Chronic consumption of rebaudioside A, a steviol glycoside, in men and women with type 2 diabetes mellitus.

This trial evaluated the effects of 16 weeks of consumption of 1000mg rebaudioside A (n=60) a steviol glycoside with potential use as a sweetener, compared to placebo (n=62) in men and women (33-75 years of age) with type 2 diabetes mellitus. Mean+/-standard error changes in glycosylated hemoglobin levels did not differ significantly between the rebaudioside A (0.11+/-0.06%) and placebo (0.09+/-0.05%; p=0.355) groups. Changes from baseline for rebaudioside A and placebo, respectively, in fasting glucose (7.5+/-3.7mg/dL and 11.2+/-4.5mg/dL), insulin (1.0+/-0.64microU/mL and 3.3+/-1.5microU/mL), and C-peptide (0.13+/-0.09ng/mL and 0.42+/-0.14ng/mL) did not differ significantly (p>0.05 for all). Assessments of changes in blood pressure, body weight, and fasting lipids indicated no differences by treatment. Rebaudioside A was well-tolerated, and records of hypoglycemic episodes showed no excess vs. placebo. These results suggest that chronic use of 1000mg rebaudioside A does not alter glucose homeostasis or blood pressure in individuals with type 2 diabetes mellitus.

The hemodynamic effects of rebaudioside A in healthy adults with normal and low-normal blood pressure.

Rebaudioside A and stevioside are steviol glycosides extracted from the plant Stevia rebaudiana Bertoni and are used in several countries as food and beverage sweeteners. This randomized, double-blind trial evaluated the hemodynamic effects of 4weeks consumption of 1000mg/day rebaudioside A vs. placebo in 100 individuals with normal and low-normal systolic blood pressure (SBP) and diastolic blood pressure (DBP). Subjects were predominantly female (76%, rebaudioside A and 82%, placebo) with a mean age of approximately 41 (range 18-73) years. At baseline, mean resting, seated SBP/DBP was 110.0/70.3mmHg and 110.7/71.2mmHg for the rebaudioside A and placebo groups, respectively. Compared with placebo, rebaudioside A did not significantly alter resting, seated SBP, DBP, mean arterial pressure (MAP), heart rate (HR) or 24-h ambulatory blood pressures responses. These results indicate that consumption of as much as 1000mg/day of rebaudioside A produced no clinically important changes in blood pressure in healthy adults with normal and low-normal blood pressure.

Microbial hydrolysis of steviol glycosides.

A review of the role of gut microbiota in the metabolism of the steviol glycosides, stevioside and rebaudioside A, indicates that they are not absorbed intact but undergo hydrolysis by the intestinal microflora to steviol. Steviol is not metabolized by the intestinal flora and is absorbed from the intestine. The rate of hydrolysis for stevioside is greater than for rebaudioside A. Recent studies using mass spectrometry have shown that steviol-16,17-epoxide is not a microbial metabolite of steviol glycosides. Bacteroides species are primarily responsible for hydrolysis via their beta-glucosidase activity. Fecal incubation studies with both human and animal mixed flora provide similar results, and this indicates that the rat is an appropriate model for studies on steviol glycosides. Given the similarity in the microbial metabolism of stevioside and rebaudioside A with the formation of steviol as the single hydrolysis product that is absorbed from the intestinal tract, the toxicological data on stevioside are relevant to the risk assessment of rebaudioside A.

Determination of conjugated linoleic acid (CLA) concentrations in milk chocolate.

The fatty acids from a series of milk-chocolate-based confectionery samples were analyzed as methyl esters by GC to determine the presence and amount of conjugated linoleic acid (CLA). A single peak corresponding to the 9-cis,11-trans isomer and ranging from less than 0.1% to nearly 0.2% of the total fatty acids, corresponding to up to 0.3 mg per g of chocolate, was observed. One of the chocolate extracts and a milk extract were subjected to silver ion HPLC and GC-MS in order to confirm the identity of the major isomer and tentatively identity minor isomers.

Chronic toxicity/carcinogenicity studies of cocoa powder in rats.

Cocoa powder (CP) was fed at levels of 0.0 (control), 1.5, 3.5 and 5.0% for 104 wk to male and female Sprague-Dawley rats derived from the F3b generation of a multigeneration study using the same CP diets. Initial methylxanthine intake was high in all treatment groups, but steadily declined until wk 26. The high dose level provided a mean methylxanthine intake of approximately 57 mg/kg body weight/day for males and 74 mg/kg body weight/day for females from wk 26 to wk 104 of the study. Compared with controls, the historical trend of methylxanthine-associated growth stimulation was evident in rats consuming diets containing 1.5% CP, while body weight was reduced in rats consuming diets containing 3.5 and 5.0% CP. Survival rates were similar in control and CP-fed rats. No evidence of treatment-related clinical disease or ocular effects was noted. An increased incidence of bilateral testicular atrophy and aspermatogenesis was present in males consuming diets containing 5.0% CP. Non-suppurative myocarditis and interstitial fibrosis of the heart were also increased in incidence in both sexes receiving diets containing 5.0% CP. The overall incidences of both pelvic dilatation and renal pelvic microcalculi were increased in most treatment groups. Although there was no difference in the incidence of benign mammary gland fibroadenomas in female rats between the control group and any CP-fed group, a marginally significant (P = 0.04) trend test was apparent. The significance of this finding is doubtful, since the incidence of this lesion in the highest dose group was well within the historical control range for this strain of rats. No evidence of carcinogenicity from dietary CP was found in either sex.

Three-generation reproductive study of cocoa powder in rats.

Male and female Sprague-Dawley rats were continuously exposed to dietary cocoa powder at levels of 0.0, 1.5, 3.5 or 5.0% for three generations. During the initial 12-wk growth periods for the F0-, F1b- and F2b-generation rats, mean methylxanthine exposures (mg/kg/day) for males/females were 30/36, 72/86 and 104/126 for the 1.5, 3.5 and 5.0% cocoa powder groups, respectively. No consistent dose-related effects on any of the monitored reproductive indices (mating, fertility, conception, gestation, viability or lactation) were noted over three generations. Minor reductions in mean body weight relative to controls at wk 12 were observed in male rats exposed to 3.5 or 5.0% cocoa powder and female rats exposed to 5.0% cocoa powder in the F1b and F2b generations. Renal tubular mineralization in the F0-generation male rats in the 5.0% cocoa powder group was the only statistically elevated histomorphological lesion observed. Plasma cholesterol concentrations in F1b-generation rats were elevated, but cocoa powder did not affect this parameter consistently across multiple generations. Thus, continuous cocoa powder consumption by rats at levels as high as 5.0% of the diet was without effect on reproductive capacity under the conditions of a standard three-generation evaluation.

Digestibility of cocoa butter and corn oil and their influence on fatty acid distribution in rats.

The comparative bioavailability of cocoa butter (a predominantly saturated fat) and corn oil (a predominantly unsaturated fat) was determined in male Sprague-Dawley rats by analysis of total fecal lipids following ad libitum feeding of purified diets containing 5, 10 and 20% cocoa butter or corn oil for 2 wk. Fecal lipid elimination was significantly increased (P less than 0.05) in each cocoa butter group when compared with the corresponding corn oil group, resulting in lower digestibility coefficients for cocoa butter (59-72%) than for corn oil (93-97%). Body weight gain and food intake data were similar among all treatment groups. Fecal fatty acid profiles in rats fed corn oil diets consisted primarily of 27-34% palmitic acid (16:0), 22-32% stearic acid (18:0) and 25-37% oleic acid (18:1). Palmitic, oleic and linoleic acids were also the primary fatty acids stored in epididymal fat tissue from corn oil-fed rats. In contrast, fecal fatty acids in animals fed cocoa butter diets consisted of 31-37% palmitic acid and 58-64% stearic acid; oleic acid was the major fatty acid stored in epididymal fat tissue. These results indicate that the decreased digestibility of cocoa butter is largely a result of its fatty acid composition. This reduced bioavailability of cocoa butter may be at least partially responsible for its previously described neutral effect on serum cholesterol.

Diet-induced alterations in theobromine disposition and toxicity in the rat.

To assess the potential influence of diet on theobromine (TBR) disposition and the development of TBR-induced thymic and testicular toxicity, male Sprague-Dawley rats were given the following diets ad libitum for 28 days: (1) semipurified (S); (2) commercial chow (Ch); (3) semipurified + 0.6% TBR (S + TBR); or (4) commercial chow + 0.6% TBR (Ch + TBR). Toxicity endpoints determined in each TBR group indicated that Ch + TBR-treated animals did not exhibit the marked reduction in body weight or testicular atrophy induced by the S + TBR diet, although thymic weight was lower regardless of diet. Metabolic studies performed after the 28-day feeding period using 5 mg/kg TBR + 10 microCi [8-14C]TBR revealed an overall inductive effect of Ch on TBR metabolism as shown by increased urinary excretion (0-24 hr) of the major TBR metabolite, 6-amino-5[N-methylformylamino]-1-methyluracil (6-AMMU), as well as 7-methylxanthine + 3-methylxanthine (7-MX + 3-MX) and 3,7-dimethyluric acid (3,7-DMU). Consumption of 0.6% TBR for 28 days in either S or Ch diets also induced its own metabolism, as shown by decreased urinary excretion of unchanged TBR and increased conversion primarily to 3,7-DMU. Fecal 14C elimination (0-24 hr) was similar between animals fed S and Ch diets, indicating no effect of control diet on TBR bioavailability. Since serum TBR concentrations and overall toxicity were lower in Ch + TBR-treated animals than in S + TBR treated animals, yet TBR bioavailability was similar, this effect was attributed to the inducing potential of the Ch diet on TBR metabolism and clearance. Investigators are cautioned to consider the potential effect of diet on metabolism when performing and evaluating toxicological studies.

Evaluation of the teratogenic potential of cocoa powder and theobromine in New Zealand White rabbits.

Studies were conducted to determine the teratogenic potential of theobromine (TBR) and cocoa powder (CP) in rabbits. TBR was given either by gavage at dose levels of 0, 25, 75, 125 or 200 mg/kg body weight/day or administered in the diet at 0, 0.0625, 0.125 or 0.1875% (approximately 0, 21, 41 or 63 mg/kg/day, respectively). CP was given at 2.5, 5.0 or 7.5% of the diet (approximately 25, 50 or 75 mg methylxanthines/kg body weight/day). The duration of exposure was from days 6 to 29 of gestation. Significant maternal mortality (40%) and reduced food consumption were observed at 200 mg TBR/kg/day. Mean foetal weights were similar to those of the control group at 25 or 75 mg TBR/kg/day, but decreases in foetal body weight and increases in various malformations and developmental variations were observed in groups given 125 or 200 mg/kg/day. Insufficient litters were available for examination in the 200-mg/kg/day dose group because of maternal toxicity/lethality (repetitive exposure by gavage to 200 mg TBR/kg approached the maternal LD50). In the dietary CP studies, three does died and three aborted, but these deaths and abortions were not treatment related. No maternal deaths occurred during dietary TBR exposure. Maternal weight gain and food consumption, and the mean number of corpora lutea were unaffected by either dietary CP or TBR. Neither foetotoxicity nor teratogenicity was associated with dietary ingestion of CP or TBR. The foetuses exposed to 0.125% or 0.1875% TBR had a significantly higher incidence of incompletely ossified or absent sternebrae, whereas exposure to 0.1875% or 7.5% CP resulted in corresponding effects on metacarpal bones, indicating a delay in osteogenesis. The predominant compound found in serum after TBR ingestion was unchanged TBR, and there was no evidence of bioaccumulation of TBR in serum during gestation. The highest levels of CP or TBR used in these studies was 38 times greater than the maximum consumption level reported for humans in marketing surveys, and corresponds to a consumption of greater than 7.5 lb milk chocolate/day.

Evaluation of the perinatal, postnatal and teratogenic effects of cocoa powder and theobromine in Sprague-Dawley/CD rats.

Studies were conducted to evaluate the effects of cocoa powder (CP) and theobromine (TBR) on perinatal and postnatal parameters and to assess their potential teratogenicity in the rat. In the peri/postnatal study, CP was given at 0, 2.5, 5.0 or 7.5% in the diet throughout gestation and lactation (postnatal day 21). In the teratology study, rats were given diets containing 0, 2.5 or 5.0% CP or 0.0675 or 0.135% TBR on days 6-19 of gestation. The CP-treated dams in the peri/postnatal study consumed significantly more food than did the controls during gestation. Weight gain was increased only in the 5.0 and 7.5% CP groups during lactation. Litter size was reduced slightly at 7.5% CP and pup survival was slightly decreased at 5.0 and 7.5% CP but none of these reductions was statistically significant. However, small but statistically significant decreases in pup body weights were noted in all treatment groups throughout lactation. In the teratology studies, rats given 2.5 or 5.0% CP or 0.0675 or 0.135% TBR consumed significantly more food than did the controls and the CP-treated dams gained significantly more weight. The percentage of pregnant dams and the mean number of corpora lutea were not affected by either CP or TBR. Foetuses exposed to 0.135 TBR had a significantly higher incidence of incompletely ossified or absent sternebrae and pubic bones, indicating a delay in osteogenesis. On the basis of the survival of treated offspring in the peri/postnatal study, these effects were not considered to be deleterious to either growth or survival. The effects are similar to those that have been reported elsewhere and been considered to indicate potential maternal or foetal toxicity that is unrelated to a specific compound/treatment. We conclude that any variations observed in these studies may be attributed to this non-specific maternal toxicity and are not related to the ingestion of either CP or TBR. The major methylxanthine found in the serum after CP or TBR ingestion was unchanged TBR and it did not bioaccumulate during gestation. The levels of CP and TBR used in these studies were more than 50 times greater than the maximum level of consumption by humans as reported in marketing surveys and were equivalent to a consumption of 10 lb of milk chocolate per day. The results indicate that neither CP nor TBR pose any health hazard to the developing foetus.

Postprandial glucose and insulin responses to various snacks of equivalent carbohydrate content in normal subjects.

To evaluate glucose and insulin responses after ingestion of snacks, we gave healthy, nondiabetic male subjects carbohydrate equivalent (25 g) snacks or isocaloric (265 kcal) snack meals in a random crossover design. Individual snacks composed of either a milk chocolate bar, granola bar, chocolate milk, peanut butter cups, yogurt, or potato chips produced similar glucose response curves. Plasma glucose concentrations were lower (p less than or equal to 0.05) at 30 and 60 min postprandially than after a corresponding oral glucose challenge. In contrast, insulin responses to the snacks exhibited a two-fold variation in peak values. Isocaloric snack meals of cereal-milk, cheese sandwich-milk, and peanut butter sandwich-chocolate milk produced glucose and insulin responses similar to individual snacks. Although glucose concentrations at 60 min fell somewhat below baseline values after each snack, clinical hypoglycemia was not evident. These data clearly indicate a similarity in glycemic response among normal individuals consuming a variety of common snacks.

High levels of methylxanthines in chocolate do not alter theobromine disposition.

Theobromine disposition was measured twice in 12 normal men, once after 14 days of abstention from all methylxanthines and once after 1 week of theobromine (6 mg/kg/day) in the form of dark chocolate. Mean theobromine t 1/2, apparent volume of distribution, and clearance after abstinence from all methylxanthines were 10.0 hours, 0.76 L/kg, and 0.88 ml/min/kg. High daily doses of chocolate for 1 week did not change these values. After subjects abstained from methylxanthines, urinary radioactivity over 72 hours after a single, oral dose of [8-14C]theobromine consisted of 42% 7-methylxanthine, 20% 3-methylxanthine, 18% theobromine, 10% 7-methyluric acid, and 10% 6-amino-5[N-methylformylamino]-1-methyluracil. A week of daily theobromine consumption in the form of dark chocolate also did not alter this urinary profile of theobromine and its metabolites. Although these results might appear to differ from other reports of inhibition of theobromine elimination after five consecutive daily doses of theobromine in aqueous suspensions, both the rate and extent of absorption of theobromine in chocolate were less than that of theobromine in solution. Relative bioavailability of theobromine in chocolate was 80% that of theobromine in solution. This reinforces the fundamental principle that both the metabolic and the therapeutic consequences of a particular chemical can differ when that chemical is given in the pure compared with the dietary form.

The cariogenic potential of several snack foods.

The use of the animal model and the Konig-Hofer program-feeding machine is a valuable tool in the research effort to identify the factors contributing to dental caries. As this methodology provides uniform host, microflora, and frequency components, the food is the variable being evaluated. The relative cariogenicity of food is dependent on variations in the composition, texture, solubility, retentiveness, and ability to stimulate saliva flow. The results of these experiments indicated the equal importance of all food-related parameters on dental caries formation. If the composition of foods was solely responsible for dental caries formation, all three of the granola bars should have had similar relative cariogenicity values. This was not true. Granola bar no. 3, which was crispier in texture and less moist, had a third the relative cariogenicity value of granola bars nos. 1 and 2. As reported by other dental researchers, the production of dental caries is food related, but not simplistic in nature. Dental caries is the outcome of complicated interactions, all of which are equally important in the progress of this disease.

Kinetics and metabolism of theobromine in male rats.

On the basis of general pharmacological information (blood cells/plasma partition, plasma protein binding) and using HPLC as the principal analytical method, we investigated the kinetics and metabolism of theobromine (a caffeine metabolite) in male rats after a single dose and after a 2 week chronic application. Doses in both conditions varied between 1 and 100 mg/kg. In in vitro and in vivo the fraction of theobromine unbound to plasma proteins averaged 0.90 over a wide range of concentrations. No significant difference was found in the pharmacokinetic profile of the drug after acute or chronic treatment at different doses except for a reduction in the absorption rate constant as the dose increased. AUC values increased in proportion to the dose. The 2 treatment schedules were also similar as regards metabolism, at least 50% of the administered dose of theobromine being excreted unchanged, and 25% as 6-amino-5-[N-methyl- formylamino ]1-methyluracil. Only at the highest doses was there a tendency for theobromine to accumulate at the expense of its major metabolite (a uracil compound).

Kinetics and metabolism of theobromine in male and female non-pregnant and pregnant rabbits.

The kinetics and metabolism of theobromine (3,7- DMX ) were investigated in male rabbits after a single oral dose and 14 days oral dosing at 1, 5, 10, 50 and 100 mg/kg/day. Female non-pregnant and pregnant rabbits were also studied after single oral doses of 1, 5 and 50 mg/kg. No significant difference was found in the pharmacokinetic profile of 3,7- DMX due to either sex, pregnancy or after chronic oral administration for 14 days. Intravenous (i.v.) administration of 3,7- DMX at 1 and 5 mg/kg resulted in calculated kinetic parameters in close agreement with oral doses. Irrespective of sex, there was a reduction in the absorption rate constant as the dose increased, coupled with a linear dose-related increase in AUC values. No qualitative difference in the metabolism of 3,7- DMX in the rabbit was observed as linked to sex, pregnancy or treatment schedule. Twenty-five percent of the administered dose of 3,7- DMX was excreted unchanged, the major metabolite being 7-methylxanthine (40%). There appeared to be a shift in the metabolic pathway at 100 mg/kg/day in the males and at 50 mg/kg/day in the females with more unchanged 3,7- DMX excreted. Only at these highest doses (100 mg/kg for males and 50 mg/kg for pregnant rabbits) was there a tendency toward accumulation.

Comparative theobromine metabolism in five mammalian species.

Biotransformation of theobromine (TBR) was compared in rats, mice, hamsters, rabbits, and dogs by assaying urinary metabolites using HPLC after oral administration of a 5 mg/kg dose containing 8-14C-TBR. Recovery of radioactivity ranged from 60-89% of the dose in urine, and from 2-38% of the dose in feces, with most material being excreted during the first 48 hr after dosing. TBR was most extensively metabolized by rabbits and male mice. The primary metabolite excreted by rats and mice was 6-amino-5-[N-methylformylamino]-1-methyluracil (6-AMMU); male mice converted TBR to this metabolite more extensively than did female mice. Rabbits and dogs metabolized TBR primarily to 7-methylxanthine (7-MX) and 3-methylxanthine (3-MX), respectively; the major metabolites excreted by hamsters were 6-AMMU and 7-MX. Overall N-demethylase activity yielding monomethyl metabolites was greatest in rabbits and lowest in rats. Ring N-demethylation at position 3 predominated over 7-N-demethylation in all species except the rat and dog. In dogs, TBR was N-demethylated primarily at position 7, while N-demethylase activity in rats was without apparent positional specificity. Oxidation of methylated xanthines to the corresponding uric acids was a relatively minor metabolic pathway in all species, but had greatest activity in mice. Oxidation of TBR to 3,7-dimethyluric acid was significantly greater in female rats than in male rats. In summary, excretion patterns of TBR and its metabolites were qualitatively similar among species, indicating that TBR is metabolized along similar pathways. Except for the excretion of small quantities of an unidentified but apparently unique metabolite by dogs, only quantitative species- and sex-related differences were observed in the metabolic disposition of TBR.

Dominant lethal testing of theobromine in rats.

Theobromine (TBR) 3,7-dimethylxanthine, the major purine alkaloid present in chocolate and cocoa products, was evaluated for dominant lethality in male Sprague-Dawley rats after administration of subacute oral doses of 0, 50, 150 and 450 mg/kg. Dominant lethal mutations were assessed weekly for a 10-week period by killing the females 13 days after the midweek of presumptive mating with treated males and counting total implants (living and dead), corpora lutea and number of pregnant females. Dead implants per total implants and preimplantation loss per total corpora lutea were evaluated statistically. No significant dose-dependent effects were observed in the % dead implants of preimplantation loss throughout the study; pregnancy rate averaged 94%. These results indicated no induction of dominant lethal mutations or adverse effects on pregnancy rate after TBR doses equivalent to 25-225 times the maximum human consumption level.

Methylxanthine composition and consumption patterns of cocoa and chocolate products.

This chapter has compiled and evaluated the current information on the methylxanthine composition of cocoa and various chocolate foods and beverages, as well as the consumption pattern for these commodities. Although the earliest recorded reference to cacao was in 1502, it was not until 1876 that milk chocolate was invented, an event that formed the backbone of the chocolate industry today. The consumption of cocoa throughout the world has been influenced by a number of factors, and the period of peak consumption occurred during the early to mid-1960s when these factors were highly favorable. The greatest consumption of cocoa in metric tons over the past 10 yr has been in the United States, although the highest per capita consumer during this period was Switzerland. The African continent has been historically the primary producer of raw cocoa, with the Ivory Coast currently being the largest individual supplier. Limited marketing survey data is available for the consumption of methylxanthines in chocolate foods and beverages. In children and teenagers, the major dietary source of caffeine was found to be tea, followed by soft drinks and coffee, respectively. Although chocolate foods and beverages ranked the lowest of these dietary sources to provide caffeine, they do constitute the major source of dietary theobromine. Cacao is the major natural source of the xanthine base theobromine. Small amounts of caffeine are present in the bean along with trace amounts of theophylline. The methylxanthine content of beans varies with the varietal type, and is influenced by the fermentation process. Chocolate liquor is a semifinished product commonly called "baking" or "cooking" chocolate. The average theobromine and caffeine content of liquors has been reported at 1.2% and 0.21%, respectively. Cocoa powder, which is prepared after removal of the cocoa butter, contains about 1.9% theobromine and 0.21% caffeine. Chocolate beverages comprise the most widely studied category of chocolate products. Hot cocoa provides 62 mg/serving of theobromine and 4 mg/serving of caffeine when prepared from commercial instant mixes. Instant cold chocolate milk mixes supply an average of 58 mg/serving of theobromine and 5 mg/serving of caffeine. The methylxanthine content of chocolate foods has received only slight attention in the literature. The methylxanthine content of sweet chocolate ranges from 0.359 to 0.628% for theobromine and 0.017 to 0.125% for caffeine.(ABSTRACT TRUNCATED AT 400 WORDS)

Analytical methods for quantitation of methylxanthines.

The methylxanthines can be determined in foods and biological systems by the chromatographic methods of TLC, GC, and HPLC. Ultraviolet spectroscopy following a separation procedure is also still used. More recently immunoassay methods have been developed. There is no single best method; the analyst must balance the features of each assay with the final requirements for data precision and reproducibility.