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-hydroxy--methylbutyrate on
muscle metabolism during resistance-exercise training
Iowa State University, Ames 50011; Metabolic Technologies Inc., Ames, Iowa 50010; MET-Rx Inc., Irvine, California 92715; and North Shore University Hospital, Manhasset, New York 11030
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
ACKNOWLEDGEMENTS
FOOTNOTES
REFERENCES
ABSTRACT 
Nissen, S., R. Sharp, M. Ray, J. A. Rathmacher, D. Rice, J. C. Fuller, Jr., A. S. Connelly, and N. Abumrad. Effect of leucine
metabolite 
-hydroxy--methylbutyrate on muscle metabolism during
resistance-exercise training. J. Appl.
Physiol. 81(5): 2095-2104, 1996.
The effects of
dietary supplementation with the leucine metabolite
-hydroxy--methylbutyrate (HMB) were studied in two experiments.
In study 1, subjects
(n = 41) were randomized among three
levels of HMB supplementation (0, 1.5 or 3.0 g HMB/day) and two protein
levels (normal, 117 g/day, or high, 175 g/day) and weight lifted for
1.5 h 3 days/wk for 3 wk. In study 2,
subjects (n = 28) were fed either 0 or
3.0 g HMB/day and weight lifted for 2-3 h 6 days/wk for 7 wk. In
study 1, HMB significantly decreased the exercise-induced rise in muscle proteolysis as measured by urine
3-methylhistidine during the first 2 wk of exercise (linear decrease,
P < 0.04). Plasma creatine
phosphokinase was also decreased with HMB supplementation
(week 3, linear decrease,
P < 0.05). Weight lifted was
increased by HMB supplementation when compared with the unsupplemented
subjects during each week of the study (linear increase,
P < 0.02). In study
2, fat-free mass was significantly increased in
HMB-supplemented subjects compared with the unsupplemented group at 2 and 4-6 wk of the study (P < 0.05). In conclusion, supplementation with either 1.5 or 3 g HMB/day
can partly prevent exercise-induced proteolysis and/or muscle
damage and result in larger gains in muscle function associated with
resistance training.
resistance training
INTRODUCTION THE ANTICATABOLIC ACTIONS of leucine and certain
metabolites of leucine such as HMB is produced from KIC by the enzyme KIC-dioxygenase and, at least in
the pig, is produced exclusively from leucine (23). Plasma
concentrations of HMB range from 1 to 4 µM but can increase 5- to
10-fold after leucine is fed (25). The cytosolic dioxygenase enzyme
differs from the mitochondrial KIC-dehydrogenase enzyme in several
aspects. The dioxygenase produces free HMB in the cytosol, whereas the
dehydrogenase enzyme produces the CoA derivative of isovaleric acid in
the mitochondria. The cytosolic dioxygenase enzyme requires iron and
molecular O2 for action, which are
not required by the mitochondrial dehydrogenase enzyme (15). The cytosolic dioxygenase enzyme is present in large amounts in the liver
compared with other tissues, including muscle, and it has a 20-fold
higher substrate concentration for half-maximal enzyme velocity (Km) than does the mitochondrial
dehydrogenase. The high substrate concentration required by the
dioxygenase enzyme compared with the liver concentration of KIC (<5
µM) suggests that HMB production in the body may be a first-order
reaction controlled by enzyme and KIC concentrations. It has been
calculated that, under normal conditions, ~5% of leucine oxidation
proceeds via this pathway (23). If humans are assumed to have enzyme
actions similar to those seen in pigs, a 70-kg human would produce from 0.2 to 0.4 g HMB/day depending on the level of dietary leucine. At leucine intakes of 20-50 g/day (which are used
therapeutically), the concentrations of leucine and KIC in the
liver increase and could result in HMB production reaching gram
quantities per day.
The objective of the first study presented was to determine whether the
administration of Ca-HMB to humans undergoing a regimen of stressful
resistance exercise would result in the slowing of exercise-induced
proteolysis. Second, because the popular literature suggests that the
intake of high-protein concentrates enhances gains in muscle function
achieved with resistance training, two levels of protein intake were
used in the present study. Normal- (117 g/day) and high-protein (175 g/day) intakes were compared to determine whether very high protein
intakes would increase muscle mass and/or muscle strength
during resistance training. A second longer study was also conducted to
determine whether the changes in body composition and strength seen
during the first study were manifest over a longer period of time.
MATERIALS AND METHODS Human Subjects
-ketoisocaproate (KIC) have been
known for 35 years (7). Both leucine and KIC are proposed
to decrease nitrogen and protein loss by inhibiting protein breakdown;
however, extensions of this in vitro work to animals and humans have
not clearly shown an anabolic effect except in situations of either severe stress or trauma in which proteolysis is greatly elevated (3, 6,
16, 18). This suggests either that KIC and leucine are active only
during periods of excessive catabolism or that a further metabolic
product of leucine (and KIC) may be variably produced depending on the
prevailing metabolic milieu and may be responsible for the
anticatabolic effects of these compounds. Based on several animal
studies, we hypothesized that the leucine metabolite
-hydroxy--methylbutyrate (HMB), produced in the body from leucine
via KIC, is responsible for the inhibitory effect on protein breakdown.
Study 1
Experimental design. Table 1 summarizes the measurements made during study 1. The experimental periods and collections are summarized as follows.
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-glutamyl transpeptidase, serum
glutamic-oxaloacetic transaminase (SGOT), and serum glutamic-pyruvic transaminase (SGPT) by Roche Biochemical Laboratories (St. Louis, MO).
Plasma was analyzed for HMB by gas chromatography-mass spectrometry (12). Plasma-free amino acids were measured by high-performance liquid
chromatography (Waters Pico Tag System).
URINE COLLECTIONS.
Two 24-h urine collections were made each week. After the total volume
of urine collected was measured, a sample was frozen at 
20°C
for later analysis of nitrogen (macro-Kjeldahl), creatinine, HMB, and
3-MH. Urine 3-MH and HMB were quantified by previously described gas
chromatography-mass spectrometry techniques (12, 13).
BODY COMPOSITION.
Estimates of whole body lean and fat masses were measured by using
TOBEC (model HA-2, EM Scan, Springfield, IL). The subjects were
scanned, and fat tissue was predicted by using equations supplied by
the manufacturer and derived from data generated by the University of
Illinois (Urbana-Champaign) (9). Scanning of all subjects in the
morning before breakfast was not logistically possible. Therefore, the
subjects were scanned between 9 A.M. and 4 P.M. Because body composition
measures are influenced by the state of hydration and food intake, an
attempt was made to more truly estimate lean body mass in the fasted
state. Fat mass measured by TOBEC was subtracted from the fasting body
weight to yield a fasting lean mass for all subjects. This assumes that only fat-free mass (FFM) will be affected by food and/or water consumption and that fat mass will remain the same regardless of fluid
and/or food intake. We hypothesized that this calculation gave
a more realistic estimate of lean tissue mass, which would have been
influenced by a fluctuation in fluid and food intake throughout the
day. This assumption was verified in a separate experiment where 12 male subjects were scanned in the morning after an overnight fast and
then scanned again in the afternoon after two meals. In the morning,
the fat mass and FFM were 24.2 ± 2.6 and 68.1 ± 2.5 kg,
respectively, and in the afternoon, fat mass and FFM were 23.8 ± 2.5 and 69.7 ± 2.4 kg, respectively. The FFM was significantly
higher in the afternoon (P < 0.001, paired t-test), but the
fat mass was not significantly different between the two times. By
using the fasting weight minus the fat mass, the corrected FFM was 68.5 ± 3.0 kg, which was not significantly different from the morning
value of 68.1 kg. Thus the assumption that food intake will not affect
fat mass was valid, and a correction to calculate fasting FFM was
appropriate.
FFM was therefore calculated by subtracting the fat mass from the
fasting body weight to measure true FFM. These calculated values vs.
the actual "fed" TOBEC values did not change the statistical interpretation of the data (HMB effect,
P < 0.09), but corrected values
showed a decrease in the FFM gained over the 3 wk compared with
uncorrected values, with the corrected values being more realistic
values.
Study 2
Thirty-two male volunteers 19-22 yr of age were selected for the study. Body weight averaged 99.3 ± 3.4 kg with a range of 72-136 kg and height was 185 ± 1.5 cm with a range of 170-198 cm. Almost all subjects were engaged in some form of exercise program before the study.Experimental Design
Body composition was measured with TOBEC as in study 1 except that all subjects were measured after an overnight fast. Measurements were made on the Friday morning before the start of the experiment and each Friday thereafter. Strength measurements were made the week before the study and consisted of three standard strength measurements: the bench press, the squat, and the hang clean. Treatments and exercise regimens were started on Monday. The exercise regimen consisted of weight training 6 days/wk, which included work on all major muscle groups, and lasted from 2 to 3 h/day. Aerobic training was also included in the exercise workout at least three times per week.No dietary control was imposed in study 2, and the subjects were instructed to eat normally. Most meals were obtained at the Iowa State University athletic-training table. In addition, a nutrient shake was available during each training session. This was available to all subjects regardless of treatment and was served in the weight-training area.
The subjects were randomly assigned to one of two supplements, one of which contained HMB, so they did not know whether they were receiving HMB. Because we found no effect of added nutrient supplementation on HMB effects in study 1, HMB was delivered in the nutrient shake identical to that used in study 1 (MET-Rx). The placebo was an orange drink mix that contained calories equal to the nutrient shake but no added protein. Thus the two groups likely had different protein intakes, although we roughly estimated the total protein intake of the placebo group to be ~180 g/day and the HMB group to be ~200 g/day.
Statistics
The general linear models procedure of the Statistical Analysis System (17) was used to statistically analyze the data. Because the major objective of the experiment was to determine the dose-responsive effect of HMB over the 3-wk period, an analysis of variance model was used that included the main effects of protein and dose-responsive linear and quadratic effects of HMB supplementation and protein by HMB interaction. Only the main effects of HMB supplementation and the main effect of protein intake are presented because no significant protein intake × HMB supplementation interactions were found. A pooled SE for HMB is given for each measurement. The SE for HMB is most indicative of the variation among HMB groups. In study 2, differences between the two treatment groups were determined with a t-test. Differences were considered significant if P < 0.05. Trends were determined for 0.05 < P < 0.11, and differences were considered not significant for P > 0.11.RESULTS
General
In study 1, two subjects withdrew during the first week of exercise because of incompatibility of the study requirements with their schedules. Compliance was >95% concerning food consumption and exercise training. One subject (3 g HMB/day and high protein) admitted to major violations of the dietary protocol on two of the sample-collection days; therefore, his blood and urine data were not included in the analysis on these days. Other minor violations in dietary protocol were 2-4 days removed from the data collections and were not considered serious enough to exclude the data collected from these subjects. One subject did not exercise for two sessions because one leg was injured. Another subject could not complete the pectoral exercise because of a previous shoulder injury but was not dropped from the study because all other exercises were done. Four subjects dropped out of the second study for personal reasons. No adverse effects were noted in either study.Study 1
Dietary intake. The calculated intakes of protein, fat, and calories are listed in Table 2. The high-protein group had 50% greater protein intake than the normal-protein subjects. Total energy intake averaged ~9.707 MJ (2,320 kcal)/day in normal-protein subjects and ~10.293 MJ (2,460 kcal)/day in high-protein subjects. This resulted in a total intake of ~10.878 additional MJ (2,600 kcal) over the 3-wk study by the high-protein subjects.
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Body composition. Body composition and weight changes are listed in Table 3. The 0-, 1.5-, and 3-g HMB groups lost 1.41, 0.26, and 0.41 kg, respectively, over the 3 wk of study (P < 0.03, negative linear effect of HMB). There was no significant effect of protein level on body weight change. The body-composition analysis showed 1-1.8 kg of fat lost over the 3-wk period. The loss of fat was not significantly different among the groups. Lean tissue gain tended to increase in a dose-responsive manner with HMB supplementation (0.4, 0.8, and 1.2 kg lean gain for 0-, 1.5-, and 3.0-g HMB groups, respectively; P < 0.11, linear).
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Muscle strength. The changes in muscle work and strength are listed in Table 4 and Fig. 1. The total work (number of repetitions × weight) at the beginning and end of the study is listed in Table 4. All subjects increased the amount of weight lifted in each exercise and the total number of abdominal efforts during the 3-wk training period (net change). No differences in the amount of weight lifted were seen between the normal- and high-protein groups for any of the exercises. HMB increased the number of abdominal exercises and the lower body weight lifted more markedly than it increased the upper body weight lifted. The increase in lower body weight lifted during the study was greater in the HMB-supplemented groups than in the unsupplemented group (P < 0.01). The unsupplemented subjects increased the number of abdominal efforts by 14% during the 3 wk, whereas both HMB-supplemented groups increased the number of abdominal efforts ~50% over the 3-wk study (P < 0.05). The total strength (combined upper and lower body weight totals) increased by 8% in the unsupplemented subjects during the 3-wk period, whereas in the 1.5- and 3.0-g HMB-supplemented groups, total strength increased by 13 and 18.4%, respectively (P < 0.02). Figure 1 graphically depicts the increase in total strength gains over the 3-wk period. Expressing the data as maximum weight lifted vs. total weight lifted (work) resulted in a similar HMB effect.
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-hydroxy--methylbutyrate (HMB). Each set of bars represents
one complete set of upper and lower body workouts.
*** P < 0.01;
** P < 0.02;
* P < 0.03 (significant linear
effect of HMB supplementation).
Muscle protein breakdown. Plasma CK and LDH levels are presented in Table 5. Plasma CK levels in subjects not supplemented with HMB increased to 15,868 U/ml after 1 wk of exercise. HMB-supplemented subjects had lower levels of CK, but because of extreme variation in the concentrations among subjects, it was not significant. By week 3, HMB supplementation had decreased plasma CK levels in a dose-responsive manner (P < 0.05). Protein intake did not affect plasma CK levels. Plasma LDH levels followed the same pattern as CK levels except for a less dramatic increase during the first week. HMB supplementation also tended to decrease plasma LDH in a dose-responsive manner in weeks 2 and 3 (P < 0.08 and P < 0.07, respectively).
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The rate of 3-MH loss in urine is also presented in Table 5. The
percent change in urinary 3-MH from the basal level is depicted in Fig.
2. One week after exercise started, urinary
3-MH increased 94% in subjects not supplemented with HMB, 85% in
subjects supplemented with 1.5 g HMB/day, and 50% in subjects
supplemented with 3 g HMB/day. In week
2 of the experiment, urinary 3-MH in control subjects
was still 27% above the basal level. However, urinary 3-MH was 4 and
15% below the basal level for the 1.5 and 3.0 g HMB/day supplemented
subjects, respectively (P < 0.001, linear effect of HMB). During the third week of the study, HMB did not have a significant effect on urinary 3-MH, although the trends continued. Expressed as a percentage of muscle broken down per day, the
unsupplemented group increased from 3% of the total muscle breakdown
per day to 6%/day, whereas the 1.5 and 3 g HMB/day groups increased
from 3 to 5.5 and 3 to 4.5%/day, respectively. High protein increased
urine volume (P < 0.05), which
resulted in a lower creatinine concentration in the urine
(P < 0.05). There was no effect of
HMB on either urine volume or creatinine concentration. The end result
was that there were no significant effects of either HMB or protein on
total urine creatinine output per day (data not shown).
Plasma amino acids. Plasma amino acids were measured during the basal period and at the end of the study. The plasma concentration of most amino acids was not significantly changed by either protein intake or HMB supplementation. However, the sum of all essential amino acids in plasma increased 32% in subjects not receiving HMB but decreased 9 and 18% in the 1.5 and 3 g HMB/day supplemented subjects, respectively (linear effect of HMB, P < 0.04). In addition, subjects consuming the high-protein diet had 26% lower plasma glycine (P < 0.05) and 25% lower plasma serine concentrations (P < 0.01). Plasma proline in the high-protein group increased by 17% during the study (P < 0.05), whereas plasma proline in the control subjects only increased 4%.
Plasma and urine HMB. Plasma HMB was measured after an overnight fast (~12 h after the last HMB consumption). Plasma HMB concentrations remained constant in subjects receiving no HMB. However, plasma HMB increased in a dose-responsive manner in the 1.5 and 3.0 g HMB/day groups, with levels increasing from basal levels of 2.8 µM to levels of 10.7 and 20.3 µM, respectively (P < 0.0001). Urine HMB was measured in the 2-day quantitative urine collections. Urine HMB varied from 10 to 30 mg of free acid equivalents per day during the basal period. Supplementation with 1.5 g HMB/day resulted in an increase to ~450-500 mg of free acid equivalent lost in urine per day over the 3-wk period (P < 0.0001). This amounted to ~43% of the HMB fed after correction for endogenous production. Supplementation with 3.0 g HMB/day increased the loss of HMB in urine to ~950-1200 mg (free acid equivalents), which again is less than one-half of that fed (P < 0.0001).
Other measurements. Plasma sodium, potassium, chloride, calcium, and phosphorus and red and white blood cell counts were unaffected by either HMB supplementation or protein intake. Also, the levels of the plasma enzymes GGT and alkaline phosphatase were unaffected by either HMB supplementation or protein intake. Values for these parameters were all within normal ranges. The plasma enzymes SGOT and SGPT showed slight but not significant increases with exercise, and there was no significant effect of HMB on these increases. There was an effect of protein intake on SGOT and SGPT. Whereas SGOT was increased in both protein groups, SGOT in the high-protein group during week 1 was increased more than in the normal-protein group (P < 0.05). SGPT decreased to basal values in the normal-protein group over the 3 wk while still remaining higher in the high-protein subjects at the end of the 3 wk (P < 0.01). No adverse reactions or other symptoms were measured relative to either HMB supplementation or protein intake. In the high-protein subjects, plasma creatinine levels decreased 12% after 3 wk (P < 0.002) when compared with the control subjects.
Study 2
The results of study 2 are presented in Table 6 and Fig. 3. Over the period of exercise, all subjects tended to increase body weight and fat weight, although there was no significant effect of supplementation on these measures. The gain in FFM depicted in Fig. 3 indicates that the HMB-supplemented subjects showed significant increase in FFM at the earliest measurements. By day 14 and through day 39, the FFM gained by the HMB-supplemented group was significantly more than in the unsupplemented group (P < 0.05). On the last day of the study, FFM was not significantly different between the groups.
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Strength measurements are also presented in Table 6. These represent 1 RMs for the bench press and the squat lift. The bench press was significantly increased by almost threefold with HMB supplementation (P < 0.01). Although the squat lift increase was numerically higher with the HMB treatment, it was not significant.
DISCUSSION
The major finding of this study is that HMB supplementation resulted in an enhancement of muscle function in humans undergoing resistance exercise. This effect was clearly shown by increases in muscle strength and is supported by increased lean tissue mass in both studies and decreased biochemical indicators of muscle damage. The most direct evidence of altered muscle metabolism by HMB was the 20% decrease in 3-MH loss in urine and a 20-60% decrease in the levels of enzymes, indicating muscle damage in the plasma. These changes suggest that HMB prevents or slows muscle damage as well as partially preventing the increase in proteolysis associated with intense muscular work. The decrease in muscle proteolysis is consistent with in vitro studies with leucine and KIC that suggest that leucine and metabolites act directly to decrease muscle proteolysis (22). This is, however, the first demonstration that the administration of either leucine or its catabolites can alter both muscle mass and strength in normal humans consuming adequate protein. The exact mechanism of the effect of HMB on muscle metabolism is not known, but at least two potential hypotheses can be put forth to explain these results.
Hypothesis 1: HMB Inhibition of Proteolytic Processes
The decrease in urine 3-MH is consistent with a decrease in muscle protein turnover. Numerous studies have shown that incubation of the muscle with either KIC or leucine inhibits proteolysis in muscle (8). This could explain the effect of HMB in the first week of exercise when unsupplemented subjects lost strength while HMB-supplemented subjects gained strength. Supporting a decrease in muscle proteolysis was the decrease in essential plasma amino acids in blood with HMB supplementation. Because these plasma amino acid values were obtained from overnight-fasted subjects, the major contributor to plasma amino acids would be endogenous muscle proteolysis. Last, there were also other indications of less muscle-specific damage in the HMB-supplemented subjects undergoing muscle-damaging heavy-resistance exercise, such as the decrease in plasma levels of CK and LDH. Lower plasma CK and LDH suggest that less inflammation and/or damage to the muscle plasma membrane may have occurred.Hypothesis 2: Participation of HMB in an Unknown Process
The metabolic function and fate of HMB are not fully understood. The data presented here suggest that over one-half of the HMB fed is metabolized in the body. Based on the known biochemistry, the most likely metabolic fate of HMB would be conversion to HMG-CoA (2). Alternatively, preliminary studies have shown that HMB may also be covalently linked in some form in the tissues. Hydrolysis with acid and base resulted in 10-100 µM HMB concentrations in tissues (24). This suggests that HMB may be part of some structural component within tissues or membranes. Although it is not known what specific chemical combinations of HMB are produced in the tissues, there are at least two possibilities. The first is through esterification to CoA derivatives or phosphorylation, which can occur with hydroxy acids and hydroxy amino acids through the hydroxyl group. The other possibility is that HMB forms a polymer or copolymer in the tissues. The chemically similar compound-hydroxybutyrate (BHB) has been found to polymerize in
plants, bacteria, and animal tissues (19). In animals, it has been
proposed that poly-BHB is a component of the calcium channel of the
cell membrane (14). Because HMB and BHB are very similar chemically and
HMB has been shown to be covalently bound in tissues, HMB could be
present in the cell as a polymer or copolymer.
The loss of almost one-half of the supplemented HMB via the urine suggests that the kidney does not actively reabsorb HMB. This is similar to the metabolism of many water-soluble vitamins and BHB in that urinary loses are proportional to blood levels. Studies with 40-kg pigs showed that feeding 2 g of HMB resulted in 200 µM plasma HMB concentrations that peaked 2 h after administration. Subsequently, plasma concentrations decreased, with a half-life between 2 and 3 h (24). Thus, in the present study, plasma concentrations of HMB could have reached 100-200 µM in the period of 2-3 h after HMB was consumed. This could have resulted in large initial losses of HMB in urine that may have diminished as plasma HMB concentrations fell. This is supported by the observation that plasma concentrations of HMB are increased 5- to 10-fold, whereas urine losses of HMB were 10- to 20-fold higher in the HMB-supplemented subjects than in the unsupplemented subjects. These data suggest that feeding HMB twice per day may not have been ideal for maximum effectiveness.
The enhancement of muscle protein metabolism by HMB in this exercise/stress model could also have relevance in other stressful situations. However, an effect of HMB could be predicted based on the effectiveness of the HMB precursors leucine and KIC in slowing protein loss during starvation (4, 11), trauma (18), and burns (1, 18). The exercise/stress model used here caused an increase in proteolysis that also occurs with chronic wasting diseases or acute stress found with burns and severe trauma. Further studies will be necessary to determine whether HMB could be useful in preventing or slowing the proteolytic process in disease.
General Effect and Effects of Protein Supplementation
The resistance-exercise regimen used in study 1 resulted in marked anabolic response with weight training, although in the second study where the frequency and length of exercise were longer, the net effects of resistance exercise without HMB supplementation appeared to be minimal. The role of protein intake in moderating this anabolic response is less clear. The popular literature and the preponderance of commercial protein products designed for exercise training suggest that a greater intake of protein is needed. This is based on the notion that muscle anabolism must increase the requirement of dietary protein. However, controlled studies defining the protein and amino acid requirements of resistance-training humans have not been extensively reported (21). This controversy is neither answered in the present study nor will it be easily answered in future clinical studies because of the myriad of potential confounding experimental conditions. These variables include intensity of training, frequency of training, the timing of training, the genetic potential for muscle growth, and the interaction of other nutrients on muscle anabolism. It should be noted that protein intake of the control group was already twice the RDA of protein intake for maintaining nitrogen balance. Thus, although there was no significant effect of protein supplementation above twice the RDA, it is unclear whether protein supplementation between the RDA and twice the RDA can benefit the anabolic response to resistance training. No significant protein intake × HMB interactions were noted relative to any of the reported measures, suggesting that the HMB effects on metabolism are additive with and independent of protein intakes.In summary, dietary supplementation of 3 g of HMB/day to humans undergoing intense resistance-training exercise resulted in an increased deposition of FFM and an accompanying increase in strength. Muscle proteolysis was also decreased with HMB, which was accompanied by lower plasma levels of muscle damage-indicating enzymes and an ~50% decrease in the concentrations of plasma essential amino acids. The mechanism by which HMB impacts muscle proteolysis and function is not currently known.
ACKNOWLEDGEMENTS
The authors thank Dr. Doug King, who performed many of the muscle biopsies during the study, and Dr. Paul Flakoll, who performed the amino acid analysis on the blood samples. The authors also acknowledge the help of Connie Coates, Ron Wilhelm, Nancy Renaud, and others who helped with the various aspects of the study and preparation of the manuscript.
FOOTNOTES
Address for reprint requests: S. Nissen, 301 Kildee Hall, Iowa State Univ., Ames, IA 50011
Received 18 August 1995; accepted in final form 1 July 1996.
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