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From The Clinical Research Centers |
Departments of Surgery (B.B.R., D.C.G., R.R.W.) and Internal Medicine (E.V.), University of Texas Medical Branch, and the Metabolism Department, Shriners Hospital (B.B.R., E.V., R.R.W.), Galveston, Texas 77550
Address all correspondence and requests for reprints to: Robert R. Wolfe, Ph.D., Shriners Hospital, Metabolism Department, 815 Market Street, Galveston, Texas 77550. E-mail: rwolfe{at}utmb.edu
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Abstract |
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Six healthy young men were studied before the treatment period and after 5 days of oral androstenedione supplementation. Muscle protein turnover parameters were compared to those of a control group studied twice as well and receiving no treatment. We measured muscle protein kinetics using a three-compartment model involving infusion of L-[ring-2H5]phenylalanine, blood sampling from femoral artery and vein, and muscle biopsies. Plasma testosterone, androstenedione, LH, and estradiol concentrations were determined by RIA.
After ingestion of oral androstenedione, plasma testosterone and LH concentrations did not change from basal, whereas plasma androstenedione and estradiol concentrations were significantly increased (P < 0.05). Compared to a control group, androstenedione did not affect muscle protein synthesis and breakdown, or phenylalanine net balance across the leg.
We conclude that oral androstenedione does not increase plasma testosterone concentrations and has no anabolic effect on muscle protein metabolism in young eugonadal men.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The purpose of this study was to assess 1) whether 5 days of oral androstenedione supplementation increases plasma testosterone concentration, and 2) whether oral androstenedione has an anabolic effect on skeletal muscle.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Six healthy male volunteers (age, 32 ± 4 yr; height, 179 ± 2 cm; weight, 77 ± 6 kg; body mass index, 23.9 ± 1.6 kg/m2) were studied in the postabsorptive state before and after androstenedione administration. The leg volume, estimated using an anthropometric approach (12), was 12.3 ± 1.6 L. Six healthy volunteers (three women and three men) were also studied twice in the postabsorptive state and used as a control group for determinations of muscle protein kinetics (age, 31 ± 3 yr; height, 168 ± 3 cm; weight, 67 ± 5 kg; body mass index, 23.7 ± 1.2 kg/m2; leg volume, 9.5 ± 0.5 L). All subjects gave informed, written consent before participating in the study, which was approved by the institutional review board of the University of Texas Medical Branch (Galveston, TX). Each subject was screened for determination of health status at the General Clinical Research Center at the University of Texas Medical Branch. Subjects were recreationally active, although none was involved in consistent resistance exercise training. None of the subjects was taking any form of medication, creatine, amino acid supplements, or anabolic steroids or was on an excessive protein diet.
Protocol
Each subject was studied twice. In the subjects given
androstenedione, the first study was performed to acquire baseline
data, and the second study was performed after 5 days (5–7 days after
the initial study) of androstenedione administration (Fig. 1a
). The six subjects included in the control
group were also studied twice. The control group was used to assess the
variability of muscle protein kinetics within the same subject on two
different occasions. The subjects did not engage in physical exercise
the day before being studied. They were asked to maintain their normal
dietary patterns, and they kept a notebook detailing their food intake
and physical activity. The night before each study the subjects were
admitted at the General Clinical Research Center of the University of
Texas Medical Branch. After 2200 h, the subjects were allowed only
water ad libitum. After an overnight fast, a Teflon catheter
was placed in a forearm vein for isotope infusion, another catheter was
placed in a wrist vein of the opposite arm for arterialized blood
sampling, and femoral arterial and venous catheters were placed. A
blood sample was drawn for background phenylalanine enrichment and
androstenedione, testosterone, estradiol, and LH determinations (Fig. 1b). A primed (2 µmol/kg), continuous infusion (0.05 µmol/kg·min)
of
L-[ring-2H5]phenylalanine
(98% enriched; Cambridge Isotope Laboratories, Woburn, MA) was then
started and maintained throughout the study. After 2 h of
infusion, an initial muscle biopsy was taken from the vastus lateralis,
approximately 20 cm above the knee, using a 5-mm Bergström biopsy
needle (Stille, Stockholm, Sweden). To measure leg blood flow, an
indocyanine green (ICG) infusion was started (0.5 mg/min) in the
femoral artery 4 h after the start of the isotope infusion and
continued for 30 min. Blood samples were collected at 10-min intervals
from the femoral vein and the heated wrist vein. Subsequently, the ICG
infusion was stopped, and four arterial and venous blood samples were
collected every 10 min for analysis of phenylalanine enrichment.
Additional blood was collected at the beginning and end of the fifth
hour of the study to measure hormone concentrations. At the end of the
study a muscle biopsy needle was inserted into the leg in the opposite
direction from the previous biopsy, and a second biopsy was taken.
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Analysis
Blood phenylalanine enrichments and concentrations were determined by GC/MS (model 5973, Hewlett-Packard Co., Palo Alto, CA) after purification of the amino acids (13) and derivatization to tert-butyldimethylsilyl derivative. Isotopic enrichments are expressed as the tracer to tracee ratio (13).
Free muscle intracellular phenylalanine enrichments were measured by GC/MS after extraction and purification as previously described (13). The enrichments of protein-bound phenylalanine were measured after hydrolysis of the extracted muscle proteins (13) using the external standard curve approach (14).
Leg blood flow was determined from blood samples collected during the
continuous infusion of ICG (15, 16). Sera from blood samples were
analyzed in a spectrophotometer with absorbance set at 
= 805
nm. The coefficient of variation of each ICG measurement (intrasubject)
was less than 5%.
Plasma androstenedione, testosterone, LH, and estradiol concentrations were determined by RIA (Diagnostic Products, Los Angeles, CA). According to the manufacturer, the cross-reactivity of androstenedione on the estradiol assay is not detectable.
Calculations
The three-compartment model of leg muscle amino acid
kinetics used in this study has been described previously (17). Use of
this model allowed us to determine the rate of utilization of
phenylalanine for muscle protein synthesis and its intracellular
appearance from muscle protein breakdown. The model assumptions are
addressed in Refs. 17, 18 . The parameters of the three-compartment
model of leg amino acid kinetics used to determine the phenylalanine
kinetics are defined as follows (Fig. 2):
phenylalanine entry into leg: Fin =
CA x BF (1); phenylalanine exit from leg:
Fout = CV x BF (2); net
balance across the leg: NB = (CA -
Cv) x BF (3); muscle inward transport:
FM,A = {[(EM -
EV)/(EA -
EM)] x CV +
CA} x BF (4); muscle outward transport:
FV,M = {[(EM -
EV)/(EA -
EM)] x CV +
CV} x BF (5); A-V shunting:
FV,A = Fin -
FM,A (6); protein breakdown:
FM,O = FM,A x
[(EA/EM) - 1] (7);
and protein synthesis: FO,M =
FM,O + NB (8). Components of the kinetic
parameters are defined as follows: CA and
CV, concentrations of phenylalanine in the artery
and vein, respectively; BF, leg blood flow; and
EA, EV, and
EM, enrichments (tracer/tracee) of phenylalanine
in the femoral artery and vein, and intracellular muscle, respectively.
The muscle protein fractional synthetic rate (FSR) was calculated using
the precursor-product model previously described (19, 20).
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Data are expressed as the mean ± SEM. Pre-post differences in hormone concentrations in the androstenedione group were assessed using the two-tailed paired t test. Differences in muscle protein kinetics between control and androstenedione were analyzed using ANOVA for repeated measures. Pairwise multiple comparisons were carried out using the t test with Bonferroni’s inequalities. Differences were considered significant at P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). The
time courses for androstenedione and testosterone concentrations after
androstenedione ingestion for one representative subject are shown in
Fig. 3.
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).
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. Phenylalanine transport into and out of
the leg did not change with time in both groups, with no differences
between the groups. There was a trend for an overall increase in muscle
protein breakdown with time (time effect, P = 0.086),
which was entirely attributable to the group receiving androstenedione
(P = 0.070, pre vs. post within the
androstenedione group; P = 0.521, pre
vs. post within the control group). Muscle protein synthesis
tended to be higher with time in both groups (P =
0.051). Although the synthesis was slightly elevated in the
androstenedione group, the change was not different from that observed
in the control group, nor was the change as large in magnitude as the
increase in breakdown. Net muscle balance of phenylalanine across the
leg, a measure of net protein deposition, did not change over time in
either group. However, net balance tended to be more negative in the
androstenedione group (time by treatment interaction; P
= 0.093), indicating an increase in net protein catabolism rather than
an improvement in anabolism as predicted (Table 3).
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).
Consistent with the data from the three-compartment model, no
differences were found between groups, and no time effect was seen in
either the treatment group (0.074 ± 0.015%/h vs.
0.077 ± 0.023%/h) or the control group (0.056 ± 0.017%/h
vs. 0.057 ± 0.008%/h).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our failure to demonstrate an effect of androstenedione on either testosterone concentrations or muscle protein synthesis is probably not due to the study design. For example, we have shown that 5 days after an im injection of 200 mg testosterone enanthate in young men whose activity level and physical characteristics were similar to those of the volunteers of the present study, plasma testosterone was significantly elevated, and muscle protein synthesis increased 2-fold (7). We have also detected an anabolic effect on muscle protein synthesis when a synthetic anabolic hormone (oxandrolone) was given orally for 5 days, even though testosterone plasma concentrations significantly declined, and oxandrolone concentrations increased only to approximately 2 ng/dL. (21). To further substantiate the validity of our study design, we have included a control group, showing that muscle protein synthesis is not affected by prior determination of the same value.
Although there was a trend for muscle protein synthesis to increase, no significant differences were found between androstenedione and control groups using two different methods (three-pool model and precursor-product approach). On the contrary, there was a trend for muscle protein breakdown to be elevated, which was entirely attributable to androstenedione intake. The trend for an increase in protein breakdown after androstenedione treatment may have been the consequence of the increase in estradiol. In fact, it has been reported that long term exposure to estrogens decreases muscle fiber size in rats (22). Overall, the trend for an increase in both protein breakdown and synthesis indicates that androstenedione tended to increase muscle protein turnover. However, the increased turnover did not lead to muscle protein anabolism, as the increase in protein breakdown exceeded the increase in protein synthesis. A similar situation takes place in catabolic states such as sepsis or burn injury, in which muscle protein breakdown is significantly elevated (23, 24), and muscle protein synthesis increases as well, although not enough to counteract the catabolic effect of increase in breakdown. The stimulation of muscle protein synthesis in this circumstance is probably due to the increased availability of intracellular amino acids secondary to accelerated rate of breakdown. The trend for an increase in muscle protein turnover may also indicate tissue remodeling and, hence, improvement in muscle function, but a recent study found no improvement in muscle function after androstenedione supplementation (25). Regardless of the mechanisms accountable for the response to androstenedione ingestion in the present study, the increase in protein turnover observed did not lead to muscle protein anabolism.
The failure of androstenedione intake to raise testosterone concentrations is somewhat surprising. A study showed that approximately 5% of circulating androstenedione is converted to testosterone (26). Considering that the daily production of testosterone is about 5 mg/day (26) and that we administered 100 mg/day androstenedione, if 5% of the oral androstenedione was converted to testosterone, we should have observed a doubling of testosterone concentrations. As this was not the case, our results could have been due to a suppression of endogenous testosterone secretion by androstenedione. However, the plasma LH concentration was not suppressed by androstenedione administration, suggesting that androstenedione per se or the derived estrogens did not inhibit the hypothalamic-pituitary-Leydig cell axis. Our results are consistent with recent data from another laboratory (25) showing that androstenedione does not increase testosterone concentrations during short term administration (100 mg/day) or when given during resistance training (300 mg/day). In addition, the same study reported no differences between the androstenedione supplement group and a placebo group after 8 weeks of resistance training for the following measures: volitional muscle strength, mean cross-sectional area of type 2 muscle fibers, and lean body mass (25). Thus, it is likely that a significant proportion of ingested androstenedione is reduced and conjugated by the liver before reaching peripheral testosterone-converting tissues. In fact, a kinetic study has shown that 5.9% of the androstenedione administered iv was converted to testosterone, whereas the corresponding value was only 1.8% when androstenedione was given via the gastrointestinal route (10). This difference was due to the fact that 89% of the orally administered androstenedione was converted to testosterone glucuronide, which was excreted through the urinary tract (10).
In conclusion, 5 days of oral androstenedione administration at double the dosage suggested by the manufacturer does not increase plasma testosterone concentration, nor does it have an anabolic effect on skeletal muscle. Our inability to demonstrate the putative beneficial effects of androstenedione on skeletal muscle protein contradicts the popular idea that androstenedione is an ergogenic aid for athletes. To the contrary, the increase in estrogens, the possible interaction or competition with androgen receptors, and the possible carcinogenic effect of prolonged androgen intake make androstenedione consumption inadvisable in healthy eugonadal men.
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Acknowledgments |
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Footnotes |
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Received June 4, 1999.
Revised July 28, 1999.
Revised October 7, 1999.
Accepted October 14, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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