio was not different from 1 (P = 0.27). Furthermore, the Bland and Altman (1986) analysis confirmed this agreement, with a mean bias in [¹³C]lysine enrichment in urine and plasma of 0.0007 (limits of agreement ranged from 0.044 to 0.045). The mean bias represents 0.23-0.35% of the range of average enrichments observed in subjects at plateau in plasma.

Flux and oxidation rates for [¹³C]phenylalanine and [¹³C]lysine are shown in Tables 2 and 3. Results from this study are compared with those reported in the literature, where the ratio of phenylalanine or lysine intake to total protein intake was kept constant.

From the 30 urine and 42 plasma samples taken from subjects participating in another study in the same laboratory (Experiment 4), the mean ratio of plasma to urine [¹³C]lysine enrichment was 0.994 ± 0.008, which was not significantly different from 1 (P = 0.49). Furthermore, the mean baseline urinary enrichment from these 30 samples was not significantly different from the mean baseline urinary enrichment of [¹³C]lysine in the five subjects who participated in Experiment 3 (0.229097 ± 0.00212 APE vs. 0.231681 ± 0.00176 APE, P = 0.36), both of which reflected the natural enrichment in the dietary source of lysine (0.228291 ± 0.00093 APE).

	DISCUSSION

Abstract Introduction Methods Results Discussion References

This investigation was prompted by the lack of indispensable amino acid requirement estimates derived by oxidation techniques in infants and children. This lack is due in part to invasive and impractical experimental designs that preclude their use in these and other vulnerable groups. The main objective of this study was to develop a 1-d, minimally invasive isotope infusion method that would produce the necessary isotopic steady-state conditions required to estimate amino acid kinetic parameters.

The goal of the first experiment was to determine the duration of a fed-state baseline period that would produce a stable V_CO2 and a constant enrichment of ¹³CO₂ in breath without prior adaptation to the experimental diet. In Experiments 2 and 3, the goals were to develop an oral, primed, equal dose infusion protocol using either L-[1-¹³C]phenylalanine or L-[1-¹³C]lysine that could produce isotopic steady state in breath and urine and thus allow for the estimation of amino acid flux and oxidation. These infusion studies were conducted under steady fed-state conditions and followed the baseline protocol determined in Experiment 1. The stable isotope L-[1-¹³C]phenylalanine has been shown to be an acceptable tracer in studies of amino acid flux and oxidation in infants and adults using urine (Wykes et al. 1992, Zello et al. 1994). L-[1-¹³C]lysine has also been used as a tracer in amino acid kinetic studies, but the use of urine to sample the arterialized plasma enrichment has not been verified for lysine. This series of experiments represents the first attempt to combine several noninvasive methods and incorporate them into an amino acid oxidation technique applicable to vulnerable populations.

A further consideration is the use of an orally administered tracer. It has been shown in both humans (Sanchez et al. 1995) and pigs (Stoll et al 1998) that both phenyalalanine and lysine are taken up in the splanchic bed (i.e., intestine plus liver), especially in the intestine (Stoll et al. 1998). For the purposes of IAAO studies of amino acid requirements, what matters is that the oxidation of the indicator (both lysine and phenylalanine have been used) is affected only by the level of the test amino acid fed (Zello et al. 1995). All of the original development and validation of the IAAO method, which was conducted in pigs, involved oral isotope administration. Therefore, although there are differences in the metabolism of intravenously vs. orally administered lysine or phenylalanine (Sanchez et al. 1995, Stoll et al. 1998), no differences are expected in the breakpoint estimate of the requirement of the test amino acid (Zello et al. 1995).

Measurement of ¹³CO₂ enrichment following administration of a ¹³C-labeled substrate occurs in the presence of a large background of naturally occurring isotope of ~1.1% ¹³C (Schoeller et al. 1980). The background ¹³C enrichment in breath must therefore be measured before isotope administration. Results from Experiment 1 show that consumption of the experimental diet increased ¹³CO₂ and V_CO2 between 0 and 225 min. A constant background ¹³C enrichment in breath and a stable rate of CO₂ production was achieved between 225 and 255 min and was maintained through to the end of the study. This suggests that five hourly meals are required to achieve a constant ¹³CO₂ enrichment. However, the change in enrichment between 225 and 255 min represented <2% of the average ¹³CO₂ enrichment at 255 min. The difference between the enrichment at 225 and 255 min was less than the measurement error associated with the isotope ratio mass spectrometer. Furthermore, the slope of the ¹³CO₂ enrichment curve measured between 225 and 360 min in each individual was not significantly different from zero. This protocol could be used in place of a longer adaptation period in populations in which adaptation to an imbalanced metabolic diet is prohibited. The effect of the macronutrient composition of an individual's usual intake on amino acid kinetic parameters is not addressed by this experiment. Therefore, at this time, the application of this baseline protocol is restricted to those populations whose habitual diets are relatively constant and similar, and in which the macronutrient composition of the experimental diet can be matched to the population's usual intake.

This oral, primed, equal dose infusion of L-[1-¹³C]phenylalanine produced isotopic steady states in urine, plasma and breath CO₂, similar to steady states produced by intravenous tracer protocols (Duncan et al. 1996, Zello et al. 1990b, 1993 and 1994). Administration of the tracer every 30 min appeared to produce a more even delivery of oral tracer into the metabolic pools than has previously been observed when the tracer is administered on an hourly basis (Basile-Filho et al. 1997). The similarity between the enrichment of ¹³C phenylalanine in plasma and urine is consistent with the results of previous studies (Wykes et al. 1990, Zello et al. 1994). The P:U ratio was not significantly different from 1, the correlation between plasma and urine enrichment (r2 = 0.99, P = 0.002) was highly significant and the mean bias in enrichment suggested good agreement between the two measurements.

Our estimates of phenylalanine flux and oxidation are similar to those reported in the literature, in which the phenylalanine intake was constant in relation to the total protein intake (Table 2) (Basile-Filho et al. 1997, Duncan et al. 1996, Lazaris-Brunner et al. 1998, Sanchez et al. 1995, Zello et al. 1993). As in a previous study, in which all subjects were female (Lazaris-Brunner et al. 1998), flux rates of the five female subjects in this study were not different from data obtained in male subjects. Comparison of the studies from this laboratory (Duncan et al. 1996, Lazaris-Brunner et al. 1998, Zello et al. 1993), in which phenylalanine and tyrosine intakes were the same as in this study [phenylalanine, 14.0 mg/(kg · d) and tyrosine, 40.0 mg/(kg · d)], revealed similar rates of phenylalanine oxidation. Flux and oxidation rates in this study were consistent with those reported by Basile-Filho et al. (1997) in which total aromatic amino acid intakes were similar [54 mg/(kg · d) vs. 39-42.3 mg/(kg · d)] and met the requirement estimates determined by oxidation techniques (Basile-Filho et al. 1997, Zello et al. 1990b). The similarity between the phenylalanine kinetic data in this study and data from the literature in which phenylalanine intakes are similar suggests that the oral, primed, equal dose infusion protocol with measurement of isotopic enrichment in urine and breath is a viable alternative for the measurement of phenylalanine kinetics in populations in which more invasive methods are contraindicated.

Oral equal dose infusion of [1-¹³C]lysine (1.6% D-isomer) produced isotopic steady states in breath ¹³CO₂ and plasma ¹³C lysine; however, urinary and plasma isotope enrichments were significantly different, with urinary enrichments 61% higher. Active transport carriers in the proximal tubules reabsorb nearly 100% of the amino acids from tubular fluids (Crim and Munro 1994, Souba and Pacitti 1992). However, because these transporters are specific to the L-isomers of the amino acids, D-isomers are not reabsorbed and are lost in the urine. Discrimination in the process of reabsorption would account for the higher urinary enrichment of ¹³C lysine compared with plasma. This conclusion was further supported by the statistically similar plasma and urinary enrichment when the ¹³C lysine tracer with <0.2% D-isomer was used.

In the [1-¹³C]lysine infusion study, the ¹³CO₂ enrichment was slightly higher between 50 and 100 min of the infusion compared with the final 120 min when isotopic steady state was achieved. This was evidence of overpriming of the lysine pool. We would therefore recommend reducing the prime of 21.89 µmol/kg to 17.1 µmol/kg.

Lysine flux and oxidation rates are shown with other results reported in the literature (Conway et al. 1980, Motil et al. 1981a, Motil et al. 1981b, Motil et al. 1994, Zello et al. 1992) in Table 3. Direct comparisons between lysine flux rates could not be made because of significant differences in lysine intake. Figure 2 depicts the relationship between lysine intake and lysine flux based on published data. At an intake of 54.06 mg/(kg · d), a lysine flux of 79.57 µmol/(kg · h) would be predicted. The measured lysine flux at that intake was 76.38 µmol/(kg · h). The similarity between measured and predicted flux values suggests that the oral, primed, equal dose infusion protocol did not alter the relationship between lysine intake and lysine flux. The results also suggest that the gender of the subjects in this study (female) did not significantly affect the relationship between lysine intake and flux. Together with the equivalent enrichment in urine and plasma, this suggests that the noninvasive protocol can be used as an alternative method for the measurement of lysine kinetics in populations in which an intravenous infusion is prohibited.

View larger version (13K): [in this window] [in a new window]   Fig 2. Linear relationship between lysine intake and lysine flux (solid line). Published (), predicted () and measured ( ) lysine flux are noted. Regression line for published lysine flux data: flux = 46.86 + 0.6(lysine intake) (r2 = 0.67, P = 0.04). Regression line (dashed line) indicates predicted lysine flux at a lower lysine intake. Lysine flux = 79.57mmol/(kg · h) predicted for an intake of 54.06 mg/(kg · d). Measured mean lysine flux = 76.38 mmol/(kg · h) for an intake of 54.06 mg/(kg · d).

In conclusion, a less invasive model to measure amino acid kinetics was developed for L-[1-¹³C]phenylalanine and L-[1-¹³C]lysine. No prior adaptation is required to an amino acid-based experimental diet. Further, with this model, a 4-h baseline period can be combined with a 4-h oral, primed, equal dose infusion protocol and measurement of isotopic enrichment in breath and urine to estimate phenylalanine and lysine kinetics, within an 8-h period. This protocol is at least 4 h shorter in duration than current oral infusion protocols and as such does not require participants to stay overnight. The ability to study amino acid kinetics on an outpatient basis with a minimally invasive protocol means that the study of children and other vulnerable populations can be greatly expanded. This minimally invasive protocol was developed in part to assess indispensable amino acid requirements in vulnerable populations by the IAAO technique. Phenylalanine and lysine were selected for study because both amino acids have been shown to be suitable indicator amino acids in animal studies (Ball and Bayley 1984 and 1986, Ball et al. 1986, Kim et al. 1983a and 1983b), and phenylalanine has been used successfully as an indicator amino acid in human studies (Duncan et al. 1996, Lazaris-Brunner et al. 1998, Zello et al. 1993) of indispensable amino acid requirements.

	FOOTNOTES

Manuscript received 4 March 1998. Initial reviews completed 27 April 1998. Revision accepted 3 July 1998.

	LITERATURE CITED

Abstract Introduction Methods Results Discussion References

• Abdalla S., Bayer E., Frank H. Derivatives for separation of amino acid enantiomers. Chromatographia 1987; 23:83-85
• Ball R. O., Atkinson J. L., Bayley H. S. Proline as an essential amino acid for the young pig. Br. J. Nutr. 1986; 55:659-668[Medline]
• Ball R. O., Bayley H. S. Tryptophan requirement of the 2.5-kg piglet determined by the oxidation of an indicator amino acid. J. Nutr. 1984; 114:1741-1746
• Ball R. O., Bayley H. S. Influence of dietary protein concentration on the oxidation of phenylalanine by the young pig. Br. J. Nutr. 1986; 55:651-658[Medline]
• Basile-Filho A., El-Khoury A. E., Beaumier L., Wang S. Y., Young V. R. Continuous 24-h L-[1-¹³C]phenylalanine and L-[3,3-²H₂]tyrosine oral-tracer studies at an intermediate phenylalanine intake to estimate requirements in adults. Am. J. Clin. Nutr. 1997; 65:473-488[Abstract/Free Full Text]
• Bell L., Jones P.J.H., Telch J., Clandinin M. T., Pencharz P. B. Prediction of energy needs for clinical studies. Nutr. Res. 1985; 5:123-129
• Bland J. M., Altman D. G. Statistical methods for assessing agreement of two methods of clinical measurement. Lancet 1986; 1:307-310[Medline]
• Conway J. M., Bier D. M., Motil K. J., Burke J. F., Young V. R. Whole-body lysine flux in young adult men: effects of reduced total protein and of lysine intake. Am. J. Physiol. 1980; 239:E192-E200[Free Full Text]
• Crim, M. C. & Munro, H. N. (1994) Proteins and amino acids. In: Modern Nutrition in Health and Disease (Shils, M. E., Olson, J. A. & Shike, M., eds.), pp. 3-35.
• Lea, Febiger, Malvern, PA. de Benoist, B., Abdulrazzak Y., Brooke O. G., Halliday D., Millward D. J. The measurement of whole body protein turnover in the preterm infant with intragastric infusion of L-[1-¹³C]leucine and sampling of the urinary leucine pool. Clin. Sci. (Lond.) 1984; 66:155-164[Medline]
• Duncan A. M., Ball R. O., Pencharz P. B. Lysine requirement of adult males is not affected by decreasing dietary protein. Am. J. Clin. Nutr. 1996; 64:718-725[Abstract/Free Full Text]
• Food and Agriculture Organization of the United Nations-Rome. (1985) Energy and Protein Requirements. World Health Organization, Geneva, Switzerland.
• Hoerr R. A., Yu Y. M., Wagner D. A., Burke J. F., Young V. R. Recovery of ¹³C in breath from NaH ¹³C O₃ infused by gut and vein: effect of feeding. Am. J. Physiol. 1989; 257:E426-E438[Abstract/Free Full Text]
• Jones P.J.H., Pencharz P. B., Bell L., Clandinin M. T. Model for determination of ¹³C substrate oxidation rates in humans in the fed state. Am. J. Clin. Nutr. 1986; 41:1277-1282[Abstract/Free Full Text]
• Kim K. I., Elliott J. I., Bayley H. S. Oxidation of an indicator amino acid by young pigs receiving diets with varying levels of lysine or threonine, and an assessment of amino acid requirements. Br. J. Nutr. 1983a; 50:391-399[Medline]
• Kim K. I., McMillan I., Bayley H. S. Determination of amino acid requirements of young pigs using an indicator amino acid. Br. J. Nutr. 1983b; 50:369-382[Medline]
• Lazaris-Brunner G., Rafii M., Ball R. O., Pencharz P. B. Tryptophan requirements in young adult women using indicator amino acid oxidation. Am. J. Clin. Nutr. 1998; 68:303-310[Abstract]
• Matthews D. E., Motil K. J., Rohrbaugh D. K., Burke J. F., Young V. R., Bier D. M. Measurement of leucine metabolism in man from a primed, continuous infusion of L-[1-¹³C]leucine. Am. J. Physiol. 1980; 238:E473-E479[Abstract/Free Full Text]
• Motil K. J., Bier D. M., Matthews D. E., Burke J. F., Young V. R. Whole body leucine and lysine metabolism studied with [1-¹³C]leucine and [alpha-¹⁵N]lysine: response in healthy young men given excess energy intake. Metab. Clin. Exp. 1981a; 30:783-791
• Motil K. J., Matthews D. E., Bier D. M., Burke J. F., Munro H. N., Young V. R. Whole-body leucine and lysine metabolism: response to dietary protein intake in young men. Am. J. Physiol. 1981b; 240:E712-E721[Abstract/Free Full Text]
• Motil K. J., Opekun A. R., Montandon C. M., Berthold H. K., Davis T. A., Klein P. D., Reeds P. J. Leucine oxidation changes rapidly after dietary protein intake is altered in adult women but lysine flux is unchanged as is lysine incorporation into VLDL-apolipoprotein B-100. J. Nutr. 1994; 124:41-51
• Patterson B. W., Hachey D. L., Cook G. L., Amann J. M., Klein P. D. Incorporation of a stable isotopically labelled amino acid into multiple human apolipoproteins. J. Lipid Res. 1991; 32:1063-1072[Abstract]
• Rosenblatt J., Chinkes D., Wolfe M., Wolfe R. R. Stable isotope tracer analysis by GC-MS, including quantification of isotopomer effects. Am. J. Physiol. 1992; 263:E584-E596[Abstract/Free Full Text]
• Sanchez M., El-Khoury A. E., Castillo L., Chapman T. E., Young V. R. Phenylalanine and tyrosine kinetics in young men throughout a continuous 24-h period, at a low phenylalanine intake. Am. J. Clin. Nutr. 1995; 61:555-570[Abstract/Free Full Text]
• SAS Institute Inc. SAS/STAT Guide For Personal Computers, 6th ed. SAS Institute, Cary, NC. Schoeller, D. A., Klein, P. D., Watkins, J. B., Heim, T. & MacLean, W. C., Jr. (1980) ¹³C abundances of nutrients and the effect of variations in ¹³C isotopic abundances of test meals formulated for ¹³C O₂ breath tests. Am. J. Clin. Nutr. 1985; 33:2375-2385[Abstract/Free Full Text]
• Souba W. W., Pacitti A. J. How amino acids get into cells: mechanisms, models, menus, and mediators. [Review]. J. Parent. Enteral Nutr. 1992; 16:569-578[Abstract/Free Full Text]
• Stoll B., Henry J., Reeds P. J., Yu H., Jahoor F., Burrin D. G. Catabolism dominates the first-pass intestinal metabolism of dietary essential amino acids in milk protein-fed piglets. J. Nutr. 1998; 128:606-614[Abstract/Free Full Text]
• Waterlow, J. C., Garlick, P. J. & Millward, D. J. (1978a) Protein Turnover in Mammalian Tissues and in the Whole Body, pp. 225-249. North Holland Publishing Co., Amsterdam, The Netherlands.
• Waterlow J. C., Golden M. H., Garlick P. J. Protein turnover in man measured with ¹⁵N: comparison of end products and dose regimes. Am. J. Physiol. 1978b; 235:E165-E174[Abstract/Free Full Text]
• Wykes L. J., Ball R. O., Menendez C. E., Ginther D. M., Pencharz P. B. Glycine, leucine, and phenylalanine flux in low-birth-weight infants during parenteral and enteral feeding. Am. J. Clin. Nutr. 1992; 55:971-975[Abstract/Free Full Text]
• Wykes L. J., Ball R. O., Menendez C. E., Pencharz P. B. Urine collection as an alternative to blood sampling: a noninvasive means of determining isotopic enrichment to study amino acid flux in neonates. Eur. J. Clin. Nutr. 1990; 44:605-608[Medline]
• Zello G. A., Marai L., Tung A. S., Ball R. O., Pencharz P. B. Plasma and urine enrichments following infusion of L-[1-¹³C]phenylalanine and L-[ring-²H₅]phenylalanine in humans: evidence for an isotope effect in renal tubular reabsorption. Metab. Clin. Exp. 1994; 43:487-491
• Zello G. A., Pencharz P. B., Ball R. O. The design and validation of a diet for studies of amino acid metabolism in adult humans. Nutr. Res. 1990a; 10:1353-1365
• Zello G. A., Pencharz P. B., Ball R. O. Phenylalanine flux, oxidation, and conversion to tyrosine in humans studied with L-[1-¹³C]phenylalanine. Am. J. Physiol. 1990b; 259:E835-E843[Abstract/Free Full Text]
• Zello G. A., Pencharz P. B., Ball R. O. Dietary lysine requirement of young adult males determined by oxidation of L-[1-¹³C]phenylalanine. Am. J. Physiol. 1993; 264:E677-E685[Abstract/Free Full Text]
• Zello G. A., Smith J. M., Pencharz P. B., Ball R. O. Development of a heating device for sampling arterialized venous blood from a hand vein. Ann. Clin. Biochem. 1990c; 27:366-371
• Zello G. A., Telch J., Clarke R., Ball R. O., Pencharz P. B. Reexamination of protein requirements in adult male humans by end-product measurements of leucine and lysine metabolism. J. Nutr. 1992; 122:1000-1008
• Zello G. A, Wykes L. J., Ball R. O., Pencharz P. B. Recent advances in methods of assessing amino acid requirements for adult humans. J. Nutr. 1995; 125:2907-2915

0022-3166/98 $3.00 ©1998 American Society for Nutritional Sciences