Determination of main tetrahydrocannabinoids by GC-MS: impact of protein precipitation by acetonitrile on solid phase extraction of cannabinoids from human serum.

The analysis of delta9-tetrahydrocannabinol (delta9-THC) and its main metabolites [11-hydroxy-delta9-tetrahydrocannabinol (11-OH-delta9-THC) and 11-nor-9-carboxy-delta9-tetrahydrocannabinol] in serum is a routine assay in forensic toxicology in the case of drivers influenced by Cannabis abuse and in other cases. Analysis of the specimen may involve protein precipitation, although there are authors who do not use this step. In this study we investigated the effect of acetonitrile as protein precipitant added to the serum on the absolute extraction recoveries of the analytes. This is very important not only from a forensic point of view, but also from the aspect of impact of delta9-THC therapy. Our results showed that in the case of spiked serum (2 ml), 80-87% extraction recovery can be achieved if 4 ml of acetonitrile is added before solid phase extraction. The second best result could be reached if no acetonitrile was added (64-73%). However, in the case of physiological sera of Cannabis consumers, no precipitation may be more advantageous in some cases. Matrix effects, which were studied by comparing the detectability and relative intensities of matrix peaks within the corresponding time windows of the analytes, were less influenced if the extraction was achieved with or without acetonitrile.

Detection of cannabigerol and its presumptive metabolite in human urine after Cannabis consumption.

Kemp et al. (1995) could detect delta9-tetrahydrocannabinol (delta9-THC), cannabinol and cannabidiol, three neutral cannabinoids, and the metabolites of delta9-THC in urine samples of Cannabis consumers. In this study we aimed to identify cannabigerol (CBG), which in its acid form is one of the main intermediate compounds of the biosynthesis of cannabinoids in hemp, in authority urine samples of proved Cannabis consumers. For this reason we applied the modified method of Kemp et al. to test for CBG, since enzymatic hydrolysis seems to be necessary for the formation of free neutral cannabinoids from conjugates. After extraction, derivatisation with N-Methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) and GC/MS analysis, peaks of characteristic fragment ions (m/z 337, 391, 377 and 460) of bis-trimethylsilyl derivative of CBG appeared at 12.48 minutes in both real sample and the urine spiked with CBG. It shows that CBG enters the body during Cannabis smoking and is excreted with urine in a conjugated form, like other neutral cannabinoids. Analysing the chromatograms of hydrolysed and trimethylsilylated extracts we checked for the presence of CBG-metabolites based on the study of Harvey and Brown (1990). We detected a compound in the Cannabis consumers' urine extracts, having fragment ions at m/z 425, 465 and 479 at the retention time of 14.19 min which is presumed to be the 4"-hydroxy-CBG or 5"-hydroxy-CBG. However, it could not be identified completely by GC/MS. This peak was absent in non-hydrolysed urine samples, indicating that it is also excreted in glucuronated form.

Different reactivities of amphetamines with N-methyl-bis(trifluoroacetamide) in heated gas chromatographic injectors.

A fast gas chromatographic mass spectrometric method has been developed earlier for the determination of amphetamine derivatives in human serum and urine. For derivatization, N-methyl-bis(trifluoroacetamide) (MBTFA) was used. Derivatization was performed using an on-line mode, since 1 microl of MBTFA and 1 microl sample extract, dissolved in toluene were injected simultaneously. In this study, the reactivity of the several amphetamine type analytes with MBTFA was investigated. MBTFA used for flash derivatization was applied undiluted on the one hand and diluted 4--4096-fold with acetonitrile on the other hand. Studying several amphetamines in the test sample spiked at the same concentrations we found that they could be divided into 3 groups based on relative target ion peak areas as a function of MBTFA dilution. Group 1, containing only primary amines showed an early increase of the relative peak areas if we increased MBTFA concentration, where group 2 (mainly N-methyl secondary amines) showed that relative peak areas started to increase intensively at higher MBTFA concentrations. Finally, MDEA as an N-ethyl secondary amine, representing group 3, showed significant increase if only slightly diluted MBTFA was used as a flash reagent. This phenomenon can be explained mainly with the less and less reactivity of amine groups in the case of groups 2 and 3, compared to group 1. These findings could help to optimise analytical methods involving flash derivatization processes.

Drugged driving in Hungary 2000-2004.

The authors analyzed the biological samples available in criminal cases that were started because of illicit and prescribed drug-impaired driving between 2000 and 2004. The result of the on-the-spot clinical test is not informative and cannot be evaluated as it is mainly affected by the simultaneous presence of alcohol. Licit or illicit drugs in the urine could be detected in 378 people out of 623 people (60.7%), whereas in 59 cases (9.5%) there was some substance present in the blood. The occurrence multiple drugs was high (36.8%). The joint use of alcohol and drugs has increased in the past few years.

Cyclodextrin (CD) complexes of cholesterol--their potential use in reducing dietary cholesterol intake.

The main objective of this investigation was to test and see if cyclodextrins, a type of molecule which form inclusion complexes, could effectively prevent the absorption of food derived cholesterol. Cyclodextrins are nontoxic and easily tolerated, having only a slight sweet taste. alpha,beta,gamma-Cyclodextrins, 2-Hydroxypropyl-beta-cyclodextrin (2-HPCD), and heptakis-O,O-dimethyl-beta-cyclodextrin (DMCD) were tested in solutions of different concentrations. These solutions were then saturated with cholesterol and the excess cholesterol was removed. The clear solutions left were then analyzed by HPLC to assess the amount of cholesterol in the solutions, and compared to a standard. DMCD gave the best results in successfully dissolving (complexing) most of the cholesterol followed by HPCD and alpha-cyclodextrin. Beta- and gamma-CD showed nearly insignificant complex formation. After administration of 10 mg of cholesterol to mice through a gastric tube, the cholesterol level increased about 125-130%, and only 15-20%, if the cholesterol was administered together with 20 mg of DMCD. That means, the DMCD formed complexes with approximately 80-85% of the cholesterol administered in the mice gastrointestinal tract.

Metabolic properties of phosphonate esters.

The object of the present work was to investigate the difference in the metabolism of the phosphonate derivatives of primary or secondary hydroxyl groups. To study the phosphorolytic cleavage of such P-O bonds, zidovudine (AZT) hexanoyloxymethyl-methylphosphonate (HOM-AZT-P), an ester of a primary OH functionality, and methyl-pivaloyloxymethyl-testosterylphosphonate (POM-T-P), an ester of a secondary OH functionality, were prepared. The actions of pure enzymes such as alkaline phosphatase and phosphodiesterase on the corresponding phosphonate compounds (AZT-P and T-P) were investigated at various pH values. The phosphonate derivative of the secondary hydroxyl group of testosterone proved completely resistant to such phosphorolytic attacks, and release of free testosterone could not be detected. The phosphonate derivative of the primary hydroxyl group of zidovudine proved resistant to phosphodiesterase, but not to alkaline phosphatase, and in this second case, release of free zidovudine could be detected.

Muscle-derived cell-mediated ex vivo gene therapy for urological dysfunction.

We have tested the feasibility of muscle-based gene therapy and tissue engineering for urological dysfunction using highly purified muscle-derived cells (MDC) that display stem cell characteristics. We then explored the potential use of these MDC as an alternative therapy for the treatment of impaired detrusor contractility. The MDC were genetically engineered to express the gene encoding beta-galactosidase and injected into the bladder walls of SCID mice. The injected bladders were harvested at various time-points after injection and assayed for beta-galactosidase activity; the presence of myofibers within the injected tissue was determined by detection of fast myosin heavy chain isoform (MyHCs). We have demonstrated that the injected MDC are capable of not only surviving in the lower urinary tract, but also improving the contractility of the bladder following an induced injury. Two potential mechanisms can be used to explain this finding. First, we have observed that some of the beta-galactosidase-expressing cells expressed alpha-smooth muscle actin, suggesting a differentiation into smooth muscle. Second, a stain for acetylcholine receptors (AChRs), which identifies the location of neuromuscular junctions, revealed that the myofibers derived from the doner cells became innervated into the bladder as early as 2 weeks after injection. These results suggest that gene therapy and tissue engineering based on MDC potentially can be used for urological dysfunction.

Botulinum toxin urethral sphincter injection resolves urinary retention after pubovaginal sling operation.

The management of prolonged urinary retention following pubovaginal sling surgery typically involves transvaginal urethrolysis for anatomical urethral obstruction. Brubaker [1] recently reported on urethral sphincter abnormalities as a cause of postoperative urinary retention following either Burch suspension or pubovaginal sling procedure. We report a case of functional urethral obstruction and detrusor acontractility following pubovaginal sling surgery that was successfully treated by botulinum A toxin urethral sphincter injection.

Botulinum toxin urethral sphincter injection resolves urinary retention after pubovaginal sling operation.

The management of prolonged urinary retention following pubovaginal sling surgery typically involves transvaginal urethrolysis for anatomical urethral obstruction. Brubaker recently reported on urethral sphincter abnormalities as a cause of postoperative urinary retention following either Burch suspension or a pubovaginal sling procedure. We report a case of functional urethral obstruction and detrusor acontractility following pubovaginal sling surgery that was successfully treated by botulinum A toxin urethral sphincter injection.

Targeted drug delivery to the central nervous system via phosphonate derivatives (anionic delivery system for testosterone).

An anionic chemical delivery system (aCDS) has been developed and applied to deliver testosterone (T) to the central nervous system (CNS). The delivery of a target compound is achieved through the use of a specific targetor moiety which is an (acyloxy)alkyl-phosphonate-type functional group. The T-aCDS readily penetrates biological membranes by passive transport due to its increased lipophilicity and enters the target organ. Hydrolytic cleavage by esterases provides a negatively charged, hydrophilic intermediate phosphonate compound (TP-), which is "locked in" the CNS and should provide sustained, site-specific release of the drug. In vitro and in vivo investigations in rats showed that methyl-pivaloyloxymethyl-17-testosterylphosphonate (T-aCDS) might function as an anionic chemical delivery system of testosterone. The concentration of T-aCDS decreased fairly rapidly in vitro. The half-lives (t1/2) in different organs are as follows: blood 4.48 min (r = 0.9388), lung 5.53 min (r = 0.9661), liver 2.82 min (r = 0.9498), and brain 7.37 min (r = 0.9972). Simultaneously with the disappearance of T-aCDS, testosterone-phosphonate (TP-) appeared as a main metabolite in increasing concentration. In vivo evaluations (tail vein 11.3 mg/kg in DMSO) found maximum T-aCDS brain levels 5-10 min after administration; they fell under the borderline of detectability (< 0.1 microgram/g) after 60 min. Maximum concentration of the decomposition product (TP-) was obtained at 30 min after administration; it did not decrease significantly during the study. Even if the phosphonate derivative of the secondary, hindered hydroxyl group in this product was fairly resistant to phosphorolytic attack, the design principle can work for other compounds.

Protein kinase C is involved in M1-muscarinic receptor-mediated facilitation of L-type Ca2+ channels in neurons of the major pelvic ganglion of the adult male rat.

We used patch clamp recording techniques to determine if muscarinic signaling mechanisms are present in dissociated autonomic neurons obtained from the major pelvic ganglion, which provides the cholinergic innervation of the urinary bladder and other pelvic organs. The M1 specific agonist, McN-A-343 (2-30 microM) enhanced Ca2+ currents in approximately 37% of neurons (by 50-80%). This enhancement was reduced by atropine (5-10 microM) or a PKC inhibitor (bisindolylmaleimide, 50-200 nM). In responsive neurons Ca2+ currents were also enhanced by the phorbol ester, phorbol-12,13-dibutyrate (50-300 nM) and the dihydropyridine agonist Bay K 8644 (5 microM) and had kinetics of activation and inactivation as expected for L-type Ca2+ channels. We conclude that in a subpopulation of MPG neurons, M1-mediated activation of PKC phosphorylates and enhances L-type Ca2+ channel activities. This muscarinic facilitatory mechanism in MPG neurons may be the same as the M1-mediated facilitation of transmitter release reported previously at the nerve terminals in the urinary bladder.

[Medical judgement on driving capability]. / A gépjármúvezetó alkalmasságának megítélése.

Driving is an activity which puts forward the question of whether the driver is able to competently fulfill the task or not. Driving is affected or altered by age and various illnesses and the driver may not always concede that his/her driving ability is affected by these. The examination of driver capability is an important target. This can be seen especially during car accidents when the driver is examined and it is discovered that he/she is aggressive, has poor vision, old age, is epileptic, or consumed alcohol or drugs.

[Outbreak of human calicivirus infection in a hospital department]. / Humán calicivírus-járvány kórházi osztályon.

Human caliciviruses (HuCV)--such as Norwalk-like and Sapporo-like viruses--members of the family Caliciviridae, are a major cause of acute non-bacterial gastroenteritis in persons of all ages worldwide. They are important pathogens in food- and waterborne diseases in which the transmission can often be traced to fecally contaminated water or foods, and spread by person-to-person contact, vomitus or airborne droplets. HuCV-associated outbreaks involving large numbers of people usually occur in settings where people congregate. Between May 9 and 24, 2000, an outbreak of acute, mild, nonbacterial gastroenteritis occurred in woman, chronic psychiatric ward of a county hospital where 35 of 143 persons (24.5%) were registered with characteristic symptoms. Reverse transcription-polymerase chain reaction (RT-PCR) assay was used for virus detection. HuCV was found in stool samples in 12 of 17 (70.6%) ill persons. This is the first HuCV-associated hospital outbreak of gastroenteritis in Hungary where HuCV was successfully detected by molecular method and its etiologic role was also supported by epidemiologic investigation.

Growth factor may decrease muscle atrophy secondary to denervation.

Despite modern microsurgical techniques, functional outcomes following brachial-plexus reconstruction and peripheral-nerve repair are usually unsatisfactory, because irreversible muscle atrophy develops before reinnervation occurs. Insulin growth factor-1 (IGF-1) has been shown to improve muscle regeneration after injury, and may have a role in muscle preservation following denervation. This study evaluated the histologic, immunohistochemical, and electrophysiologic differences between normal and denervated muscle over an 8-week time period, and also evaluated the effects of injecting IGF-1 into denervated muscle. Denervated mice gastrocnemius muscles demonstrated a decrease in muscle diameter, a decrease in muscle weight, early nuclear proliferation, and a decrease in fast twitch and maximum tetanic strength, compared to normal gastrocnemius muscle up to 8 weeks following denervation. Four weeks after denervated muscle was injected with IGF-1 at time zero, however, relative preservation of muscle diameter and weight, and maintenance of electrophysiologic contractile properties were observed. These preliminary data suggest that IGF-1 may prevent muscle atrophy secondary to denervation.

Facilitation of transmitter release in the urinary bladders of neonatal and adult rats via alpha1-adrenoceptors.

Age-dependent changes in the effects of the alpha1-adrenoceptor agonist, phenylephrine were investigated on neurally evoked contractile responses and basal tone in smooth muscle strips from rat urinary bladder. Phenylephrine facilitated the neurogenic contractions in both neonatal and 7-month-old adult rats. However, phenylephrine increased the basal tone in adult but not neonatal rats. In adult rats, phenylephrine-induced facilitation of neurally evoked contractions occurred before and after the block of cholinergic contractions with 1 microM atropine. In adult rats, the phenylephrine facilitation was reduced at stimulation parameters (20 Hz, 80 shocks and maximal voltage) which activated muscarinic receptor mediated facilitation of acetylcholine release. The results indicate that pre-synaptic alpha1-adrenoceptors facilitate the release of both acetylcholine and the non-cholinergic non-adrenergic transmitter. In summary, alpha1-adrenoceptor-mediated facilitation is less expressed when muscarinic M1 receptor mediated facilitation is functioning; pre-junctional alpha1-adrenoceptors are present in the bladder of both neonatal and adult rats, whereas post-junctional alpha1-adrenoceptors are expressed only in older adult rats.

Botulinum toxin urethral sphincter injection to restore bladder emptying in men and women with voiding dysfunction.

PURPOSE: Botulinum toxin injection into the external urinary sphincter in spinal cord injured men with detrusor-sphincter dyssynergia has been reported. We expand the clinical use of botulinum toxin for a variety of bladder outlet obstructions and to decrease outlet resistance in patients with acontractile detrusor but who wish to void by the Valsalva maneuver. MATERIALS AND METHODS: Prospective treatment was performed for voiding dysfunction in 8 men and 13 women 34 to 74 years old. The reasons for voiding dysfunction included neurogenic detrusor-sphincter dyssynergia in 12 cases, pelvic floor spasticity in 8 and acontractile detrusor in 1 patient with multiple sclerosis who wished to void by the Valsalva maneuver. Using a rigid cystoscope and a collagen injection needle, a total of 80 to 100 units of botulinum A toxin (Botox) were injected into the external sphincter at the 3, 6, 9 and 12 o'clock positions. RESULTS: Preoperatively 19 of 21 patients were on indwelling or intermittent catheterization. After botulinum A injection all but 1 patient were able to void without catheterization. No acute complications, such as general paralysis or respiratory depression, occurred and none of the patients had dribbling or stress urinary incontinence. Postoperative post-void residual decreased by 71% and voiding pressures decreased on average 38%. Of the 21 patients 14 (67%) reported significant subjective improvement in voiding. Followup ranges from 3 to 16 months, with a maximum of 3 botulinum A injections in some patients. CONCLUSIONS: Urethral sphincter botulinum injection should be considered for complex voiding dysfunction. Encouraging improvement without complications were seen in most of our patients. We have expanded the use of botulinum toxin to treat pelvic floor spasticity and also women.

Smooth muscle and parasympathetic nerve terminals in the rat urinary bladder have different subtypes of alpha(1) adrenoceptors.

Neurally evoked contractions and release of (3)H- acetylcholine (ACh) during electrical field stimulation were measured in rat urinary bladder strips. The alpha(1) agonist phenylephrine (PE, 2-8 microM) increased the amplitude of neurally evoked contractions, facilitated the release of ACh and increased the baseline tone of the bladder strips. The PE-induced facilitation of the contractions did not significantly change during a prolonged exposure to PE (120 min), whereas the PE-induced rise in baseline tone gradually decreased to 65% of the initial value. Low concentrations of specific alpha(1A) antagonists, 5-methyl urapidil (5-MU), REC15/2739 and WB-4101 competitively inhibited the facilitation of the neurally-evoked contractions (pA(2:) 8.77; 9.59 and 9.62, respectively), whereas higher concentrations of 5-MU (IC(50): 48 nM) were required to suppress the PE-rise in baseline. WB-4101 (100 microM) inhibited the PE-induced facilitation of ACh release. The irreversible alpha(1B) antagonist chloroethyl-clonidine (CEC, 10-50 microM) inhibited the PE-evoked rise in base line tone, but did not affect the PE-induced facilitation of the neurally evoked contractions nor the facilitation of ACh release. However, CEC increased the area and amplitude of the neurally-evoked contractions by 261+/-33 and 47.2+/-8.4%, respectively. Atropine significantly inhibited the CEC evoked increase in area and amplitude of the electrically evoked contractions (76.5+/-4.8 and 40.8+/-3%, respectively) indicating that CEC facilitated the cholinergic responses of the electrically stimulated bladder strips. It is concluded that alpha(1A) and CEC sensitive alpha(1B) and/or alpha(1D) adrenoceptors are expressed in the rat bladder in different locations. On the cholinergic nerve terminals alpha(1A) adrenoceptors mediate prejunctional facilitation, whereas postjunctional alpha(1B)/alpha(1D) adrenoceptors mediate smooth muscle contraction.

Distribution of inorganic elements in human autopsy tissue.

In the practice of forensic medicine, we find many of cases of death where the actual cause is not determinable with autopsy, histological or toxicological examinations. In these cases of death, we can consider cardiac dysfunction of unknown origin, in the background of which such a physiologic cardiac insufficiency occurs that cannot be detected with the previously mentioned methods. The dysfunction is possibly associated with a significant change in certain inorganic elements, primarily in the conduction system of the heart. In the absence of published data, our goal was to determine the concentration of inorganic elements in the specialized rhythm determining muscle cell groups in the heart: sinus node (SN), atrioventricular node (AV), septum (SE), left ventricle anterior wall (LVAW). With microwave technology we destroyed the muscle tissue and measured the concentration of ions (Na, Mg, K, Ca, Mn, Fe, Cu, Zn, P, S) using Inductive Completed Plasma Atom Emission Spectrometry (ICP-AES) equipment. Of the 24 cases examined, the average ion concentrations in microgram/g were the following; Sinus: Na 2602 +/- 493, Mg 120 +/- 24, K 1787 +/- 347, Ca 244 +/- 41, Mn 0.129 +/- 0.011, Fe 58 +/- 12, Cu 2.171 +/- 0.46, Zn 10.4 +/- 2.027, P 1147 +/- 227, S 2301 +/- 245; Septum: Na 1452 +/- 315, Mg 243 +/- 56, K 3269 +/- 689, Ca 105 +/- 26, Mn 0.17 +/- 0.05, Fe 74 +/- 16, Cu 3.557 +/- 0.952, Zn 25.75 +/- 8.4, P 2764 +/- 494, S 3001 +/- 523; Av: Na 2614 +/- 517, Mg 242 +/- 40.2, K 2010 +/- 395, Ca 271 +/- 27.3, Mn 0.13 +/- 0.029, Fe 54 +/- 12, Cu 2.369 +/- 0.297, Zn 15 +/- 3.2, P 1625 +/- 291, S 2535 +/- 346; Lvaw: Na 1340 +/- 201, Mg 250 +/- 37, K 3659 +/- 532, Ca 88 +/- 22, Mn 0.175 +/- 0.05, Fe 76 +/- 19, Cu 3.62 +/- 0.58, Zn 27.13 +/- 3.1, P 3025 +/- 441, S 3140 +/- 440.

Use of growth factors to improve muscle healing after strain injury.

Muscle injuries represent a large number of professional and recreational sports injuries. Muscle strains habitually occur after an eccentric contraction, which often leads to an injury located in the myotendinous junction. Treatment varies widely, depending on the severity of the trauma, but has remained limited mostly to rest, ice, compression, elevation, antiinflammatory drugs, and mobilization. The authors' research group aims to develop new biologic approaches to improve muscle healing after injuries, including muscle strains. To achieve this goal, the authors investigated several parameters that will lead to the development of new strategies to enhance muscle healing. The authors first evaluated natural muscle healing after strain injuries and showed that muscle regeneration occurs in the early phase of healing but becomes impaired with time by the development of tissue fibrosis. Several growth factors capable of improving muscle regeneration were investigated; basic fibroblast growth factor, insulin-like growth factor, and nerve growth factors were identified as substances capable of enhancing muscle regeneration and improving muscle force in the strained injured muscle. The current study should aid in the development of strategies to promote efficient muscle healing and complete recovery after strain injury.

Review article: effects of the 5-HT3 receptor antagonist alosetron on neuromuscular transmission in canine and human intestinal muscle.

BACKGROUND: Currently, therapeutic treatments for irritable bowel syndrome fail to produce significant clinical results. We hypothesized that alosetron, a selective 5-HT3 antagonist, may provide symptomatic relief in irritable bowel syndrome patients through a decrease in the amplitude of gastrointestinal contractions. AIM: To determine the in vitro effect of alosetron on neuromuscular transmission in the canine and human jejunal and colonic muscularis externa. RESULTS: Alosetron diminished electrical field-stimulated (EFS) contractions recorded from muscles of the canine and human small and large intestines. Mechanistically, the diminished EFS response could be explained by the ability of alosetron to decrease the fractional release of 14C-choline radiolabelled acetylcholine evoked by EFS from human jejunal muscle. The inhibition of EFS contractions was not limited to atropine-sensitive events, as non-cholinergic excitatory EFS evoked contractions were also inhibited. Additionally, alosetron at high concentrations (> 30 microM) directly altered bethanechol stimulated contractions. CONCLUSION: Caution must be used in the interpretation of these data because significant alterations in EFS-induced contractions were only observed with large pharmacological concentrations of alosetron, and the response was not selective for cholinergically-mediated excitatory neuromuscular transmission.