Medical Hypotheses (1999) 52(5), 407416 1999 Harcourt Brace & Co. Ltd Article No. mehy.1997.

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Pyruvate and hydroxycitrate/carnitine may synergize to promote reverse electron transport in hepatocyte mitochondria, effectively uncoupling the oxidation of fatty acids
M. F. McCarty, J. C. Gustin
NutriGuard Research, Encinitas, CA, USA

Summary In a recent pilot study, joint administration of pyruvate, hydroxycitrate (HCA), and carnitine to obese subjects was associated with a remarkable rate of body-fat loss and thermogenesis, strongly suggestive of uncoupled fatty-acid oxidation. Hepatocytes possess an uncoupling mechanism reverse electron transport that enables fasting ketogenesis to proceed independent of respiratory control. Electrons entering the respiratory chain at the coenzyme Q (CoQ) level via FAD-dependent acyl coA dehydrogenase, can be driven up the chain by the electrochemical proton gradient to reduce NAD+; if these electrons are then shuttled to the cytoplasm, returning to the respiratory chain at the CoQ level, the net result is heat generation at the expense of the proton gradient, enabling the uncoupled flow of electrons to oxygen. Pyruvates bariatric utility may stem from its ability to catalyze the rapid transport of high-energy electrons from mitochondria to the cytoplasm, thus stimulating electron shuttle mechanisms. By enabling rapid mitochondrial uptake of fatty acids and thus disinhibiting hepatocyte ketogenesis, HCA/carnitine should initiate reverse electron transport: concurrent amplification of electron shuttle mechanisms by pyruvate can be expected to accelerate this reverse electron transport, thereby decreasing the electrochemical proton gradient. As a result, hepatocytes may be able to convert fatty acids to CO2 and heat with little net generation of ATP. These considerations suggest that it may be feasible to render hepatocytes functionally equivalent to activated brown fat, such that stored fat can be selectively oxidized in the absence of caloric restriction. Other measures which enhance the efficiency of hepatocyte electron shuttle mechanisms may increase the efficacy of this strategy.

ROLE OF REVERSE ELECTRON TRANSPORT IN KETOGENESIS Recent anecdotial clinical experience strongly suggests that pyruvate administration (4 g orally t.i.d.) interacts synergistically with hydroxycitrate (HCA) and carnitine to promote rapid fat loss and marked thermogenesis (see Appendix). HCA/carnitine has been recommended as a diet aid on the basis of its presumed ability to promote the transport of free fatty acids (FFAs) into hepatic mitochondria (13). This transport step is believed to be rate-limiting for
Received 2 September 1997 Accepted 15 October 1997 Correspondence to: Mark F. McCarty MD, NutriGuard Research, 1051 Hermes Avenue, Encinitas, CA 92024, USA

hepatic ketogenesis (4,5). More specifically, the conversion of cytoplasmic fatty acyl coA to fatty acyl carnitine via the enzyme carnitine palmitoyl transferase I (CPT) is pace-setting for fatty acid transport into mitochondria and thus for ketogenesis (4,5). HCA the chief acid found in fruits of the genus Garcinia disinhibits CPT by suppressing synthesis of malonyl coA, a key allosteric inhibitor of this enzyme (6); it achieves this by acting as a potent competitive inhibitor of citrate lyase (79), whose activity in hepatocytes is required for the cytoplasmic generation of acetyl coA, the biosynthetic precursor of malonyl coA. Carnitine is the essential cofactor for CPT activity, and ordinary non-fasting hepatocyte levels of this coenzyme appear to be subsaturating, such that provision of extra carnitine accelerates hepatocyte ketogenesis when CPT is activate (4). Thus, it has been 407

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proposed that joint administration of HCA and carnitine will be clinically useful as a strategy for disinhibiting and activating CPT, thereby stimulating ketogenesis. Although the process of ketogenesis reduces both NAD+ and FAD, the rate of ketogenesis appears to be substantially independent of respiratory control (1013), i.e. it can proceed at a high rate even when hepatocyte metabolism is generating ADP at a low rate. The most reasonable explanation for this phenomenon has been offered by Berry and co-workers (11,13). They suggest that, during ketogenesis, high-energy electrons enter the respiratory chain at a greater rate than hepatocytes can generate ADP, resulting in an electron glut and an increased electrochemical proton gradient. Under these circumstances, electrons entering the chain at the level of coenzyme Q (via the FAD-dependent acyl coA dehydrogenase reaction) can be pushed up the respiratory chain to NAD dehydrogenase, which transfers them to NAD+. These electrons can then be shipped to the cytosol via the malate/aspartate shuttle and, after reducing NAD+ or NAD(P) in the cytosol, can then re-enter the mitochondrial respiratory chain at the coenzyme (CoQ) level via the glycerol-3-phosphate shuttle. The reverse electron transport step in this cycle is driven by the electrochemical proton gradient of the mitochondrial inner membrane which diminishes as a result or, alternatively, by conversion of ATP to ADP via the mitochondrial ATP synthase. After two turns of this cycle, the electrochemical proton gradient will be sufficiently diminished to enable the electrons to pass down the respiratory chain from CoQ to oxygen, without any obligate coupling to ATP synthesis. (Alternatively, the two ADPs generated by two turns of the cycle will enable the coupled transport of these electrons from CoQ to oxygen.) This mechanism thus would effectively uncouple the transfer of electrons from FADH2 to oxygen during ketogenesis. This reverse electron transport mechanism is well documented in mitochondria and sub-mitochondrial particles in vitro (1418); its role in vivo is still speculative, but it seems to offer a very plausible explanation for the mysterious uncoupling of hepatic ketogenesis. PYRUVATE PROMOTES ELECTRON SHUTTLE MECHANISMS The well-documented bariatric benefits of pyruvate (1924) remain unexplained. We propose that administration of pyruvate enhances the efficiency of the shuttle mechanisms required for reverse electron transport, by serving as a biosynthetic precursor for key substrates of these shuttles namely oxaloacetate and dihydroxyacetone phosphate; it is also conceivable that pyruvate has an as-yet-uncharacterized allosteric effect on the activity or synthesis of enzymes which catalyze the shuttles.
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When concurrent administration of HCA/carnitine disinhibits hepatic ketogenesis, flooding hepatic mitochondria with reducing equivalents and initiating reverse electron transport, the pyruvate-mediated activation of shuttle mechanisms may substantially amplify the rate of reverse electron transport, such that not only ketogenesis, but also Krebs cycle activity is at least partially uncoupled, enabling the complete oxidation of FFAs to CO2 with little net production of ATP and a substantial release of heat. Thus, we propose that, under these circumstances, electrons transferred to NAD+ from the Krebs cycle can be shuttled to enter the respiratory chain at the CoQ level, and that a portion of these electrons will then be subjected to reverse electron transport thereby diminishing the electrochemical proton gradient and enabling uncoupled respiration. The efficient conversion of pyruvate to mitochondrial oxaloacetate may be crucial to pyruvates utility. The rapid production of acetyl coA during ketogenesis can be expected to strongly activate pyruvate carboxylase, whereas pyruvate dehydrogenase will be inhibited (l,2). Furthermore, the generation of ketone bodies from excess acetyl coA will promote pyruvate transport into mitochondria. Thus, pyruvate administered during ketogenesis should be rapidly converted to mitochondrial oxaloacetate. Owing to the high redox potential in ketogenic mitochondria, and the fact that reduction of oxaloacetate is highly exergonic, much of this oxaloacetate will be quickly reduced to malate, which readily exits mitochondria. In this way, pyruvate can be expected to catalyze the transfer of high energy electrons from mitochondria to the cytosol. The resulting decrease in mitochondrial NADH should stimulate the Krebs cycle and promote reverse electron transport. Provided that efficient enzymatic machinery is in place to transfer electrons from cytoplasmic malate to dihydroxyacetone phosphate, and to oxidize the resulting glycerol-3-phosphate at the mitochondrial inner membrane, the exported electrons can be quickly returned to CoQ in the respiratory chain, enabling further reverse electron transport. Indeed, if one were to logically devise an optimal method for expediting the transport of excess high-energy electrons from mitochondria, one could probably do no better than to administer an agent which, like pyruvate, promotes the rapid intramitochondrial generation of oxaloacetate. (A Trojan horse such as pyruvate is required to increase mitochondrial oxaloacetate levels, since oxaloacetate itself cannot cross the mitochondrial inner membrane.) This may hold the key to pyruvates bariatric utility even in the absence of reverse electron transport, pyruvate should catalyze the shuttle-mediated discounting of high-energy electrons, whereby electrons flow from mitochondrial NADH through the cytoplasm to enter the respiratory chain at CoQ.
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Figure 1 demonstrates how it would be theoretically conceivable for reverse electron transport to completely uncouple hepatocyte oxidative metabolism. If ketogenesis and Krebs cycle activity transfer reducing equivalents to NAD+ and FAD at rates x and y, respectively, then a net reverse electron transport from CoQ to NAD+ at a rate of 2(x+y) would collapse the electrochemical proton gradient sufficiently (or generate sufficient ADP) to enable respiration in the absence of net ATP production, i.e. FFAs (and any other substrate oxidized by hepatic mitochondria) could be converted to CO2 and heat at a rate independent of hepatocyte metabolic activity. In practice, respiration will be partially coupled, as hepatocytes have metabolic needs that generate ADP thus, it cannot be expected that reverse electron transport will ever achieve a rate as high as 2(x+y). Nonetheless, a considerable degree of uncoupling can be anticipated if reverse electron transport can be substantially boosted. Electron flow during reverse electron transport may well proceed in an oscillatory fashion. Reverse electron flow will gradually collapse the electrochemical proton gradient, slowing the reverse flow but accelerating forward flow to oxygen. The increased forward flow, to the degree that it is uncoupled, will recharge the proton gradient, slowing forward flow but promoting an increased rate of reverse flow. (And so, to quote the King, et cetera, et cetera, et cetera.) Pyruvate supplementation should also have thermogenic effects that drive the coupled oxidation of FFAs by stimulating the futile cycle pyruvate oxaloacetate phospho-enol-pyruvate pyruvate, utilizing one net ATP (or if malic enzyme is induced, the cycle pyruvate oxaloacetate malate pyruvate), and by acting as substrate for gluconeogenesis, which requires 2ATP equivalents plus an NADH per molecule of pyruvate converted to glucose. The activation of pyruvate carboxylase by HCA/camitine should promote these mechanisms. The mitochondrial oxaloacetate generated from

pyruvate will also aid Krebs cycle activity, and the pyruvate-stimulated production of malate will accelerate mitochondrial uptake of anionic substrates such as pyruvate and phosphate. An increase in hepatocyte malate levels is typically seen in response to measures exercise and gluconeogenic hormones that boost the respiratory capacity of hepatocyte mitochondria (25). It can be anticipated that, even with the concurrent use of pyruvate, a proportion of the acetyl coA generated from fatty acids will not be oxidized, but rather exported from the liver as ketone bodies, committed to oxidation in peripheral tissues. This peripheral oxidation of ketones will presumably be coupled, but will in any case complete the selective oxidation of fats, such that a low respiratory quotient favorable to fat loss is achieved. Ketosis can also suppress adipocyte lipolysis (26); while this may slow the rate of fat loss, it should have a favorable impact on cardiovascular risk factors. Some of the electrons evolved during hepatocyte fatty acid oxidation will not flow to oxygen, but rather will be used to convert acetoacetate to beta-hydroxybutyrate. SHUTTLE FUNCTION AS A KEY TO THERMOGENESIS It undoubtedly is not coincidental that the thermogenic hormones thyroxine and (in rats) high-dose DHEA potently induce the expression of two enzymes in hepatocytes the mitochondrial glycerol-3-phosphate dehydrogenase and the cytoplasmic malic enzyme which can be rate-limiting for electron shuttle mechanisms (27,28); cafeteria-feeding, which is potently thermogenic, has an analogous effect (13,30). Catecholamines also thermogenic for hepatocytes activate mitochondrial glycerol3-phosphate dehydro-genase by increasing hepatocyte intracellular calcium levels (31). Even in the absence of reverse electron transport, the transfer of electrons from mitochondrial NADH to respiratory chain CoQ via these shuttle mechanisms effectively lowers the P:O ratio from 3 to 2 thus partially uncoupling hepatic oxidative metabolism. Clearly, however, the greatest degree of uncoupling can be anticipated when an up-regulation of shuttle mechanisms is allied with a disinhibition of ketogenesis that initiates reverse electron transport. We propose that this is precisely what happens when pyruvate is administered in conjunction with HCA/carnitine. Alternate means of up-regulating shuttle function might be expected to synergize with these measures. Whereas high-dose DHEA is not suitable for clinical use (owing to its ability to give rise to excessive levels of sex hormones), Lardy and colleagues have recently demonstrated that 7-hydroxy or 7-keto derivatives of DHEA which to some degree are produced endogenously, and do not give rise to sex-hormone activity are actually more potent than
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Fig. 1 Uncoupling of hepatocyte respiration via reverse electron transport.

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DHEA itself in inducing thermogenic shuttle enzymes (as well as fatty acyl coA oxidase) in rat hepatocytes (32,33). If these agents prove to be effective in this regard in humans, without unacceptable side-effects or toxicities (such as are seen with thyroid hormone), they may well have a bright future in bariatric medicine, alone or in conjunction with the techniques suggested here. Folkers has demonstrated that many individuals are functionally deficient in CoQ, in the sense that addition of CoQ to their mitochondria can enhance the oxidation of succinate by succinate dehydrogenase (34). Could CoQ levels likewise sometimes be rate-limiting for the glycerol-3-phosphate shuttle and reverse electron transport? If so, supplemental CoQ might sometimes enhance the efficacy of the measures recommended here. Some years ago, an open study by Van Gaal provided evidence that correction of documented CoQ deficiency (as assessed by succinate dehydrogenase activity) might accelerate weight loss in dieters (35). If pyruvates bariatric utility is primarily reflective of the fact that it is an efficient precursor for mitochondrial oxaloacetate (as noted above, the possibility that it has direct inductive effects cannot presently be discounted), it is possible that aspartic acid will have comparable activity. Aspartate is avidly transported into mitochondria and de-aminated to yield oxaloacetate, as a key portion of the malate-aspartate shuttle; as noted, oxaloacetate cannot directly enter mitochondria, but can do so when disguised as aspartate. If aspartate is in fact effective in this regard, this may be of some practical importance as aspartates are currently far less expensive than pyruvates. (It may be noted that the process of de-amination and the subsequent conversion of NH4+ to urea will expend an additional 4 ATPs but also requires reduction of NAD+.) Lactic acid, likewise inexpensive, is readily converted to pyruvate and thus might be considered as an alternative to this compound. However, the oxidation of lactate to pyruvate would be expected to increase cytoplasmic redox potential, which could slow the rate of electron transport from mitochondria to the cytoplasm. It should be noted that Stankos original testing of pyruvate and dihydroxyacetone as remedies for ethanolinduced fatty liver (36), was rooted in the fact that these compounds can function as electron acceptors whereas lactate is clearly an electron donor. However, there are alternative pyruvate precursors the amino acids serine and glycine that do not mediate reductions when converted to pyruvate. Alanine, however, may be inappropriate for this purpose, as it is an inhibitor of pyruvate kinase. With regard to the demonstrated utility of dihydroxyacetone as an aid to thermogenesis in animals (19), it should be noted that phosphorylation of this compound yields the oxidized component of the glycerol-3Medical Hypotheses (1999) 52(5), 407416

phosphate shuttle. Thus, dihydroxyacetone appears to be a preferable (though much more expensive) alternative to glycerol. The latter compound appears to have a temporary anorexic activity in rat (37), although controlled clinical evaluations of its impact as an adjuvant to lowcalorie diets have failed to confirm efficacy in this regard (38,39). Hepatocyte levels of dihydroxyacetone phosphate can be increased most conveniently with fructose; the liver-specific fructokinase produces fructose-1-phosphate, which is then cleaved by aldolase to yield glyceraldehyde and dihydroxyacetone phosphate. While fructose alone is clearly not a diet aid, it would be interesting to determine whether catalytic amounts might stimulate reverse electron transport in the context of the measures recommended here. Dihydroxyacetone phosphate levels should also be increased somewhat by gluconeogenic prescursors including pyruvate and aspartate. ACTIVATION OF PYRUVATE KINASE WITH METFORMIN AND BIOTIN The drug metformin may also have utility as an adjuvant in this weight-loss strategy (3). By activating flux through pyruvate kinase (40,41), metformin may enhance endogenous generation of pyruvate, decrease the loss of administered pyruvate to gluconeogenesis, and concurrently enhance the thermogenic impact of the futile cycles centered on pyruvate. The tendency of metformin to promote weight loss and decreased appetite in diabetics is well known (4245). Its use in conjunction with ketogenic techniques, in diabetics or borderline diabetics, may in any case be desirable to prevent excessive stimulation of gluconeogenesis by accelerated hepatic FFA oxidation (40,44,4651). An alternative (or adjunctive) approach to activating pyruvate kinase may be offered by high-dose biotin. In rodent studies, pharmacological doses of biotin can produce significant increases in the expression of glucokinase in both hepatocytes and pancreatic cells (5256); activation of guanylate cyclase by supraphysiological concentrations of biotin may mediate this effect (57,58). Increased activity of glucokinase in hepatocytes can be expected to increase pyruvate kinase activity both by enhancing the level of its allosteric activator, fructose1,6,-dipkosphate, and by stimulating increased synthesis of the enzyme (5962). An elevation of glucokinase should aid electron shuttle function by increasing the concentration of dihydroxyacetone phosphate, and can be expected to promote thermogenic futile cycles involving glycolytic intermediates (61). A further benefit of biotin could be that, by increasing glucokinase activity in cells, it may act to offset a potential adverse effect of high-dose HCA on glucose-stimulated insulin secretion (63). A recent report that biotin lessens weight gain (presumably, fat
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gain) in a congenitally obese strain of rat despite slightly increasing food intake suggests that biotin does indeed have thermogenic potential (64). Since 3 mg t.i.d. has been shown to aid glycemic control in human diabetics (65), this dose schedule is likely to be adequate for increasing hepatocyte glucokinase activity clinically. A THERMOGENIC ROLE FOR GLUCAGON In the pilot study described in the Appendix, patients were advised to perform fasting aerobic exercise and to eat substantial amounts of protein. Each of these measures can be expected to increase glucagon activity. Glucagon promotes intramitochondrial fatty acid transport and ketogenesis, both by decreasing the synthesis of malonylcoA (via suppression of acetyl-coA carboxylase activity) and by decreasing the potency of malonyl-coA as an allosteric inhibitor of carnitine palmitoyl transferase (66,67); thus, measures which enhance glucagon activity have been recommended as adjuvants to HCA/carnitine (2). Glucagons ability to stimulate pyruvate carboxylase and thus promote gluconeogenesis may simply reflect allosteric activation by the increased levels of acetyl-coA stemming from accelerated fatty acid oxidation (68); glucagon also promotes mitochondrial uptake of pyruvate (25,69). Mitochondria from glucagon-treated animals have greater respiratory activity ex vivo (70); this is due in part to increased efficiency of electron flow from NADH through NADH dehydrogenase to CoQ (71). It seems not unlikely that, conversely, glucagon could increase the efficiency of reverse flow from CoQ to NADH during ketogenesis, though experimental demonstration of this appears to be lacking. Homeostatically, it would make good sense for glucagon to increase the efficiency of reverse electron transport, thereby promoting more rapid ketogenesis. Glucagon acts as a signal of carbohydrate deficiency, inducing the conversion of the bodys chief stored substrates, protein and fat, to glucose and ketones (respectively), thus helping to fuel the central nervous system during starvation. Efficient ketogenesis is evidently crucial in this regard, as it enables sparing of tissue protein. When insulin levels are low, glucagon infusion has a marked thermogenic impact in humans (72); stimulation of gluconeogenesis and ureagenesis undoubtedly contributes to this effect. As these processes are driven by ATP, they can be expected to promote the coupled oxidation of hepatic lipid. A high protein intake will activate these processes, both by stimulating glucagon output, and by providing substrate for them. In the context of the measures recommended here, the chief benefit of these endergonic processes may be that they stimulate electron flow from the Krebs cycle, for which ADP is an allosteric activator.
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OVERVIEW There may be a certain logical inevitability to the ideas proposed here. The clinical results described in the Appendix can only credibly be attributed to a substantial uncoupling of fatty acid oxidation. Since HCA/carnitine can be expected to have its most profound impact on the liver, this is likely to be the chief site of this uncoupling. The other major thermogenic organ in man is skeletal muscle; since pyruvate has been shown to enhance exercise endurance (73,74), it seems most unlikely that pyruvate promotes uncoupling in muscle. The liver is known to have a mechanism for uncoupled oxidative metabolism reverse electron transport which should be stimulated by HCA/carnitine. It is therefore logical to suspect that an amplification of this mechanism is responsible for the uncoupling observed during joint administration of pyruvate and HCA/carnitine. Pyruvates role as an efficient precursor for mitochondrial oxaloacetate suggests that pyruvate administration will indeed aid reverse electron transport. It is very clear to us that the results reported in the Appendix demonstrate a marked synergism between pyruvate and HCA/carnitine. When used in conjunction with low-calorie diets or an overfeeding regimen, pyruvate (at 1530 g daily) diminished body fat by about 1 kg over 21 days relative to placebo (2224); in the context of a low-fat weight-maintaining regimen in hyperlipidemic subjects, about half of whom were obese, the incremental fat loss associated with pyruvate (2244 g daily) was 0.4 kg over six weeks (75). In a double-blind study in which overweight volunteers received HCA/carnitine/ chromium picolinate while asked to avoid fatty foods, eat more fiber, and get more physical activity, the additional fat loss attributable to the supplement was 0.6 kg over four weeks (76). Used alone, either of these techniques appears to have some utility, but they should rightly be construed as adjuvants to traditional weight-management techniques. The results reported here with the combined use of these measures are clearly far greater than the sum of their individual efficacies and, if confirmable, suggest a new and definitive approach to obesity control. If indeed it is possible to at least partially uncouple FFA oxidation in the liver, this organ might reasonably be compared to a vast slab of activated brown fat. Under these circumstances, unprecedented rates of selective fat loss can be anticipated. The liver evidently can only degrade FFAs that reach it. Thus, these methods should have their most rapid impact on fat stores such as visceral adipocytes that are relatively lipolytically active. Indeed, diets and exercise regimens that successfully achieve fat loss tend to have a disproportionate impact on visceral fat stores (7780). A reduction in lipolytically active fat stores can
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be expected to be of practical benefit with respect to the insulin resistance syndrome (syndrome X) and its frequent sequel, type II diabetes (8185). Conversely, the subcutaneous fat stores which would be more gradually responsive to the measures suggested here presumably the gynoid depots are precisely those which have minimal impact on health (and, if desired, could be targeted for liposuction). The ability of hepatocytes to achieve reverse electron transport, and thus decouple ketogenesis from the metabolic energy needs of hepatocytes, is by no means an evolutionary fluke. Clearly, ketogenesis is crucial to meeting the energy needs of the central nervous system while enabling protein sparing during prolonged fasts; it would be most counterproductive if sluggish hepatocyte metabolism in fasted animals were to impede the efficiency of ketone generation. The central thesis here is that it may prove possible perhaps via pyruvate, HCA, and carnitine to amplify reverse electron transport in hepatocytes, thus substantially uncoupling hepatocyte oxidative metabolism and promoting the efficient conversion of portal FFAs to CO2 and heat. In vitro studies, in which hepatocytes are exposed concurrently to pyruvate, HCA, carnitine, and FFAs, should readily determine whether these proposals have validity. In the meantime, clinical efforts along the lines described in the Appendix should continue, and attempt to verify not only the efficacy, but also the safety of these measures. In regard to safety considerations, it may be noted that the strategy suggested here utilizes only nutrients, a natural metabolite, and a food compound (HCA) traditionally consumed in Indian cuisine none of which has shown any evident toxicity in previous clinical experience. Also, with respect to the increased gallstone formation commonly observed with rapid-weight-loss diets (8689), this appears to be largely attributable to the gallbladder stasis associated with semi-starvation (8991); since the methods proposed here do not require caloric restriction, there is little reason to suspect that they will induce gallstones.

APPENDIX Summary of an informal pilot trial A short pilot study was conducted to test the impact of a supplement regimen, in conjunction with dietary and exercise advice, on fat loss and physique modification in overweight volunteers. The daily regimen provided the following, to be consumed in three equal doses: calcium pyruvate, 12 g; HCA 1.5 g; L-carnitine 250 mg; chromium (as chromium picolinate) 600 mcg. The supplements were to be consumed in the morning (prior to exercise or eating), with lunch, and at bedtime. Participants were
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asked to walk at least 20 min each morning, on an empty stomach; they were asked to walk at a brisk pace at which they could converse normally. Dietary advice consisted of recommendations to eat a high-protein, low-fat diet (30% and 10% of calories, respectively) in an amount that provided 0.751 g protein per pound of lean mass daily (or 100 g daily for women with lean mass under 100 lb). These recommendations corresponded to total daily caloric intakes ranging from 13003100 kcal daily, dependent on body size; the average recommended intake was around 2000 kcal daily. This was to be consumed in numerous small meals daily, and no calories were to be ingested in the 3 h prior to bedtime. An ample intake of water was also suggested, and fiber-rich foods were recommended. Body composition was evaluated at baseline and at weekly intervals thereafter using Futrex 5000, a new infrared technique for quantifying the depth of underlying subcutaneous fat (92,93). All measurements were made by the junior author (JCG) on the dominant arm, using a contact point on the anterior aspect of the exterior midline of the biceps, halfway between the antecubital fossa and acromion (as recommended by the Futrex manual.) On each occasion (except the last), measurements were taken in triplicate and averaged. (Unfortunately, time constraints prevented triplicate measurements during the final evaluation. This probably had little impact on the final results, as JGs replicate measurements typically vary by less than 2%). Heights and weights were also determined, thus enabling calculation of body-mass indexes (BMIs). A total of 23 volunteers the majority Samoan-American appeared for enrollment. They were admitted in three groups on 6/28/97, 7/2/97, and 7/5/97; final evaluation for all subjects was on 7/26/97. Three subjects did not return after baseline evaluation, and one subject did not return for final evaluation. Two subjects refused to be weighed at baseline (insisting on reporting their own weights) and therefore could not be properly assessed. One subject developed a rash after three days, and pyruvate was discontinued. There were thus 16 participants who were evaluable. The initial body weights of these subjects ranged from 69 to 231 kg, with a mean weight of 117 kg. Percentage body fat ranged, from 26% to 54%, with a mean value of 41%. The average BMI was 39.3 (range 26.263.7). Most subjects tolerated the regimen quite well. As noted, one subject developed a rash and pyruvate discontinued. The largest enrolled subject (initial weight 231 kg), who also lost the most weight and fat, had a prior history of gout, and experienced an attack of gout during the final week which prevented him from walking. One subject noted borborygmus and gas. Most subjects reported feeling warm during the study.
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In the first week, three subjects reported sweating and/or subjective feelings of heat (including a woman who at baseline had complained that she was always cold). Since this was thought to be a possible sign of increased thermogenesis, the other subjects were queried as to whether they were experiencing sensations of warmth, and the subjects were virtually unanimous in affirming this. One subject (again, the largest one) noted profuse sweating and indicated that he needed to turn a fan on himself at bedtime to enable himself to get to sleep. The other virtually unanimous subjective response was of considerably increased physical energy. Self-reported compliance with the dietary and exercise recommendations was excellent in many subjects, but others confessed to occasional junk-food binges, a failure to achieve the suggested intake of protein, or sporadic adherence to morning walking exercise. Self-reported compliance with the supplement regimen in general was quite good, although a few subjects noted that they had missed several doses. Participants were free-living and, with the exception of a whey protein supplement provided to several subjects, their food was self-chosen. Exercise was not monitored. Thus, the conditions of the study were closer to real world application than is the case in most carefully supervised clinical studies. Furthermore, no subjects were excluded from the final analysis owing to sporadic (or in a few instances, non-existent) compliance with either dietary, exercise, or supplementation recommendations. Since subjects were enrolled for varying periods of time (34 weeks), results are reported as average weight loss and average fat loss per week. In the entire group of 16 subjects, average weekly weight loss was 1.5 kg; average weekly fat loss was 2.3 kg. This evidently implies a weekly gain of lean mass averaging 0.8 kg. The largest subject achieved, within 24 days, a weight loss of 11.8 kg and, remarkably, Futrex analysis indicated a fat loss of 22.7 kg. If this subject is excluded from the analysis as atypical, the average weekly weight and fat losses in the remaining 15 subjects were 1.4 kg and 2.0 kg, respectively. Since grossly obese subjects have often been noted to lose significant weight initially when their diet is regulated (preventing binges on favorite. foods), a separate analysis was made of the five subjects with initial weight under 200 lb (91 kg). In these subjects, average weekly weight loss and fat loss averaged 1.3 kg and 1.8 kg respectively not greatly different from the group as a whole. The subject with the lowest body weight, as well as the subject with the lowest initial percentage body fat, achieved rates of fat loss of 2.0 and 2.7 kg per week, respectively. Thus, benefit does not appear to be contingent on severe obesity. To ascertain the regularity of response, it is appropriate
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to note the poorest response in the 16 subjects. A 74-kg woman lost 3.2 kg of weight and 4.5 kg of fat over four weeks. During the final week, she had been traveling to Australia, and had been completely noncompliant with the diet and exercise recommendations. It is notable that, in every subject, lean mass increased during the study. Thus, response was quite different from that seen during very-low-calorie dieting. The subjective increase in energy (which helped some subjects comply with the recommended exercise) and in body warmth, is also hardly typical of response during calorie deprivation. Perhaps the most telling indication of the success of the regimen was the mob scene which greeted the investigators at the final evaluation dozens of friends and relatives of the volunteers had shown up, demanding to be included in the study! The authors are acutely aware that a study of longer duration would have been more meaningful particularly since it is important to confirm durability of response to this regimen. Unfortunately, the businessman sponsoring the study (not affiliated with Nutrition 21) refused to provide pyruvate capsules beyond the four-week point, when he became apprised of the fact that pyruvate had already been patented for use as a diet aid. If one assumes that 1 lb of fat corresponds to about 3500 kcal, whereas 1 lb of lean averages 700 kcal, the average daily calorie deficit was approximately 2370 kcal (2100 kcal if the heaviest subject is excluded form analysis). Given the fact that the subjects were not subjected to severe caloric restriction (indeed, a few complained that they were being asked to eat too much!), and were asked to do only walking exercise of modest duration, these responses are likely to be unprecedented, and are strongly suggestive of a dramatic increase in thermogenesis a view consistent with the many reports of heat and sweating. Even if skeptics were to disregard the Futrex analyses as unreliable, the average monthly weight loss approximately 6.5 kg is exceptional in the context of a regimen that does not include severe caloric restriction, draconian exercise, or drugs. Although not discussed in the body of this paper, the nutrient chromium picolinate was included in the supplement regimen. In the two largest controlled studies to date evaluating the impact of this nutrient on body composition in overweight subjects (400800 mcg chromium daily), a significant incremental fat loss (relative to placebo) averaging 0.4 kg per month was observed (94,95). Presumably, the supplemental chromium may have had an additive impact on the fat loss achieved during the present study. However, whether it might also have had a synergistic interaction with the other compounds administered (for example, by down-regulating insulin secretion, allowing glucagon to work more effectively),
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obviously cannot be determined from this study, but requires consideration in future studies. As a scientific study, the pilot trial described above has many overt deficiencies. Clearly, confirmatory studies are urgently needed using double-blind design, a more accurate technique for body composition assessment (i.e. immersion densitometry or DEXA), more extended duration, assessment of blood parameters to monitor safety, and respiratory analysis or the double-labeled water technique to quantify metabolic rates and respiratory quotients. However, despite the fact that the current study was virtually an exercise in guerilla nutrition conducted not for science per se but to aid in product development the results are so striking that they seem very likely to point the way to a new and important approach to modification of body composition. Whether or not the theoretical mechanism suggested in the body of this paper proves to have validity, the authors are convinced that the joint administration of pyruvate, HCA, L-carnitine, and chromium picolinate, in adequate and appropriate doses, has very considerable potential as a novel strategy for physique modification. We urge experienced well-trained bariatric scientists (which the authors cannot claim to be) to attempt replication of methods described here; frankly, we do not expect properly skeptical scientists to believe the remarkable and counterintuitive results reported above until they have seen it with their own eyes. That being said, we nonetheless affirm that we have attempted to be scrupulously honest in collecting, analyzing, and reporting the data from this informal study. With regard to safety considerations, it is conceivable that the rash experienced by one subject reflected a trace contaminant in the 12 g of pyruvate consumed daily. An allergic reaction to the Garcinia extract used (50% HCA as the calcium salt) is also a possibility, but seems unlikely since the rash cleared while Garcinia intake continued. The flare-up of gout experienced by one subject might be attributable to ketosis, which reduces renal clearance of uric acid (96,97); the high protein intake might also have been a contributory factor. No subjects complained of abdominal distress suggestive of gallstones. REFERENCES
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