Volatile scent chemicals in the urine of the red fox, Vulpes vulpes.

The red fox is a highly adaptable mammal that has established itself world-wide in many different environments. Contributing to its success is a social structure based on chemical signalling between individuals. Urine scent marking behaviour has long been known in foxes, but there has not been a recent study of the chemical composition of fox urine. We have used solid-phase microextraction and gas chromatography-mass spectrometry to analyze the urinary volatiles in 15 free-ranging wild foxes (2 female) living in farmlands and bush in Victoria, Australia. Foxes here are routinely culled as feral pests, and the urine was collected by bladder puncture soon after death. Compounds were identified from their mass spectra and Kovats retention indices. There were 53 possible endogenous scent compounds, 10 plant-derived compounds and 5 anthropogenic xenobiotics. Among the plant chemicals were several aromatic apocarotenoids previously found in greater abundance in the fox tail gland. They reflect the dietary consumption of carotenoids, essential for optimal health. One third of all the endogenous volatiles were sulfur compounds, a highly odiferous group which included thiols, methylsulfides and polysulfides. Five of the sulfur compounds (3-isopentenyl thiol, 1- and 2-phenylethyl methyl sulfide, octanethiol and benzyl methyl sulfide) have only been found in foxes, and four others (isopentyl methyl sulfide, 3-isopentenyl methyl sulfide, and 1- and 2-phenylethane thiol) only in some canid, mink and skunk species. This indicates that they are not normal mammalian metabolites and have evolved to serve a specific role. This role is for defence in musteloids and most likely for chemical communication in canids. The total production of sulfur compounds varied greatly between foxes (median 1.2, range 0.4-32.3 µg 'acetophenone equivalents'/mg creatinine) as did the relative abundance of different chemical types. The urinary scent chemistry may represent a highly evolved system of semiochemicals for communication between foxes.

Scent Chemicals of the Tail Gland of the Red Fox, Vulpes vulpes.

Like all animals, the red fox uses chemical signals for social communication. The supracaudal or tail gland smells of violets, attributed to the presence of carotenoid degradation products, or apocarotenoids, which commonly occur as aromatics in flowers. We have more fully characterized the scent chemistry of the fox tail gland. Volatile chemicals were analyzed by gas chromatography-mass spectrometry (GC-MS) and identified from their electron ionization mass spectra and Kovats retention indices. The 3 previously reported apocarotenoids were confirmed, and many additional compounds found. These include the apocarotenoids ß-cyclocitral, ß-homocitral, ß-ionone, cyclic ß-ionone, ß-ionone-5,6-epoxide, α-ionene, α-ionone, 2,6,6-trimethylcyclohexanone (IUPAC 2,2,6-), 2,6,6-trimethyl-2-cyclohexen-1-one, 6-methyl-5-hepten-2-one (sulcatone), and geranyl acetone. Notably, sulcatone is a semiochemical in several species. 3,3-Dimethyl-2,7-octanedione was identified as a probable apocarotenoid which is likely to be a significant fox scent chemical. The γ-lactone of 4-hydroxyhexadecanoic acid (hexadecan-4-olide) was also found, one of a group of known mammalian signaling compounds. This rich mixture of volatile apocarotenoids implies an adequate consumption of plant carotenoids, which are known to be necessary for optimal health. Dietary carotenoids color the skin and feathers of some birds, used as a visual signal to conspecifics, and the floral aroma of the fox tail gland may provide an olfactory signal to other foxes.

Biologically meaningful scents: a framework for understanding predator-prey research across disciplines.

Fear of predation is a universal motivator. Because predators hunt using stealth and surprise, there is a widespread ability among prey to assess risk from chemical information - scents - in their environment. Consequently, scents often act as particularly strong modulators of memory and emotions. Recent advances in ecological research and analytical technology are leading to novel ways to use this chemical information to create effective attractants, repellents and anti-anxiolytic compounds for wildlife managers, conservation biologists and health practitioners. However, there is extensive variation in the design, results, and interpretation of studies of olfactory-based risk discrimination. To understand the highly variable literature in this area, we adopt a multi-disciplinary approach and synthesize the latest findings from neurobiology, chemical ecology, and ethology to propose a contemporary framework that accounts for such disparate factors as the time-limited stability of chemicals, highly canalized mechanisms that influence prey responses, and the context within which these scents are detected (e.g. availability of alternative resources, perceived shelter, and ambient physical parameters). This framework helps to account for the wide range of reported responses by prey to predator scents, and explains, paradoxically, how the same individual predator scent can be interpreted as either safe or dangerous to a prey animal depending on how, when and where the cue was deposited. We provide a hypothetical example to illustrate the most common factors that influence how a predator scent (from dingoes, Canis dingo) may both attract and repel the same target organism (kangaroos, Macropus spp.). This framework identifies the catalysts that enable dynamic scents, odours or odorants to be used as attractants as well as deterrents. Because effective scent tools often relate to traumatic memories (fear and/or anxiety) that cause future avoidance, this information may also guide the development of appeasement, enrichment and anti-anxiolytic compounds, and help explain the observed variation in post-traumatic-related behaviours (including post-traumatic stress disorder, PTSD) among diverse terrestrial taxa, including humans.

Lipids of the Tail Gland, Body and Muzzle Fur of the Red Fox, Vulpes vulpes.

The tail gland of the red fox (Vulpes vulpes) secretes lipids containing volatile terpenes used in social communication. We have analysed lipids extracted from fur of the tail gland, body (flanks) and muzzle of foxes. GC-MS showed a novel group of iso-valerate and tiglate monoesters of alkane-1,2-diols (C18:0-22:0). There was also a larger group of Type II diesters in which a second, longer chain, fatty acid (FA) was attached to the free alcohol group. LC-MS showed the full range of diol diesters, mostly C36:0-50:0, with smaller amounts of the corresponding mono-unsaturated tiglate esters. An additional group of diesters with higher MW (C49:0-62:0) containing two long-chain FA was present in the lipids of body and muzzle fur. After saponification and GC-MS, 98 fatty acids were characterized as their methyl esters. Apart from the C5 FA, most were saturated n-, iso-, anteiso- or other methyl-branched FA (C12:0-28:0) whose structures were determined by a combination of their mass spectra and Kovats retention indices. Several FA have not previously been found in nature or in vertebrates. Thirty-four alkane-1,2-diols were found as their TMS derivatives, mostly n-, iso- or anteiso-isomers of C16:0-25:0. The tail gland had the greatest amount of wax esters, from a greater variety of FA and diols, but lacked the esters with two long-chain FA. These findings show that fox skin lipids comprise mono- and di-esters of alkane-1,2-diols, and exhibit enormous complexity due to the diversity of their constituent FA, diols and the many possible isomers of their esters.

Injection of Pharmaceuticals Designed for Oral Use: Harms Experienced and Effective Harm Reduction Through Filtration.

Several pharmaceutical products are liable to 'abuse' or use outside their prescription, which frequently involves their injection. Examples are slow-release forms of morphine and oxycodone, and sublingual buprenorphine. During injection preparation, the drug is extracted into water, after crushing and heating the tablet if considered necessary. Since these products are designed for oral administration, they can contain excipients (ingredients other than the drug) which are poorly soluble, resulting in suspension of particles in the injection solution. Injected particles are able to produce medical complications such as the blockage of small blood vessels leading to ischaemia (inadequate blood flow) and tissue damage. Filtration can be used to remove particles from the suspension; including bacteria if the porosity is small enough (0.2 µm). However, filters are liable to blockage when overloaded, especially if the pore size is small. This problem can be minimised by using a larger pore size (e.g. 5-10 µm), but the resulting filtrate will contain many residual small particles. The use of two filters, coarse and fine, either sequentially or in a double membrane device, enables removal of the majority of particles as well as bacteria, although not quite meeting pharmaceutical standards for safe injection. Although not yet evaluated by a clinical trial, this highly effective filtration process would be expected to greatly reduce the risk of vascular and related complications, as well as non-viral infections. Careful technique ensures that drug is not lost by filtration, a priority for most drug consumers. Practical issues that affect acceptability of filtration by injecting drug users, including ease of use and cost, will need to be considered. However, given the laboratory evidence demonstrating the effectiveness of filters it is time to consider these tools as essential for safe injection as sterile needles/syringes for the world's approximately 16 million people who inject drugs.

Isolation and unnatural death of elderly people in the aging Japanese society.

PURPOSE: Japanese society has reached an unprecedented level of aging, with elderly people accounting for 25.1% of the population in October 2013. These changes have created concerns regarding deaths among the elderly. In this study, we compared recent forensic autopsy cases with cases from about 20 years ago, with the goal of understanding the context of death among the elderly within Japanese society today. METHODS: We investigated the forensic autopsy records of 297 people aged 65 years or above. In order to examine the effect of residential circumstances, we classified these cases into two groups: people who lived alone (group A) and those who lived with their family (group B). Forty-five of these autopsy cases were conducted about 20 years ago (1989 to 1993) and 252 cases were recent (2009 to 2013). The cases were limited to people who had been found dead or in a critical situation at home. We investigated the first finder, the period of time elapsed between death and discovery, and the cause of death. RESULTS: The incidence of the first finder being a family member was more than 20% greater in group B compared with group A. The proportions of cases for which it took more than three days for someone to find the body or an abnormal situation were about 14% and 7% in groups A and B, respectively, 20 years ago, and about 48% and 19% among the recent cases. These proportions were significantly higher among the recent cases. Among recent cases, a post-mortem elapsed time of more than 3 days occurred more often in group A than group B (p=0.0002). None of the older cases had an unknown cause of death in either group. However, among the recent cases from both groups, 20-30% of cases resulted in unknown causes of death. The incidences of unknown causes of death were significantly higher among the recent cases in both groups (p=0.015) and in group B alone (p=0.037). The incidences of murder cases were significantly lower in group B among the recent cases (p=0.0022). DISCUSSION: Elderly people who live alone are not easily found or aided when in critical situations, and they may only be discovered after death. Prolongation of the postmortem interval (PMI) results in the deterioration of the corpse making determination of cause of death problematic. The results of this study suggest that there are three factors that isolate elderly people and increase the difficulty in determining their cause of death: reduced communication with family members, reduced communication with neighbors or the community, and the increasing prevalence of the nuclear family. In group B, the prolonged discovery time and the increased incidence of unknown causes of death suggest reduced communication with family members, even though the incidence of being found by a family member was higher than in group A. The murder rate was significantly lower in group B, which may suggest that cases of domestic murder were overlooked. Support for a safe life and peaceful ending for the elderly requires a system based on three factors: remote monitoring to ensure safety, the establishment of elderly groups providing mutual support, and increased visits from welfare workers. Understanding the circumstances of the elderly who die alone is beneficial to countries facing an aging society with weakened family or community structures, and who hope for better support for the elderly.

Triacylglycerol estolides, a new class of mammalian lipids, in the paracloacal gland of the brushtail possum (Trichosurus vulpecula).

The paracloacal glands are the most prevalent scent glands in marsupials, and previous investigation of their secretions in the brushtail possum (Trichosurus vulpecula) has identified many odorous compounds together with large amounts of neutral lipids. We have examined the lipids by LC-MS, generating ammonium adducts of acylglycerols by electrospray ionisation. Chromatograms showed a complex mixture of coeluting acylglycerols, with m/z from about 404 to 1048. Plots of single [M + NH4](+) ions showed three groups of lipids clearly separated by retention time. MS-MS enabled triacylglycerols and diacylglycerol ethers to be identified from neutral losses and formation of diacylglycerols and other product ions. The earliest-eluting lipids were found to be triacylglycerol estolides, in which a fourth fatty acid forms an ester link with a hydroxy fatty acid attached to the glycerol chain. This is the first report of triacylglycerol estolides in animals. They form a complex mixture with the triacylglycerols and diacylglycerol ethers of lipids with short- and long-chain fatty acids with varying degrees of unsaturation. This complexity suggests a functional role, possibly in social communication.

Detection of Human Polyomavirus DNA Using the Genome Profiling Method.

BACKGROUND: In the field of forensic medicine, it is very difficult to know prior to autopsy what kind of virus has infected a body. OBJECTIVE: We assessed the potential of the genome profiling (GP) method, which was developed in the field of bioengineering, to identify viruses belonging to one species. METHOD: Two species in the same family, JC and BK viruses, were used in this study. Using plasmid samples, we compared the findings of molecular phylogenetic analysis using conventional genome sequencing with the results of cluster analysis using the random PCR-based GP method and discussed whether the GP method can be used to determine viral species. RESULTS: It was possible to distinguish these two different viral species. In addition to this, in our trial we could also detect the JC virus from a clinical sample. CONCLUSION: This method does not require special reagent sets for each viral species. Though our findings are still in the trial period, the GP method may be a simple, easy, and economical tool to detect viral species in the near future.

Filtration of crushed tablet suspensions has potential to reduce infection incidence in people who inject drugs.

INTRODUCTION AND AIMS: The medical complications of injecting preparations from crushed tablets can be severe, and most can be attributed to the injection of insoluble particles and micro-organisms. Previously we have shown that most of the particles can be removed by filtration, but it was not known whether bacteria could also be filtered in the presence of a high particle load. This study aims to determine the feasibility of filtration to remove bacteria from injections prepared from tablets. DESIGN AND METHODS: Injections were prepared from crushed slow-release morphine tablets, in mixed bacterial suspensions of Staphylococcus aureus, Streptococcus pyogenes and Pseudomonas aeruginosa. The injection suspensions were passed through syringe filters of porosity 0.45 or 0.20 µm, or combined 0.8 then 0.2 µm, and the bacterial load was counted. RESULTS: Bacterial concentrations in unfiltered injections were 2.5-4.3 × 10(6) colony forming units mL(-1) . Both the 0.20 and 0.45 µm filters blocked unless a prefilter (cigarette filter) was used first. The 0.2 µm filter and the combined 0.8/0.2 µm filter reduced the bacteria to the limit of detection (10 colony forming units mL(-1) ) or below. Filtration through a 0.45 µm filter was slightly less effective. DISCUSSION AND CONCLUSIONS: Use of a 0.2 µm filter, together with other injection hygiene measures, offers the prospect of greatly reducing the medical complications of injecting crushed tablets and should be considered as a highly effective harm reduction method. It is very likely that these benefits would also apply to other illicit drug injections, although validation studies are needed.

Tracing Jomon and Yayoi ancestries in Japan using ALDH2 and JC virus genotype distributions.

BACKGROUND: According to the dual structure model, the modern Japanese ethnic population consists of a mixture of the Jomon people, who have existed in Japan since at least the New Stone Age, and the Yayoi people, who migrated to western Japan from China around the year 300 bc Some reports show that the Yayoi are linked to a mutation of the aldehyde dehydrogenase 2 gene (ALDH2). Recent viral studies indicate two major groups found in the Japanese population: a group with the CY genotype JC virus (JCV) and a group with the MY genotype JCV. It is unclear whether either genotype of the JC virus is related to the Jomon or Yayoi. In this study, we attempted to detect JCV genotypes and ALDH2 mutations from the DNA of 247 Japanese urine samples to clarify the relationship between the dual structure model and the JCV genotype through ALDH2 mutation analysis and JCV genotyping. FINDINGS: The ALDH2 polymorphism among 66 JC virus-positive samples was analyzed, and it was found that the ALDH2 variant is significantly higher in the population with CY genotype JCV (51.5 %) than in the population with the MY genotype (24.2 %) (p < 0.05). CONCLUSION: From these findings, it may be inferred that the ALDH2 mutation, which is related to the Yayoi, is related to CY genotype JCV. When the Yayoi migrated to the Japanese archipelago, they brought the ALDH2 mutation as well as the CY genotype JCV.

The utilization of stable isotope analysis for the estimation of the geographic origins of unidentified cadavers.

The number of unidentified cadavers is increasing worldwide and the effective methods which reveal their geographic origin are not well known. This study reports on the utilization of δ(18)O, δ(13)C, δ(2)H and δ(15)N ratios gained through stable isotope analysis of urine samples collected from eight locations: Chiba, Japan; Fuzhou, China; and Denpasar, Indonesia in our pilot study with data from healthy volunteers from five further locations from healthy volunteers: Melbourne and Perth, Australia; Qingdao, China; Turku, Finland and Oklahoma, USA. This study posits that the utilization of δ(18)O and δ(2)H is more feasible than δ(13)C and δ(15)N stable isotope ratios in differentiating or estimating the origin of human samples. Secondly, this study demonstrated that the δ(18)O and δ(2)H stable isotope ratios of urine samples from eight locations differed significantly.

A trial of the utilization of stable isotope analysis for the estimation of the geographic origins of unidentified cadavers.

The number of unidentified cadavers is increasing worldwide and the effective methods which reveal their geographic origin are not well known. In this study, we analyzed the urine stable isotope ratio of hydrogen and oxygen collected from three locations: Chiba (Japan), Fuzhou (China), and Denpasar (Indonesia) from healthy volunteers. In addition, analysis of the effect of drinking bottled water on stable isotope ratios found in urine, and the comparison of the stable isotope ratios of urine and saliva, were conducted. Statistically significant differences in δ(2)H and δ(18)O values from the three locations were found. In this pilot study, urine δ(18)O values became increasingly similar to those of bottled drinking water during an eight-day period of drinking only bottled water. In a separate pilot study significant differences in δ(18)O, δ(13)C, and δ(15)N values from urine and saliva were found, but not in δ(2)H values. In all three studies, although the number of samples was limited, the results suggest that with further research, stable isotope analysis from urine samples might be used to identify the origins of unidentified corpses, assist in determining the length of time an individual has been in a given area and distinguish between body fluids.

Mechanisms for eliminating monoterpenes of sagebrush by specialist and generalist rabbits.

Pygmy rabbits (Brachylagus idahoensis) are one of only three vertebrates that subsist virtually exclusively on sagebrush (Artemisia spp.), which contains high levels of monoterpenes that can be toxic. We examined the mechanisms used by specialist pygmy rabbits to eliminate 1,8-cineole, a monoterpene of sagebrush, and compared them with those of cottontail rabbits (Sylvilagus nuttalli), a generalist herbivore. Rabbits were offered food pellets with increasing concentrations of cineole, and we measured voluntary intake and excretion of cineole metabolites in feces and urine. We expected pygmy rabbits to consume more, but excrete cineole more rapidly by using less-energetically expensive methods of detoxification than cottontails. Pygmy rabbits consumed 3-5 times more cineole than cottontails relative to their metabolic body mass, and excreted up to 2 times more cineole metabolites in their urine than did cottontails. Urinary metabolites excreted by pygmy rabbits were 20 % more highly-oxidized and 6 times less-conjugated than those of cottontails. Twenty percent of all cineole metabolites recovered from pygmy rabbits were in feces, whereas cottontails did not excrete fecal metabolites. When compared to other mammals that consume cineole, pygmy rabbits voluntarily consumed more, and excreted more cineole metabolites in feces, but they excreted less oxidized and more conjugated cineole metabolites in urine. Pygmy rabbits seem to have a greater capacity to minimize systemic exposure to cineole than do cottontails, and other cineole-consumers, by minimizing absorption and maximizing detoxification of ingested cineole. However, mechanisms that lower systemic exposure to cineole may come with a higher energetic cost in pygmy rabbits than in other mammalian herbivores.

Effects of filtration on the presence of particulate and oxycodone content of injections prepared from crushed OxyContin® tablets.

It is common for injecting drug users (IDU) to prepare injections by crushing tablets which are not designed for parental administration. The injection of insoluble tablet excipients can lead to serious local and systemic medical complications. The aim of the study was to investigate the effectiveness of various types of filters in removing harmful insoluble particles from the injections prepared using crushed oxycodone tablets. Injections were prepared from a sustained-release oxycodone tablet formulation. The filtration of tablet extracts was carried out following procedures used by IDU using makeshift filter and commercially available filters. Particulate contamination and oxycodone content were analysed using light microscopy and spectrophotometer. Unfiltered extracts contained hundreds of thousands of particles of sufficient size to cause harms. Cigarette filters removed large particles but failed to remove small particles. The combination of cigarette filter and syringe filter (0.45 µm or 0.22 µm) reduced the particle count by 90 - 95%. A double membrane syringe filter (0.8/0.2 µm) removed more than 99% of the particles. Recovery of oxycodone was more than 95% with the tested syringe filters. Particulate contamination in injections prepared from crushed tablets can be effectively removed using a combination process of cigarette filter and syringe filters, or a 0.8/0.2 µm syringe filter. Compared to other filters, the 0.8/0.2 µm syringe filter did not block, the filtration was quick and easy to perform, and did not retain oxycodone. The use of a 0.8/0.2 µm syringe filter can provide an important harm reduction measure for IDU.

Scent chemicals of the brushtail possum, Trichosurus vulpecula.

The common brushtail possum (Trichosurus vulpecula) is the most widespread browsing marsupial in Australia, where it occupies woodland, agricultural, and urban environments. Following its introduction into New Zealand in the 19th century it has become a major feral pest, threatening native forests. The adaptability of the possum is thought to be due in part to its social organization, in which chemical communication is important. Possums have cloacal glands and exhibit related marking behavior. This study sought to characterize the chemicals involved in scent marking. Swabs were taken of the cloacal region of 15 possums (5 females, 10 males) from north-eastern Tasmania and analyzed by gas chromatography-mass spectrometry. There was a large number of compounds present, including 81 branched and unbranched, and saturated and unsaturated, fatty acids (C(4)-C(15)) and alcohols (C(6)-C(26)); 27 esters of 2,6- and 2,7-dimethyloctanol; 29 esters of formic acid; 39 sulfur compounds including S(8) and a series of dialkyl disulfides, trisulfides, and tetrasulfides (C(4)-C(10)); and several alkylglycerol ethers. Many of these cloacal compounds are new to biology. There was considerable individual variability in the relative amounts of compounds found, and no evident sex differences, although the study was not designed to test this. This pattern suggests that these compounds may be acting collectively as a signature mixture of semiochemicals, carrying information on the individual, its kinship, and physiological and social status. This is the first detailed description of putative semiochemicals in any marsupial species.

Administration of rotenone enhanced voluntary alcohol drinking behavior in C57BL/6J mice.

Rotenone, a commonly used lipophic pesticide, is a high-affinity mitochondrial complex I inhibitor. The aim of this project is to study the causal relationship between changes of brain monoamine levels and drinking behavior in rotenone-treated mice. In the first experiment, we investigated the effects of acute exposure to rotenone (20 mg/kg, p.o.) on the 8-h time limited-access alcohol drinking behavior and brain monoamine levels in C57BL/6J mice at 0, 2, 8 and 24 h. Dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC) and 5-hydroxyindoleacetic acid (5HIAA) levels in the nucleus accumbens (ACC), caudate-putamen (C/P) and lateral hypothalamus (LH) of rotenone-treated mice were decreased at 2 and/or 8 h. Rotenone-exposed mice showed a suppression of voluntary alcohol intake at 4 and 8 h, but total daily alcohol intake did not differ significantly between the two groups. The effects of chronic exposure to rotenone (1, 5, 10 and 20 mg/kg, p.o. for 30 days) on the alcohol drinking behavior and monoamine levels of rotenone-exposed mice (10 mg/kg, p.o.) were investigated in the second experiment. The mice treated with rotenone showed increases in alcohol drinking behavior. Levels of DA and 5-HT in the ACC and C/P of chronic rotenone-treated mice were decreased, while the ratios of DOPAC to DA in the ACC and C/P and of 5HIAA to 5-HT in the ACC, C/P and DRN were increased significantly. Tyrosine hydroxylase immunoreactivity of chronic rotenone-treated mice (10 mg/kg, p.o.) slightly were decreased in both the striatum and the substantia nigra. Ethanol and acetaldehyde metabolism was not significantly different between mice treated with rotenone (10 mg/kg, p.o.) and controls. It was suggested that rotenone-treated mice had increased alcohol drinking behavior associated with increases in the DA turnover ratios of ACC and striatum to compensate for the neural degeneration.

Locational characteristics of the increasing number of forensic autopsy cases in Kyoto, Japan.

In Japan, the definition of unnatural death is not prescribed in law. However, a legal judgment recently defined unnatural death as all deaths, excluding natural deaths and deaths from diseases. Legally, unnatural deaths must be reported to the police. In the case of a reported death being considered as suspicious by the police, a forensic autopsy is required. The number of autopsies and the autopsy rate in Japan and Kyoto has increased over the last 10 years. Using data collected from 221 autopsy cases between 2008 and 2010 in Kyoto, Japan, the characteristics of locations where autopsy cases were discovered were analysed to identify reasons for the increase in autopsy numbers. It was found that factors including amount of human interaction and socioeconomic factors may help to explain the statistically significant correlations found.

The use of moxibustion scars in the identification of individuals in forensic medicine.

The use of moxibustion today does not only take place in Eastern Asia, but recently also in Europe and the United States. Moxibustion scars are often seen in autopsy cases in Japan. However, no reports of the use of moxibustion in autopsy cases have been reported. This paper reports on an autopsy case in which moxibustion scars were used to extrapolate on the past symptoms of the deceased, date of therapy and the location of the moxibustion clinic which administered the moxibustion.

Effect of filtration on morphine and particle content of injections prepared from slow-release oral morphine tablets.

BACKGROUND: Injections of mixtures prepared from crushed tablets contain insoluble particles which can cause embolisms and other complications. Although many particles can be removed by filtration, many injecting drug users do not filter due to availability, cost or performance of filters, and also due to concerns that some of the dose will be lost. METHODS: Injection solutions were prepared from slow-release morphine tablets (MS Contin) replicating methods used by injecting drug users. Contaminating particles were counted by microscopy and morphine content analysed by liquid chromatography before and after filtration. RESULTS: Unfiltered tablet extracts contained tens of millions of particles with a range in sizes from < 5 microm to > 400 microm. Cigarette filters removed most of the larger particles (> 50 microm) but the smaller particles remained. Commercial syringe filters (0.45 and 0.22 microm) produced a dramatic reduction in particles but tended to block unless used after a cigarette filter. Morphine was retained by all filters but could be recovered by following the filtration with one or two 1 ml washes. The combined use of a cigarette filter then 0.22 microm filter, with rinses, enabled recovery of 90% of the extracted morphine in a solution which was essentially free of tablet-derived particles. CONCLUSIONS: Apart from overdose and addiction itself, the harmful consequences of injecting morphine tablets come from the insoluble particles from the tablets and microbial contamination. These harmful components can be substantially reduced by passing the injection through a sterilizing (0.22 microm) filter. To prevent the filter from blocking, a preliminary coarse filter (such as a cigarette filter) should be used first. The filters retain some of the dose, but this can be recovered by following filtration with one or two rinses with 1 ml water. Although filtration can reduce the non-pharmacological harmful consequences of injecting tablets, this remains an unsafe practice due to skin and environmental contamination by particles and microorganisms, and the risks of blood-borne infections from sharing injecting equipment.

Papyriferic acid, an antifeedant triterpene from birch trees, inhibits succinate dehydrogenase from liver mitochondria.

Papyriferic acid (PA) is a triterpene that is secreted by glands on twigs of the juvenile ontogenetic phase of resin producing tree birches (e.g., Betula neoalaskana, B. pendula) and that deters browsing by mammals such as the snowshoe hare (Lepus americanus). We investigated the pharmacology of PA as a first step in understanding its antifeedant effect. After oral administration to rats, PA and several metabolites were found in feces but not urine, indicating that little was absorbed systemically. Metabolism involved various combinations of hydrolysis of its acetyl and malonyl ester groups, and hydroxylation of the terpene moiety. The presence of a malonyl group suggested a possible interaction with succinate dehydrogenase (SDH), a mitochondrial enzyme known to be competitively inhibited by malonic acid. The effect of PA on the oxidation of succinate by SDH was examined in mitochondrial preparations from livers of ox, rabbit, and rat. In all three species, PA was a potent inhibitor of SDH. Kinetic analysis indicated that, unlike malonate, PA acted by an uncompetitive mechanism, meaning that it binds to the enzyme-substrate complex. The hydrolysis product of PA, betulafolienetriol oxide, was inactive on SDH. Overall, the evidence suggests that PA acts as the intact molecule and interacts at a site other than the succinate binding site, possibly binding to the ubiquinone sites on complex II. Papyriferic acid was potent (K(iEIS) ranged from 25 to 45 microM in the three species) and selective, as malate dehydrogenase was unaffected. Although rigorous proof will require further experiments, we have a plausible mechanism for the antifeedant effect of PA: inhibition of SDH in gastrointestinal cells decreases mitochondrial energy production resulting in a noxious stimulus, 5-HT release, and sensations of nausea and discomfort. There is evidence that the co-evolution of birches and hares over a large and geographically-diverse area in Northern Europe and America has produced marked differences in the formation of PA by birches, and the tolerance of hares to dietary PA. The present findings on the metabolic fate and biochemical effects of PA provide a rational basis for investigating the mechanisms underlying differences among populations of hares in their tolerance of a PA-rich diet.