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Clinical Studies |
Pennington Biomedical Research Center and Louisiana State University School of Medicine; and the Departments of Psychology (D.G.) and Experimental Statistics (R.M.), Louisiana State University, Baton Rouge, Louisiana 70808
Address all correspondence and requests for reprints to: Jennifer C. Lovejoy, Ph.D., Pennington Biomedical Research Center, 6400 Perkins Road, Baton Rouge, Louisiana 70808-4124. E-mail: lovejoj{at}mhs.pbrc.edu
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Abstract |
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Introduction |
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The catabolic effects of thyroid hormone are well known. Elevations in T3 (either endogenous or exogenous) result in hypermetabolism, negative N and calcium balance, loss of body protein stores, and loss of body fat (4). Previous studies have also reported dietary effects on thyroid hormone levels. In a study of six men living on a metabolic unit for 105 days, serum levels of both T3 and T4 were influenced by energy balance (5). Furthermore, the carbohydrate content of the diet influences levels of endogenous thyroid hormones during overfeeding (6). Whether diet composition influences thyroid hormone status at neutral energy balance during T3 administration is unknown.
The purpose of the present study was to characterize the effects of induction of a catabolic state by treatment for 9 weeks with low doses of T3 on body composition, N balance, and protein turnover in ambulatory young men. Our primary goals were to identify a dose of T3 that would increase N and calcium excretion without producing adverse side-effects and to examine the time course of metabolic responses to T3. We hypothesized that healthy individuals would initially lose body protein stores in response to T3 treatment, but would compensate for these changes over time. Secondly, we examined whether the effects of T3 treatment differed in subjects eating a high vs. a low fat diet. We also hypothesized that a high fat diet would attenuate some of the effects of T3 treatment by promoting body fat storage over fat oxidation and that differences in thyroid hormone levels might be observed on the two diets.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Eight Caucasian male subjects entered for the 77 day in-patient study. During the run-in period, one subject was identified as having a seizure disorder and was dropped from the study. Therefore, data are reported for 7 subjects only. The age of the subjects was 26.0 ± 2.2 yr (mean ± SE), and initial body mass index (BMI) was 22.9 ± 1.4 kg/m2. All subjects had normal values for laboratory measures of blood chemistry, endocrine function, and blood count. Additionally, they had normal cardiac function (by electrocardiogram and echocardiogram) and normal psychological profiles. Written informed consent was obtained from each subject, and the protocol was approved by the Louisiana State University institutional review board.
Experimental design
The experimental design is shown in Fig. 1
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During a run-in period of 3 weeks, all subjects were adapted to the
experimental diets and the 24-h collection procedures. Baseline testing
was also performed during this period. The men lived at a Metabolic
Research Unit for the duration of the protocol, but were permitted to
go to work or school during the day (lunch, medications, and collection
containers were packed in an insulated bag). Although this protocol did
not allow for strict control of physical activity, the men were not
permitted to exercise or engage in sports activities outside the
Metabolic Unit. A treadmill was provided on the ward for subjects who
wished to exercise during the study, and the amount of exercise was
kept constant throughout the protocol. Subjects were randomly assigned
to receive either a high fat (50% energy as fat, 35% as carbohydrate)
or a low fat (20% energy as fat, 65% as carbohydrate) diet for the
duration of the protocol. Protein was held constant in both diets at
15% of the total calories. Each diet consisted of a 5-day rotating
menu, and our food analysis laboratory validated the macronutrient
content of samples of the daily diets. The diets were designed to be
isocaloric, and total intake was adjusted individually with a goal of
weight maintenance throughout the study.
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4.6 nmol/L). All subjects but two received
dose reduction to either 62.5 or 50 µg/day T3, in most
cases due to higher than desirable levels of serum T3. The
timing of dose reduction differed among subjects, with most subjects
being reduced to 62.5 µg/day after approximately 2 weeks of
treatment. N balance
All food and fluids consumed were measured, and all urine and stools were collected for balance studies. Analysis of creatinine excretion verified the accuracy of daily urine collections. Fecal collection periods were 7 days, separated by administration of an indigestible fecal marker (carmine red dye). Corrections for fecal losses were carried out by quantitating the nonabsorbable marker polyethylene glycol (10% solution; 10 mL, three times daily, with meals). Blood N loss was calculated, and skin and sweat losses were estimated (8, 9). Values reported are based on creatinine-corrected urinary and polyethylene glycol-corrected stool N.
Body composition
Body composition (lean and fat tissue, as well as bone mass) was determined during the run-in period and after 6 and 9 weeks of T3 treatment by dual energy x-ray absorptiometry (QDR2000, Hologic, Waltham, MA). Additionally, seven-site skinfold measures were performed using Lange calipers.
24-h energy expenditure (EE)
Twenty-four-hour EE was measured in an open circuit, whole room, indirect calorimeter. The calorimeter is equipped with a futon bed, a desk, washroom facilities, television/video cassette recorder, refrigerator, motion detectors, and a treadmill. The interior measures 10 x 14 x 8 ft for a total volume of 27,000 L. Subjects entered the calorimeter at 0800 h and remained there until 0700 h the next day. An activity schedule was imposed which included two 45-min periods of treadmill walking. When not exercising or eating, subjects spent their free time watching television, working at the desk, reading, or talking on the telephone. Sleeping during the day was discouraged, and lights were out from 2300–0630 h.
O2 consumption and CO2 production were measured using a Magnos 4G magnetopneumatic O2 analyzer and a Uras 3G infrared CO2 analyzer (Applied Automation, Bartlesville, OK). The analyzers were calibrated daily, and propane burning tests were conducted weekly to verify the precision and accuracy of the calorimeters (>99% for both O2 and CO2). From these values, the daily EE as well as fat and carbohydrate oxidation were calculated according to the method of Jequier et al. (10). Twenty-four-hour urinary N excretion was used for determination of nonprotein respiratory quotient. Sleeping EE was calculated from the lowest sustained metabolic rate between 0200–0500 h, extrapolated to 24 h. Exercise EE was calculated using the area under the curve above baseline during the two exercise periods.
Protein turnover (leucine kinetics)
After an overnight fast, a forearm vein was cannulated for infusion of solutions, and a superficial hand vein in the contralateral arm was cannulated in a retrograde direction and kept open by normal saline infusion. The hand with the sampling vein was kept warm using a heating pad to provide arterialized venous blood samples (11). After baseline blood and breath samples were obtained, a primed (4.8 µmol/kg) continuous (0.06 µmol/kg·min) infusion of L-[1-13C]leucine was given. In addition, a 0.087 mg/kg NaH13CO3 bolus was given to prime the bicarbonate pool (12). Blood and breath samples were obtained every 15 min from 120–180 min, and urine was collected to measure N excretion. O2 consumption and CO2 production were measured using a Sensormedics 2900Z metabolic cart (Yorba Linda, CA).
Plasma [13C]leucine and 
-ketoisocaproic acid
enrichment with 13C were analyzed by gas
chromatography-mass spectrometry, using chemical ionization and
selected ion monitoring (13). The 13CO2
enrichment in expired air was measured using an automated trapping box
and a Finnigan MAT 252 gas isotope ratio mass spectrometer (Finnigan
MAT, Bremen, Germany). Rates of leucine oxidation, incorporation into
body protein, and leucine breakdown were calculated as described by
Motil et al. (14). The rate of 13CO2
released by oxidation of labeled leucine was calculated from the
CO2 production rate and the 13CO2
enrichment in expired air at isotopic steady state, using 0.81 as a
correction for the fraction of 13CO2 released
on oxidation of [1-13C]leucine, but retained in the
bicarbonate pool, and the enrichment of -ketoisocaproic acid, the
immediate precursor for the oxidative decarboxylation of leucine.
Leucine incorporation was calculated from the difference between
leucine flux and leucine oxidation.
Analytical methods
N was determined in daily urinary composites, 7-day fecal composites, and 7-day food composites. Urinary and fecal N were measured by chemiluminescence using a model 703C pyrochemiluminescent system (Antek Instruments, Houston, TX) equipped with an automatic sample injector and a Spectra Physics computing integrator (Spectra Physics, San Jose, CA). The method correlates well with the Kjeldahl method for total N content and has been found to be an effective and reliable monitor of N balance (15). Food N was determined using a Perkin-Elmer model 2410 N analyzer (Norwalk, CT). Thyroid hormones were measured on an Abbott IMx analyzer, using either a microparticle enzyme immunoassay (T3 and ultrasensitive TSH) or a fluorescence polarization immunoassay (T4; Abbott Laboratories, Abbott Park, IL).
Statistical analysis
Data were analyzed using SAS for Windows (SAS Institute, Cary,
NC). Univariate statistics were calculated to determine means and
SEs and to assess the normality of the data. Changes over
time with treatment for measures performed at baseline and 9 weeks were
determined by calculating the difference from baseline for each subject
and assessing whether the changes were significantly different from
zero using a paired, two-tailed Student’s t test. For
measures determined at baseline, 6 weeks, and 9 weeks, a repeated
measures ANOVA was used to assess changes over time, and
post-hoc multiple comparisons were performed using the Tukey
or Dunnett’s procedures. Comparisons between the two diets were made
using a two-tailed Student’s t test. Analysis of N balance
data was performed on the 7-day pooled data using a repeated measures
ANOVA based on the maximum likelihood method. < 0.05 was
considered significant.
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Results |
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. Blood levels of T3 after 1–2
days of treatment ranged from 3.4–5.4 nmol/L, representing, on the
average, a 340% increase above baseline. As most subjects reached
T3 levels slightly above the nominal peak serum
T3 level of 4.6 nmol/L, all but two had their doses
reduced. After dose reduction, mean levels of T3 reached a
plateau of 3.3 ± 0.3 nmol/L by 1 month of treatment and remained
around this level for the duration of the study (200% above
baseline). Initial serum T3 levels showed little
oscillation from morning to evening with the five divided doses per day
(5.4 ± 0.3 at 0800 h vs. 5.5 ± 0.4 nmol/L
at 2000 h; coefficient of variation = 7%). Mean
T4 levels were reduced by nearly 50% after 2 weeks of
treatment and reached a nadir of 36.5 ± 9.6 nmol/L by 9 weeks.
There was some variability in the reduction in serum T4
levels. T4 suppressed by 50% or more by 2 weeks of
treatment in five of the seven subjects while in two subjects
T4 was never decreased by more than 25%. Serum TSH was
also suppressed from a mean baseline level of 1.50 ± 0.32 mU/L to
undetectable levels (<0.03 mU/L) by 1 week in all subjects and
remained suppressed throughout the study.
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. N balance was
slightly, but not significantly, below zero during the 3-week baseline
period (mean range, -0.6 to -2.2 g/day). During the first 3 weeks of
T3 treatment, subjects exhibited a significantly negative N
balance. Specifically, N balance during the second and third weeks of
T3 treatment was significantly lower than the average
baseline N balance (P < 0.05; Fig. 3). Subsequently,
mean N balance tended to return toward zero, although five of the
subjects remained in negative N balance for the duration of the study.
There was a particularly large range of N balance values for the final
week (week 9), with some individuals exhibiting quite negative (-37.8
g/day) and others quite positive (+11.1 g/day) N balance. Despite this
variance, there was a significant difference between the pooled data
from the second through fourth weeks of treatment compared to data from
the fifth through ninth weeks (P = 0.002).
Additionally, there was a significant linear trend in the postbaseline
data (P = 0.004), reflecting the fact that N balance
went from more negative toward neutral over the course of
treatment.
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Subjects lost both lean and fat mass during T3 treatment,
resulting in significant differences from baseline at both 6 and 9
weeks. There were also significant decreases from baseline in triceps,
subscapular, suprailiac, and abdominal skinfold thickness, but the
change in the biceps skinfold was of borderline significance
(P = 0.07; data not shown). Despite the decrease in
LBM, total EE and sleeping EE were both significantly increased by
T3 treatment (Table 1). There were no significant effects
of T3 treatment on respiratory quotient (RQ; data not
shown), probably because of confounding diet effects.
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. Although leucine oxidation tended to be higher after
9 weeks of treatment (P = 0.07), there were no
statistically significant differences in any measure of protein
breakdown between the beginning and the end of the study, paralleling
the effects observed for N balance and LBM at these times.
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although the differences between high and low fat diets were not
statistically significant. Diet had little influence on LBM, N balance,
or 24-h EE. Diet also did not alter 24-h RQ, although baseline RQ was
significantly different between the groups and may have masked dietary
effects (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The desired T3 levels were reached or exceeded within 1
weeks, and there were only slight oscillations in T3 levels
measured at different times of the day. In most cases, the dose of
T3 used in the present study had to be reduced from the
original 75 µg/day suggested by Reed et al. (7) to reach
the target level. A computer model of the dose-response curve to oral
T3 published by Reed et al. (7) showed that
serum T3 levels remain under 300 ng/dL (4.8 nmol/L) at
doses up to 80 µg/day. In contrast, our data show that a dose of 75
µg/day produces serum T3 levels over 400 ng/dL (6.1
nmol/L) in most subjects, suggesting that the previous model is at
least one third too high. Subsequent data from our laboratory
demonstrated that a single loading dose of 100 µg/day, followed by a
50 µg/day maintenance dose, consistently produces the desired rapid
increase in serum T3, with maintenance at 4.8 nmol/L or
slightly less (Lovejoy, J. C., Smith S. R., and Bray G. A., unpublished
data). In addition to the changes in serum T3, we observed
that TSH fell abruptly whereas T4 declined more slowly. Two
subgroups were differentiated based on T4 response, one
exhibiting the expected decline of 50% by 2 weeks, and the other
exhibiting a lesser decline (20%), suggesting higher persistent
release of T4 from the thyroid.
Frank hyperthyroidism is known to be associated with increased catabolism and loss of LBM (16). The present study suggests that the body may be able to partially compensate over time for the catabolic effects of more moderate increases in T3. Although both lean and fat mass were decreased by T3 treatment, as has been seen previously, N balance showed an initial decline, but returned to neutral and, in some individuals, even positive N balance by 9 weeks. This finding is similar to that of Wilson and Lamberts (17), who reported that T3 treatment in obese patients, while promoting weight loss, did not cause a deterioration in N balance. It is also similar to the few findings available from long term space flight, which suggest that astronauts partially compensate for the initial negative N balance in space (18). We observed significant changes in most aspects of 24-h EE with thyroid hormone treatment, which have been noted previously in as little as 2 weeks (19). These data suggest that studies of shorter duration (<6 weeks) should be sufficient to examine some metabolic alterations during experimental hyper-thyroidism.
Differences in dietary composition had minimal effects on metabolic measures, with the exception of a tendency toward attenuation of the loss of body fat caused by T3 with the high fat diet. High fat diets are known to promote obesity (20, 21), whereas low fat diets promote weight loss (21). Thus, our observation that a high fat diet prevents some degree of body fat loss is not totally surprising, although, again, this question has not been examined in hyperthyroid subjects consuming an isocaloric diet. In contrast, a number of studies have examined the effects of T3 administration in individuals consuming very low calorie diets for the purpose of weight loss (22, 23). In these studies, the loss of both fat and fat-free mass was accelerated by T3 administration.
Interestingly, we failed to observe changes in protein turnover and synthesis, which would be expected with elevated thyroid hormone levels. This result may be due to the timing of the leucine kinetics studies, which were performed at baseline and after 9 weeks of treatment (a point when changes in N balance were minimal). Changes occurring at shorter (<6 weeks) intervals may have been missed by this protocol. Previous studies with higher doses of T3 (100 µg/day) detected significant changes in whole body leucine oxidation and protein breakdown after 2 weeks of treatment (24).
One limitation to this study was the small number of subjects, particularly given the variability of some of the measures, which gave low statistical power for some variables. Despite this limitation, we were able to detect significant changes in N balance, muscle and fat mass, and EE variables. It is also possible that our treatment method using a set T3 dose, rather than a dose based on body weight, might have resulted in greater variability in the response to treatment, reducing significant changes. Limited data are available on the variability of response associated with doses given per unit body weight; thus we opted to use a previously published paradigm of experimental T3 treatment (7).
In summary, the present model of experimental hyperthyroidism indicates that healthy men are able to compensate over time for mild increases in serum T3 concentrations by decreasing N losses. There are persistent changes in lean and fat masses as well as measures of EE in this paradigm, however. The dose of thyroid hormone used did not produce clinically significant objective or subjective adverse effects in these young men despite accelerated catabolism. Thus, we suggest that exogenous T3 administration at TSH-suppressive doses may provide a useful addition to the antiorthostatic hypokinesia model of simulated space flight, as well as other experimental and clinical conditions of increased catabolism.
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Acknowledgments |
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Footnotes |
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Received July 29, 1996.
Revised August 29, 1996.
Revised October 25, 1996.
Accepted November 27, 1996.
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References |
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