Towards a replacement therapy for stimulant betel quid dependence: A proof of concept study.

Stimulant betel quid (SBQ) containing Piper betle leaf (L), green unripe Areca catechu nut (AN) and the alkalizing agent, slaked lime, is an addictive, carcinogenic stimulant, with no pharmacotherapy, chewed by millions of people in the Asia/Pacific region. We compared the in vivo physiological profile of chewing (1) non-stimulant P. betle leaf+AN (LAN), (2) SBQ utilizing slaked lime and (3) a novel SBQ utilizing Mg(OH)2 , as an alkalizing agent, by measuring physiological parameters of intoxication and these were correlated with in vitro levels of alkaloids measured by UHPLC-MS/MS. Chewing LAN, which contains high levels of arecoline, had no stimulatory physiological effect. Chewing SBQ containing slaked lime or novel SBQ containing Mg(OH)2 , induced equivalent stimulatory physiological responses. In vitro, slaked lime hydrolyzed muscarinic esters in LAN while Mg(OH)2 did not. The physiological stimulation induced by chewing both SBQ and the lack of physiology to chewing LAN can be explained by changes in lipid solubility of phytochemicals induced by mouth pH during chewing of basic SBQ or acidic LAN. Since antiquity people have added slaked lime to SBQ to enhance absorption of phyto-chemicals across oral membranes to stimulate physiology. The same physiological changes can be induced by substituting slaked lime for less physically and chemically destructive bases. If attitudes regarding SBQ dependence can advance towards the more progressive attitudes already used to help smokers quit tobacco, modern chemistry has the potential to make chewing SBQ safer and quitting programs may become more accessible and efficacious.

Rapid green analytical methodology for simultaneous biomonitoring of five toxic areca nut alkaloids using UHPLC-MS/MS for predicting health hazardous risks.

Areca nut (AN) is a fundamental component of betel quid (BQ), an addictive and carcinogenic mixture chewed by hundreds of millions of people in India-Asia-Pacific. Chewing of BQ is associated with oral cancers due to specific carcinogenic alkaloids (arecaidine, guvacine, guvacoline, arecoline, N-Nitrosoguvacoline) in AN. To predict the hazardous health risks of short and long-term chewing of BQ, it is crucial to identify five toxic AN alkaloids in saliva and urine of BQ chewers. This study reports a green analytical methodology comprising in-syringe assisted vortex-induced salt-enhanced liquid-liquid microextraction coupled with ultra-HPLC-MS/MS for simultaneous biomonitoring of five AN alkaloids in saliva and urine. The analytical method validation results exhibited good linearities between 0.05 and 1000 ng mL-1 with r2 > 0.9930. The detection and quantification limits were between 0.01 and 1.5 and 0.05-5 ng mL-1. Relative recoveries ranged between 87.9% and 110.1% with RSD < 9.1% for saliva samples, 81.5-115.1% with RSD < 9.7% for urine samples. The results indicated the successful identification and real-time monitoring of concentrations of five target AN alkaloids in saliva and urine of BQ chewers and demonstrated the utility of this technique as an efficient analytical protocol for routine biomonitoring of levels of toxic AN alkaloids from BQ chewers and to predict the exposure level and its harmful health risk.

Intoxication and substance use disorder to Areca catechu nut containing betel quid: A review of epidemiological evidence, pharmacological basis and social factors influencing quitting strategies.

AIM: We present a systematic review of substance use disorder (SUD) to Areca catechu nut (AN) and AN containing betel quid (ANcBQ) with emphasis on dependence resulting from chewing of tobacco-free ANcBQ. We examined pharmacology of intoxication and addiction, and factors influencing quitting strategies. METHODS: Epidemiological publications of SUD were included according to PRISMA criteria. Pharmacological publications were retrieved from the PUBMED database and websites of the WHO, United Nations, and Sigma-Aldrich. RESULTS: Nine epidemiological studies show clear evidence of abuse and dependence in tobacco-free ANcBQ and/or ANcBQ+Tobacco chewers. Dependency is greater if ANcBQ contains tobacco. In both groups higher dependency scores were positively correlated with higher frequency of chewing. Dependency on AN+Lime is associated with altered brain morphology, resting state brain activity, neurochemistry and deterioration of working spatial memory. ANcBQ contains a complex mixture of neuroactive compounds that have the potential to act directly upon all major cerebral neurotransmitter systems. Of these compounds, only arecoline (muscarinic agonist) has been the focus of limited pharmacological investigation. In animal studies, arecoline increases dopamine transmission in the mesocorticolimbic circuit and this action may be one factor contributing to ANcBQ dependency in humans. Societal and familial acceptance of ANcBQ consumption is paramount for commencement and persistence of chewing. CONCLUSIONS: ANcBQ SUD remains an orphan disease. The limited understanding of pharmacological basis of intoxication and SUD determines there are no pharmacological replacement therapies for ANcBQ SUD. The addictive properties of ANcBQ coupled with social acceptance of ANcBQ chewing limits the effectiveness of counseling-based quitting programs.

Characterization of the psychological, physiological and EEG profile of acute betel quid intoxication in naïve subjects.

Betel quid use and abuse is wide spread in Asia but the physiological basis of intoxication and addiction are unknown. In subjects naïve to the habit of betel quid intoxication, the psychological and physiological profile of intoxication has never been reported. We compared the effect of chewing gum or chewing betel quid, and subsequent betel quid intoxication, on psychological assessment, prospective time interval estimation, numerical and character digit span, computerized 2 choice tests and mental tasks such as reading and mathematics with concurrent monitoring of ECG, EEG and face temperature in healthy, non-sleep deprived, male subjects naïve to the habit of chewing betel quid. Betel quid intoxication, dose dependently induced tachycardia (max 30 bpm) and elevated face temperature (0.7°C) (P<0.001) above the effects observed in response to chewing gum (max 12 bpm and 0.3°C) in 12 subjects. Gross behavioral indices of working memory such as numerical or character digit span in 8 subjects, or simple visual-motor performance such as reaction speed or accuracy in a two choice scenario in 8 subjects were not affected by betel quid intoxication. Betel quid intoxication strongly influenced the psychological aspects of perception such as slowing of the prospective perception of passage of a 1 minute time interval in 8 subjects (P<0.05) and perceived increased arousal (P<0.01) and perceived decreased ability to think (P<0.05) in 31 subjects. The EEG spectral profile recorded from mental states associated with open and closed eyes, and mental tasks such as reading and eyes closed mental arithmetic were significantly modified (P<0.05) relative to chewing gum by betel quid intoxication in 10 subjects. The prevalence of betel quid consumption across a range of social and work settings warrants greater investigation of this widespread but largely under researched drug.

Seasonality and fasting effect in raccoon dog Nyctereutes procyonoides serum leptin levels determined by canine leptin-specific enzyme-linked immunosorbent assay.

Leptin is an adipocyte-derived peptide hormone that acts on the brain and regulates food intake and energy balance. Several previous reports have suggested that overwintering raccoon dogs Nyctereutes procyonoides are able to control their adiposity efficiently, but the contribution of leptin to weight regulation in these animals remains unclear. To study the seasonality of overwintering raccoon dogs as well as the effects of fasting on them, serum leptin levels were investigated using a newly established canine leptin-specific enzyme-linked immunosorbent assay (ELISA) kit. Of the nine animals studied, five were fed and four were fasted (deprived of food for 2 months in winter). Blood samples and body fat weights were monitored once a month throughout the experimental period (July 2007-March 2008). Leptin concentrations obtained by ELISA were significantly higher than and had a positive correlation with those obtained by previously used multispecies radioimmunoassay (RIA) kits. Moreover, ELISA showed a clearer correlation between the body fat weight and leptin levels compared with RIA, suggesting the efficacy of canine leptin-specific ELISA kit for leptin estimation in raccoon dogs. Autumnal fattening was observed in both groups of animals, but the wintertime loss of adipose tissue was more obvious in the fasted group. Serum leptin concentrations determined by ELISA showed seasonal changes without significant differences between the fed and fasted animals. Therefore, high levels of leptin may be responsible for the suppression of feeding behavior in raccoon dogs before winter.

Overwintering strategy of wild free-ranging and enclosure-housed Japanese raccoon dogs (Nyctereutes procyonoides albus).

The raccoon dog, Nyctereutes procyonoides, is a canid with a passive overwintering strategy in northern Europe. However, the behaviour and physiology of the Japanese subspecies, N. p. albus, which has fewer chromosomes than the other subspecies, remain unknown. We measured body temperature, body composition and blood biochemistry of wild free-ranging and fasted enclosure-housed N. p. albus during boreal winter in Hokkaido, Japan. Body temperature of N. p. albus decreased from 38 degrees C in autumn to 35.9-36.7 degrees C while maintaining a circadian rhythm in late February (n = 3). A transient 18-36% decrease in resting heart rate occurred when body temperature was low (n = 2). Despite a 33-45% decrease in body weight due to winter fasting, circulating glucose, total protein and triglyceride levels were maintained (n = 4). Serum urea nitrogen dropped by 43-45% from autumn to spring, suggesting protein conservation during fasting. The overwintering survival strategy of N. p. albus in central Hokkaido is based upon large changes in seasonal activity patterns, winter denning and communal housing without the large decrease in body temperature that is characteristic of subarctic animals exhibiting hibernation or torpor.

Mammalian cerebral metabolism and amino acid neurotransmission during hibernation.

This report demonstrates that during the torpor phase of hibernation, hamsters utilize (14)C and (13)C glucose in torpor-specific brain metabolic pathways. Microdialysis of (14)C glucose into the striatum rapidly induced a steady state labeling of extracellular fluid (ECF) lactate and labeling of tissue GABA, glutamate, glutamine, and alanine in ipsilateral and contralateral striata. The same tissue metabolites were labeled in cortex, hypothalamus, and brainstem after microdialysis of (14)C lactate into the lateral ventricle. Serine, aspartate, glycine, taurine, tyrosine, and methionine were not synthesized from glucose or lactate during torpor. ECF levels of amino and organic acids were low and unchanging during torpor and increased late during arousal to cenothermia. Labeled intracellular (14)C GABA and glutamate were not communicated to the striatal ECF or ventricular space during torpor. (13)C NMR demonstrated rapid formation of lactate and functional tricarboxylic acid cycles in GABAergic and glutamatergic neurons, and enrichment of glutamine and alanine after i.v. (13)C glucose. Large changes in tissue levels of amino acids occur prior to or during entrance into torpor but not during torpor. It is proposed that cerebral intracellular dehydration, the enlargement of ECF and the biochemistries associated with brain water homeostasis may have a role in regulating hibernation.

Brain ECF antioxidant interactions in hamsters during arousal from hibernation.

Warming from hibernation to cenothermia involves intense metabolic activity and large fluxes in regional blood flow and volume. During this transition, levels of the antioxidants, ascorbate (AA), urate and glutathione (GSH) in brain tissue, extracellular fluid (ECF) and plasma change substantially. Striatal ECF was sampled and manipulated using very slow perfusion microdialysis to examine the mechanisms that influence the changing profile of striatal ECF AA, urate and GSH levels during arousal from hibernation to cenothermia in Syrian hamsters (Mesocricetus auratus). Omission of glucose from the perfusate had no effect upon the respective decrease, increase and transient increase in striatal ECF levels of AA, GSH and urate observed during arousal from hibernation to cenothermia. In contrast, inhibition of xanthine dehydrogenase/oxidase (XOR) activity by reverse dialysis with oxypurinol, itself a free radical scavenger, decreased ECF urate and preserved ECF AA levels. This suggests that some ECF AA is oxidized by free radical products of XOR flux and/or by other free radical producing processes activated during the transition from hibernation to cenothermia. Local supplementation of ECF AA, GSH and cystiene had no effect upon the profile of transient increase of ECF urate observed during arousal from hibernation. The production of free radicals by XOR and the disappearance of AA from the ECF continues for at least 2h immediately after the hamster has attained cenothermia. The hamster, immediately after arousal from hibernation, can be utilized as a natural model to study free radical production and effective scavenging at cenothermia.

Brain antioxidant levels in hamsters during hibernation, arousal and cenothermia.

Warming from hibernation to cenothermia involves intense metabolic activity coincident with large fluxes in blood flow and is considered to be a period of oxidative stress during which utilization of endogenous antioxidants prevents pathology. Very slow flow brain microdialysis enabled temperature independent sampling of the brain striatal extracellular fluid (ECF) during hibernation, arousal and cenothermia in Syrian hamsters (Mesocricetus auratus). Brain tissue and dialysates were analyzed to provide the first profile of changes in ECF levels of ascorbate (AA), glutathione (GSH) and urate during hibernation and the transition to cenothermia. Brain tissue content of AA and GSH was unchanged between hibernation and cenothermia; however, arousal was associated with substantial oxidation of AA from the brain ECF and plasma compartments. ECF GSH increased during arousal. Brain tissue urate content was decreased 50% during hibernation. ECF urate levels were unchanged in hibernation and cenothermia but transiently increased 100% during arousal. These experiments demonstrate that arousal from hibernation is a suitable experimental model for examination of the mechanisms by which non-pathological tissue integrity is maintained in the face of the generation of free radicals during increasing metabolism, temperature and cerebral reperfusion.

Chemical polymerization of m-phenylenediamine, in the presence of glucose oxidase, produces an enzyme-retaining electrooxidisable polymer used to produce a biosensor for amperometric detection of glucose from brain dialysate.

A new procedure involving chemical polymerization of a monomer of m-phenylenediamine (m-ppd) containing glucose oxidase (GOx) and subsequent electro-synthesis of the functional GOx containing polymer onto platinum needle electrodes (PTNE) was used for the amperometric analysis of glucose concentration in brain dialysates. Monomer solutions of o-phenylenediamine (o-ppd) and m-ppd were polymerized by low concentrations of glutaraldehyde (GA) and precipitated from solution. The 1,3 position of the amines on the benzene was amenable to stable polymerization by GA but polymerization of o-ppd (1,2 position) by GA was unstable and degraded. Polymerization of m-ppd appears to proceed by dehydration synthesis. GA induced polymerization of m-ppd polymer in the presence of GOx produced a polymer with strongly bound, functional GOx. This GOx-m-ppd polymer formed a stable matrix that was effectively employed in flow injection analysis (FIA) of glucose. If maintained under O(2) free atmosphere after chemical polymerization, the GOx-m-ppd polymer retained the ability to be electropolymerized. PTNE coated with GOx-m-ppd polymer by repeated dip/amperometry produced stable, sensitive amperometric glucose sensors with good interference exclusion properties and long shelf life. Scanning EM demonstrated that amperometry modified the structure of the GOx-m-ppd on the PTNE surface. GOx-m-ppd PTNE glucose sensors and bare PTNE were placed in a radial flow cell and FIA was employed for the simultaneous measurement of glucose and ascorbic acid, respectively, from dialysates of brain tissue.

Perfusate oxygen and carbon dioxide concentration influence basal microdialysate levels of striatal glucose and lactate in conscious rats.

O(2) concentration ([O(2)]) in air equilibrated solutions at room temperature is three fold higher than that in brain extracellular fluid (ECF), and CO(2) concentration ([CO(2)]) is 100 times lower. Using microdialysis the ECF is routinely dialyzed against glucose free isotonic perfusates containing 200 microM O(2) and 10 microM CO(2). In conscious rats, 2 days after probe implantation, decreasing perfusate [O(2)] from 200 to 68 microM (physiologic level) for 60 min, while maintaining a low [CO(2)] (10 microM), increased striatal dialysate glucose and lactate by 12% and 33%, respectively. The same protocol on the third day essentially had no effect on monoamine metabolites. Decreasing [O(2)] concurrent with increasing [CO(2)] to 1.3 mM (physiologic level) for 60 min increased glucose and lactate by 17% and 37%, respectively. This study demonstrates for dialysis studies of glucose and lactate, perfusates that mimic physiologic ECF [O(2)] and [CO(2)] are more appropriate.

Hyperoxia caused by microdialysis perfusion decreased striatal monoamines: involvement of oxidative stress.

Due to complex influence, such as utilization and permeability of arterial vessels to oxygen, there is a considerable difference of oxygen tension between extracellular fluid and perfusate usually used in microdialysis (30-60 Torr versus 145 Torr). Dialysate dopamine and monoamine metabolites-3,4-dihydroxyphenylacetic acid, homovanillic acid and 5-hydroxyindoleacetic acid were measured under different kinds of oxygen tension solutions (145, 72, 48 Torr). In the acute and anesthetized group, dopamine, 3,4-dihydroxyphenylacetic acid, 5-hydroxyindoleacetic acid and homovanillic acid increased 72, 93, 86 and 65%, respectively when changing the perfusate from 145 Torr to near physiological 48 Torr, while in chronic and conscious group, carried out 72 h after surgery, these compounds showed obscure increases (only homovanillic acid produced a significant change of 14%). The different effect of perfusate oxygen tension on dialysate levels of monoamines in anesthetized and conscious rats might be caused by oxidative stress triggered by hyperoxia combined with anesthesia and surgical trauma.

Cyclic voltammetry characterization of metal complex imprinted polymer.

Polymer capable of specific binding to Cu(2+)-2, 2'-dipyridyl complex was prepared by molecular imprinting technology. The binding specificity of the polymer to the template (Cu(2+)-2, 2'-dipyridyl complex) was investigated by cyclic voltammetric scanning using the carbon paste electrode modified by polymer particles in phosphate buffer solution. Factors that influence rebinding of the imprinted polymer were explored. The results demonstrated that cyclic voltammetry was an efficient approach to explore interactions between template and imprinted polymers.

Continuous on-line measurement of cerebral hydrogen peroxide using enzyme-modified ring-disk plastic carbon film electrode.

An amperometric method suitable for the continuous on-line measurement of cerebral hydrogen peroxide from a microdialysate has been successfully performed for the first time by using an enzyme-modified ring-disk plastic carbon film electrode (PCFE) in a thin-layer radial flow cell. PCFE consists of a ring electrode modified with horseradish peroxidase to detect H2O2 at 0.0 V (vs Ag/ AgCl) and a disk electrode coated with ascorbate oxidase (AOx) to preoxidize ascorbic acid (AA) and thus suppress interference via direct oxidation. Analytes in solution (brain dialysates or standards) are mixed on-line with a phosphate-buffered solution containing dissolved oxygen and chelating agent, EDTA. The buffered solution is used to provide the O2 necessary for the AOx catalytic reaction, stabilize the changes in dialysate pH that are associated with the in vivo formation of H2O2, and remove heavy metal ion impurities and thus suppress reactions between AA and H2O2. This procedure enables trace levels of H2O2 to be readily monitored, virtually interference-free from physiological levels of AA, uric acid, electroactive neurotransmitters and their principle metabolites, in a continuous-flow system.