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Article

Prior Protein Intake May Affect Phenylalanine Kinetics Measured in Healthy Adult Volunteers Consuming 1 g Protein · kg^-1 · d^-1

^a Departments of Nutritional Sciences and ^b Paediatrics, University of Toronto, ^c The Research Institute, The Hospital for Sick Children, Toronto, Ontario, Canada, M5G 1X8 and ^d Department of Agricultural, Food and Nutritional Sciences, University of Alberta, Edmonton, Canada, T6G 2P5

	ABSTRACT

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Study of the amino acid metabolism of vulnerable groups, such as pregnant women, children and patients, is needed. Our existing protocol is preceded by 2 d of adaptation to a low ¹³C formula diet at a protein intake of 1 g · kg^-1 · d^-1 to minimize variations in breath ¹³CO₂ enrichment and protein metabolism. To expand on our potential study populations, a less invasive protocol needs to be developed. We have already established that a stable background ¹³CO₂ enrichment can be achieved on the study day without prior adaptation to the low ¹³C formula. Therefore, this study investigates phenylalanine kinetics in response to variations in prior protein intake. Healthy adult subjects were each fed nutritionally adequate mixed diets containing 0.8, 1.4 and 2.0 g protein · kg^-1 · d^-1 for 2 d. On d 3, subjects consumed an amino acid-based formula diet containing the equivalent of 1 g protein · kg^-1 · d^-1 hourly for 10 h and primed hourly oral doses of L-[1-¹³C]phenylalanine for the final 6 h. Phenylalanine kinetics were calculated from plasma-free phenylalanine enrichment and breath ¹³CO₂ excretion. A significant quadratic response of prior protein intake on phenylalanine flux (P = 0.012) and oxidation (P = 0.009) was identified, such that both variables were lower following adaptation to a protein intake of 1.4 g · kg^-1 · d^-1. We conclude that variations in protein intake, between 0.8 and 2.0 g · kg^-1 · d^-1, prior to the study day may affect amino acid kinetics and; therefore, it is prudent to continue to control protein intake prior to an amino acid kinetics study.

KEY WORDS: • phenylalanine • dietary protein • humans • amino acids • stable isotopes

	INTRODUCTION

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Stable isotopes have been used extensively to study amino acid kinetics and to determine amino acid requirements in vivo (Fuller & Garlick 1994 , Waterlow 1996 , Young et al. 1989 , Zello et al. 1995 ). Recently, we developed a new experimental model of indicator amino acid oxidation for measuring amino acid kinetics and assessing indispensable amino acid requirements (Zello et al. 1995 ). This model is based on the concept that when an indispensable amino acid is limiting for protein synthesis, all other amino acids are in relative excess and will therefore be oxidized. These methods were originally developed for the study of amino acid requirements in young pigs (Ball and Bayley 1984 ) using radioisotopes. We adapted this model for the measurement of amino acid kinetics in adult humans (Duncan et al. 1996 , Lazaris-Brunner et al. 1998 , Zello et al. 1993 ) using stable isotopes. To date, most of the studies were conducted in healthy adult volunteers. The work described in this paper is part of a series of studies we have been carrying out in order to simplify our current methods and make them applicable to a wider range of study populations.

Briefly, our original protocol involves a 2-d adaptation period to a low ¹³C formula diet at a protein intake of 1 g · kg^-1 · d^-1, followed by measurement of amino acid kinetics over an 8-h period using a stable-isotope tracer infusion in the fed state (Zello et al. 1990a ), sampling either plasma (Zello et al. 1990c ) or urinary (De Benoist et al. 1984 , Wykes et al. 1990 , Zello et al. 1994 ) free amino acid enrichments. In comparison to previous methods employed to measure amino acid kinetics and establish requirements using stable isotopes, this experimental protocol is relatively noninvasive and the study period is short-term (Zello et al. 1995 ). Our latest paper describes the measurement of lysine and phenylalanine kinetics following oral infusion of the isotope (Bross et al. 1998 ). By using this protocol, we have the potential to study groups that have not been investigated, such as infants, children and nonhealthy subjects. However, it would be advantageous to be able to complete the measurements in a single day, without prior adaptation to the formula diet. This is of particular importance in patient groups for whom a period of dietary control may impede treatment and cause undue stress.

The formula diet consists of a mixture of free amino acids, an amino nitrogen-free liquid formula and protein-free cookies, allowing precise modification of the amino acid, protein and energy content of the diet (Zello et al. 1990b ). The diet provides adequate energy and protein with a constant ¹³C content, thus allowing a stable breath and whole body ¹³C enrichment to be established. An individual's ¹³C enrichment is determined by their typical dietary intake of ¹³C, which varies significantly (Schoeller et al. 1980 ). A low and stable background ¹³C enrichment is essential in order to accurately determine increments in enrichment due to the administration of ¹³C-labeled amino acids. In healthy adult subjects, a stable breath ¹³C enrichment can be achieved after four, hourly formula meals, indicating a stable background enrichment in the free amino acid pool (Bross et al. 1998 ). Thus, apparently no prior adaptation to a low ¹³C intake is necessary as long as the baseline enrichments in breath and plasma are measured following a minimal period in the fed state on the formula diet. For the 2 d prior to the study day, this has meant that it is possible to use a more acceptable source of dietary protein for the population being studied, rather than the amino acid-based formula diet. This improved study protocol was successfully implemented to establish a requirement for dietary tyrosine in children with phenylketonuria (Bross 1997 ). As part of the treatment of this inborn error of metabolism, these children are maintained on a controlled protein intake, similar to the intake of protein on the study day. However, to study other vulnerable groups, whose diet is not controlled, it is necessary to establish whether adaptation to a constant protein intake is indeed necessary.

The aim of this study was to investigate the effects, if any, of variations in the intake of protein on the 2 d prior to measuring amino acid kinetics, thus helping to determine whether it is necessary to control dietary protein intake prior to investigating amino acid metabolism at an intake of 1 g protein · kg^-1 · d^-1 on the study day. Hoerr et al. (1993) and Motil et al. (1994) showed that adaptation to a different protein intake is complete within 48 h. Therefore, our objective was to investigate L-[1-¹³C]phenylalanine kinetics, at an adequate protein intake of 1 g · kg^-1 · d^-1, following 2 d of adaptation to different protein intakes, over a range considered to be representative of usual intakes in this adult population. We chose to investigate the metabolism of L-[1-¹³C]phenylalanine because this is used as the indicator amino acid for the majority of our oxidation studies.

	SUBJECTS AND METHODS

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Subjects.

Six healthy adult volunteers participated in the study on an outpatient basis in the Clinical Investigation Unit at The Hospital for Sick Children (HSC),⁴Toronto, Canada. Subject characteristics are detailed in Table 1. None of the subjects had a history of recent weight loss, endocrine disorders or medication use. The design and aims of the study, as well as potential risks involved, were fully explained to each subject and written consent was obtained. All procedures during the study were approved by the University of Toronto Human Experimental Committee and the Human Subjects Review Committee of HSC. Subjects received financial compensation for their participation.

Each subject was randomly assigned to each of three dietary protein levels of 0.8, 1.4 and 2.0 g · kg^-1 · d^-1. Each study consisted of 2 d of the prescribed protein intake followed by a single study day for the measurement of phenylalanine kinetics, using L-[1-¹³C]phenylalanine, at a protein intake of 1g · kg^-1 · d^-1. Study periods were separated by at least 1 wk, with subjects completing all three studies within 2 mo.

Diet and energy intakes.

Dietary intakes at the three protein levels were prescribed by a registered dietitian based on the usual food intake of the individual subjects as assessed by a brief diet history. For each of the dietary intakes, protein was provided from the same foods in order to maintain constant proportions in the intake of dietary amino acids. Energy intakes were based on the FAO/WHO/UNU predictive equations (FAO/WHO/UNU 1985 ), multiplied by an activity factor of 1.7. Subjects were provided with a detailed diet prescription, a set of diet scales and a measuring cup. All diets were agreed upon by the subjects prior to each dietary period, and subjects were encouraged to space the dietary intake throughout the day as three main meals and intermittent snacks. No other food or beverages, including diet products containing artificial sweeteners, were consumed during the 2-d adaptation period. Compliance with the diet was assessed post-hoc from analysis of plasma urea (Kesteloot and Joossens 1993 ) and the urinary urea-to-creatinine ratio (Middendorf et al. 1986 ) of spot samples taken on the morning of the study day (Lee and Arroyave 1966 ).

On the study day the diet was provided as a low ¹³C experimental formula developed for amino acid kinetic studies (Zello et al. 1990b ). Energy intakes were prescribed as above. The protein intake of 1 g · kg^-1 · d^-1 was provided entirely as a crystalline amino acid mixture based on the composition of intact egg protein, in keeping with our usual study protocol to minimize variation in the dietary intake of ¹³C on the study day. The amount of L-[1-¹³C]phenylalanine given during the study day was subtracted from the dietary provision of phenylalanine. The total intake of phenylalanine was 14 mg · kg^-1 · d^-1 with a tyrosine intake of 40 mg · kg^-1 · d^-1 to ensure adequacy, as determined previously (Zello et al. 1990a ).

The total duration of each isotope study was 10 h. The experimental diets were prepared in the research kitchen of HSC and portioned into 10 hourly meals each providing one twelfth of the total daily intake of protein and energy. Subjects had free access to water, but no other food or beverage was taken throughout the length of the study day.

Tracer protocol.

The tracers used in this study included NaH¹³CO₃ (99 atom percent) and L-[1-¹³C]phenylalanine (99 atom percent), purchased from Merck, Sharp and Dohme (Montreal, Quebec) and Mass Trace (Woburn, MA), respectively. Isotope solutions were prepared in deionized water and stored at -20°C. Before dispensing, the isotope solutions were sterilized through a 0.22 µm Millipore filter (Millipore Corporation, Bedford, MA). Oral priming doses of NaH¹³CO₂ (0.176 mg/kg) and L-[1-¹³C]phenylalanine (0.664 mg/kg) were given with the fifth hourly meal. Simultaneously, an hourly oral dosing protocol of L-[1-¹³C]phenylalanine (1.2 mg · kg^-1 · d^-1) was commenced and continued throughout the remaining 6 h of the study.

Sample collection and analysis.

We showed that urine can be used as a means of determining plasma amino acid enrichments (Wykes et al. 1990 , Zello et al. 1994 ). We chose to sample the plasma pool directly, rather than via the urine, since there was no restriction on intravenous access in this healthy adult population. Baseline samples of breath CO₂ and plasma were collected at 30, 20 and 10 min before the initiation of the isotope protocol. As predicted (Bross et al. 1998 ), a background (baseline) isotopic steady state was achieved in all subjects within 4 h of commencing feeding. Samples were also collected at isotopic steady state, during the period 210 to 330 min following the commencement of the isotope protocol, at 20-min intervals. Breath samples were collected for 7 min through a ventilated mask and passed through a vacuum extraction system (Pump VB0025, Vortex Blower; Spencer Turbine Company, Windsor, CT). Breath CO₂ was collected by trapping in 1 mol/L NaOH solution. To ensure complete trapping, respiratory gases were bubbled through 10 mL of 1 mol/L NaOH in a modified reflux condenser at a rate of 500 mL/min. The resulting Na₂CO₃ solution was then injected into a vacutainer (Vacutainer Brand 6441; Becton Dickenson Inc., Mississauga, Ontario, Canada) and stored at -20°C. Carbon dioxide production was measured twice during the study day for 20 min, using an indirect calorimeter (2900 Computerized Energy Measurement System; Sensormedics, Yorba Linda, CA).

The enrichment of ¹³C in breath CO₂ was measured on a dual inlet isotope ratio mass spectrometer (Vacuum Generator Micromass 602D, Cheshire, England) using techniques described previously (Jones et al. 1985 ). Breath ¹³CO₂ Enrichments were expressed as atoms percent excess over a reference standard of compressed CO₂ gas.

At the beginning of the study day, a 21-gauge needle was inserted into a superficial dorsal vein in the right hand. This remained in situ throughout the length of the study day. The 2-mL samples of arterialized venous blood were collected into heparinized syringes (Aspirator^TM; Marquest Medical Products, Englewood, CO) and placed on ice. Arterialized venous blood was obtained by heating the hand inside a thermostatic chamber maintained at 60°C (Zello et al. 1990c ). Plasma was extracted following centrifugation at 4°C at 1500 x g for 20 min and stored at -20°C until analysis.

Plasma-free [¹³C]phenylalanine enrichment was measured by gas chromatography–[selected ion monitoring-negative chemical ionization]–mass spectrometry (Hewlett-Packard 5890 Series; GC, Mississauga, Ontario; VG Trio-2 quadropole MS system, Cheshire, England). Free amino acids in 200 µL of plasma were derivatized according to he method described by Patterson et al. (1991) to their heptafluorobutyryl n-propyl esters. Selected-ion chromatograms were obtained by monitoring mass-to-charge ratios of 383 and 384 for [¹³C]phenylalanine corresponding to the unenriched (m) and enriched (m + 1) peaks, respectively. The areas under the peaks were integrated by a digital DECp 450₂LP computer, using a Lab-Base program (VG Biotech, Altringham, England).

Estimation of isotope kinetics.

Phenylalanine kinetics were calculated according to the stochastic model of Matthews et al. (1980) , previously employed by Zello et al. (1990a) . Isotopic steady state in the metabolic pool was represented by plateau in free-[¹³C]phenylalanine in plasma and ¹³CO₂ in breath, plateau being defined by the absence of a significant slope, assessed by linear regression analysis. The mean breath ¹³CO₂ enrichments of the three baseline and the five plateau samples were used to determine atoms percent excess above baseline at isotopic steady state. The mean ratios of the enriched peak (m + 1) to the unenriched (m) for both the baseline and plateau samples were used to calculate molecules percent excess.

Phenylalanine flux (µmol · kg^-1 · h^-1) was measured during isotopic steady state from the dilution of the L-[1-¹³C]phenylalanine infused into the plasma metabolic pool. The rate of ¹³CO₂ released by phenylalanine tracer oxidation (F¹³CO₂: µmol ¹³CO₂ · kg^-1 · h^-1) was calculated according to Matthews et al. (1980) from the expiration of ¹³CO₂, using a factor of 0.82 to account for the ¹³CO₂ retained in the body because of bicarbonate fixation (Hoerr et al. 1989 ). The rate of phenylalanine oxidation (µmol · kg^-1 · h^-1) was calculated from the F¹³CO₂ and the plasma free phenylalanine enrichment (Matthews et al. 1980 ).

Data analysis.

Repeated measures analysis of variance was performed on the results for urinary urea to creatinine ratio and plasma urea to determine compliance with the dietary regimen (Proc GLM, SAS 6.12 for Windows; SAS Institute Inc., Cary, NC). Analysis of variance was also performed to assess the effect of prior protein intake on phenylalanine flux, tracer oxidation and phenylalanine oxidation (Proc GLM, SAS 6.12 for windows, SAS Institute Inc.), with post-hoc testing by Duncan's multiple-range test. Where an effect was identified, the data were analyzed for both linear and quadratic responses (Proc Mixed, SAS 6.12 for Windows, SAS Institute Inc.). Differences were considered significant at P 0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

 
The subjects' usual protein intake, estimated by diet history, ranged from 1.0 to 1.5 g · kg^-1 · d^-1. Both the urinary urea to creatinine ratio (P = 0.035) and plasma urea (P = 0.001) results were higher for each subject with increasing prior protein intake as shown in Table 2, indicating a degree of compliance with the prescribed dietary regimen.

View larger version (15K): [in this window] [in a new window]   Figure 1. Label oxidation (F13CO2) in healthy adult subjects in response to 2 d prior protein intake. Regression analysis showed no significant effect of prior protein intake on F13CO2 (P = 0.104). Phe, phenylalanine.

View larger version (17K): [in this window] [in a new window]   Figure 2. Phenylalanine (Phe) flux in healthy adult subjects in response to 2 d prior protein intake. Regression analysis showed a significant quadratic response of prior protein intake on phenylalanine flux (P = 0.012), such that flux was lower following 2-d adaptation to a protein intake of 1.4 g · kg-1 · day-1 than following a prior protein intake of 0.8 or 2.0 g · kg-1 · day-1.

	DISCUSSION

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

Previous studies provided limited information about the effect of acute changes in protein intake on amino acid kinetics. Motil et al. (1994) showed that, in response to changes in protein intake from 1.0 g · kg^-1 · day^-1 to either 0.4 or 1.5 g · kg^-1 · day^-1, alterations in the peak ¹³C enrichment of CO₂ following a bolus dose of ¹³C-leucine are complete after 2 d. Hoerr et al. (1993) also showed that 80% of the changes in both leucine and lysine flux, in response to changes in protein intake, occur within 1 d. The study detailed here shows that in healthy adult volunteers the intake of protein on the preceding 2 d may have an effect on amino acid kinetics measured on the study day. Prior protein intakes were controlled at 0.8, 1.4 and 2.0 g · kg^-1 · day^-1. These intakes were chosen to ensure an intake at or above requirement for all subjects and to encompass wide variation in daily protein intakes. Phenylalanine kinetics were measured at a protein intake of 1 g · kg^-1 · d^-1, according to our existing protocol. Adequate energy intakes were maintained throughout the study period to avoid any effect on protein metabolism. A significant quadratic response was identified such that results for phenylalanine flux (P = 0.012) and oxidation (P = 0.009) were higher following protein intakes of both 0.8 and 2.0 g · kg^-1 · day^-1, compared to 1.4 g · kg^-1 · d^-1.

Both plasma urea and the urinary urea-to-creatinine ratio responded to increments in protein intake, as shown in Table 2 , indicating compliance with the dietary prescription for the 24 h before the study (Kesteloot and Joossens 1993 , Middendorf et al. 1986 ). These samples were taken on the morning of study d 3 until 4 h after commencement of the formula diet and are therefore partially influenced by the intake of protein on the study day. In another study, samples from normal individuals were taken 3.5 h following consumption of a standard protein meal (0.4 g/kg) indicating stability in both plasma urea levels at 5.4 ± 0.3 mmol/L and urinary urea-to-creatinine ratio at 30.8 ± 2.1 µmol/µmol, without any restriction on the intake of protein on the preceding 2 d (Cheema-Dhadi & Halperin, 1993 ). This paper also indicates that there is minimal diurnal variation in the urinary urea-to-creatinine ratio in normal individuals consuming a regular diet and carrying out normal daily activities.

The observation of a significant quadratic response may be interpreted merely as a statistical finding, resulting from natural variation in the estimation of amino acid kinetics. In this study there does not appear to be higher within-subject variability in comparison to previous studies (Lazaris-Brunner et al. 1998 , Zello et al. 1993 ), which might have been expected if prior protein intake did indeed alter phenylalanine kinetics. Slightly higher within-subject variation may also be expected due to the less-stringent study conditions. Apart from the changes in prior protein intake, this study differs from these previous studies in that the isotopes were administered orally and the subjects were adapted to a mixed diet, rather than an experimental formula, of the 2 d prior to the study day. Also, the timing of the menstrual cycle and the use of oral contraceptives in our female volunteers were not controlled for, as was done by Lazaris-Brunner et al. (1998) . Zello et al. (1993) found within-subject coefficient of variation (CV) of 3 to 16% for flux rates in adult males at intakes above their individual requirement for the test amino acid. Lazaris-Brunner et al. (1998) found within-subject CV of 1 to 9% in adult females at intakes above requirement for the test amino acid. The within-subject CV in flux for the present study ranged from 12 to 19% in males and 2 to 11% in females. These appear to be comparable to our earlier data from amino acid oxidation protocol and, hence, suggest that the quadratic response observation may simply be a statistical finding.

Alternatively, a biochemical mechanism could be sought to explain this response. Differences in phenylalanine kinetics, measured at a protein intake of 1.0 g · kg^-1 · d^-1, may indicate that there was a carryover effect of the adaptation to the previous protein intake. The increase in phenylalanine flux shown with a prior protein intake of 2.0 g · kg^-1 · d^-1 could be due to adaptation to the higher intake of protein. According to Motil et al. (1994) two-thirds of the increase in flux in response to a higher protein intake can be attributed to a greater rate of oxidation. In the study presented here, following 2 d of consuming 2.0 g of protein · kg^-1 · d^-1, phenylalanine oxidation as also increased in response to this higher prior intake of protein. Therefore, there was a carryover effect on oxidation of the high intake of protein prior to the study day.

The quadratic response shown for the effect of prior protein intake on phenylalanine oxidation may be due directly to the response identified in the flux results. No significant effect was seen for F¹³CO₂, the rate of excretion of the label in breath CO₂ and a marker of the oxidation of the labeled amino acid. The rate of phenylalanine oxidation is calculated from the estimates of phenylalanine flux and F¹³CO₂. Calculation of the rate of oxidation involves greater error than the independent measurement of its components, flux and F¹³CO₂. Amino acid requirements from IAAO studies may be estimated from F¹³CO₂ results alone, rather than from calculated oxidation results (Duncan et al. 1996 , Zello et al. 1993 ). Therefore, it may not be necessary to adapt subjects to a constant protein intake when only measurements of label oxidation are required.

Following the lowest protein intake in the present study (0.8 g · kg^-1 · d^-1), higher rates of phenylalanine flux and oxidation were observed at the study intake of 1 g protein · kg^-1 · d^-1 than following a prior protein intake of 1.4 g · kg^-1 · d^-1. This observation is not consistent with the concept of adaptation to the prior intake of protein. Hoerr et al. (1993) fed a diet deficient in protein (0.1 g · kg^-1 · d^-1) for 1 wk and the following day measured leucine and lysine fluxes at a higher protein intake (1.5 g · kg^-1 · d^-1). Both leucine and lysine fluxes were only 80% of expected at this protein intake, indicating adaptation to the previously low intake. However, the metabolic response to such a variation in intake (0.1 vs. 1.5 g · kg^-1 · d^-1) may not be comparable to the variations in intakes of protein in this study.

In conclusion, protein intake on the 2 d prior to the study day appears to influence the rate of phenylalanine kinetics measured on the study day. The mechanism by which prior protein intake affects amino acid kinetics cannot be explained biochemically, and may be merely a statistical finding. It is unlikely that the number of subjects involved in this study is too small, as we have repeatedly shown that amino acid requirements can be defined from the response in amino acid oxidation following intravenous isotope infusion from just six subjects. More recently, such a response was also observed using oral administration of the isotopes (Bross 1997 ). At present, it is prudent that protein intake be controlled for 2 d prior to studies of phenylalanine metabolism.

	FOOTNOTES

 
¹ To whom correspondence should be addressed.

¹ Supported by Medical Research Council of Canada, Grant MT 10321; Mead Johnson Canada, Protein-Free Powder.

² The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked ''advertisement'' in accordance with 18 USC section 1734 solely to indicate this fact.

³ Abbreviations used: CV, coefficient of variation; HSC, The Hospital for Sick Children; IAAO, indicator amino acid oxidation.

Manuscript received May 19, 1998. Initial review completed July 7, 1998. Revision accepted November 2, 1998.

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