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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2006-0570
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The Journal of Clinical Endocrinology & Metabolism Vol. 91, No. 8 3158-3164
Copyright © 2006 by The Endocrine Society

The Response of Luteinizing Hormone Pulsatility to 5 Days of Low Energy Availability Disappears by 14 Years of Gynecological Age

Anne B. Loucks

Department of Biological Sciences, Ohio University, Athens, Ohio 45701

Address all correspondence and requests for reprints to: Anne B. Loucks, Department of Biological Sciences, Ohio University, Athens, Ohio 45701. E-mail: loucks{at}ohiou.edu.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Context: The incidence of menstrual disorders declines during adolescence. The mechanism responsible is unknown.

Objective: The objective of the study was to test the hypothesis, formulated a priori, that the dependence of LH pulsatility and ovarian function on energy availability declines with gynecological age (years since menarche).

Design: The study was a controlled experiment repeated in two menstrual cycles, performed 2001–2004.

Setting: The study was conducted at a university laboratory and general clinical research center.

Participants: The study population consisted of healthy, habitually sedentary, young women of normal body composition with 5–8 yr (adolescents, n = 9) and 14–18 yr (adults, n = 10) of gynecological age recruited by advertisement from approximately 9000 women aged 18–34 yr in a college community. Samples were similar in age of menarche, length of menstrual cycle and luteal phase, body size and composition, aerobic capacity, and dietary intake. None were withdrawn due to adverse effects.

Interventions: Interventions included energy availabilities of 45 and 10 kcal/kg of fat-free mass per day for 5 d in the early follicular phases of separate menstrual cycles in random order.

Main Outcome Measures: LH pulsatility, estradiol, and luteal phase length were measured.

Results: Low energy availability reduced LH pulse frequency in adolescents (P < 0.01) but not adults (P = 0.39), did not increase LH pulse amplitude in either group (both P = 0.13), and suppressed 24-h mean LH in adolescents (P = 0.01) but not adults (P = 0.72). Estradiol was unaffected (both P = 0.48), but the subsequent luteal phase was shorter in adolescents (P < 0.01).

Conclusions: In women of normal body composition, the response of LH pulsatility and ovarian function to 5 d of low energy availability disappears by 14 yr of gynecological age.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
PROBLEMS WITH MENSTRUATION are a leading reason for adolescents to visit physicians (1). The progressive decline in the incidence of menstrual disorders such as anovulation and short luteal phase during the first decade after menarche (Fig. 1Go) (2) as age-specific marital fertility rates are rising (3) has long been attributed to the gradual maturation of the hypothalamic-pituitary-ovarian axis (4), but the mechanism of this maturation is not known.


Figure 1
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  		FIG. 1. Participants were regularly menstruating older adolescents with 5–8 yr and young adults with 14–18 yr of gynecological age, age ranges selected a priori to be well within the age ranges with high and low incidences of anovulation and short luteal phase in 14,741 menstrual cycles of 523 women (2 ).

 
Ovarian function depends on the pulsatile secretion of GnRH from the hypothalamus and the consequent and clinically assessable pulsatile secretion of LH from the pituitary (5). In amenorrheic athletes, LH pulsatility is disrupted, whereas pituitary responsiveness to GnRH is increased, indicating that their amenorrhea is of hypothalamic origin (5, 6). Menstrual disorders in female athletes have been attributed to low body fatness, but effects of metabolic challenges on LH pulsatility occur far more rapidly than changes in adiposity (7), and most studies find no difference in body composition between amenorrheic and eumenorrheic athletes (8). These disorders have also been attributed to the stress of exercise, but exercise has no suppressive effect on LH pulsatility in healthy young women [mean (SD) = 8.7 (3.3) yr of gynecological age (time since menarche)] beyond the impact of its energy cost on energy availability, operationally defined as dietary energy intake minus exercise energy expenditure, so that apparent effects of exercise on LH pulsatility can be prevented by dietary supplementation (9, 10). The induction of amenorrhea in monkeys by intensifying their exercise regimen without altering their diets (11) can also be reversed by supplementing their diets without moderating their exercise (12). In exercising young women [gynecological age = 8.3 (5.4) yr], LH pulsatility is disrupted abruptly below a threshold of energy availability at approximately 30 kcal/kg of fat-free mass (FFM) per day (13).

Some other investigators have failed to detect a disruptive effect of fasting on LH pulsatility (14, 15), but their participants were up to 35 and 40 yr of age. Therefore, the primary purpose of this experiment was to test the hypothesis that the dependence of LH pulsatility and ovarian function on energy availability declines with gynecological age.

Two types of mechanisms have been hypothesized to mediate the influence of energy availability on the GnRH pulse generator (7): certain hormones have been hypothesized to signal information to the GnRH pulse generator about the availability of metabolic fuels for the body as a whole, and the function of the GnRH pulse generator has been hypothesized to depend directly on the availability of metabolic fuels to the brain itself. Therefore, the secondary purpose of this experiment was to determine whether any age-related decline in the response of LH pulsatility to energy availability is associated with effects on selected metabolic hormones and substrates.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Healthy, habitually sedentary, regularly menstruating women of normal body composition were recruited through posters and newspaper advertisements from approximately 9000 women 18–34 yr of age in the local college community. All volunteers signed consent forms and received a full verbal and written description of the nature of the experiment, its associated risks and benefits, and their ability to withdraw from the experiment at any time. The same 9-d protocol used in the past (13) was approved by the Institutional Review Boards of Ohio University, The Ohio State University, and the Surgeon General of the Department of the Army. All procedures were conducted in accordance with the guidelines in The Declaration of Helsinki.

Screening

Older adolescents with 5–8 yr and young adults with 14–18 yr of gynecological age (time since menarche) were screened for participation as previously described (13). These nonoverlapping age ranges were selected a priori to be well within the age ranges with high and low incidences, respectively, of anovulation and short luteal phase in 14,741 menstrual cycles of 523 women (2) (Fig. 1Go). All participants were required to have luteal phases longer than 11 d as determined by an immunoradiometric assay of LH in daily urine samples because we had previously found more extreme effects of low energy availability on LH pulsatility in women with luteal phases only 11 d long (13). (Women with shorter luteal phases had been excluded from that experiment.)

Participants were enrolled by the investigator and allocated to groups by gynecological age without blinding. The demographic characteristics of the participants are shown in Table 1Go. By design, adolescents and adults differed greatly in gynecological age and calendar age (both P < 0.001) but not in age of menarche, the lengths of the menstrual cycle, and luteal phase, in any parameter of body size or composition, maximal aerobic capacity, or habitual dietary energy intake (all P ≥ 0.10). All subjects had been regularly menstruating for at least the previous 3 months. The narrow range of menstrual cycle length and the normal luteal phase length of the participants reduced the likelihood that results were confounded by preexisting menstrual disorders.


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  		TABLE 1. Demographic characteristics of the participants

 
Treatments

Energy availability was defined operationally as dietary energy intake minus exercise energy expenditure. Contrasting energy availability treatments were administered for 5 d in the early follicular phases of menstrual cycles separated by at least 2 months to allow for recovery from blood loss and wash out physiological effects of exercise and dietary restriction (Table 2Go). Treatments were administered in random order to distinguish treatment effects from fixed effects of prior participation in the experiment. The sequence of energy availability treatments (10 or 45 kcal/kg FFM per day first) administered to each subject was determined before the first treatment by the flip of a coin by the investigator. The sequence was not concealed from either the participant or the investigator.


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  		TABLE 2. Energy availability treatments applied to the adolescents and adults

 
Energy intake, expenditure, and availability were normalized to FFM to control the energy available to actively metabolizing tissue despite individual differences in body size and composition. In both treatments, participants performed X = 15 kcal/kg FFM per day of exercise, above habitual energy expenditure during the same hours, at 70% of aerobic capacity by walking up a grade on a motorized treadmill ergometer. A commercially available clinical dietary product (Ensure Plus; Ross Products Division, Abbott Laboratories, Columbus, OH) was used to control dietary energy intake at either I = 60 or 25 kcal/kg FFM per day to provide energy availabilities of A = I – X = 45 and 10 kcal/kg FFM per day, respectively, in which I is energy intake, X is expenditure, and A is availability. For these subjects, these energy availabilities amounted to approximately 2000 and approximately 450 kcal/d.

Diet and exercise treatments were accurately and precisely administered (Table 2Go). By design, the normalized controlled dietary energy intake (CDI), energy availability (EA), and 24-h energy balance (24EB) were much lower during the 10 than during the 45 kcal/kg FFM per day treatment in both groups (all P < 0.001). 24EB was extremely negative during the 10 kcal/kg FFM per day treatment (both P < 0.001) and indistinguishable from zero during the 45 kcal/kg FFM per day treatment (both P > 0.39). By design, the normalized controlled energy expenditure (CEE) during exercise and energy expenditure net of nonexercise energy expenditure during exercise (EEE) were within 1 kcal/kg FFM per day during each treatment in both groups (all P > 0.15). A few statistically significant but physiologically negligible differences between the intensities (submaximal workload and percent of maximum heart rate) and durations of exercise were necessary for individuals with less exercise tolerance to achieve these objectives. Normalized 24-h energy expenditure (24EE), determined as previously described (13), was also within 1 kcal/kg FFM per day in both treatments and groups (all P > 0.44). At each energy availability, normalized controlled dietary energy intake, energy availability, and 24EB were also within 1 kcal/kg FFM per day in both groups (all P > 0.09). Consequently, low energy availability reduced body weight similarly (P = 0.89) in the adolescents [mean (95% confidence interval [CI]) = –1.5 (–2.3 to –0.7) kg] and adults [–1.5 (–2.0 to –1.1) kg] (both P < 0.001). As intended, differences between the treatments administered to the two groups could have had only negligible influence on our results.

Blood and urine sampling

Beginning on the second to sixth day of the menstrual cycle according to whether a participant’s follicular phase in the screening cycle had been shorter or longer, participants provided a urine and a blood sample between 0730 and 0830 h on 3 pretreatment days and 5 treatment days. After the exercise session on the fifth treatment day, participants were admitted to a General Clinical Research Center at which the same energy availability was administered as blood samples were drawn at 10-min intervals and all urine was collected for 24 h. Participants then collected daily urine samples for the next 10–14 d.

Assays

Blood and urine samples were processed and assayed for LH, estradiol, glucose, ß-hydroxybutyrate (ßHOB), insulin, cortisol, GH, IGF-I, IGF-I binding proteins (IGFBP)-1 and IGFBP-3, T3, and leptin. We used the same assays as previously (13), except for LH (ICN Biomedicals, Inc., Aurora, OH), ß-HOB (Wako Diagnostics, Richmond, VA) and GH (Advantage; Nichols Institute Diagnostics, San Clemente, CA). Intraassay and interassay coefficients of variation for these assays were as follows: LH, 4 and 9% at 8.5 IU/liter; estradiol, 6 and 15% at 90 pmol/liter; glucose, 1 and 3% at 4.6 mmol/liter; ßHOB, 6 and 10% at 1.9 mmol/liter; insulin, 3 and 8% at 26 pmol/liter; cortisol, 3 and 5% at 380 nmol/liter; GH, 1 and 1% at 2 µg/liter; IGF-I, 4 and 11% at 280 ng/ml; IGFBP-1, 5 and 6% at 39 ng/ml; IGFBP-3, 1 and 4% at 3650 ng/ml; T3, 4 and 11% at 1.6 nmol/liter; and leptin, 6 and 13% at 7.6 ng/ml, respectively.

Data analysis

As previously described in more detail (13), daily urine samples collected after participants were discharged from the General Clinical Research Center were assayed for LH, and the length of the luteal phase was calculated as the number of days from the LH surge to the onset of bleeding in the next menses. The time series of LH concentrations over 24 h was analyzed for pulse frequency, pulse amplitude, and 24-h mean concentration using the validated, objective pulse detection algorithm Cluster (16). A 2 x 1 pulse configuration was used with up and down T ratios of 2.5 to detect pulses in relation to the measurement error of the data set being analyzed. Measurement error was determined as a function of LH concentration in a separate assay of 20 replicates at 14 concentrations. Missing data were linearly interpolated between adjacent values. No values were undetectable. Effects of energy availability on LH pulsatility and metabolic substrates and hormones were calculated as previously described (13).

Statistical methods

The objective of this experiment was to test the following null hypotheses.

H01. The effects of 5 d of low energy availability (10 vs. 45 kcal/kg FFM per day) on LH pulsatility (frequency, amplitude, and 24-h transverse mean) and ovarian function (estradiol and luteal phase length) are no smaller in adults than adolescents.

H02. The effects of low energy availability on metabolic substrates (glucose and ßHOB) and hormones (insulin, cortisol, GH, IGF-I, IGFBP-1, IGFBP-3, the ratios IGF-I to IGFBP-1 and IGF-I to IGFBP-3, T3, and leptin) are no different in adults than adolescents.

All data sets were tested for nonnormality, heteroscedasticity, and outliers before hypothesis tests were performed. Outliers were rejected and data sets were transformed as necessary. Single-sided tests were used to test H01 because the directions of interest in this hypothesis were known in advance (13). Two-sided tests were used to test H02 because the directions of interest in this hypothesis were not known.

A sample of size 9 was chosen to detect a difference (d) of 1.5 SD between effects of low energy availability on LH pulsatility in the adolescents and adults with 100{alpha} = 5% and 100ß = 10% probabilities of types I and II error rates, respectively, in single-sided, two sample Student’s t tests of H01, as indicated in the following calculation (17):

n = 2 * (tdf=16{alpha}=0.05 + tdf=16ß=0.10)2 * (SD/d)2 = 2 * (1.75 + 1.34)2 * (1/1.5)1/2 = 8.4 -> 9


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Effects on LH pulsatility and ovarian function

LH pulse profiles of a representative adolescent and adult after the two energy availability treatments are shown in Fig. 2Go. After 45 kcal/kg FFM per day, there were no differences between groups in LH pulse frequency over 24 h or during the feeding and fasting phases of the 24-h day in LH pulse amplitude or 24-h transverse mean LH. Therefore, LH pulsatility parameters after 45 kcal/kg FFM per day are pooled in Table 3Go.


Figure 2
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  		FIG. 2. LH pulse profiles. Twenty-four hour LH pulse profiles of a representative adolescent and adult after energy availability treatments of 45 and 10 kcal/kg FFM per day. Asterisks indicate LH pulses. The filled bar indicates when lights were turned off. Arrows indicate meals.

 

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  		TABLE 3. LH pulsatility at 45 kcal/kg FFM per day and effects of low energy availability (10 kcal/kg FFM per day) on it

 
Treatment effects on absolute LH pulsatility are also listed in Table 3Go. Relative effects on LH pulse frequency, amplitude, and transverse mean over the whole 24-h day are illustrated in Fig. 3Go. Low energy availability suppressed LH pulse frequency in the adolescents (P < 0.01) but not the adults (P = 0.39) and did not increase LH pulse amplitude in either the adolescents or the adults (both P = 0.13). The reduction in frequency without an increase in amplitude reduced the 24-h transverse mean LH in the adolescents (P = 0.01) but not the adults (P = 0.72).


Figure 3
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  		FIG. 3. Treatment effects on LH pulsatility. Effects [mean (95% CI)] of percentage changes relative to values at 45 kcal/kg FFM per day. Effects on frequency and 24-h mean were significant in adolescents and different from those in adults. **, P < 0.01; ***, P < 0.001.

 
Estradiol levels measured at 0800 h on the pretreatment days were indistinguishable between the adolescents [mean (95% CI) = 90 (75–106) pmol/liter] and adults [90 (66–113) pmol/liter; P = 0.96]. After the follicular phase had progressed for 5 d at an energy availability of 45 kcal/kg FFM per day, these concentrations increased but were still indistinguishable [adolescents 148 (96–200) and adults 149 (88–210) pmol/liter; P = 0.98]. Low energy availability had no effect on estradiol in either the adolescents [–1 (–50 to 49) pmol/liter; P = 0.48] or adults [–1 (–58 to 55) pmol/liter; P = 0.48]. Nevertheless, the luteal phase after the low energy availability treatment was shorter (P < 0.01) in the adolescents [mean (95% CI) = 11.4 (9.7–13.2) d] than the adults [14.1 (13.2–15.0) d].

Effects on metabolic substrates and hormones

The levels of metabolic substrates and hormones after the 45 kcal/kg FFM per day treatment are shown in Table 4Go. As expected, levels of GH, IGF-I, and the ratio IGF-I to IGFBP-1, an index of free IGF-I, were lower in the adults. Unexpectedly, insulin levels were also lower in the adults during the feeding phase of the day. Otherwise, the metabolic substrates and hormones after the 45 kcal/kg FFM per day treatment were similar in the two groups.


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  		TABLE 4. Metabolic substrates and hormones at 45 kcal/kg FFM per day

 
Absolute effects of low energy availability on metabolic substrates and hormones are listed in Table 5Go, and relative effects are illustrated in Fig. 4Go. All metabolic substrates and hormones were altered by low energy availability in both groups, and most responses were similar in the two groups. In the adolescents, the suppression of insulin during the feeding phase of the day was larger in absolute terms but smaller in relative terms (P < 0.001). Because their insulin was higher during the feeding phase of the day after the 45 kcal/kg FFM per day treatment, insulin responses of the adolescents to the 10 kcal/kg FFM per day treatment reduced their insulin levels during the feeding phase of the day to levels [adolescents mean (95% CI) = 51 (41–61) pmol/liter] indistinguishable from those in the adults [44 (38–51) pmol/liter; P = 0.23]. Both absolute and relative effects on GH were greater in the adults, especially during the feeding phase of the day. The effect on the ratio IGF-I to IGFBP-1 was smaller in the adults. Because the ratio was lower in the adults after the 45 kcal/kg FFM per day treatment, the IGF-I to IGFBP-1 responses to the 10 kcal/kg FFM per day treatment led to ratios in the adolescents [3 (1–5)] and adults [2 (1–3)] that were also indistinguishable (P = 0.12).


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  		TABLE 5. Effects of low energy availability (10 kcal/kg FFM per day) on metabolic substrates and hormones

 

Figure 4
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  		FIG. 4. Treatment effects on metabolic substrates and hormones over 24 h. Effects [mean (95% CI)] on glucose (5GLU), ßHOB (B/50), insulin (INS), cortisol (F), GH (GH/2), IGF-I, T3, (T3), and leptin (LEP) are shown as percentage changes relative to values at 45 kcal/kg FFM per day. Effects on glucose are multiplied by 5, ßHOB divided by 50, and GH divided by 2 for graphical clarity. All effects were significant (all P < 0.01), and all were similar between the two groups (P > 0.05) except GH (*, P = 0.03).

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
In this experiment the response of LH pulsatility to 5 d of low energy availability disappeared between 8 and 14 yr of gynecological age, despite predominantly similar responses of metabolic substrates and hormones. Because the adolescents and adults in this experiment were similar in demographic characteristics other than gynecological age (Table 1Go) and the energy availability treatments administered to the two groups were very similar (Table 2Go), our results can be reliably attributed to the planned contrast in gynecological age. To our knowledge, this is the first experimental evidence that the dependence of LH pulsatility on energy availability declines with gynecological age.

Effects of gynecological age on the response of LH pulsatility to energy availability

Previously we presented the first experimental evidence that LH pulsatility is more extremely disrupted by low energy availability in certain identifiable young women (13). Five days at 10 kcal/kg FFM per day suppressed LH pulse frequency by approximately 60% in regularly menstruating young women [gynecological age (mean SD) = 8.3 (5.4) yr] whose luteal phases were 11 d long under energy-balanced conditions but by only approximately 20% in those with longer luteal phases (13). (Women with shorter luteal phases had been excluded from that experiment.) We have now shown that LH pulsatility is not affected at all by this treatment in another identifiable group of young women, those older than 14 yr of gynecological age.

The degree of energy restriction in this experiment was extreme (–78%), but its duration was short (5 d) and the adolescents had normal luteal phases under energy-balanced conditions. The resulting 28% suppression of LH pulsatility in the adolescents was insufficient to lower estradiol in the midfollicular phase or induce anovulation, but it did shorten their subsequent luteal phase. This did not happen in the adults.

Most observational studies of undernourished athletes (e.g. Refs. 6 and 18, 19, 20, 21, 22) and other women (23) have found amenorrhea and eumenorrhea in those with younger and older gynecological ages, respectively. The results of most prospective experiments that have attempted to disrupt reproductive function in premenopausal women by reducing their energy availability would also be explained by a declining responsiveness of the GnRH pulse generator to low energy availability. Fasting (14, 15) and exercise training (24, 25, 26) have had little if any effect in gynecologically mature women, whereas dietary restriction (9) and exercise training (10, 13, 27) have disrupted reproductive function in younger women. In a 1-yr-long experiment, LH pulsatility and ovarian function were unaffected in 17 women [gynecological age (mean SD) = 17.8 (4.3) yr] who increased their running mileage up to 70 miles/wk without increasing their dietary energy intake (26).

Nevertheless, some amenorrheic athletes are older than 14 yr of gynecological age (18, 28, 29), and reproductive function has been disrupted experimentally by low energy availability in lean women in that age range (30, 31, 32). Therefore, LH pulsatility in our adults may have become responsive to low energy availability, if their energy deficiency had been prolonged enough to substantially reduce their fat stores.

Potential mechanisms mediating the influence of energy availability on LH pulsatility

Despite the qualitatively different effects of low energy availability on LH pulsatility and ovarian function in our adolescents and adults, the effects of low energy availability on metabolic substrates and hormones in this experiment did not provide persuasive evidence for either the metabolic signaling or the metabolic fuels hypothesis about how the influence of low energy availability on GnRH and LH pulsatility is mediated. Considerable evidence implicates central glucose as a key regulator of GnRH and thereby LH pulsatility (33). In a previous experiment on young women [gynecological age = 8.3 (5.4) yr], we found the dose-response effects of low energy availability on LH pulsatility to closely resemble those on plasma glucose (13). In this experiment, low energy availability had similar effects on LH pulse frequency and plasma glucose in the adolescents but not in the adults.

Many metabolic hormones have been hypothesized to signal information about energy availability to the GnRH pulse generator, but conflicting evidence has been presented for all of them (34, 35). If the signaling hypothesis is true and if any of the hormones measured in this experiment are involved, then the sensitivity of the central nervous system to these signals must decline during adolescence because the effects of low energy availability on these hormones were so similar in our two groups. Furthermore, such desensitization, if it occurs, would not seem to be mediated by estradiol because estradiol levels were virtually identical in the two groups.

To our knowledge, GH has never been hypothesized to signal information about energy availability to the GnRH pulse generator, but it is tempting to interpret the larger GH response to low energy availability in our adults as support for the metabolic fuels hypothesis. GH promotes lipolysis in adipose tissue during periods of energy deficiency (36). In rats, long-chain fatty acids (LCFA) cross the blood brain barrier (37) and accumulate in certain arcuate nucleus neurons, opening KATP channels, reducing neuron firing rate, and suppressing glucose production and feeding (38). Three days of LCFA overfeeding induces neuronal LCFA oxidation, preventing LCFA accumulation and these anorexic effects (39). Our results suggest that such neurons may also regulate GnRH pulsatility in women.

Even without a difference in energy mobilization there may still be more energy available to the brain in adults than adolescents with the same energy availability. During adolescence positive calcium balance declines to zero at 14 yr of gynecological age (40), much like the decline in the incidence of menstrual disorders (2). The lower levels of GH, IGF-I, and the ratio IGF-I to IGFBP-1 at 45 kcal/kg FFM per day in our adults reflect the monotonic decline in somatotrophic drive after linear growth stops in the midteens (41). Thus, scarce metabolic fuels may be preferentially directed toward peripheral tissues and away from the brain in growing adolescents.

Implications for research and clinical practice

This experiment demonstrates that the maturation of the human female reproductive axis consists, at least in part, of a decline in the responsiveness of LH pulsatility to low energy availability and stimulates new questions about whether that decline results from changes in signaling pathways, fat mobilization, or the rate of somatic growth. Investigators of the regulation of reproductive function should analyze data from adolescents and adults separately, and physicians treating adolescent menstrual disorders should consider advising behavior modifications to increase energy availability.


   Acknowledgments
 
Dr. P. Cadamagnani, Dr. W. Myles, Dr. W. Malarkey, D. Murray, J. R. Thuma, T. Wiese, E. T. Wolke, and the nursing staff of the General Clinical Research Center contributed to data collection.


   Footnotes
 
This work was supported by Grant DAMD 17-95-1-5053 from the U.S. Army Medical Research and Material Command (Defense Women’s Health and Military Medical Readiness Research Program); the General Clinical Research Branch, Division of Research Resources, National Institutes of Health Grant M01 RR00034; and Ross Laboratories. The content of the information reported in this paper does not necessarily reflect the position or the policy of the Government, and no official endorsement should be inferred. The funders played no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; or preparation, review, and approval of the manuscript.

First Published Online May 23, 2006

Abbreviations: CDI, Controlled dietary energy intake; CEE, controlled energy expenditure; CI, confidence interval; EA, energy availability; 24EB, 24-h energy balance; 24EE, 24-h energy expenditure; EEE, energy expenditure during exercise; FFM, fat-free mass; ßHOB, ß-hydroxybutyrate; IGFBP, IGF-I binding protein; LCFA, long-chain fatty acids.

Received March 13, 2006.

Accepted May 12, 2006.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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