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Am J Physiol Endocrinol Metab 287: E489-E496, 2004; doi:10.1152/ajpendo.00262.2003
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Phase of menstrual cycle affects lysine requirement in healthy women

Wantanee Kriengsinyos,1,3 Linda J. Wykes,4 Laksiri A. Goonewardene,5 Ronald O. Ball,1,5 and Paul B. Pencharz1,2,3,5

Departments of 1Nutritional Sciences and 2Paediatrics, University of Toronto, Toronto M5S 3E2; 3The Research Institute, The Hospital for Sick Children, Toronto, Ontario M5G 1X8; 4School of Dietetics and Human Nutrition, McGill University, Montreal, Quebec H9X 3V9; and 5Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Alberta T6G 2P5, Canada

Submitted 12 June 2003 ; accepted in final form 1 April 2004


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The aim of this study was to investigate whether the phases of the menstrual cycle affect lysine requirement in healthy adult females, as determined by the indicator amino acid oxidation (IAAO) method. Five healthy females with regular menstrual cycles were studied at seven graded levels of lysine intake, in random order, with an oral [13C]phenylalanine tracer protocol in both the follicular and luteal phases. A total of 14 studies were conducted for each subject. Breath and plasma samples were collected according to the standard IAAO protocol. Serum 17{beta}-estradiol and progesterone concentrations were measured on each IAAO study day. The rate of release of 13CO2 from [13C]phenylalanine oxidation (F13CO2) was measured, and a two-phase linear regression crossover model was applied to determine lysine requirement. F13CO2 was higher during the luteal phase (P < 0.001) and was positively associated with serum concentrations of 17{beta}-estradiol and progesterone. The F13CO2 data were adjusted for subjects and sex hormones and used to define breakpoints for lysine requirements. The lysine requirement of healthy females in the luteal phase was 37.7 mg·kg–1·day–1 and higher (P = 0.025) than that of females in the follicular phase (35.0 mg·kg–1·day–1). At all lysine intake levels, plasma amino acids were lower and phenylalanine oxidation was higher in the luteal relative to the follicular phase. Therefore, we reason that the higher lysine requirement observed in the luteal phase is probably due to higher amino acid catabolism.

indicator amino acid oxidation

STUDIES ON THE REQUIREMENT of indispensable amino acids (IDAAs) in women, especially using the advanced carbon oxidation technique, are scanty. In fact, there has been only one study conducted specifically on women by use of the indicator amino acid oxidation (IAAO) technique to determine tryptophan requirement in women during the follicular phase (23). A few studies looked at IDAA requirements using both male and female subjects together in the experiments (2, 12, 27, 28, 35). Two of the five studies (2, 12) were conducted in female subjects during the 7–10 days after the onset of menstrual bleeding, whereas the other three studies (27, 28, 35) did not control for menstrual phases of the female subjects. The lack of female studies is possibly due to the complexity imposed on the experimental design by including the menstrual cycle.

Some evidence exists regarding metabolic changes associated with the menstrual cycle. Increased basal metabolic rate (37) and 24-h energy expenditure (40), higher nitrogen excretion (7), and lower plasma amino acid concentrations (29) have been reported in women in the luteal vs. the follicular phase of the menstrual cycle. The use of tracer techniques has produced conflicting results regarding protein or amino acid turnover during the different phases of the menstrual cycle. Using [15N]glycine with an ammonia end product, Garrel et al. (15) reported that there was no difference in whole body protein turnover during the fed state between the two phases of the menstrual cycle. However, later the same group [Lariviere et al. (22)] reported that leucine turnover during the fasted state in the follicular phase was lower than in the luteal phase. In addition, it was found that leucine oxidation (22) and tryptophan catabolism (18) increased during the luteal phase. Fluctuations in sex hormones provide an appealing explanation for the metabolic differences observed between the two phases of the menstrual cycle. Progesterone and estrogen levels are higher during the luteal compared with the follicular phase. Toth et al. (38) reported positive relationships between estradiol and rate of leucine appearance and between estradiol and leucine oxidation.

Presently, it has been accepted that the IAAO technique is a suitable method to define amino acid requirements in humans (34). Changes in amino acid turnover and metabolism that occur during the menstrual phase may affect amino acid requirement; however, this issue has never been investigated. Because of limited data that exist on amino acid requirements in women and no clear evidence of differences in amino acid requirements between the sexes, currently the dietary reference intake (DRI) for amino acid requirements in women is set the same as those of men. Furthermore, no consideration was given to the phases of the menstrual cycle (13). Whether this current DRI is appropriate or not is not known. Therefore, the aim of the present study was to investigate whether menstrual cycle phase in women is an important factor in defining IDAA requirements. Lysine requirements in healthy women in both the follicular and luteal phases were studied by using the IAAO method. Furthermore, because L-[1-13C]phenylalanine was used as an IDAA, whether the rate of metabolism of phenylalanine differs between the two phases of the menstrual cycle was also investigated. To answer this question, it was important that all of the female subjects be studied at several levels of lysine intake in both the follicular and luteal phases of the menstrual cycle. Therefore, the less invasive IAAO method with oral tracer protocol (4) was selected and used in the present study to minimally burden subjects and be able to complete the studies in a reasonable time frame.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Study subjects. Five healthy adult females participated in the study on an outpatient basis in the Clinical Investigation Unit at The Hospital for Sick Children, Toronto, Canada. Their characteristics are described in Table 1. All subjects were apparently healthy with regular menstrual cycles during the previous 12 mo. Their regular menstrual length was obtained by interviews and from individual subjects' records of the 2–3 mo preceding their participation in the study. None of the subjects used any oral contraceptive pills or other birth control devices that would affect their sex hormone profiles during the previous 2 yr. None of the subjects had a history of unusual dietary practices, recent weight loss, or endocrine disorders or were on any medications before or during the study. The nature, purposes, and the possible risks of the study were fully explained to each subject before she gave her written consent to participate. The study protocol was approved by the Research Ethics Board of The Hospital for Sick Children. All volunteers were remunerated for their participation.


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  		Table 1. Characteristics and energy intakes of adult females participating in the study

 
Experimental design. Each subject was studied at seven graded levels (10, 25, 30, 35, 40, 45, and 60 mg·kg–1·day–1) of lysine test intake, in random order, in both the follicular and luteal phases of the menstrual cycle (14 studies in total). For the follicular phase, the study was conducted on days 3–7 immediately after the 1st day of menstrual bleeding and for the luteal phase 4–7 days before the onset of next menstrual bleeding. The IAAO study day of the luteal phase was set on the basis of each subject's length of the menstrual cycle, and the length and regularity of the cycle were determined in the initial interview. The phase of the cycle was reconfirmed by measuring the concentrations of 17{beta}-estradiol and progesterone on each IAAO study day. One or two IAAO studies per subject were conducted during each phase for each menstrual cycle. Overall, it took 5–6 mo per subject to complete all lysine levels in both phases.

Subjects were weighed on each IAAO study day to ensure accurate prescription of diets and isotopes, and to confirm weight maintenance throughout the study. Each study consisted of a 2-day adaptation period to a prescribed diet in accordance with their energy requirement. The diet provided 1.0 g protein·kg–1·day–1 and was followed by a single study day on which phenylalanine (Phe) kinetics were measured with the use of L-[1-13C]Phe and a crystalline amino acid intake of 1.0 g·kg–1·day–1. Amino acid concentrations were also measured at the end of each IAAO study day.

Dietary intake and experimental diet. Dietary intake during the adaptation period (2 days before IAAO study) was provided in the form of milk shakes (Scandishake; Scandipharm, Birmingham, AL), with added carbohydrate (Caloreen; Nestle Clinical Nutrition, North York, ON, Canada), protein (Promod; Ross Laboratories, Columbus, OH) and 3.25% homogenized milk to tailor the formula to the exact energy and protein intake required. The diet was given as three meals and snacks spread throughout the day as normally consumed by each subject. Energy intake was based on each subject's resting metabolic rate (RMR) after a 12-h overnight fast, as determined by continuous, open-circuit, indirect calorimetry (2900 Computerized Energy Measurement System-Paramagnetic; Sensormedics, Yorba Linda, CA) and multiplied by an activity factor of 1.7. RMR was measured at least twice, in both the follicular and luteal phases. When a significant difference was observed, the highest RMR was used in the calculation of dietary energy intake given to individual subjects. No other food or beverages were consumed except water, clear tea, or clear coffee. Subjects also consumed a daily multivitamin supplement (Centrum; Whitehall-Robins, Mississauga, ON, Canada) throughout the study.

The experimental diet used during each IAAO study consisted of a liquid formula and protein-free cookies as previously developed for amino acid kinetic studies (42). The study protocol on each IAAO is depicted in Fig. 1. The diet was divided into eight isocaloric, isonitrogenous meals representing one-twelfth of the subject's daily requirements. Hourly meals were consumed to ensure metabolic steady state in the fed condition (14, 44). The diet provided 37% of energy from fat, 53% from carbohydrate, and 10% from protein. The main source of energy came from a flavored, nonprotein liquid formula diet [Protein-Free Powder, product 80056 (Mead Johnson, Evansville, IN); Tang (Kraft, Don Mills, ON, Canada); Koolaid (Don Mills)]. The protein intake was provided at a level of 1 g·kg–1·day–1 supplied as a crystalline L-amino acid mixture based on the amino acid composition of egg protein. The only kinds of amino acid that diverged from this profile were Phe, lysine, and alanine. The intake of the IDAA Phe was fixed at 15 mg·kg–1·day–1 (which included the amount of L-[1-13C]Phe administered during the tracer) with an excess tyrosine intake (40 mg·kg–1·day–1). This level of Phe intake was previously determined to meet the requirements of 95% of adult men when tyrosine was in excess (43). Lysine was provided at seven graded levels, as mentioned earlier. Alanine intake was adjusted to maintain a constant nitrogen intake.



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  		Fig. 1. Protocol for determining phenylalanine (Phe) oxidation in female subjects on each indicator amino acid oxidation study day. 1The experimental diet was a liquid formula (crystalline amino acid diet with randomly graded levels of lysine intake at 10, 25, 30, 35, 40, 45 and 60 mg·kg–1·day–1) and protein-free cookies. The diet was provided hourly for 8 h. Each meal was isocaloric and isonitrogenous and represented 1/12 of each subject's daily requirement. 2Priming doses of L-[1-13C]Phe and NaH13CO3 were started at the 5th meal, and then a simulated continuous dose of L[1-13C]Phe was commenced simultaneously and continued hourly throughout the remaining 4 h of study. 3Two baseline plasma and breath samples were collected at 15 and 45 min before the isotope protocol began. Four plateau plasma and breath samples were collected at isotopic steady state every 30 min during the period 150–240 min after initiation of the isotope protocol. 4CO2 production rate (CO2) was measured by indirect calorimetry after 4 h of consuming the experimental diet.

 
Tracer protocol. Two tracers, NaH13CO3 (99%) and L-[1-13C]Phe (99%; Cambridge Isotope Laboratories, Woburn, MA), were used in this study. Isotopic and optical purity of L-[1-13C]Phe was verified by the manufacturer of the isotopes with gas chromatography-mass spectrometry (GC-MS) and nuclear magnetic resonance. The enrichment and enantiomeric purity of the L-[1-13C]Phe were reconfirmed by GC-MS of the n-propyl, heptofluorobutyramide derivative (32), using a chiral column (ChirasilVal, R symbol; Alltech Associates, Deerfield, IL). The measured fractional molar abundance of L-[1-13C]Phe was 97.5%. This value was used in the calculation of Phe turnover. The tracers were prepared in deionized water and stored at –20°C until used.

Priming doses of NaH13CO3 (2.071 µM/kg) and L-[1-13C]Phe (3.995 µM/kg) were started at the fifth meal. Four-hour adaptation to experimental diet was needed to ensure sufficient time for 13CO2 background equilibration in expired air (4). A simulated continuous dose of L-[1-13C]Phe (7.991 µM·kg–1·h–1) was commenced simultaneously and continued every hour throughout the remaining 4 h of the study.

Sample collection. Two baseline and four plateau blood samples (3 ml each) were collected in heparinized tubes for the analysis of [13C]Phe enrichment in plasma. Another 5 ml of clotted blood were collected from each study for the analysis of 17{beta}-estradiol and progesterone. Blood was collected from a 20-gauge needle inserted into a superficial dorsal or antecubetal vein. The line was kept patent by administering isotonic normal saline between blood samplings. To obtain arterialized venous blood, the arm was placed inside a thermostatic chamber maintained at 60°C for 15 min before the blood was sampled (45). Baseline arterialized blood samples were collected at 45 and 15 min before the initiation of the isotope infusion. Plateau arterialized blood samples (isotope steady state) were collected during the period of 150–240 min after the initiation of the isotope protocol, at 30-min intervals. At 240 min, blood was also collected for the analysis of amino acid concentrations. Blood samples were centrifuged at 1,500 g for 10 min at 4°C; plasma was removed and stored at –20°C for subsequent analysis.

Baseline and plateau breath samples were collected simultaneously with blood samples in Haldane-Priestley tubes (Venoject; Terumo Medical, Elkton, MD), using a collection mechanism that permits the removal of dead-space air. Breath samples were stored at room temperature pending analysis. Expired CO2 production and O2 consumption rates were measured using an indirect calorimeter (2900 Computerized Energy Measurement System; Sensormedics) 4 h after the experimental diet was consumed on each study day. Both breath and plasma enrichment achieved a satisfactory isotopic steady state similar to that we have previously reported (19, 36). The slope of the plateau enrichment in breath and plasma samples on each study day was not significantly different from zero.

Analytical procedure. Expired 13CO2 enrichment was measured by a continuous-flow isotope ratio mass spectrometer (CF-IRMS 20/20; PDZ Europa, Cheshire, UK) and was expressed as atom percent excess (APE) over a reference standard of compressed CO2 gas. After isolation by cation exchange (Dowex 50W-X8, 100–200 mesh H+; Bio-Rad Laboratories, Richmond, CA), plasma Phe was derivatized to its n-propyl ester, heptofluorobutyramide derivative (32). The enrichment of plasma [13C]Phe was measured using methane negative chemical ionization GC-MS (Hewlett-Packard 5890 series GC; Hewlett-Packard 5988A MS system, Mississauga, ON, Canada). Selected-ion chromatograms were obtained by monitoring [M + HF–] ions at mass-to-charge ratios (m/z) 383 for L-Phe and 384 for L-[1-13C]Phe. Isotopic enrichment in molecules percent excess (MPE) was calculated from peak area ratios at isotopic steady state and baseline.

Serum concentrations of 17{beta}-estradiol and progesterone were analyzed by radioimmunoassay (RIA, Toronto, ON, Canada). The intra- and interassay coefficients of variation for estrogen were 7.0 and 8.1%, respectively, and those for progesterone were 8.8 and 9.7%, respectively. Plasma free amino acids were separated using a cation exchange column, as mentioned earlier, using norleucine as an internal standard. Plasma amino acid concentrations were measured by reverse-phase high-performance liquid chromatography (Summit HPLC system; Dionex, Sunnyvale, CA; operated under HPLC pump model P580A LPG and UV/VIS Detector UVD 170S) of their phenylisothiocyanate derivatives (adapted from Pico Tag; Waters, Milford, MA) (1). The areas under the amino acid peaks were integrated using Chromeleon software version 6.2 (Dionex).

Estimation of isotope kinetics. The model used to study Phe metabolism was a simplified single-pool model (39)

where Q is Phe flux (µmol·kg–1·h–1); S is the rate of Phe nonoxidative disposal, a measure of the rate of Phe incorporation into body protein; O is the rate of Phe oxidation; B is the rate of Phe released from body protein; and I is the rate of exogenous Phe intake. Phe flux (Q) used in the simplified single-pool model was calculated from the dilution of the L-[1-13C]Phe infused into the body amino acid pool at isotopic steady state. The isotopic enrichment of plasma Phe was used to represent that of intracellular precursor. The calculation is done with the following equation (25)

where i is the rate of [1-13C]Phe infused (µmol·kg–1·h–1), and Ei and Ep are the isotopic enrichments as MPE of the infusate and plasma Phe at isotopic plateau. The –1 removes the contribution of the isotope infusion to the flux.

The rate of Phe oxidation (O) was calculated as

where F13CO2 represents the rate of 13CO2 released by Phe tracer oxidation (µmol13CO2·kg–1·h–1) and calculated by the equation

where FCO2 is the CO2 production rate (ml/min), ECO2 is the 13CO2 enrichment in expired breath at isotopic steady state (APE), and W is the actual body weight (kg) of the subject on each study day. The constants 44.6 µM/ml and 60 min/h convert FCO2 to µM/h and the factor 100 changes APE to a fraction. The factor 0.82 accounts for 13CO2 retained in the body because of bicarbonate fixation (17). The factor of CO2 recovery used in the calculation of the rate of Phe oxidation in the follicular and luteal phases was assumed to be equal. This assumption was based on a study of Lariviere et al. (22), who reported no difference in CO2 recovery between the two phases of the menstrual cycle during the fasted state.

Statistical analysis. All variables were analyzed with a repeated-measures design using the mixed-model procedure of SAS (24). The analysis of variance (ANOVA) model included the level of lysine treatment as a fixed effect, phase (follicular and luteal) as a fixed repeated effect, and the subject within lysine treatment as a random effect. The subject within lysine effect was used as the error term to test lysine treatment. The dependent variables F13CO2, Phe flux, and oxidation were adjusted for 17{beta}-estradiol and progesterone by using covariance, and adjusted means will be presented throughout the article. The variance-covariance matrix chosen was based on Schwarz's Bayesian criterion. The Kenword-Roger method was used to determine denominator degrees of freedom (SAS, version 8.1; SAS Institute, Cary, NC). The significance for fixed effects was reported, and the least square means were considered different at P < 0.05.

The adjusted F13CO2 values were used to determine breakpoints for each phase of the menstrual cycle. The breakpoint represented the lysine requirement, and the estimates were calculated by using the nonlinear (NLIN) procedure of SAS (41). The variation of the mean breakpoint was calculated as 95% confidence interval (CI), using Fieller's theorem (46). The statistical difference between the two breakpoints was ascertained by using the comparison of a two-sample t-test (31).

The relationship between variables considered independent was determined by the Pearson's product-moment correlation coefficient. The association was further evaluated using forward stepwise regression to determine which hormones and physiological variables explained variation in F13CO2. Analyses performed to explain this variation were separated into two categories, i.e., one addressing the data above the breakpoint and the other below the breakpoint. The possible predictor variables, including 17{beta}-estradiol, progesterone, and CO2 production, were analyzed by stepwise regression. All analyses were done using SAS statistical software (SAS, version 8.2). Differences were considered significant at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
There were no changes in body weight (P = 0.95) or body composition (P = 0.87) of subjects during the 5–6 mo of study (data not shown). Neither body weight nor body composition was different between phases of the menstrual cycle (P = 0.46 and 0.52, respectively). The concentrations of 17{beta}-estradiol and progesterone were elevated in all subjects during the luteal phase (Table 2). There was no difference in the mean concentrations of these sex hormones among all levels of lysine intake (P = 0.09 for 17{beta}-estradiol and 0.93 for progesterone). A wide range in 17{beta}-estradiol was observed in both phases of the menstrual cycle, with coefficients of variation within each subject ranging from 14.4 to 34.2% and from 15.4 to 45.6% for the follicular and luteal phases, respectively. Progesterone concentrations were relatively low (1–4 nmol/l) and constant in the follicular phase, whereas higher concentrations (7–66 nmol/l) were observed in the luteal phase, and the coefficient of variation within the subjects ranged from 18.6 to 82.2%.


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  		Table 2. 17{beta}-Estradiol and progesterone concentrations of female subjects measured on each IAAO study day (at all levels of lysine test intake) during follicular and luteal phases

 
Because of high variation of sex hormones during the menstrual cycle, it was important to adjust all metabolic variables for both 17{beta}-estradiol and progesterone. Mean adjusted Phe flux, nonoxidative Phe disposal (NOPD), and Phe release from proteolysis (BPhe) (Table 3) were not different (P > 0.05) among lysine test intakes in both the follicular and luteal phases. Lysine intake, however, had a significant effect on Phe oxidation (P = 0.0032) during both phases. Oxidation rates of Phe were higher (P = 0.003) at lysine intake of 10 mg·kg–1·day–1 compared with other levels. No interaction (P > 0.05) between phases of the menstrual cycle and levels of lysine intake was observed in any of the variables.


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  		Table 3. Effect of lysine intake on phenylalanine kinetics adjusted for 17{beta}-estradiol and progesterone concentrations in follicular and luteal phases of the menstrual cycle

 
The rate of 13CO2 release from Phe tracer oxidation (F13CO2) in both the follicular and luteal phases decreased as lysine intake increased from 10 to 35 mg·kg–1·day–1 and remained relatively stable thereafter (Fig. 2). The F13CO2 response appeared to be elevated at a lysine intake of 60 mg·kg–1·day–1 in the luteal phase. However, the F13CO2 value at that point was not different from that of lysine intake of 40 and 45 mg·kg–1·day–1 (P = 0.20 and 0.18, respectively). Mean F13CO2 at each level of lysine test intake was 13–24% higher during the luteal phase compared with the follicular phase (P = 0.02).



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  		Fig. 2. Effect of lysine intake on production of 13CO2 (F13CO2) from oxidation of L-[1-13C]Phe after adjusting for subjects and sex during follicular and luteal phases of the menstrual cycle. Values are means of adjusted F13CO2 ± SD at 7 tested lysine intakes. Breakpoint (value of lysine intake where residual variance is the smallest) analysis represented lysine requirement and was calculated using the nonlinear procedure of SAS. All adjusted F13CO2 (35 values for each phase of the menstrual cycle) were used to the breakpoint. Linear regression equations for estimated lysine requirement are: y = 0.5143 – 0.00373x and y = 0.3682 + 0.000447x for follicular phase; y = 0.6615 – 0.00591x and y = 0.3052 + 0.00355x for luteal phase. Breakpoint of 37.7 obtained during the luteal phase is significantly higher than that (35.0) obtained during the follicular phase (P = 0.025). 95% Confidence intervals of breakpoints were 22.1, 47.9, and (31.8, 43.6) for follicular and luteal phases, respectively.

 
The breakpoint estimates of the adjusted F13CO2 data, which represent the mean lysine requirement, occurred at a dietary lysine intake of 35.0 and 37.7 mg·kg–1·day–1 for the follicular and luteal phases, respectively (Fig. 2). The breakpoint estimate obtained in the luteal phase (37.7 mg·kg–1·day–1) was higher than that obtained in the follicular phase (35.0 mg·kg–1·day–1; P = 0.025). The 95% CIs were estimated to be (22.1, 47.9) and (31.8, 43.6) mg·kg–1·day–1 for the follicular and luteal phases, respectively. The adjusted Phe oxidation data showed the same pattern as the F13CO2 data. The breakpoint estimates, derived for the Phe oxidation in both the follicular and luteal phases, were a little lower than those obtained for the F13CO2 data, but wider ranges of 95% CI of the breakpoint were observed in both phases of the menstrual cycle (data not shown).

Plasma lysine concentration increased in response to increasing lysine intake (Fig. 3). The mean plasma lysine concentrations either in the follicular or luteal phase increased in linear fashion from lysine intakes of 10–60 mg·kg–1·day–1. However, lysine concentrations in the luteal phase were 71–83% of those in the follicular phase. Concentrations of other amino acids at each level of lysine test intakes during each phase of the menstrual cycle did not differ (P > 0.01). Concentrations of other amino acids and total amino acids were 10–25% lower in the luteal compared with the follicular phase (data not shown).



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  		Fig. 3. Lysine concentrations of women during follicular and luteal phases at each level of lysine intake. Data are means ± SD; n = 5. Those with different superscripts in each menstrual cycle phase differ, P ≤ 0.05.

 
O2 consumption and CO2 production rates increased by ~5 and 7%, respectively, in the luteal phase compared with the follicular phase (data not shown). There was a positive correlation between CO2 production rate and 17{beta}-estradiol (r = 0.37, P = 0.002) and progesterone (r = 0.35, P = 0.003) concentrations. In addition, positive correlations between 17{beta}-estradiol and F13CO2 (r = 0.37, P = 0.002), progesterone and F13CO2 (r = 0.53, P < 0.0001), and 17{beta}-estradiol-to-progesterone ratio and F13CO2 (r = –0.38, P < 0.0001) were observed. The coefficient of variation of 17{beta}-estradiol during the follicular phase (33.6%) was higher than that during the luteal phase (20.7%). The coefficient of variation of progesterone was even higher in both the follicular (48.5%) and luteal (43.4%) phases.

Table 4 shows the independent predictors of F13CO2 as determined by stepwise regression. Progesterone concentration (P < 0.0001) and the level of lysine intake (P = 0.0002) together explained 53% of the variation in F13CO2 response at lysine intake below the breakpoint, with progesterone concentration alone explaining 31%. Variation in F13CO2 above the breakpoint explained by CO2 production was 14% but increased to 22% when combined with the progesterone concentration.


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  		Table 4. Relationship and stepwise regression analysis between F13CO2 and sex hormones, CO2, and lysine intake

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
This study provided evidence that, although an absolute value of Phe oxidation was observed by phases of the menstrual cycle, estimates of lysine requirements derived from the regression breakpoints from each phase were not affected by the absolute value of Phe oxidation. Therefore, differences in lysine requirements were partly attributed to the physiological phases and not to the IAAO method itself. Mean lysine requirement based on F13CO2 was 35.0 and 37.7 mg·kg–1·day–1 in the follicular and luteal phases, respectively, and the difference between these two breakpoints was significant (P = 0.025). To the best of our knowledge, this is the first study to determine the amino acid requirement in women during both the follicular and luteal phases of the menstrual cycle. An earlier study that employed a similar method to that of the present study (IAAO) determined the tryptophan requirement in women (23); however, it was conducted only in the follicular phase. Experiments using both men and women have been reported in some studies from the Massachusetts Institute of Technology (MIT) and Millward's group (2, 12, 27, 28, 35). However, none of these studies was done on women in the follicular and luteal phases, and some studies did not control for the phases of the menstrual cycle (27, 28, 35).

Serum progesterone and 17{beta}-estradiol concentrations in the present study were elevated ~17- and 3-fold, respectively, in the luteal compared with the follicular phase. This observation is consistent with the finding of an earlier study, which documented an increase in progesterone and estradiol levels in the luteal phase vs. the follicular phase by factors of 10.7 and 2, respectively (3). Fluctuations in sex hormones appeared to affect F13CO2, since a weak-to-moderate positive correlation between sex hormones (both 17{beta}-estradiol and progesterone) and F13CO2 was observed in our study. It suggests that an increase in either 17{beta}-estradiol or progesterone is associated with an increase in F13CO2 during the luteal phase. Recently, Toth et al. (38) reported a positive correlation between estradiol and leucine oxidation. In support of this finding, results from the stepwise analysis demonstrated that the progesterone concentration was the strongest predictor of F13CO2. Taken together, it is suggested that progesterone has a greater impact on amino acid catabolism than 17{beta}-estradiol in the luteal phase. Progesterone has been shown to act catabolically when injected into healthy men, as evidenced by a reduced plasma amino acid concentration (20, 21). Conversely, ethinyl estradiol therapy has no effect on the leucine turnover of the whole body in hypogonadal, prepubescent females (26). However, the response to estrogen in these girls may not be the same as in adult healthy women. Hence, further research to address this issue is warranted.

Phe oxidation and F13CO2 in the luteal phase relative to the follicular phase were on average 15 and 18% higher, respectively. In agreement with this finding, higher oxidation in other amino acids has been observed in women during the luteal compared with the follicular phase. Lariviere et al. (22) reported an increase in leucine oxidation by 15% in women during the luteal phase. Similarly, a higher catabolism of tryptophan via the kynurenine pathway in the luteal phase has also been documented (18). Although Lariviere et al. reported an increase in leucine flux in the luteal phase, no change in Phe flux between the two phases of the menstrual cycle was observed in the present study. This disparity in findings may be due to differences in study design employed by the two studies. Lariviere et al. conducted their study in the fasted state, whereas the present study was conducted in the fed state. It is possible that an increased amino acid turnover in the luteal phase occurred during the fasted state but not during the fed state. A sufficient amino acid supply from dietary intake during the latter state might have alleviated the amino acid turnover. Garrel et al. (15) also reported no significant menstrual cycle effects on whole body protein turnover. They used a single oral dose of [15N]glycine and urinary ammonia end product measured randomly over the course of the menstrual cycle.

The higher lysine requirement estimate in the luteal phase is probably due to the increase in amino acid oxidation in the luteal phase relative to the follicular phase. Although weight gain, water retention, and change in food intake may occur during the luteal phase (6, 33), these variables are unlikely to affect our estimation of lysine requirement. There were no changes in body weight and body composition over the 6 mo in any of the female subjects who participated in this study. In addition, the experimental diet was strictly controlled throughout the study, in which only levels of lysine intake were varied according to the study design.

Because of the limited number of subjects used in the present study (n = 5), a wide range in the 95% CI for lysine requirement was observed either during the follicular (22.1, 47.9) or luteal (31.8, 43.6) phases. High interindividual variability in Phe oxidation is likely one explanation for this observation. A much wider 95% CI for lysine requirement in the follicular phase was observed compared with that in the luteal phase. A higher coefficient of variation of sex steroid hormones, especially 17{beta}-estradiol concentration, was observed in the follicular phase, although this was adjusted for in our study. Studying a larger sample could be one solution, since by doing so smaller standard errors, and thus narrower CI ranges, would likely result. However, it would be more expensive and difficult to enlist more subjects into this study given the nature of the study protocol in which an individual female subject has to be studied in both the follicular and luteal phases of the menstrual cycle for approximately 6 mo.

There are a few physiological possibilities that may be related to the increase in lysine requirement in the luteal phase. First is an elevation in either the whole body protein synthesis or the obligatory amino acid loss. Second, the whole body protein breakdown might decrease and therefore contribute less endogenous lysine. However, the rate of Phe released from protein breakdown (derived from Phe flux and Phe intake; Table 3) in the present study was not affected by the phases. Under normal physiological conditions, the luteal phase is characterized by the high vascularization of uterus endometrium and the formation of the corpus luteum, whereas the follicular phase occurs when the endometrium increases its thickness (16). Whether the protein synthesis between the two phases of the menstrual cycle is different has never been studied directly. There is no evidence that an increase in lysine requirement observed in this study could be translated into an increase in protein synthesis, since there was no difference in the calculated NOPD (an index of protein synthesis) between the two menstrual phases (Table 3). However, increased Phe oxidation was clearly observed in the luteal phase, suggesting that increased lysine requirement in the luteal phase may be due to an increase in amino acid obligatory loss. This is confirmed by the observation of decreases in plasma lysine and total plasma amino acid concentrations to 71–83% in the luteal relative to the follicular phase (Fig. 3). This finding was in agreement with reports of a fall in plasma amino acids at midcycle (29) and during the luteal phase (8, 9, 29).

The increase in Phe oxidation in the luteal phase observed in this study was associated with a 5–8% increase in O2 consumption and CO2 production, measured 4–6 h after hourly meals were consumed. This finding is in agreement with other studies that reported an increase in RMR and energy expenditure in the luteal phase (22, 37, 40). The mechanism whereby this variation exists in women is not known. The 18% increase in F13CO2 in the luteal phase could be explained, at least in part, by the ~7% increase in CO2 production. The mechanism for the higher Phe oxidation, as measured by F13CO2, occurring in the luteal phase remains unresolved. A previous study has reported that tyrosine transaminase activity increased in women taking oral contraceptives (30). Administration of oral contraceptives can increase the levels of estrogen and progesterone in women. Therefore, it is possible that the tyrosine transaminase activity may have increased during the luteal phase, a period when endogenous estrogen and progesterone levels are elevated compared with the follicular phase. To unravel the actual mechanisms, future studies should look at the activity and regulatory changes in the Phe-degrading enzymes and the uptake rate of amino acid substrate in the major oxidation sites throughout both the follicular and the luteal phases.

An increased Phe oxidation and lysine requirement estimate in the luteal phase observed in the present study leads to the question whether other IDAAs provided in the experimental diet were limited and possibly affected Phe kinetics. Nevertheless, this is unlikely to be the case, as the amounts of IDAAs in the experimental diet were in accordance with the standard egg protein. In this reference diet, the IDAAs are more than double the amount present in the new recommended amino acid intake by the Food and Nutrition Board (2002) (13).

Lysine requirements for women obtained in the present study were 35.0 and 37.7 mg·kg–1·day–1 during the follicular and luteal phases, respectively. These numbers are somewhat higher than the current lysine requirement (31.0 mg·kg–1·day–1) of healthy adults (both sexes) proposed by the Food and Nutrition Board (13). The lysine requirement of 31.0 mg·kg–1·day–1 has been set on the basis of the average lysine requirement obtained from several tracer studies (ranging from 26.6 to 36.9 mg·kg–1·day–1), which were conducted in male subjects. It is possible that lysine requirements of women may be higher than those of men, especially during the luteal phase as indicated in our study. Because of the limited data that exist at the present time, it is inappropriate to do any comparison of lysine requirements between the sexes in this study. Hence, further studies directed to this issue are needed.

In conclusion, the present study provided evidence that the phases of the menstrual cycle affected phenylalanine oxidation and lysine requirements. Phenylalanine oxidation and lysine requirement were shown to be significantly higher during the luteal phase despite significant between-subject variability. Future studies with larger numbers are warranted to obtain better estimates of population variance.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This research was conducted with financial support from Grant MT 10321 of the Canadian Institutes of Health Research (CIHR). W. Kriengsinyos's financial support from a Nestle Nutrition Scholarship (Switzerland), the University of Toronto (Connaught Scholarship), and the Hospital For Sick Children's Research Training Committee studentship is gratefully acknowledged.


   ACKNOWLEDGMENTS
 
We thank the volunteers for their help in making this study possible. Thanks also go to Karen Chapman for coordinating the activities in the Clinical Investigation Unit of the Hospital for Sick Children (HSC), and Linda Chow (Department of Nutrition and Food Service, HSC) for preparing the protein-free cookies. We acknowledge the statistical expertise of Dr. Paul Corey and Eshetu Atenafu-Mshri and the technical expertise of Mahroukh Rafii. We appreciate the kind donation of Mead Johnson (Canada) of protein-free powder for the experimental diets, and Whitehall Robins of multivitamin supplements.

Current address of W. Kriengsinyos: Institute of Nutrition, Mahidol University, Salaya, Phutthamonthon, Nakhon-Pathom 73170, Thailand


   FOOTNOTES
 

Address for reprint requests and other correspondence: P. B. Pencharz, Div. of Gastroenterology & Nutrition, The Hospital for Sick Children, 555 University Ave., Toronto, ON M5G 1X8 Canada (E-mail: paul.pencharz{at}sickkids.ca).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 RESULTS
 DISCUSSION
 GRANTS
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