Effects of Ractopamine HCl Stereoisomers on Growth, Nitrogen
Retention, and Carcass Composition in Rats1
E. A. Ricke*, D. J. Smith†,2, V. J. Feil†, G. L. Larsen†, and J. S. Caton*
*Department of Animal and Range Sciences, North Dakota State University, Fargo 58105
and †USDA-ARS Biosciences Research Laboratory, Fargo, ND 58105
ABSTRACT: The objectives of this study were to
determine the effects of ractopamine HCl (RAC)
stereoisomers (RR, RS, SR, and SS) on performance,
carcass composition, and nitrogen retention in growing
female rats. Forty-eight rats (eight rats/treatment)
were treated with 0 or 320 mg/d of RAC or with 80 mg/d
of the RR, RS, SR, or SS stereoisomers of ractopamine.
Rats had free access to feed and water before and
during the experiment. Ractopamine and
stereoisomers were delivered via i.p. implanted os-
motic pumps for 14 d, and rats were then slaughtered.
Control rats were fitted with osmotic pumps contain-
ing saline. Ractopamine increased ( P < .05) feed
intake ( d 1 to 6); body weight; carcass CP; and intake,
apparent absorption, retention, and retained:intake
Key Words: Ractopamine, Growth, b-Adrenergic Agonists
1999 American Society of Animal Science. All rights reserved.
Introduction
Phenethanolamine b-adrenergic agonists are, by
definition, chiral compounds. That is, molecular asym-
metry exists at the hydroxylated carbon b- to an
aliphatic nitrogen and a- to a substituted benzyl group
common to all phenethanolamine b-agonists (Figure
1). On a practical level, this means that most
commercial phenethanolamine drugs are mixtures of
stereoisomers unless stereospecific synthetic or purifi-
cation techniques are used during their manufacture.
Cimaterol (U.S. Pharmacopeia, 1997), clenbuterol
(Abou-Basha and Aboul-Enein, 1996), ractopamine
1Mention of trademark or proprietary products does not consti-
tute a guarantee or warranty of the product by the USDA and does
not imply its approval to the exclusion of other products that may
also be suitable.
2To whom correspondence should be addressed: P.O. Box 5674,
University Station (phone: 701/239-1238; fax: 701/239-1430; E-mail:
smithd@fargo.ars.usda.gov).
Received October 21, 1997.
Accepted October 14, 1998.
701
ratio of CP on d 1 to 6 of the study. Ractopamine
decreased ( P < .05) carcass lipid and visceral lipid.
Rats dosed with the RR stereoisomer responded
similarly to rats dosed with RAC, except for carcass
lipid. Carcass lipid was decreased ( P < .01) by RAC
relative to controls, but it was not different from
controls in rats treated with the RR isomer. Compared
with controls, BW, carcass CP, and CP retention were
increased by the RR stereoisomer, and visceral lipid
was decreased. The RS isomer also decreased visceral
lipid ( P < .10), but variables measured in rats dosed
with the RS, SR, and SS isomers generally did not
differ from controls. Results of this study indicate that
the RR isomer of RAC is responsible for a majority of
the leanness-enhancing effects of RAC in rats.
J. Anim. Sci. 1999. 77:701–707
(U.S. Pharmacopeia, 1997), salbutamol (Aboul-Enein
et al., 1981), and zilpaterol (U.S. Pharmacopeia,
1997) are leanness-enhancing agents (Anderson et
al., 1990; Casey et al., 1997a,b) that are stereoiso-
meric mixtures. To our knowledge, the only leanness-
enhancing b-agonist that was developed as a single
stereoisomer and tested in livestock is L-644,969
(Zhang and Grieve, 1995; Zhang et al., 1995).
For direct-acting phenethanolamine b-adrenergic
agonists, biologically active stereoisomers are
levorotatory. Dextrorotatory stereoisomers are either
inactive or are considerably less potent than the
levorotatory isomer (Ruffolo, 1991). There are few
data published regarding the leanness-enhancing ef-
fects of b-adrenergic agonist stereoisomers.
Ractopamine HCl ( RAC; Figure 1 ) is a
phenethanolamine b-adrenergic agonist that contains
two chiral carbons and exists as a mixture of four
stereoisomers (Smith et al., 1993; U.S. Pharmacopeia,
1997). Our objectives for this study were to determine
the effects of individual ractopamine stereoisomers on
growth rate, protein retention, and body composition
of growing rats.
702 RICKE ET AL.
Figure 1. Ractopamine HCl. Asterisks indicate the
locations of chiral carbons in ractopamine. The chiral
carbon a- to the phenol ring and b- to the aliphatic
nitrogen is common to all phenethanolamine b-adrener-
gic agonists. Two conformations can exist at each chiral
carbon (R or S); therefore, ractopamine exists as a
mixture of four stereoisomers.
Materials and Methods
Synthesis of Ractopamine HCl Stereoisomers
Stereoisomers of ractopamine HCl were prepared as
described by Anderson et al. (1987, 1988) with
several modifications. d,l-1-Methyl-3-(4-benzylox-
yphenyl)propyl amine was prepared from methyl-
2-(4-benzyloxyphenyl) ethyl ketone via its oxime.
Hydroxylamine HCl (191.8 g, 2.76 mol) was refluxed
for 2 h with 350 g (1.38 mol) of the ketone in 1.5 L of
methanol and 500 mL of pyridine. Approximately one-
half of the solvents were removed by distillation, and
400 mL of water was added. The solution was cooled,
and 373 g of product was collected by filtration;
melting point ( m.p.) was 130 to 136°C. The crude
oxime (269 g, 1 mol) was dissolved in 1 L of dry
tetrahydrofuran ( THF) and added slowly to a suspen-
sion of 100 g of lithium aluminum hydride in 1 L of
THF. After the addition was completed, the mixture
was refluxed for 1 h, cooled, and the complex was
decomposed by the slow addition of 250 mL of water.
The solution was filtered, the solvents were removed,
and the residue was distilled at .05 torr (6.67 Pa) to
yield 180 g of d,l-1-methyl-3-(4-benzyloxyphenyl)
propyl amine. The S isomer of the amine was resolved
by reaction with an equimolar amount of ( S ) - ( + )
mandelic acid and recrystalized from methanol: m.p.
170 to 172°C, [a]D +31.73. The solvent was removed
from the mother liquor, the resulting solid was
converted to the free base, and it was reacted with
( R ) - ( −) mandelic acid. Recrystalization from
methanol yielded the R isomer: m.p. 170 to 173°C, [a]D
−31.59.
The borane-dimethyl sulfide reaction described in
the patent literature proved to be troublesome.
Formation of the des-hydroxyl compound (dehydrated
at the benzylic carbon) was consistently observed but
was formed to a variable extent depending on the
reaction. Rate of addition and temperature during
borane-dimethyl sulfoxide addition did not correlate
with the extent of formation. The des-hydroxyl com-
pound could be removed by recrystallization, albeit
with a considerable loss of product. Removal of the
des-hydroxyl compound at this stage was vital because
ractopamine stereoisomers could not be recrystallized
from the des-hydroxyl compound after the debenzyla-
tion step. Samples of ractopamine stereoisomers that
were contaminated with the des-hydroxyl compound
could be purified using preparative HPLC with C-18
packing and an isocratic mobile phase consisting of
84% .05 M NaCl and 16% CH3CN.
The hydrochloride salt of the ractopamine
stereoisomers could be prepared by the addition of
ethereal hydrogen chloride (an excess of acid would
result in decomposition) or by the addition of an
excess of 2.5 N HCl and removal of water and excess
acid by freeze-drying. The following specific rotations
[a]25D were obtained in methanol: RR, −22.0; SS,
+21.3; RS, −47.0; and SR, +45.0. The specific rotation
of the des-hydroxyl formed in the RR preparation was
+5.5; thus, small amounts of this impurity would
significantly affect the specific rotation of the final
product.
Chemical identities of the individual stereoisomers
were confirmed using 1H- and 13C-nuclear magnetic
resonance spectroscopy, (+)-ion fast atom bombard-
ment mass spectrometry, Fourier-transformed in-
frared spectroscopy, and optical rotation.
Determination of Stereochemical Purity
Stereochemical purities of individual ractopamine
HCl stereoisomers were determined by derivatization
with [ ( 4 S-cis)-2,2-dimethyl-5-isothiocyanato-4-
phenyl-1,3-dioxane; PHEDIT] followed by reverse-
phase HPLC. The PHEDIT reagent was synthesized
as described by Desai and Gal (1992). Individual
stereoisomers of ractopamine HCl were dissolved in
methanol ( 2 mg/mL), and 500-mL aliquots of each
stereoisomer were transferred to conical glass reaction
vessels. Methanol was evaporated using a centrifugal
evaporator (Savant Instruments, Farmingdale, NY),
and the residue was dissolved in 100 mL of a water:
acetonitrile:triethylamine (50:50:3) solution. Each
reaction vessel was capped, placed on a block heater,
and heated to 60°C. After the reaction vials had
reached 60°C, 50 mL of a 60 mg/mL solution of the
PHEDIT reagent (dissolved in acetonitrile) was
added to each vial. After a 30-min reaction period,
reactions were removed from the heating block and
diluted with 400 mL of acetonitrile. Chromatographic
analysis was conducted as described by Smith et al.
(1995).
Ractopamine Stereoisomer Study
Seventy 100-g female Sprague-Dawley rats (Harlan
Sprague-Dawley, Madison, WI) were housed in in-
dividual stainless steel cages. Daily body weights were
 RACTOPAMINE HCL STEREOISOMERS IN RATS
measured and recorded. Rats were provided ad libitum
access to feed and water for 7 d and were housed
under a 12 h light-dark cycle. Diets consisted of
ground (2-mm screen) rat chow (Purina Rat Chow
no. 5012, Purina Mills, St. Louis, MO; CP ≥ 22%, fat ≥
4%, crude fiber ≤ 5%, ash ≤ 8%, and added minerals ≤
8%). Forty-eight rats were selected based on unifor-
mity relative to a target body weight of 150 g and were
randomly assigned to six treatments: control ( C) ,
ractopamine ( RAC) , or RR, RS, SS, or SR
stereoisomers of RAC. Control rats were implanted
with mini-osmotic pumps containing sterile saline.
Surgical techniques were approved by the Institu-
tional Animal Care and Use Committee and followed
the procedure outlined by Smith and Paulson (1994).
Mini-osmotic pumps implanted into the RAC-treated
rats contained 26.7 mg/mL of RAC; mini-osmotic pumps
implanted into the RR-, RS-, SR-, and SS-treated rats
contained 6.7 mg/mL of the RR, RS, SR, and SS
stereoisomers, respectively. The target dose for RAC-
treated rats was 320 mg/d and was 80 mg/d for the RR,
RS, SR, and SS stereoisomer-treated rats.
Rats were surgically implanted with mini-osmotic
pumps designed to release .5 mL/h (Alzet model 2002;
226 mL mean fill volume; Alza, Palo Alto, CA). Rats
were anesthetized with 5% halothane in O2 ( 1 L/min)
and nitrous oxide ( 1 L/min). Anesthesia was main-
tained using 2.5% halothane; nitrous oxide and O2
delivery rates were reduced to .4 L/min. Osmotic
pumps were implanted as described by Smith and
Paulson (1994). Two rats died during surgery because
of over-anesthesia, but they were replaced with
preselected substitute rats. Surgical implantation of
the pumps into the remaining rats was unremarkable.
Immediately after completion of implantation of the
mini-osmotic pumps, rats were placed in individual
metabolism cages designed to allow for the separate
collection of urine and feces. Urine was collected into
50-mL conical tubes containing 1 mL of 6 N HCl.
Urine and feces were gathered every 48 h and kept
frozen until they were analyzed. Rats were given ad
libitum access to feed and water during the study
period, and all rats completed the study. On d 14 of
the study, rats were killed by asphyxiation with CO2
and were immediately dissected to remove the osmotic
pumps and to separate the carcass from the viscera.
Viscera consisted of the gastrointestinal tract, in-
traperitoneal adipose tissue, pancreas, and spleen.
Carcasses consisted of head, hide, bone, tail, muscle,
heart, lungs, kidneys, liver, and reproductive tracts.
Viscera and carcasses were placed in labeled bags and
kept frozen until they were analyzed.
Frozen carcasses were individually ground through
an electric meat grinder two times and mixed
thoroughly. Carcasses were refrozen and lyophilized.
Dried carcasses were blended to uniformity using a
Waring blender. Viscera were blended to a slurry in a
Waring blender, frozen, and then lyophilized.
703
Fecal samples were dried at 50°C in a drying oven
overnight and then allowed to cool. Fecal samples
were combined so that samples collected on d 1 to 6
and 7 to 12 were pooled within each rat. Pooled fecal
samples were then ground through a 2-mm screen and
were stored. Duplicate fecal and carcass samples were
dried in a 100°C drying oven to determine dry matter
(AOAC, 1990; method 950.46). Viscera data are
expressed on a dry matter basis obtained from
lyophilization.
Lipid content of carcasses and gastrointestinal
tracts was determined by Soxhlet extraction (AOAC,
1990; method 960.39). Feces, gastrointestinal tracts,
and carcasses were analyzed for CP according to
AOAC (1990; method 981.10). Urine samples were
pooled as described for feces and analyzed for CP as
described by AOAC (1990; method 981.10).
Verification of RAC release from the pumps was
accomplished by placing primed pumps in 170 mL of
continuously stirred physiological saline and deter-
mining the appearance of RAC in the saline over time
by HPLC. One-milliliter samples of the contents of
each vial were removed daily and frozen prior to
analysis by HPLC. Tandem Nova-Pak C-18 HPLC
columns (100 × 8 mm; Waters Associates, Milford,
MA) were attached to Gilson (Middleton, WI) pumps
and a Gilson ultraviolet detector set at 254 nm. The
RAC was eluted from the column using a mobile phase
consisting of .05 M ammonium acetate (pH 4.5) and
acetonitrile. Acetonitrile was increased linearly from 5
to 25% of the mobile phase during the 40-min
chromatographic run.
Data were analyzed as a completely randomized
design. Significant F-tests served as an indication to
separate means using the method of least significant
difference (Cochran and Cox, 1957).
Results
Syntheses
Adequate quantities of each stereoisomer were
chemically synthesized with difficulty. Loss of product
and purity in the borane reduction and debenzylation
steps hampered the efficacy of the reaction. Final
chemical purity of each stereoisomer was greater than
97% as assessed by HPLC, and stereochemical purity
of each isomer was greater than 98%.
Stereoisomer Growth Trial
Feed intake (Table 1 ) was increased ( P < .02),
relative to controls, by RAC and by the RR
stereoisomer during d 1 to 6, but feed intake was not
altered by either RAC or any of the stereoisomers
during d 7 to 12 of the growth trial. When feed intake
was expressed as a percentage of BW, no treatment
differences were observed over the entire study. Live
704 RICKE ET AL.

Table 1. The effects of intraperitoneal administration of ractopamine HCl (RAC) and individual RAC
stereoisomers (RR, RS, SR, and SS) on feed intake, BW, and gain:feed in rats (n = 8/group)

Ractopamine Stereoisomer
Item Control RAC RR RS SR SS SE Pa
Intake, g/d
Days 1 to 6 14.3bc 15.4d 14.8cd 14.2bc 14.3bc 14.0b .31 .02
Days 7 to 12 14.4 14.7 14.9 13.9 14.7 14.6 .40 .60
Intake, % BW
Days 1 to 6 8.2 8.7 8.5 8.1 8.1 8.3 .20 .32
Days 7 to 12 7.5 7.3 7.3 7.2 7.4 7.5 .13 .56
Body weight, g
Day 6 168b 177d 174bcd 170bc 176cd 169b 2.64 .07
Day 12 193b 203cd 204d 194b 197bc 195b 2.86 .02
Gain:feed
Days 1 to 6 .42bc .45c .46c .46c .41b .39b .02 .09
Days 7 to 12 .29 .23 .28 .27 .27 .31 .02 .31
Average daily gain, g
Days 1 to 6 6.50bcd 7.25c 6.85bc 6.02d 6.13bd 6.02d .33 .05
Days 7 to 12 4.17 3.45 3.98 3.79 4.00 4.48 .38 .55
Net gain, g
Days 1 to 6 39.0bcd 43.5c 41.1bc 36.1d 36.8bd 36.1d 1.95 .05
Days 7 to 12 25.0 20.8 23.9 22.8 24.0 26.9 2.30 .55
aProbability value for treatment F-test.
b,c,dMeans within a row with differing superscripts differ ( P < .10).

BW was increased ( P < .07) by RAC and the SR
stereoisomer during d 1 to 6 of the study, but by d 12
only the RAC and RR treatments had caused signifi-
cant ( P < .02) increases compared to controls. Neither
RAC nor individual stereoisomers increased the effi-
ciency of feed utilization for d 1 to 6 or d 7 to 12 of the
study relative to controls, although this variable was
greater ( P < .09) for the RAC and RR treatments than
for the SR and SS treatments on d 1 to 6. No
treatment effects were observed for ADG or net BW
gain during d 1 to 6 or 7 to 12 relative to controls.
During d 1 to 6, the ADG and BW gain were greater
for RAC treatments than for the RS, SR, or SS
stereoisomers.
Table 2 shows the effects of RAC and individual
ractopamine stereoisomers on body composition of
rats. No differences among treatments were observed
for gross body weight ( d 14), carcass weight, or
visceral weight. Carcass CP was increased ( P < .01)
by the RAC and RR stereoisomer treatments. The RS,
SR, and SS stereoisomers had no effect on carcass
protein. Carcass lipid content (Soxhlet extraction)
was decreased by RAC ( P < .01), but not by any of the
stereoisomers. Visceral crude protein was not affected
by treatment. Visceral lipid content, however, was
substantially reduced ( P < .01) by RAC and the RR
and RS stereoisomers. There was no effect of either
the SR or SS stereoisomers of ractopamine on visceral
lipid.
Effects of RAC and of ractopamine stereoisomers on
CP retention of rats are shown in Table 3. Crude
protein intake was increased on d 1 to 6 by RAC

Table 2. Influence of intraperitoneal administration of ractopamine HCl (RAC) and ractopamine stereoisomers
(RR, RS, SR, and SS) on carcass and visceral protein, lipid, and weights in rats (n = 8/group)

Ractopamine stereoisomer
Item Control RAC RR RS SR SS SE Pa
BW, d 14, g 209 217 213 208 213 213 3.6 .45
Carcass wt, gb 177 184 182 175 180 180 2.8 .29
Visceral wt, gc 22 25 21 21 22 22 1.0 .13
Carcass CP, %d 66d 70e 69e 66d 64d 66d 1.1 .01
Carcass lipid, %b 27de 24f 26df 27de 29e 29e .8 .01
Visceral CP, %c 53 53 55 54 53 53 1.0 .47
Visceral lipid, %c 32e 21f 21f 22f 32e 31e 1.5 .01
aProbability value for treatment F-test.
bCarcass consisted of the head, hide, bone, tail, muscle, heart, lungs, kidneys, liver, and reproductive tracts.
cViscera consisted of the gastrointestinal tract, intraperitoneal adipose tissue, pancreas, and spleen.
d,e,fMeans within a row with different superscripts differ ( P < .10).
 RACTOPAMINE HCL STEREOISOMERS IN RATS 705
Table 3. The effects of intraperitoneal administration of ractopamine HCl (RAC) and individual RAC
stereoisomers (RR, RS, SR, and SS) on crude protein retention in rats (n = 8/group)

Ractopamine stereoisomer
Crude protein, g/d Control RAC RR RS SR SS SE Pa
Intake
Days 1 to 6 2.92b 3.24c 3.13bc 2.91b 3.01bc 2.96bc .07 .02
Days 7 to 12 3.05 3.12 3.16 2.95 3.11 3.09 .08 .60
Fecal
Days 1 to 6 .76 .81 .78 .72 .74 .78 .03 .20
Days 7 to 12 .84 .88 .89 .79 .87 .81 .03 .13
Urinary
Days 1 to 6 1.95 1.93 1.85 1.94 1.92 1.91 .06 .88
Days 7 to 12 1.51 1.45 1.61 1.51 1.62 1.76 .12 .51
Apparent absorption
Days 1 to 6 2.16b 2.44d 2.35cd 2.20b 2.26bc 2.19b .06 .03
Days 7 to 12 2.22 2.24 2.27 2.16 2.24 2.28 .07 .83
Retention
Days 1 to 6 .22b .51c .51c .26b .35b .28b .06 .01
Days 7 to 12 .70 .79 .65 .65 .63 .52 .09 .41
Retained:intake
Days 1 to 6 .065b .155c .161c .087b .116bc .095b .022 .01
Days 7 to 12 .229 .256 .205 .222 .202 .172 .028 .42
Absorbed:intake
Days 1 to 6 .737 .751 .752 .754 .753 .737 .008 .45
Days 7 to 12 .725 .717 .719 .732 .721 .739 .007 .22
aProbability value for treatment F-test.
b,c,dMeans within a row with differing superscripts differ ( P < .10).

compared to controls ( P < .02). There were no
treatment effects on CP intake on d 7 to 12 of the
study. Urinary and fecal CP excretion were not
affected by treatment. Apparent CP absorption was
increased ( P < .03) by RAC and the RR stereoisomer
during the initial 6 d of the study but was not affected
( P > .83) on d 7 to 12. Retention of CP was also
increased ( P < .10) by the RAC and RR treatments
compared to the other treatments during the initial 6
d of the study, but, similar to CP absorption, it was
not affected on d 7 to 12. Efficiency of CP utilization
was also increased by RAC and the RR stereoisomer
during the initial 6 d of the study but was not
influenced by treatment during d 7 to 12. There were
no treatment effects on the efficiency of CP absorption
for either the 1- to 6- or 7- to 12-d study periods.
Discussion
b-Adrenergic agents generally promote increased
rates of weight gain, efficiency of feed utilization,
carcass leanness, and dressing percentage of test
animals (Anderson et al., 1990). Data from this study
are in agreement with those of Smith and Paulson
(1994) indicating that i.p. administered ractopamine
HCl is effective at increasing the growth performance
of rats. Additionally, results from dose titration trials
conducted at our laboratory indicate that ractopamine
HCl increases carcass leanness in rats (unpublished
observations). These results are consistent with the
effects of b-adrenergic “repartitioning” or leanness-
enhancing agents in other species.
Performance data (feed intake, BW, and gain:feed)
have indicated that significant responses in rats were
likely to occur at RAC levels at 320 mg/d (Smith and
Paulson, 1994). In swine, significant improvements in
gain:feed are observed at levels as low as 5 ppm of
dietary RAC, but significant effects on feed intake
were not observed until dietary RAC was at 20 ppm
(Watkins et al., 1990). As discussed by Smith and
Paulson (1994), a dietary level of 20 ppm of RAC for
rats the size used in this study represents an oral
equivalent of roughly 320 mg RAC per day assuming a
feed intake of roughly 16 g/d.
In contrast to results from Smith and Paulson
(1994), RAC-treated rats in this study did not have
increased ADG relative to controls; however, BW was
increased by RAC. In swine, ADG is not consistently
increased by RAC. Bark et al. (1992) and Watkins et
al. (1990) observed significant improvements in ADG
with dietary RAC, but Yen et al. (1990) and Aalhus et
al. (1990) did not. In turkeys, Wellenreiter and
Tonkinson (1990) observed significant increases in
weight gain after treatment with 11, 22, or 44 ppm of
dietary RAC.
Carcass lipid was reduced, and carcass CP was
increased by RAC, consistent with its leanness-
enhancing effects in other species (Bergen et al., 1989;
Anderson et al., 1990; Dunshea et al., 1998). In
addition, RAC reduced visceral lipid in rats. Effects of
RAC on visceral lipid have been measured in a
706 RICKE ET AL.
diversity of species, and the effect in rats is not
unique. For example, RAC reduced mesenteric fat in
catfish (Mustin and Lovell, 1993) and decreased the
proportion of leaf fat in swine (Adeola et al., 1990;
Watkins et al., 1990), but did not reduce kidney,
pelvic, and heart fat in feedlot steers (Carroll et al.,
1990).
Collectively, the performance, body composition,
and nitrogen retention data are consistent with the
hypothesis that the RR stereoisomer of RAC is
responsible for its leanness-enhancing effect in rats.
Such results are consistent with the report by Ruffolo
(1991) indicating that for direct-acting b-adrenergic
agonists the levorotatory stereoisomer is responsible
for biological effects. Both the RR and RS
stereoisomers of ractopamine HCl are levorotatory,
suggesting that each of these isomers might be active,
or that a combination of the two isomers might be
necessary for biological activity. The RR stereoisomer
mimicked the effect of RAC on feed intake, body
weight, carcass CP, visceral lipid, apparent CP
absorption, CP retention, and retained CP:intake CP.
The RS stereoisomer had significant effects on
reducing visceral lipid content but did not mimic the
effects of RAC for any of the other variables.
Nevertheless, the reduction in carcass lipid of the RS
stereoisomer might be expected because the RS isomer
is levorotatory. In addition, the RS isomers of
phenethanolamine b-adrenergic agonists closely
related to ractopamine were lipolytic in mice (Yen et
al., 1983, 1989) and reduced the accumulation of body
fat in rats (Shaw et al., 1981). The fact that the RS
isomer did not provide the full range of effects
observed with RAC might be a function of the
administered dose, species, or differing rates of
individual stereoisomer biotransformation. For exam-
ple, if the RS isomer were cleared more rapidly than
the RR isomer, it would not be expected to have the
same potency as the RR isomer. Alternatively, if the
SR and SS stereoisomers of RAC compete with the RS
stereoisomer for biotransformation enzymes, the clear-
ance of the RS stereoisomer in RAC might be slower
than when administered singly.
The leanness-enhancing b-adrenergic agonist L-
644,969 (Webster et al., 1995; Zhang et al., 1995) is
the RR stereoisomer of 6-amino-a-{[(1-methyl-3-
phenylpropyl)amino]methyl}-3-pyridine methanol di-
hydrochloride (Zhang and Grieve, 1995; Zhang et al.,
1995). Presumably the most active stereoisomer of the
possible four was produced for investigational pur-
poses. The fact that the RR conformation of ractopa-
mine was the most active at mimicking the effects of
RAC is therefore consistent with other known lean-
ness-enhancing agents.
In a broader sense, the results of this study indicate
that data from studies investigating the mechanisms
of b-adrenergic action at the cellular and receptor level
could be misinterpreted because test systems (cells,
cell membranes, tissues, etc.) have been tested with
mixtures of stereoisomers. Structure activity studies
with b-adrenergic agonists (Ruffolo, 1991) clearly
indicate that the biological activities of phenethanola-
mine stereoisomers differ widely. When elucidating
cellular and molecular mechanisms of action, simplic-
ity is undermined when mixtures of stereoisomers are
used because competition among stereoisomers may be
occurring at receptors, target enzymes, or binding
proteins (Ariëns, 1984). This competition may result
in ambiguous data that are difficult to interpret. For
example, receptor affinities, binding constants, rates
of enzyme activation, and other measures may
represent the action of only one isomer in a mix, and
that isomer may have to compete with others for the
binding site. When delineating mechanisms of drug
action, the physiological response(s) to the “whole”
drug must be considered, but an understanding of the
collective actions of individual components (i.e.,
stereoisomers or metabolites) is critical to a mechanis-
tic understanding of the drug. That is, the physiologi-
cal effects of individual stereoisomers and metabolites
must each be considered in the context of the overall
action of the drug. The use of isomerically pure
compounds might simplify the process of elucidating
the unknown cellular and molecular mechanisms of
action of b-adrenergic leanness-enhancing agents.
Implications
This study provides evidence that the RR
stereoisomer of ractopamine is the leanness-enhancing
isomer of ractopamine HCl. This finding is consistent
with scientific evidence that the levorotatory
stereoisomers of direct-acting b-adrenergic agonists
are responsible for their biological effects. Studies
probing the mechanisms of b-adrenergic agonist-
induced “repartitioning” might be simplified by the
use of isomerically pure compounds.
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