Mutation Research 579 (2005) 149–162

Bioefficacy of a novel calcium–potassium salt of

(−)-hydroxycitric acid
Bernard W. Downs a , Manashi Bagchi a , Gottumukkala V. Subbaraju b ,
Michael A. Shara c , Harry G. Preuss d , Debasis Bagchi a,c,∗
a InterHealth Research Center, Benicia, CA, USA
b Laila Impex Research Center, Vijayawada, India
c Department of Pharmacy Sciences, Creighton University Medical Center, 2500 California Plaza, Omaha, 68178 NE, USA
d Department of Physiology and Biophysics, Georgetown University Medical Center, Washington, DC, USA

Received 24 November 2004; received in revised form 16 February 2005; accepted 16 February 2005
Available online 1 August 2005

Abstract

Obesity is associated with cardiovascular disease, diabetes and certain forms of cancer. Popular strategies on weight loss
often fail to address many key factors such as fat mass, muscle density, bone density, water mass, their inter-relationships and
impact on energy production, body composition, and overall health and well-being. (−)-Hydroxycitric acid (HCA), a natural
plant extract from the dried fruit rind of Garcinia cambogia, has been reported to promote body fat loss in humans without
stimulating the central nervous system. The level of effectiveness of G. cambogia extract is typically attributed solely to HCA.
However, other components by their presence or absence may significantly contribute to its therapeutic effectiveness. Typically,
HCA used in dietary weight loss supplement is bound to calcium, which results in a poorly soluble (<50%) and less bioavailable
form. Conversely, the structural characteristics of a novel Ca2+ /K+ bound (−)-HCA salt (HCA-SX or Super CitriMax) make it
completely water soluble as well as bioavailable. An efficacious dosage of HCA-SX (4500 mg/day t.i.d.) provides a good source
of Ca2+ (495 mg, 49.5% of RDI) and K+ (720 mg, 15% of RDI). Ca2+ ions are involved in weight management by increasing
lipid metabolism, enhancing thermogenesis, and increasing bone density. K+ , on the other hand, increases energy, reduces
hypertension, increases muscle strength and regulates arrhythmias. Both Ca and K act as buffers in pH homeostasis. HCA-SX
has been shown to increase serotonin availability, reduce appetite, increase fat oxidation, improve blood lipid levels, reduce
body weight, and modulate a number of obesity regulatory genes without affecting the mitochondrial and nuclear proteins
required for normal biochemical and physiological functions.
© 2005 Elsevier B.V. All rights reserved.

Keywords: Obesity; (−)-Hydroxycitric acid; Calcium; Potassium; Weight loss; BMI; Lipid metabolism; Regulatory genes

∗ Corresponding author. Tel.: +1 402 280 2956; fax: +1 402 280 1883.
E-mail address: debsis@creighton.edu (D. Bagchi).

0027-5107/$ – see front matter © 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.mrfmmm.2005.02.021
150 B.W. Downs et al. / Mutation Research 579 (2005) 149–162

1. Introduction redistribution of body mass. Strategies that promote

healthy natural body mass redistribution may induce
Obesity in America has reached epidemic propor- different and unexpected patterns in weight change,
tions. More than 65% of the adult population is con- and may provide more sustainable and successful long-
sidered to be overweight with a body mass index term weight management than previous methods.
(BMI) of 25 or greater. Another 15 million Americans The obesity epidemic has generated a never-ending
who exhibit a BMI ≥ 30 are classified as morbidly or array of weight loss strategies. In addition to drug ther-
severely obese, which is associated with cardiovascu- apy, numerous procedures suggested to promote weight
lar disease, diabetes and certain forms of cancer. In loss include: reduced caloric intake (such as meal
recent years, obesity has been attributed to chronically replacement products), special weight loss diets (i.e.
exceeding the existing dietary guidelines of a western grapefruit diet or cabbage soup diet), increased caloric
type diet. The current food guide pyramid was devel- expenditure (via exercise and/or stimulants), reduced
oped to create guidelines for healthy dietary habits fat diets, increased water consumption, increased fiber
that emphasizes reduced fat and increased carbohydrate consumption, reduced carbohydrate consumption, and
consumption. This paradigm is a part of nutrition sci- glycemic indexing (i.e. Atkins’ Diet, The South Beach
ence curriculums and influences the direction of food Diet, The Zone Diet, The Cave Man Diet, etc.), appetite
production strategies and consumer food consumption suppression, fat blockers, fat burners, starch/sugar
patterns. However, emerging evidence indicates that blockers, surgery (i.e. liposuction or stomach reduc-
chronic intake of low-fiber, high-carbohydrate and low- tion), acupuncture, hypnotism, support groups and
fat foods may have contributed to the overemphasis on body wraps, all supported by products, programs and/or
carbohydrate consumption, which has resulted in an services designed to help the consumer achieve desired
epidemic of syndrome X or insulin resistance disorder. results. Rapid weight loss products and programs dom-
Syndrome X is a common metabolic disorder affect- inate the focus of marketers and consumers alike. Fur-
ing more than 75 million Americans where the body thermore, rapid weight loss is potentially unhealthy
becomes resistant to its own insulin. When insulin is not and disturbs metabolic set-point homeostasis, induc-
functioning properly, the body compensates by releas- ing undesirable rebound weight gain consequences.
ing more insulin resulting in hyperinsulinemia, obe- Commercialized procedures to induce weight loss
sity, hyperlipidemia, cardiovascular disease, hyperten- routinely measure results by a common bathroom
sion, hypoglycemia, and adult onset diabetes mellitus weight scale; a poor indicator of healthy changes in
[1–4]. In light of this emerging evidence, fundamental body composition. As a specific volume of body fat
changes to the food guide pyramid are imminent and weighs less than the same volume of muscle or bone,
new strategies to readdress the consequences of exces- a preoccupation with weight loss results, as measured
sive refined carbohydrate consumption are emerging. by the numbers on the bathroom scale, can promote
Emphasis on weight loss neglects considerations for potentially unhealthy weight loss behaviors and pro-
other important body composition criteria (e.g. mus- mote inappropriate weight loss expectations. As such,
cle mass and bone density) needed to influence body successful tactics that improve total body composition
mass redistribution strategies. It would be expected that (fat: muscle: bone ratios) may fluctuate between mild
improved body composition would be evidenced by weight gain and loss, but ultimately should result in
fat loss as well as concomitant improvement in denser a sustainable loss of inches, which is still desirable in
muscle and bone mass [5,6]. contrast to unhealthy weight loss alternatives.
The current obesity epidemic has resulted in com- Concerns about exaggerated unsubstantiated
mercialization of weight loss programs and products benefit claims, undesirable side effects and federal
that exploit the urgent need for rapid weight loss or that regulatory intervention regarding dietary supplements
result in loss of healthy muscle mass. Such methods are are increasing. Consequently, the search intensifies for
in contradiction to those which produce healthy, long- products that promote healthy body composition, body
term successful and sustainable improvements in body mass redistribution, but do not stimulate the central
composition. Gradual reductions in total body weight nervous system, elevate blood pressure, promote
are more desirable as that process reflects healthier hypertension, cause nervousness, interfere with sleep
 B.W. Downs et al. / Mutation Research 579 (2005) 149–162 151

and/or block nutrient absorption (i.e. fat, starch and Loe et al. [17] developed a novel gas chromatogra-
sugar-blockers). Outside of significant and permanent phy mass spectrometric (GC–MS) technique to accu-
lifestyle changes, previously acceptable conventional rately identify and quantify plasma HCA levels in blood
methods alone, or in combination, have not resulted in humans, using [U-(13)C]citrate (CA*) as internal
in sustained success. In some cases, these regimens standard to account for losses associated with the
resulted in undesirable side effects and/or serious isolation, derivatization, and measurement of HCA.
health consequences, such as the use of ephedrine and HCA and CA* were derivatized with BSTFA + 10%
phenylpropanolamine (PPA), which have prompted TMCS and analyzed using PCI/GC/MS (CA*, m/z
regulatory scrutiny and intervention. 471; and HCA, m/z 553). Plasma HCA concentration
was measured over a 3.5 h period in fasting and fed
subjects following ingestion of 2 g of HCA-SX (60%
2. (−)-Hydroxycitric acid (HCA), a natural Ca2+ /K+ salt of HCA as HCA-SX) in four healthy
extract from Garcinia cambogia subjects (three males and one female, non-smokers).
Their plasma HCA concentration ranged from 0.8 to
HCA has been reported to safely promote weight 8.4 ng/mL 30 min and 2 h after ingestion, respectively.
loss in laboratory animals and humans without stim- These results demonstrate that when taken on an empty
ulating the central nervous system [7–9]. Extensive stomach 30–120 min before a meal, HCA-SX is well
animal and cell culture studies show that the effects absorbed, with peak absorption at 2 h following admin-
of HCA are due to its dose-dependent ability to com- istration. At 4 h, HCA-SX levels were 50% lower than
petitively inhibit ATP citrate lyase, the citrate cleav- at 2 h, but remained in the blood stream for up to 9 h.
age enzyme. Inhibition of this enzyme decreases the HCA-SX taken in combination with a full meal resulted
transformation of citrate into acetyl CoA, an essen- in significantly lower HCA levels than when taken
tial building block for fat, cholesterol and triglyceride on an empty stomach. This simple method requiring
biosynthesis [9–16]. Numerous animal studies have minimal sample preparation is able to measure trace
used intravenous HCA administration, which circum- amounts of HCA with accuracy and precision [17].
vents the important influence of digestion on bioavail-
ability, an essential consideration for oral consumption.
A review of the literature on the effects of HCA in 4. Human clinical studies
humans reveals inconsistent results. One reason for
these inconsistencies is the differences in the composi- A number of studies on HCA, singly and in com-
tional characteristics of the extract that influences the bination with other natural ingredients, have demon-
bioavailability of HCA. Commercially, HCA is avail- strated beneficial effects for weight loss and fat loss
able as Na+ salt, Ca2+ salt, K+ salt, Mg2+ salt, Ca2+ /K+ [15–20]. Following are the summaries of the human
double salt and Mg2+ /K+ double salt. The solubility clinical studies conducted on different forms of HCA
of these compounds, which can affect bioavailability, and formulations.
ranges from less than 50 to 100%. A randomized, placebo-controlled, double-blind
study was conducted in 54 male and female subjects
(age: 21–55 years; degree of obesity: 15–45% over-
3. Bioavailability of HCA-SX weight) [18]. This 8 weeks study consisted of group
A (n = 30) given Lipodex-2TM (500 mg rind of G.
In order to be effective, orally ingested HCA must be indica and 100 ␮g elemental chromium as chromium
absorbed properly in the body. Studies to determine the nicotinate) three times a day, and group B (n = 24)
effectiveness of HCA as a weight management agent who received placebo. All subjects were given same
are inconclusive if it is not bioavailable and the evi- dietary instructions consisting of a low-fat, low-sugar,
dence of bioavailability is not demonstrated. To date, low-Na+ , and high-fiber diet plan (1200 kcal diet/day)
only one systematic study has been conducted on the and were encouraged to drink 64 oz of water/day.
bioavailability of HCA in a novel 100% Ca2+ /K+ salt Twenty-two subjects in group A and 17 subjects in
of 60% HCA (HCA-SX). group B completed the study. The average weight loss
152 B.W. Downs et al. / Mutation Research 579 (2005) 149–162

per subject (n = 23) in group A was 5.06 kg (11.14 lbs), in weight reduction is highly significant (p < 0.001).
while under the identical clinical conditions group B The composition of the weight loss, determined with
subjects (n = 17) incurred an average weight loss of NIR technique, show that 87% of the weight loss in
1.91 kg (4.2 lbs). One subject with multiple allergies the HCA group is due to fat loss, while corresponding
experienced itching around the mouth, and was figure in the placebo group is 80%. Blood pressure,
advised to discontinue the study [18]. total cholesterol, and hip and waist circumferences
Ramos et al. [19] conducted a randomized, double- were significantly reduced in both groups. A statisti-
blind, placebo-controlled trial on 35 healthy subjects cally significant difference between the groups in favor
for 8 weeks. Forty subjects started the program, while of the HCA group was seen in all these parameters
35 completed the study. Three subjects dropped due (p < 0.001). Appetite scores during the study, using
to influenza and two subjects in the placebo group visual analog scales, were significantly reduced in the
due to lack of positive results. The subjects were ran- HCA group, but not in the placebo group (p < 0.001).
domly divided into placebo group (n = 17; 4 males The tolerability of the treatments was excellent. Two
and 13 females; age 38.7 + 12.3 years; % overweight subjects stopped the treatment due to stomach pain,
52.4 + 20.5; BMI 33.2 ± 4.4 kg/m2 ) and G. cambo- one in the HCA group and one in the placebo group
gia extract group [n = 18; 5 males, 13 females; age [20]. Girola et al. [21] conducted a randomized, double-
35.3 + 11.8 years; % overweight 50.7 + 20.8; BMI blind, placebo-controlled trial in 150 obese subjects
32.6 + 4.3 kg/m2 ]. The placebo group received 500 mg for 4 weeks. Group 1 (n = 50) received a hypocaloric
capsule of placebo before each meal, while the second diet plus 1 capsule/day of dietary integrator (cap-
group received 500 mg lyophilized extract of G. cam- sules containing chitosan 240 mg, G. cambogia extract
bogia (GCE) in a similar form daily for 8 weeks. At 55 mg and chromium 19 (j.g), group 2 (n = 50) received
the beginning of study and every 2 weeks throughout a hypocaloric diet and 2 capsules/day, and group 3
the study all subjects underwent a physical examina- (n = 50) received a hypocaloric diet and a placebo. Fol-
tion and blood chemistry analysis. Both groups were lowing completion of the study, group 1 demonstrated
placed on recommended diets providing 1000, 1200, or a weight loss of 7.9%, total cholesterol reduction of
1500 kcal depending on their theoretical ideal weight 19.8%, LDL cholesterol reduction of 22.9%, triglyc-
(TIW). At the end of the study, total cholesterol sig- erides reduction of 18.3% and HDL cholesterol aug-
nificantly decreased by 18%, triglyceride levels sig- mentation of 9.0%. Group 2 exhibited a weight loss
nificantly decreased by 26% in the HCA group as of 12.5%, total cholesterol reduction of 28.7%, LDL
compared to the control group. Average weight loss cholesterol reduction of 35.1%, triglycerides reduc-
after 8 weeks was 1.3 ± 0.9 kg in the placebo group tion of 26.6% and HDL cholesterol augmentation by
and 4.1 ± 1.8 kg (p < 0.001) in the GCE group. The 14.1%. In the placebo group (group 3), weight reduc-
GCE group also experienced reduced appetite start- tion was 4.3%, total cholesterol reduction was 10.7%,
ing from the first day of administration. Two subjects LDL cholesterol reduction was 15.2%, triglycerides
treated with GCE stated that they experienced slight reduction was 13.2% and HDL cholesterol augmenta-
headaches and nausea, while one subject in the control tion was 6.3%. Adverse events were reported in 6.4% of
group experienced similar symptoms. No other adverse subjects in the placebo group (nausea and/or constipa-
reactions were observed [19]. tion), in 6.1% of subjects in group 1 (nausea); and 2.1%
A randomized, placebo-controlled, double-blind of subjects in group 2 (headache) without any statisti-
study was conducted in 60 obese subjects (44 females cally significant difference between these three groups.
and 16 males) [20]. All subjects were on a low-fat No pathologic or clinically significant changes in blood
diet of 1200 kcal/day and were instructed to exercise chemistry or hematological assay were observed [21].
three times a week for 8 weeks. Subjects were given Rothacker and Waitman [22] evaluated the effec-
either placebo (n = 30) or HCA (n = 30) t.i.d. 30 min tiveness of a HCA and natural caffeine combination
before meals. The daily dose of HCA was 1320 mg/day. in weight loss in a randomized, placebo-controlled,
The HCA group significantly lost an average body double-blind study for 6 weeks. Fifty obese subjects
weight of 6.4 kg (p < 0.001), while the placebo group (BMI 27–33) enrolled in the study, while 48 completed
lost an average body weight of 3.8 kg. The difference the study. Subjects were randomized to treatment with
 B.W. Downs et al. / Mutation Research 579 (2005) 149–162 153

either a proprietary combination containing 400 mg and following 3 days of HCA treatment, RQ was not
G. cambogia extract (≈50% HCA), 25 mg natural significantly lowered during rest (Protocol A) nor dur-
caffeine (guarana and green tea) or 20 ␮g elemental ing exercise (Protocol B) compared with the placebo
chromium as chromium polynicotinate, or identical treatment. Treatment with HCA did not affect EE,
appearing placebo caplet. Subjects (mostly females) either during rest or during moderately intense exer-
were instructed to take two caplets three times per day, cise. Furthermore, the blood substrates measured were
30 to 60 min before meals. All subjects were instructed not significantly different between treatment groups
to follow the same 1200 kcal high fiber diet. Herbal under the fasting conditions of this study. These results
supplement group (n = 25) exhibited a mean weight do not support the hypothesis that HCA alters the short-
change of −4.0 ± 3.5 kg while the placebo group term rate of fat oxidation in the fasting state during rest
(n = 23) exhibited −3.0 ± 3.1 kg. End point revealed or moderate exercise, with doses likely to be achieved
directional differences between treatment groups in in humans while subjects maintain a typical Western
favor of the HCA group (p = 0.30). No serious adverse diet (approximately 30–35% total calories as fat) [24].
events were reported [22]. Mattes and Bormann [25] assessed the effects
Heymsfield et al. [23] conducted a randomized, of HCA on appetitive variables in a randomized,
placebo-controlled, double-blind study to evaluate the placebo-controlled, double-blind, parallel group study
antiobesity potential of a G. cambogia extract (GCE). in 89 mildly overweight female subjects (age: 18–65
One hundred thirty-five overweight men and women years). Forty-two subjects were given 400 mg caplets
(BMI approximately 32 kg/m2 ) enrolled in the study of G. cambogia extract 30–60 min before each meal
while 84 subjects completed the study. Subjects were three times a day (total dose: 2.4 g/day; total dose
randomized or receive either GCE (n = 66; 1500 mg of of HCA: 1.2 g/day); 47 subjects were given placebo
HCA/day) or placebo (n = 69), and both groups were for 12 weeks. Weight and body composition were
prescribed a high-fiber, low-energy (1200 kcal) diet. assessed at baseline and every other week. Participants
The treatment period was 12 weeks. Body weight was were counseled to adhere to a 1200 kcal exchange diet
evaluated every other week and fat mass was measured that contained 30% of energy from fat. Although both
at week 0 and 12. Both the placebo and GCE groups groups lost body weight, the active group (G. cambo-
lost weight. However, no significant differences were gia group) achieved a significantly greater reduction
observed. No significant adverse effects were reported (7.0 ± 3.1 kg versus 2.4 + 2.9 kg). No adverse events
[23]. were reported. No effects of the HCA were observed
Kriketos et al. [24] conducted a double-blind, on appetitive variables [25].
placebo-controlled, randomized, crossover study In a study conducted by van Loon et al. [26] the
involving 3 days of HCA (3.0 g/day) or placebo effects of acute (−)-hydroxycitrate supplementation on
supplementation in sedentary adult male subjects substrate metabolism was assessed at rest and during
(n = 10, age: 22–38 years, BMI: 22.4–37.6 kg/m2 ). exercise in a randomized controlled trial in humans.
The effect of HCA supplementation on metabolic Ten cyclists [age: 24 + 2 years, weight: 73 + 2 kg, max-
parameters with or without moderately intense exer- imal oxygen uptake: 4.95 + 0.11 L/min, maximal work
cise was studied over four laboratory visits. Two of output (W:max): 408 + 8 W]. Subjects were studied at
the four visits involved no exercise (Protocol A) with rest and during 2 h of exercise at 50% W:max on two
and without HCA treatment, while the remaining occasions. Both 45 and 15 min before exercise and
two visits included a moderately intense exercise 30 and 60 min after the start of exercise, 3.1 mL/kg
regimen (Protocol B; 30 min at 40% maximal aerobic body wt of an HCA solution (19 g/L) or placebo was
fitness (VO2max ) and 15 min at 60% VO2max ) with and ingested. Total fat and carbohydrate oxidation rates
without HCA treatment. Energy expenditure (EE, by were assessed. Blood samples were collected at 15-
indirect calorimetry) and respiratory quotient (RQ) min intervals at rest and every 30 min during exer-
were measured for 150 min following an overnight cise. Plasma HCA concentrations increased after HCA
fast. Blood samples were collected for the deter- ingestion up to 0.39 + 0.02 mmol/L (82.0 ± 4.8 mg/L).
mination of glucose, insulin, glucagon, lactate, and However, no significant differences in total fat and
beta-hydroxybutyrate concentrations. In a fasted state carbohydrate oxidation rates were observed between
154 B.W. Downs et al. / Mutation Research 579 (2005) 149–162

trials. Accordingly, plasma glucose, glycerol, and weight and the waist-hip ratio. Thus, GCE is useful in
fatty acid concentrations did not differ between trials. reducing body fat accumulation, especially visceral fat
Plasma lactate concentrations were significantly lower accumulation. No adverse effects were noted [28].
in the HCA than in the placebo trial after 30 min of exer- Westerterp-Plantenga and Kovacs [29] evaluated the
cise, but at the end of the exercise period they did not effect of HCA on energy intake and satiety in a random-
differ between trials. In conclusion, HCA, even when ized, placebo-controlled, single-blind study in twenty-
provided in large quantities, does not increase total fat four overweight, healthy, dietary unrestrained subjects
oxidation in vivo in endurance-trained humans [26]. (12 males and 12 females; BMI: 27.5 ± 2.0 kg/m2 ; age:
Kovacs et al. [27] assessed the effects of 2- 37 ± 10 years). In this 6-week trial, subjects consumed
week ingestion of HCA combined with medium-chain three times for 2 weeks 100 mL tomato juice (placebo)
triglycerides (MCT) on satiety and food intake in 21 and, separated by a 2 week wash-out period, 100 mL
normal to moderately obese subjects (7 males and 14 tomato juice with 300 mg HCA (HCA-SX). After 2
females; age: 43 ± 10 years; BMI: 27.6 ± 2.0 kg/m2 ). weeks, 24 h energy intake (El), appetite profile, hedo-
The study consisted of three intervention periods of 2 nics, mood and possible change in dietary restraint
weeks separated by washout periods of 2 or 6 weeks in were assessed. Prevention of degradation and bio-
a randomized, cross-over, placebo-controlled, double- availability was documented. Twenty-four hour El was
blind study. Subjects consumed three self-selected decreased by 15–30% (P < 0.05) with HCA treatment
meals and four isoenergetic snacks daily with either no compared to placebo, without changes in the appetite
supplementation (Placebo), with 500 mg HCA (HCA), profile, dietary restraint, mood, taste perception and
or 500 mg HCA and 3 g MCT (HCA + MCT). Each hedonics, while body weight tended to decrease. Sati-
intervention period ended with a test day, consisting ety was sustained [29].
of a standardized breakfast and ad libitum lunch and Lim et al. [30] conducted a randomized, placebo-
dinner. There was significant body weight loss during controlled study to determine whether short-term
the 2 weeks of intervention (placebo: −0.5 ± 0.3 kg, HCA ingestion increases fat oxidation during exercise
p < 0.05; HCA: −0.4 ± 0.2 kg, p < 0.05; HCA + MCT: in athletic human volunteers. A. Subjects were
−0.7 ± 0.2 kg, p < 0.01), but the reduction was not dif- administered 250 mg of HCA or placebo for 5 days,
ferent between the treatments. No adverse events were after each time performing cycle ergometer exercise
reported [27]. at 60% VO2max for 60 min followed by 80% VO2max
Hayamizu et al. [28] assessed the effects of long- until exhaustion. Blood was collected and expired
term administration of G. cambogia extract (GCE) on gas samples were analyzed at rest and every 15 min.
visceral fat accumulation in humans. A randomized, The respiratory exchange ratio was significantly lower
double-blind, placebo-controlled trial was conducted in HCA trial than in the control trial (p < 0.05). Fat
in forty subjects (BMI 25–35 kg/m2 ) for 8 weeks. Sub- oxidation was significantly increased by short-term
jects were randomized to either a GCE (n = 20, 1000 mg administration of HCA, and carbohydrate oxidation
HCA per day) or a placebo group (n = 20). Each was was significantly decreased (p < 0.05) during exercise,
subjected to a computed tomography (CT) scan at the presumably resulting in increasing the cycle ergometer
umbilical level before and after the treatment period, exercise time to exhaustion after 1 h of 60% VO2max
and blood samples were taken to measure the clinical performance with increasing fat oxidation, which
laboratory data every 4 weeks. As for a higher visceral spares glycogen utilization during moderate intensity
fat area (VFA) in the subjects (with an initial VFA over exercise in athletes [30].
90 cm2 ), both the VFA and VFA/SFA (subcutaneous fat In another randomized, double-blind, placebo-
area) ratio was measured. In the GCE group, both the controlled study Lim et al. [31] assessed whether HCA
VFA and VFA/SFA significantly decreased, compared ingestion increases fat utilization during exercise in six
to the placebo group (p < 0.01 and p < 0.05, respec- untrained female subjects. Subjects ingested 250 mg
tively). Triacylglycerol was also reduced significantly HCA or placebo capsule for 5 days and then partic-
in higher VFA subjects in the GCE group, compared ipated in a cycle ergometer exercise. Subjects cycled
to the initial levels (p < 0.05), but there were no sig- at 40% VO2max for 1 h and then the exercise inten-
nificant difference between the groups in loss of body sity was increased to 60% VO2max until exhaustion
 B.W. Downs et al. / Mutation Research 579 (2005) 149–162 155

on day 5 of each experiment. HCA decreased the res- SX), group B was given a combination of HCA-SX
piratory exchange ratio (RER) and carbohydrate oxi- 4667 mg, 400 ␮g elemental chromium as niacin-bound
dation during 1 hour of exercise. In addition, exer- chromium (NBC) and 400 mg GSE, and group C was
cise time to exhaustion was significantly enhanced given a placebo daily in three equally divided doses
(p < 0.05). These results suggest that HCA increases fat 30–60 min before meals. In addition, subjects received
metabolism, which may be associated with a decrease 2000 kcal/day diet and underwent a 30 min/day super-
in glycogen utilization during the same intensity exer- vised walking program, 5 days/week for 8 weeks. In
cise and enhanced exercise performance [31]. group A, body weight and BMI decreased by 6.3%,
Preuss et al. [8] conducted a randomized, placebo- food intake was reduced by 4%, total cholesterol, LDL
controlled, double-blind pilot study to determine the and triglycerides levels were reduced by 6.3, 12.3
efficacy of HCA-SX and a combination of HCA- and 8.3%, respectively, while HDL and serotonin lev-
SX, niacin-bound chromium and Gymnema sylvestre els increased by 10.7 and 40%, respectively (Fig. 1).
extract (GSE) in weight management in thirty obese Serum leptin levels were decreased by 36.6%, and the
subjects (aged 21–50 years; BMI > 26 kg/m2 )(Fig. 1). enhanced excretion of urinary fat metabolites, includ-
Subjects were randomly divided into three groups ing malondialdehyde (MDA), acetaldehyde (ACT),
(10 subjects/group): group A was given 4667 mg formaldehyde (FA) and acetone (ACON), increased
HCA-SX (60% Ca2+ /K+ salt of HCA as HCA- by 125–258%. Under identical conditions, group B

Fig. 1. Effects of HCA-SX and HCA-SX formula administration on body weight, BMI, serum leptin, serotonin, LDL, HDL, triglycerides, and
total cholesterol levels in 30 human subjects.
156 B.W. Downs et al. / Mutation Research 579 (2005) 149–162

reduced body weight and BMI by 7.8 and 7.9%, respec- and efficacy; and would account for profound dif-
tively, food intake was reduced by 14.1%, total choles- ferences in results. Varying the number and kinds
terol, LDL, and triglyceride levels were reduced by 9.1, of co-ingredients is also a confounding factor. Fur-
17.9 and 18.1%, respectively, while HDL and sero- ther, omitting details about product specifications,
tonin levels increased by 20.7 and 50%, respectively qualitative and quantitative analyses, other product
(Fig. 1). Serum leptin levels decreased by 40.5% and components and characteristics (aside from HCA)
enhanced excretion of urinary fat metabolites increased makes it virtually impossible to accurately evaluate,
by 146–281%. Group C (placebo) reduced body weight compare and explain results from one clinical study to
and BMI by only 1.6 and 1.7%, respectively, food the another. Finally, adequate or optimum dosage plays
intake was increased by 2.8%, and LDL, triglycerides an important role in demonstrating efficacy. Large
and total cholesterol decreased by 0.8, 0.2 and 0.8%, variations in the dosage were used in the different
respectively, while HDL were reduced by 4.1%. Serum clinical studies employing HCA. Sullivan et al. [15]
leptin levels were increased by 0.3%, and excretion of indicated that higher levels of HCA are required to
urinary fat metabolites did not change in MDA, ACT produce significant lipogenesis and weight loss, and
and FA, and marginally increased in the case of ACON that divided daily doses are significantly more effective
(Fig. 1). Results demonstrate that HCA-SX, and to a than single daily doses. Extrapolation of a therapeutic
greater degree the combination of HCA-SX, NBC and animal dose indicated a human daily dose of 4500 mg
GSE, are effective in reducing body weight and pro- of HCA-SX or 2700 [7,8,32]. This dose was further
moting healthy cholesterol levels. No adverse effects supported by dose-dependent HCA-SX-induced opti-
were observed [8]. mum serotonin release from rat brain cortex [7,8,32].
In a follow-up randomized, placebo-controlled, Without such information, given a valid study method,
double-blind study, Preuss et al. [32] evaluated the the level of effectiveness of a G. cambogia extract, by
effects of HCA-SX and a combination of HCA-SX default, has been attributed solely to HCA, when in fact
plus NBC and GSE in 60 obese subjects (ages other components by their presence or absence can sig-
21–50, BMI > 26 kg/m2 ). Subjects were randomly nificantly contribute to or detract from the therapeutic
divided into three groups. Group A was administered efficacy.
4667 mg HCA-SX, group B was administered a
combination of 4667 mg HCA-SX, 400 ␮g elemental
chromium as NBC and 400 mg GSE, while group 5. HCA-SX, serotonin regulation and appetite
C was given placebo daily in three equally divided suppression
doses 30–60 min before meals. All subjects received
a 2000 kcal diet/day and participated in a supervised In our earlier in vitro studies we have demon-
walking program for 8 weeks. At the end of 8 weeks, strated HCA-SX-induced increased serotonin release
body weight and BMI decreased by 5–6% in both and serotonin receptor reuptake inhibition (SRRI)
treatment groups A and B. Food intake, total choles- in isolated rat brain cortex [7], and suggested that
terol, low-density lipoproteins, triglycerides and serum serotonin regulation may be a major mechanism of
leptin levels were significantly reduced in both groups, appetite suppression by HCA-SX. In subsequent in
while high-density lipoprotein levels and excretion vivo studies, we found elevated serotonin levels in
of urinary fat metabolites increased in both groups. the brain tissues of male and female rats (unpub-
A marginal or non-significant effect was observed lished data). These findings were further corroborated
in all parameters in group C (placebo). Results in the human clinical trials conducted by Preuss et
demonstrate that HCA-SX, and to a greater degree the al. [8,32]. Thus, serotonin regulation may be a major
combination of HCA-SX, NBC and GSE, are effective mechanism of appetite suppression by HCA-SX. In a
in reducing body weight and promoting healthy very recent study, HCA-SX caused a significant reduc-
cholesterol levels. No adverse effects were observed tion of basal neuropeptide Y (NPY) concentrations in
[32]. the hypothalamic tissues, further establishing a role
Differences in the composition and potency of for HCA-SX as an appetite suppressant (unpublished
GCE or HCA can affect pH, solubility, bioavailability data).
 B.W. Downs et al. / Mutation Research 579 (2005) 149–162 157

6. HCA-SX and obesity regulatory genes Table 1

Genes upregulated following HCA-SX supplementation
Obesity is an energy-balance disorder in which Genes/regulatory proteins Function
certain genes that are programmed to resist loss of PDZ domain containing 1 Serotonin receptor
body fat prevail. This programmed genetic predispo- signaling
sition is responsible for down-regulating the resting 5-Hydroxytryptamine (serotonin) receptor 2A Serotonin receptor
5-Hydroxytryptamine (serotonin) receptor 2B Serotonin receptor
metabolic rate in response to dietary and caloric restric-
5-Hydroxytryptamine (serotonin) receptor 3a Serotonin receptor
tion, which is significantly disrupted following rapid 5-Hydroxytryptamine (serotonin) receptor 4 Serotonin receptor
weight loss regimens [33]. Over-consumption of food 5-Hydroxytryptamine (serotonin) receptor 7 Serotonin receptor
(excess energy intake) is a normal consequence con- Prepoenkephalin, related sequence Neuropeptide
tributing to weight gain and obesity. A resistance to signaling
Prostagladin d synthase (PGDS) Lipid metabolism
the hormone leptin also characterizes common obesity.
Aldolase B Carbohydrate
Insulin has been shown to increase leptin secretion by metabolism
25% [34]. Ample evidence demonstrates that insulin Fructose-1,6-biphosphatase 1 Glycolysis
resistance is also a primary contributor to obesity, sug- Lipocalin 2 Transporter
gesting that insulin resistance-induced hyperinsuline- Low-density lipoprotein receptor-related Lipid metabolism
protein 2
mia can provoke leptin resistant hyperleptinemia with
a consequential increase in fat synthesis and storage in
adipocytes, a characteristic sequel of Syndrome X. Fur-
thermore, adipocytes from fatter animals secrete more nal obesity in humans [37]. These findings corroborate
leptin and a correlation between intracellular ATP con- with our previous in vitro and in vivo studies demon-
centration and the rate of leptin secretion appears to strating HCA-SX’s ability to enhance serotonin release
exist [34]. As such, leptin concentration correlates pos- and availability in the brain vicinity and act as a mild
itively with percent body fat. A low resting metabolic serotonin receptor re-uptake inhibitor (SRRI) [7,8,32].
rate for a given body size and composition, a low rate HCA-SX has also been shown to modulate a sig-
of fat oxidation, and low levels of physical activity are nificant number of genes including prostaglandin d
risk factors for weight gain and common traits of obese synthase (PGDS), aldolase B, lipocalin 2, fructose-1,6-
individuals [35]. biphosphatase 1 and low-density lipoprotein receptor-
The effects of low-dose oral HCA-SX was investi- related protein 2, which play a prominent role in
gated on the body weight and abdominal fat transcrip- lipid metabolism, carbohydrate metabolism, glycol-
tome in rats [36]. HCA-SX restricted body weight gain ysis, and cell communication (Table 1) [36]. It is
in rats and lowered abdominal fat leptin expression. worthwhile to mention that PGDS serves as a lig-
High-density microarray analysis of 9960 genes and and for the nuclear receptor peroxisome proliferator-
ESTs present in the fat tissue identified a specific set activated receptor (PPAR-␥), and PPAR-␥ signaling
of genes sensitive to dietary HCA-SX [36]. causes metabolic changes ultimately leading to obe-
Functional characterization of HCA-SX sensitive sity [38,39]. These findings corroborate with our clin-
genes revealed that up-regulation of genes encoding ical studies demonstrating HCA-SX influence on the
serotonin receptors represents a distinct effect of HCA- reduction on LDL, triglycerides and total cholesterol,
SX on appetite suppression (Table 1) [36]. HCA-SX marginal increase in the HDL level, enhanced excretion
up-regulated a significant number of genes associ- of urinary fat metabolites including malondialdehyde,
ated with serotonin receptor and neuropeptide signal- acetaldehyde, formaldehyde and acetone, in conjunc-
ing, which demonstrate its ability to suppress appetite tion with body weight loss and BMI reduction [8,32].
(Table 1) [36]. Stimulation of serotonergic receptors Mitochondrial/nuclear proteins necessary for funda-
reduces feeding and perhaps enhances the satiating mental support of the tissue were not affected by HCA-
effect of food. Furthermore, regulation of 5-HT2A gene SX, which demonstrated the safety of HCA-SX [36].
plays an essential role in body weight management These findings further demonstrate that HCA-SX is
through the regulation of cortisol secretion [37]. Also, safe, efficacious and capable of regulating a significant
polymorphism of the 5-HT2A genes causes abdomi- number of obesity regulatory genes.
158 B.W. Downs et al. / Mutation Research 579 (2005) 149–162

7. Structural characterization of HCA-SX 0.05 M Na2 SO4 in water (pH 2.3, adjusted with sulfu-
ric acid) at 25 ± 2 ◦ C. The retention time for HCA was
A typical compositional analysis of HCA-SX noted at 4.78 min, which was reconfirmed by spiking
(Fig. 2A) contains approximately 60% (−)-hydroxy- with an authentic standard of HCA (Wako, Japan).
citric acid (HCA) in its free form, less than 1.0% Ultraviolet spectrum of HCA-SX was recorded on a
HCA in its lactone form, approximately 11% Ca2+ and Varian Cary 50 UV–vis spectrometer (Mulgrave, Vic-
14–17% K+ . HCA-SX was characterized by injecting toria, Australia). HCA-SX in water showed a shoulder
a 20 ␮L solution of HCA-SX (sample concentration at λmax 210 nm.
1.6 mg/mL) in water (pH 2.1, adjusted with sulfuric Infrared spectrum of HCA-SX was recorded on a
acid) on a Shimadzu HPLC (Tokyo, Japan) equipped Perkin-Elmer Spectrum BX FT-IR spectrometer (Nor-
with LC-10AT pumps, SCL-10A system controller, walk, CT, USA) (KBr pellet, cm−1 ) and showed
SIL-10A auto injector, SPD-M10AVP detector (detec- absorptions at 3403 (OH), 1599 (asymmetric C O
tor was set at 210 nm) and CLASS-M10A software, and stretching band), 1397 (symmetric C O stretching
a 5 ␮Altima C18 column (250 mm × 4.6 mm) (Alltech band), 1296 (C O stretch), 1099, 1062 (alcoholic
Associates, Inc., Deerfield, IL, USA) at a flow rate of C O absorption), 906, 837 (C C stretching), 627 cm−1
1 mL/min in an isocratic mode using a mobile phase of (C C bending).

Fig. 2. (A) Structure of HCA-SX, (B) 1 H nuclear magnetic resonance spectrum (300 MHz, D2 0) of HCA-SX, (C) 13 C nuclear magnetic resonance
spectrum (75 MHz, D2 0) of HCA-SX, and (D) mass spectrum of HCA-SX under electrospray ionization (ESI), negative ion mode conditions.
 B.W. Downs et al. / Mutation Research 579 (2005) 149–162 159

Proton NMR spectrum (300 MHz, D2 O) was strates that diets rich in Ca+ and K+ produce a potent
recorded on a JEOL JNM-LA (Lambda) NMR spec- antihypertensive effect [40]. Angelos et al. reported
trometer (Tokyo, Japan) and the chemical shift values that rat hearts perfused with glucose, insulin and
were expressed in ppm. 1 H NMR data of HCA-SX K+ had significantly higher ATP, creatine phosphate
showed H-2 at δ 3.98 s, H-4a at δ 2.53 d (J = 16.5 Hz) and NADP(+), and lower AMP and inosine levels
and H-4b at δ 2.69 d (J = 16.5 Hz) (Fig. 2B). compared to controls after 30 min of reperfusion.
Carbon-13 NMR spectrum (75 MHz, D2 O) was Reperfusion improved post-ischemic recovery of con-
recorded on a JEOL JNM-LA (Lambda) NMR spec- tractile function and the myocardial bioenergetic state
trometer (Tokyo, Japan) and the chemical shift values [41].
were expressed in ppm. 13 C NMR spectrum of HCA- Most neurons use glucose for energy, but glucose
SX exhibited C-1, C-5 and C-6 at δ 181.0, 179.7 and sensing neurons in the brain also use glucose for signal-
178.7 ppm, and C-2, C-3 and C-4 at δ 42.4, 79.8 and ing to regulate neuronal firing and transmitter release.
77.1, respectively (Fig. 2C). Glucose responsive neurons increase their firing rate
Mass spectrum was recorded on an Agilent 1100 with rising brain glucose levels similar to pancreatic
Series LC/MSD (Palo Alto, USA) under electrospray beta-cells. The release of these neurons is crucial to
ionization (ESI), negative ion mode conditions. In elec- the effector systems, which regulate energy home-
trospray ionization, the mass spectrum contains abun- ostasis [42]. Deriaz et al. [43] showed that chronic
dant pseudomolecular ions (M + 1, M + 23, etc., in pos- changes in serum K+ concentrations were significantly
itive ion mode and M − 1 in negative ion mode). These correlated with changes in energy expenditure. Span-
ions were used to confirm the molecular weight of the swick et al. [44,45] demonstrated that hypothalamic
compounds. The peak at m/z 207 (M − 1) in the mass K+ (ATP) channel function is crucial for physiologi-
spectrum of HCA-SX corresponds the pseudomolec- cal regulation of food intake and body weight. A low
ular ion of hydroxycitric acid (molecular weight 208) K+ intake has been shown to alter serum K+ levels,
(Fig. 2D). K+ homeostasis and suppress cellular energy expendi-
ture [46]. Insulin action depends on the energy level
of target cells and K+ (ATP) channels are believed
8. Roles of Ca2+ and K+ in weight management to influence glucose transport due to their role in
energy homeostasis in the insulin target tissues [47].
Potassium (K+ ) and calcium (Ca2+ ) are important Nazar et al. showed that supplementation of Ca2+ , K+
ions in a number of metabolic pathways influencing and Na+ phosphates increased resting metabolic rate
energy expenditure, leptin metabolism and weight in two groups of overweight women (double-blind,
control. K+ is a major mineral in the body and the placebo-controlled, cross-over) on a low energy diet
recommended daily intake is 3500 mg. Severe K+ defi- by 12 and 19%. The study found that mineral sup-
ciency causes cardiac arrthymias, muscle weakness plementation improved thyroid plasma T3 levels and
and glucose intolerance, while moderate deficiency T4 to T3 ratio. The study found that mineral sup-
leads to increased blood pressure and salt sensitivity, plementation in obese patients on a low-energy diet
an increased risk of kidney stones and increased bone enhanced RMR irrespective of the rate of weight loss
turnover. Inadequate K+ intake may also increase the [48].
risk of cardiovascular disease, particularly stroke, and Dietary Ca2+ also plays a crucial role in regulat-
may disturb intracellular pH homeostasis. K+ and Ca2+ ing energy metabolism. High Ca2+ diets have been
flux may play important roles in coupling intracellular shown to inhibit fat synthesis and storage in adipocytes
energy production to leptin secretion. Restriction of and reduce weight gain during over-consumption of
Na+ intake is a common dietary recommendation in an energy-rich diet. High Ca2+ intake was shown to
the treatment of Syndrome X disorders. However, increase lipolysis and preserve thermogenesis during
meta-analyses indicate that increased Ca2+ and K+ caloric restriction, accelerating weight loss. In con-
intake should be the focus of dietary recommendations, trast, low Ca2+ diets have been shown to inhibit body
rather than restriction of Na+ in the management of fat loss [49,50]. Several clinical studies of Ca2+ intake
such disorders as hypertension. Evidence demon- were found to be associated with reduced weight in
160 B.W. Downs et al. / Mutation Research 579 (2005) 149–162

Fig. 3. Mechanisms of HCA-SX in obesity regulation.

all groups. The Ca2+ -treated subjects in a controlled 9. Conclusion

trial exhibited significant weight loss across nearly 4
years of observation [51]. In addition, other data from A novel Ca2+ /K+ salt of HCA (HCA-SX) has
six observational studies and three controlled trials thus shown to be effective in obesity management as
were evaluated to determine the relationship between evidenced by lowering of body fat and BMI, increased
Ca2+ intake and body fat. Analysis reveals that higher excretion of fat metabolites, enhanced leptin and sero-
intake of Ca2+ is consistently associated with body tonin metabolism, improved blood lipid levels and net
weight loss and reduced weight gain. Heaney et al. body weight reduction. HCA-SX was demonstrated to
[52] demonstrated that a 300 mg increment of regu- perform through a number of mechanisms including
lar Ca2+ intake is associated with 1 kg less body fat ATP citrate lyase inhibition (a building block for fat,
loss in children and 2.5–3.0 kg lower body weight in cholesterol and triglyceride biosyntheses), appetite
adults [53]. Furthermore, Heaney et al. [52] estimated suppression by enhancing serotonin release and avail-
that while Ca2+ intake explains only a fraction of the ability in the brain vicinity, fat oxidation, maintaining
variability in weight gains, increased Ca2+ intake could healthy lipid profile, and triggers genetic signaling
reduce the prevalence of overweight and obesity sig- to downregulate obesity (Fig. 3). Other studies using
nificantly [53]. A related review by Teegarden reports HCA or GCE routinely attribute results only to the
that Ca2+ may play a key role in reducing the inci- effects of HCA, apparently ignoring the contributions
dence of obesity and prevalence of insulin resistance of other important components and salient features.
syndrome [54]. The most obvious reason for adequate Efficacious dosage, higher bioavailability, appropriate
Ca2+ intake by postmenopausal women during a weight and regular diet, and moderate exercise coupled with
loss regimen is to reduce the risk of bone demineral- synergistic influences of Ca2+ and K+ , can significantly
ization disorders, like osteoporosis [55]. This evidence enhance the therapeutic effects of HCA in obesity
supports the notion that adequate intake of dietary regulation.
K+ and Ca2+ enhances energy production, leptin and
insulin metabolism, and satisfies particular nutrient
needs of important pathways required for healthy sus-
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