Structure elucidation of thioketone analogues of sildenafil detected as adulterants in herbal aphrodisiacs

Two analogues of sildenafil were detected in herbal dietary supplements marketed as aphrodisiacs. Both compounds were identified as thioketone analogues of sildenafil in which the carbonyl group in the pyrimidine ring of sildenafil was substituted with a thiocarbonyl group. The first compound was identified as thiosildenafil, a compound that has recently been reported as an adulterant in health supplements. The structure of the second compound was established using LC–MS, UV spectroscopy, ESI-MSⁿ, NMR and a hydrolytic process. A detailed study of the hydrolysis products of sildenafil, thiosildenafil, and the second unknown compound proved that the second compound, named thiomethisosildenafil, had a structure analogous to sildenafil in which the N-methylpiperazine moiety had been replaced with 2,6-dimethylpiperazine and the oxygen atom of the carbonyl group in the heterocyclic ring had been replaced with a sulfur atom. Under the hydrolytic reaction conditions employed in this study, thioketones hydrolyze to ketones (e.g., thiosildenafil → sildenafil), making this a valuable technique for the structure elucidation of thiosildenafil analogues. Ten herbal dietary supplements, each as a capsule dosage form, were found to contain 8–151 mg of thiomethisosildenafil per capsule, and one herbal dietary supplement was found to contain 35 mg of thiosildenafil per capsule.     

Dietary supplement adverse events: Report of a one-year poison center surveillance project

Background

The safety and efficacy of dietary supplements is of growing concern to regulators, health-care providers and consumers. Few scientific data exist on clinical effects and potential toxicities of marketed products. Harmful supplements may not be identified for months or years with existing adverse event monitoring mechanisms. Retrospective review of poison center statistics to capture supplement-associated toxicity also has limitations.

Methods

We collaborated with the FDA Center for Food Safety and Nutrition (CFSAN) to conduct a 1-year prospective surveillance study of dietary supplement-related poison control center calls in 2006. Prompt follow-up of symptomatic cases, laboratory analysis of implicated dietary supplements, and causality assessment by a case review expert panel were performed.

Results

Of 275 dietary supplements calls, 41% involved symptomatic exposures; and two-thirds were rated as probably or possibly related to supplement use. Eight adverse events required hospital admission. Sympathomimetic toxicity was most common, with caffeine products accounting for 47%, and yohimbe products accounting for 18% of supplement-related symptomatic cases. Suspected drug-herb interactions occurred in 6 cases, including yohimbe co-ingested with buproprion (1) and methamphetamine (3), and additive anticoagulant/antiplatelet effects of NSAIDs taken with fish oils (1) and ginkgo (1). Laboratory analysis identified a pharmacologically active substance in 4 cases; supplement toxicity was ruled unlikely when analytical testing was negative in 5 cases.

Conclusion

Most supplement-related adverse events were minor. Clinically significant toxic effects were most frequently reported with caffeine and yohimbe-containing products. Active surveillance of poison control center reports of dietary supplement adverse events enables rapid detection of potentially harmful products, which may facilitate regulatory oversight.     

Analytical methods for determination of magnoflorine and saponins from roots of Caulophyllum thalictroides (L.) Michx. using UPLC, HPLC and HPTLC

Analytical methods including HPLC, UPLC and HPTLC are presented for the determination of major alkaloid and triterpene saponins from the roots of Caulophyllum thalictroides (L.) Michx. (blue cohosh) and dietary supplements claiming to contain blue cohosh. A separation by LC was achieved using a reversed phase column, PDA with ELS detection, and ammonium acetate/acetonitrile gradient as the mobile phase. Owing to their low UV absorption, the triterpene saponins were detected by evaporative light scattering. The eight triterpene saponins (cauloside H, leonticin D, cauloside G, cauloside D, cauloside B, cauloside C, cauloside A and saponin PE) and the alkaloid magnoflorine could be separated within 35 min using HPLC method and within 8.0 min using UPLC method with detection limits of 10 μg/mL for saponins and 1 μg/mL for magnoflorine. The detection wavelength was 320 nm for magnoflorine and ELS detection was used for the eight saponins. The methods were also successfully applied to analyze different dietary supplements. For the products claiming to contain blue cohosh, there was a significant variability in the amounts of triterpene saponins detected. Calculations based on the analysis results for dietary supplements showed that maximum daily intake of alkaloid and saponins vary with the form (solids/liquids) and recommended doses according to the products label. Intakes varied from 0.57 to 15.8 mg/day for magnoflorine and from 5.97 to 302.4 mg/day for total saponins. LC-mass spectrometry coupled with electrospray ionization (ESI) method is described for the identification and confirmation of nine compounds in plant samples and dietary products. A HPTLC method was also developed for the fast chemical fingerprint analysis of C. thalictroides samples.     

Chronic toxicity and carcinogenicity studies of chromium picolinate monohydrate administered in feed to F344/N rats and B6C3F1 mice for 2 years

Trivalent chromium (Cr(III)) has been proposed to be an essential element, which may increase sensitivity to insulin and thus participate in carbohydrate and lipid metabolism. Humans ingest Cr(III) both as a natural dietary constituent and in dietary supplements taken for weight loss and antidiabetic effects. Chromium picolinate (CP), a widely used supplement, contains Cr(III) chelated with three molecules of picolinic acid and was formulated in an attempt to improve the absorption of Cr(III). In order to examine the potential for CP to induce chronic toxicity and carcinogenicity, the NTP conducted studies of the monohydrate form (CPM) in groups of 50 male and female F344/N rats and B6C3F1 mice exposed in feed to concentrations of 0, 2000, 10,000 or 50,000 ppm for 2 years; exposure concentrations were selected following review of the data from NTP 3-month toxicity studies. Exposure to CPM did not induce biologically significant changes in survival, body weight, feed consumption, or non-neoplastic lesions in rats or mice. In male rats, a statistically significant increase in the incidence of preputial gland adenoma at 10,000 ppm was considered an equivocal finding. CPM was not carcinogenic to female rats or to male or female mice.     

Detection of Δ6-methyltestosterone in a "dietary supplement" and GC-MS/MS investigations on its urinary metabolism

Since a few years more and more products have appeared on the market for dietary supplements containing steroids that had never been marketed as approved drugs, mostly without proper labeling of the contents. Syntheses and few data on pharmacological effects are available dated back mainly to the 1950s or 1960s. Only little knowledge exists about effects and side effects of these steroids in humans.The present study reports the identification of Δ6-methyltestosterone in a product named “Jungle Warfare”, which was obtained from a web-based supplement store.The main urinary metabolites, 17α-hydroxy-17β-methylandrosta-4,6-dien-3-one (Δ6-epimethyl-testosterone), 17α-methyl-5β-androstane-3α,17β-diol (3α,5β-THMT), and 17β-methyl-5β-androstane-3α,17α-diol, as well as the parent compound excreted after a single oral administration were monitored by GC-MS/MS. Δ6-Epimethyltestosterone and 3α,5β-THMT served for long-term detection (still present in the 181-189 h urine). 17α-Methyltestosterone and its 17-epimer were not detected in the urines (LOD 0.3 ng/mL). The highest concentrations were found in the 14-20.5 h urine for Δ6-epimethyltestosterone (600 ng/mL), and 3α,5β-THMT (240 ng/mL) and in the 36-44.5 h urine for 17β-methyl-5β-androstane-3α,17α-diol (7 ng/mL).For reference methyltestosterone and epimethyltestosterone were dehydrogenated with chloranil. The characterization of the products was performed by GC-MS(/MS) and NMR.     

Hepatotoxicity associated with weight loss or sports dietary supplements,including OxyELITE Pro™ — United States, 2013

In September 2013, the Hawaii Department of Health (HDOH) was notified of seven adults who developed acute hepatitis after taking OxyELITE Pro™, a weight loss and sports dietary supplement. CDC assisted HDOH with their investigation, then conducted case‐finding outside of Hawaii with FDA and the Department of Defense (DoD). We defined cases as acute hepatitis of unknown etiology that occurred from April 1, 2013, through December 5, 2013, following exposure to a weight loss or muscle‐building dietary supplement, such as OxyELITE Pro™. We conducted case‐finding through multiple sources, including data from poison centers (National Poison Data System [NPDS]) and FDA MedWatch. We identified 40 case‐patients in 23 states and two military bases with acute hepatitis of unknown etiology and exposure to a weight loss or muscle building dietary supplement. Of 35 case‐patients who reported their race, 15 (42.9%) reported white and 9 (25.7%) reported Asian. Commonly reported symptoms included jaundice, fatigue, and dark urine. Twenty‐five (62.5%) case‐patients reported taking OxyELITE Pro™. Of these 25 patients, 17 of 22 (77.3%) with available data were hospitalized and 1 received a liver transplant. NPDS and FDA MedWatch each captured seven (17.5%) case‐patients. Improving the ability to search surveillance systems like NPDS and FDA MedWatch for individual and grouped dietary supplements, as well as coordinating case‐finding with DoD, may benefit ongoing surveillance efforts and future outbreak responses involving adverse health effects from dietary supplements. This investigation highlights opportunities and challenges in using multiple sources to identify cases of suspected supplement associated adverse events. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.     

Polychlorinated biphenyls (PCBs) contamination and aryl hydrocarbon receptor (AhR) agonist activity of Omega-3 polyunsaturated fatty acid supplements: Implications for daily intake of dioxins and PCBs

Omega-3 polyunsaturated fatty acid (n-3 PUFA) rich oils derived primarily from fish are frequently consumed as supplements. Due to the tendency of persistent organic pollutants (POPs) to accumulate in exposed organisms, n-3 PUFA supplements can contain sufficient POPs to present a risk to consumers. Here we investigated PCB concentrations and aryl hydrocarbon receptor (AhR) agonist activity in 17 n-3 PUFA supplements available in Canada. PCBs ranged from <0.8 to 793 ng g⁻¹ oil, with salmon- and seal-derived products yielding the highest values. AhR agonist activity from a reporter gene assay ranged from 1.3 to 72.2 pg TEQ g⁻¹ oil, with salmon and tuna yielding the highest values. When consumed at the recommended doses and as a supplement to the average Canadian diet, seal-derived oil can contribute to exceedance of the tolerable daily intake of 20 ng PCBs kg-BW⁻¹ day⁻¹, and salmon-, tuna-, and sea herring-derived oils can contribute to exceedance of the tolerable daily intake limit of 2.3 pg TEQ kg-BW⁻¹ day⁻¹. The beneficial properties of fish and n-3 PUFA supplements, and the results of this study suggest that it is prudent to consume supplements derived from small, cold-water fatty fish. Further research will be necessary to draw firm conclusions.     

Modulation of CYP1A1 activity by a Ginkgo biloba extract in the human intestinal Caco-2 cells

Ginkgo biloba is a widely consumed dietary supplement. Some dietary active compounds modulate the activity of biotransformation enzymes inside the enterocytes and more interestingly of cytochrome P-450 1A1 (CYP1A1). This enzyme is of a particular interest because of its implication in the metabolism of some exogenous pro-carcinogens or endogenous molecules. In the present work, we have used Caco-2 cells to study the effect of a standard reference material of a Ginkgo biloba extract (GBE) (10-400 μg/ml), as well as of its major individual active compounds (kaempferol, quercetin, isorhamnetin, ginkgolides and bilobalide), alone or in mixtures, at realistic intestinal concentrations, on the induction of CYP1A1 activity, in the presence or absence of benzo[a]pyrene (B[a]P) (0.1 μg/ml), a well-known CYP1A1 inducer. 3-O-rutinosides of kaempferol, quercetin and isorhamnetin were also tested. We have demonstrated a strong induction (p < 0.005) of CYP1A1 activity and a slight, but significant (p < 0.005), decrease of this activity in the presence of B[a]P by the GBE at the realistic exposure level of 100 μg/ml. The inductive effect was explained, in part, by quercetin and kaempferol after 24 h exposure while unknown compounds seem to be responsible for the strong CYP1A1 induction observed after 6 h exposure. The inhibitory potency of flavonols on CYP1A1 activity in presence of B[a]P was much stronger for the aglycones than for the 3-O-rutinosides, explaining the slight effect observed with the GBE, mainly composed of glycosylated flavonoids. These results indicate that GBEs may disturb intestinal CYP1A1 activity and, in turn, affect the metabolism of other compounds. The present paper thus highlights the necessity to take these side effects into account when administrating Ginkgo biloba herbal supplements.     

Toxicology studies with N-acetylglycine

N-acetylglycine (NAGly) has been identified as a minor constituent of numerous foods. The current paper reports the outcome of in vitro and in vivo genotoxicity, acute oral and repeated dose dietary toxicology studies conducted with NAGly. No evidence of genotoxicity was observed with NAGly in vitro bacterial tester strains or in vivo bone marrow micronucleus studies conducted in mice. No mortalities or evidence of adverse effects were observed in Sprague–Dawley rats following acute oral gavage with NAGly at a dose of 2000 mg/kg of body weight or following repeated dose dietary exposure to NAGly at targeted doses of 100, 500, or 1000 mg/kg of body weight/day for 28 days. No biologically significant or test substance related differences were observed in body weights, feed consumption, or clinical pathology response variables in any of the treatment groups. Based on these results it was concluded that NAGly is not genotoxic or acutely toxic. Further, the no-observed adverse-effect-level (NOAEL) for systemic toxicity from repeated dose dietary exposure to NAGly was 898.9 mg/kg of body weight/day for male rats and 989.9 mg/kg of body weight/day for female rats.     

Effect of dietary inclusion of salt substitutes “Obu-Otoyo” on some biochemical indices in rat

“Obu-Otoyo” in folklore has been used as salt substitutes in diet of patients for the management of hypertension with no report on its toxicological effects. Hence, this study sought to investigate the effect of dietary inclusion of two types of the salt substitutes (Obu-Otoyo): salt A and salt B on some biochemical indices in rats. The mineral content of the salt substitutes was determined and the salt substitutes were fed to normal rats as dietary inclusion (0.1–1.0%). The dietary inclusion of the salt substitutes caused a significant (P < 0.05) increase in plasma activities of alkaline phosphatase (ALP), alanine transaminase (ALT), aspartate transaminase (AST), total bilirubin, and atherogenic lipids content (total cholesterol, LDL-cholesterol and triglyceride) with a concomitant decrease in the HDL-cholesterol content when compared with the control. Furthermore, the mineral determination of the salt substitutes revealed the presence of some toxic metals. The alterations in plasma activities of liver function enzymes and lipid profile in rat fed dietary inclusions of “Obu-Otoyo” may be related to the high content of some toxic/heavy metals in the salt substitutes. Therefore, this finding indicates that Obu-Otoyo is toxic to rat.     

Dietary exposure to essential and toxic trace elements from a Total diet study in an adult Lebanese urban population

This study assesses, by the Total diet study approach, the adequacy of micronutrient intake (Co, Cu, Fe, Mn, Ni, Zn) and the dietary exposure of a Lebanese adult urban population to two toxic elements (Cd, Pb). The foods that made up the average ‘total diet’ were derived from a previous individual consumption survey. A total of 1215 individual foods were collected, prepared and cooked prior to analysis. Analytical quantification was performed using inductively coupled plasma mass spectrometry. Average daily intakes of Co (11.4 μg/day), Cu (1104.19 μg/day), Fe (13.00 mg/day), Mn (2.04 mg/day), Ni (126.27 μg/day) and Zn (10.97 mg/day) were below toxicological reference values and were found to satisfy nutritional recommendations, except for manganese in men and iron in women. Average dietary exposure to Pb and Cd represented 3.2% and 21.7% of the respective provisional tolerable weekly intakes. Estimates of dietary intakes of iron appeared to be inadequate for 63% of adult women. These findings should constitute a current measure of assessing the adequacy and safety of foods consumed in Lebanon and may be a basis for future monitoring studies.     

Mission impossible? Regulatory and enforcement issues to ensure safety of dietary supplements

Dietary supplements are widely used across all ages and user groups and constitute a considerable business sector in most developed countries. Hazards relating to concentration, composition, individual contaminants and supplement interactions present an increasing public health concern. The aim of this paper is to review the literature for reported supplement contaminations (occurs in ca 25% of supplements, with anabolic steroids being the most common) and complement these findings with notifications logged in the EU’s Rapid Alert System for Food and Feed (RASFF) through imports or market surveillance, typically logged for poor quality control issues. Notifications in the RASFF have steadily increased by sixfold for supplements in the past 7 years with the USA and China being the major transgressors. Finland and Italy lead in detections, mainly notifying unpermitted substances and contaminants in sexual-enhancing or weight-loss supplements. This paper highlights the paucity of enforcement. Regulating supplements as a foodstuff and not a medicine, coupled with the fact that a significant proportion of the supplement market is distributed via the Internet (hence absent from routine border control and surveillance), make ensuring and enforcing safety a very challenging task. The need for better quality control, compliance and public awareness is evident.     

Seventy day safety assessment of an orally ingested, l-glutamine-containing oat and yeast supplement for horses

We describe a safety assessment of an oral supplement designed to nutritionally support the gastrointestinal system of horses. The supplement comprised a mixture of essential (l-threonine) and conditionally essential (l-glutamine) amino acids, polar lipids, oat bran rich in beta glucans and yeast extract. Young (1–2 years) horses of both sexes were allocated to control (n = 7) and treatment groups (n = 7) and studied for 9 weeks. Horses in the treatment group received the supplement daily for 8 weeks. After 8 weeks of supplementation, horses were studied for one additional week. Outcome measures included body mass, weight gain, results of clinical examination, hematology and plasma chemistry. There were no adverse events associated with supplementation and horses in both groups showed normal weight gain, clinical signs, hematology and chemistry. l-Glutamine, which is not yet listed as GRAS, was considered with respect to its potential for nutritional support and safety when ingested orally. It is concluded that this oral supplement, when ingested by horses at twice the recommended daily level, was safe and does not pose a health risk when used in accordance with good feeding practice.     

Inorganic cobalt supplementation: Prediction of cobalt levels in whole blood and urine using a biokinetic model

Soluble cobalt (Co) supplements with recommended daily doses up to 1000 μg Co/day are increasingly being marketed to consumers interested in healthy living practices. For example, some athletes may consider using Co supplements as blood doping agents, as Co is known to stimulate erythropoesis. However, the distribution and excretion kinetics of ingested Co are understood in a limited fashion. We used a Co-specific biokinetic model to estimate whole blood and urine Co levels resulting from oral exposure or ingestion of Co in amounts exceeding typical dietary intake rates. Following 10 days of Co supplementation at a rate of 400 to 1000 μg/day, predicted adult Co concentrations range from 1.7 to 10 μg/L in whole blood, and from 20 to 120 μg/L in urine. Chronic supplementation (?1 year) at a rate of 1000 μg Co/day is predicted to result in blood levels of 5.7 to 13 μg/L, and in urine levels from 65 to 150 μg/L. The model predictions are within those measured in humans following ingestion of known doses. The methodology presented in this paper can be used to predict urinary or blood Co levels following acute or chronic occupational incidental ingestion, medicinal therapy, supplemental intake, or other non-occupational exposures.     

Detection of undeclared erectile dysfunction drugs and analogues in dietary supplements by ion mobility spectrometry

An ion mobility spectrometry (IMS) method was developed to screen for the presence of undeclared synthetic erectile dysfunction (ED) drugs or drug analogues in herbal dietary supplements claiming to enhance male sexual performance. Ion mobility spectra of authenticated reference materials including three FDA approved drugs (sildenafil citrate, tadalafil, vardenafil hydrochloride trihydrate) and five previously identified synthetic analogues (methisosildenafil, homosildenafil, piperidenafil, thiosildenafil, thiomethisosildenafil) were measured to determine their reduced ion mobilities (K₀). All eight compounds exhibited reduced mobilities between 0.8257 and 1.2876 cm²/(V s). Twenty-six herbal products were then screened for the presence of these compounds, and 15 of the 26 products tested positive for the presence of ED drug or drug analogue adulterants based on their reduced ion mobilities. IMS results were compared against the results obtained from an independent LC/MS reference method for the identical samples. Herbal dietary supplements containing adulterants were classified with 100% accuracy and most of the adulterants were correctly identified by a comparison of the K₀ of the adulterant to the K₀ of the authenticated reference material. The results demonstrate that IMS is a viable method for screening herbal dietary supplements for the presence of ED drug or drug analogue adulterants.     

Health-, medication- and dietary supplement-related behaviors and beliefs relatively unchanged during the COVID-19 pandemic lockdown

BackgroundThe lockdown imposed to counter the coronavirus disease 2019 (COVID-19) pandemic has evoked an unprecedented phenomenon that could affect health behaviors and beliefs.ObjectiveTo examine how medication-, dietary supplement- and health-related behaviors, beliefs and other psychological constructs changed in Polish online health service users during the COVID-19 pandemic lockdown.MethodsA one-time online survey accessed through a health service website was completed before and during the pandemic lockdown by separate samples of respondents. The survey examined beliefs about medicines and dietary supplements, consumption of dietary supplements, trust and contact with their advertisements, sources of dietary supplement knowledge as well as perceived health, diet, physical activity and smoking, among other things.ResultsThe study included 1560 participants. Most examined outcomes remained unchanged over COVID-19 pandemic lockdown. Beliefs that the dietary supplement quality is well controlled became significantly more pronounced during the lockdown (adjusted ratio of estimates 1.16, 95%CI 1.06–1.27, p = 0.001). Fewer people reported having contact with dietary supplement advertisements (adjusted odds ratio 0.59, 95%CI 0.43–0.83, p = 0.002).ConclusionsThe results may help understand some health-related issues associated with COVID-19 pandemic lockdown and may be used to shape aspects of health-related policy.     

Safety evaluation of Monascus purpureus red mould rice in albino rats

Monascus purpureus MTCC 410-fermented rice (red mould rice) is one of the food supplements to lower blood–lipid levels and monacolins have been proven to be the main active constituents in red mould rice (RMR). In this study, we have assessed the safety of RMR by conducting toxicological studies in albino rats. Acute and sub-chronic toxicity studies were conducted on both sexes of albino rats. Feeding acute doses of RMR at 0.5, 1.0, 2.5 and 5.0 g/kg body weight to rats did not cause any symptoms of toxicity or mortality. Similarly, dietary feeding of RMR at 2.0%, 4.0%, 8.0% and 12.0% level (w/w) for 14 weeks did not produce any significant changes in food intake or gain in body weight of the experimental rats compared to control rats. There were no significant differences in the relative weight of vital organs, hematological parameters, macroscopic and microscopic changes in vital organs and serum clinical enzyme levels between the experimental and control groups. Moreover, the rats fed with RMR showed a significant reduction in cholesterol and triglyceride levels in both serum and liver. The results showed that toxicity studies with RMR of M. purpureus did not cause any toxic effects in albino rats.     

National survey of dietary supplement policies in acute care facilities.

PURPOSE: Policies on the use of dietary supplements in acute care facilities were studied. METHODS: A national online survey was conducted in 2004 to determine institutional policies and practices related to the use of dietary supplements. Directors of pharmacy in acute care settings were asked about the role of the pharmacy and therapeutics committee, the use of a nonformulary request process, the use of informed consent, requirements for written orders, mechanisms to identify dietary supplements, recording of supplement use in the pharmacy database, recording of supplement use in the medication administration record, prohibitions on dietary supplement use, storage of dietary supplements, reporting of adverse events, and the use of published references. RESULTS: Usable responses were received from 302 (25.4%) of 1189 pharmacy directors. Policies on dietary supplements were developed and implemented in 62% of facilities, with 38% of respondents indicating that no such policy existed. Policies most commonly required a written order by an authorized prescriber, documentation of use in the medication administration record, and a mechanism for identification of dietary supplements by a health care practitioner prior to use. Thirty percent of respondents with policies reported that they prohibited dietary supplement use, and 9% of those without policies prohibited use in practice. Most institutions allowed the use of a patient's own supply of supplements if ordered by an authorized prescriber. Supplements were most commonly stored at the nurses' station or in a patient drawer. The most commonly used reference was the Internet. Pharmacists had concerns about the consistency of dietary supplement formulations, the lack of FDA review of supplements, and the difficulty of identifying supplements and distinguishing reputable manufacturers. CONCLUSION: A survey of pharmacy directors in acute care facilities revealed that many had no formal policy regarding dietary supplements and had not implemented planning for such a policy.     

Assessment of usnic acid toxicity in rat primary hepatocytes using C isotopomer distribution analysis of lactate, glutamate and glucose

The lichen metabolite usnic acid (UA) has been promoted as a dietary supplement for weight loss, although cases of hepatotoxicity have been reported. Here we evaluated UA-associated hepatotoxicity in vitro using isolated rat hepatocytes. We measured cell viability and ATP content to evaluate UA induced cytotoxicity and applied ¹³C isotopomer distribution measuring techniques to gain a better understanding of glucose metabolism during cytotoxicity. The cells were exposed to 0, 1, 5 or 10 μM UA concentrations for 2, 6 or 24 h. Aliquots of media were collected at the end of these time periods and the ¹³C mass isotopomer distribution determined for CO₂, lactate, glucose and glutamate.The 1 μM UA exposure did not appear to cause significant change in cell viability compared to controls. However, the 5 and 10 μM UA concentrations significantly reduced cell viability as exposure time increased. Similar results were obtained for ATP depletion experiments. The 1 and 5 μM UA doses suggest increased oxidative phosphorylation. Conversely, oxidative phosphorylation and gluconeogenesis were dramatically inhibited by 10 μM UA. Augmented oxidative phosphorylation at the lower UA concentrations may be an adaptive response by the cells to compensate for diminished mitochondrial function.