Development of a new multi-analyte assay for the simultaneous detection of opioids in serum and other body fluids using liquid chromatography-tandem mass spectrometry.

A liquid chromatography-tandem mass spectrometry method using electrospray ionization in positive ionization mode was developed for the simultaneous detection of multiple opioid-type drugs in plasma. The presented assay allows the quantitative determination of alfentanil, buprenorphine, codeine, desomorphine, dextromethorphan, dextrorphan, dihydrocodeine, dihydromorphine, ethylmorphine, fentanyl, hydrocodone, hydromorphone, methadone, morphine, naloxone, naltrexone, oxycodone, oxymorphone, pentazocine, pethidine, pholcodine, piritramide, remifentanil, sufentanil, and tramadol as well as the metabolites 6-monoacetylmorphine, bisnortilidine, morphine-3-glucuronide, morphine-6-glucuronide, naltrexol, norbuprenorphine, norfentanyl, norpethidine, nortilidine, and O-desmethyltramadol. Serum and blood samples were purified by solid-phase extraction. The analytes were separated on a phenyl-hexyl (100mm) column by formic acid/acetonitrile gradient elution using an UPLC 1290 Infinity coupled with a 6490 Triple Quadrupole mass spectrometer. The limits of detection ranged from 0.02 to 0.6ng/mL and the lower limits of quantification ranged from 0.1 to 2.0ng/mL. The calibration curves were linear between Calibration Levels 1-6 for all 35 substances. Recovery rates ranged between 51 and 88% for all compounds except alfentanil, bisnortilidine, pethidine, and morphine-3-glucuronide. The matrix effect ranged from 86% for ethylmorphine to 105% for desomorphine. Using the validation procedure proposed by the German Society of Toxicological and Forensic Chemistry, acceptable precision and accuracy data for almost all analytes were obtained. The method was successfully applied to 206 authentic serum samples provided by the palliative and intensive care units of the University Medical Center and the police authorities. Furthermore, a suspected fatal intoxication is demonstrated by an analysis of the sufentanil in post mortem body fluids and tissues.

[Tibial press-fit fixation of flexor tendons for reconstruction of the anterior cruciate ligament]. / Tibiale Press-fit-Fixierungen von Beugesehnen zur Rekonstruktion des vorderen Kreuzbandes.

BACKGROUND: Press-fit fixation of hamstring tendon autografts for anterior cruciate ligament reconstruction is an interesting technique because no hardware is necessary. This study compares the biomechanical properties of press-fit fixations to an interference screw fixation. METHODS: Twenty-eight human cadaveric knees were used for hamstring tendon explantation. An additional bone block was harvested from the tibia. We used 28 porcine femora for graft fixation. Constructs were cyclically stretched and then loaded until failure. Maximum load to failure, stiffness and elongation during failure testing and cyclic loading were investigated. RESULTS: The maximum load to failure was 970±83 N for the press-fit tape fixation (T), 572±151 N for the bone bridge fixation (TS), 544±109 N for the interference screw fixation (I), 402±77 N for the press-fit suture fixation (S) and 290±74 N for the bone block fixation technique (F). The T fixation had a significantly better maximum load to failure compared to all other techniques (p<0.001). CONCLUSION: This study demonstrates that a tibial press-fit technique which uses an additional bone block has better maximum load to failure results compared to a simple interference screw fixation.

Expression and distribution of key enzymes of the cyclic GMP signaling in the human clitoris: relation to phosphodiesterase type 5 (PDE5).

The clitoris contributes to the normal female sexual response cycle. A significance of cyclic guanosine monophosphate (GMP) has been assumed in the control of clitoral vascular smooth muscle. As only a few investigations on the physiology of the vascular and non-vascular clitoral tissue have been carried out, knowledge on the mechanisms controlling this particular female genital organ is still vague. It has been suggested that human clitoral corpus cavernosum smooth muscle is regulated by nitric oxide (NO)/cyclic GMP and related key enzymes, such as NO synthases (NOSs) and the phosphodiesterase type 5 (PDE5). The present study evaluated in the human clitoris, by means of immunohistochemistry, the expression and distribution of key enzymes of the cyclic GMP pathway, such as the endothelial NOS, PDE2, PDE11 and cyclic GMP-dependent protein kinase type I (cGKI) in relation to the PDE5. Immunohistochemistry revealed the presence of PDE2, PDE5 and cGKI in the smooth muscle wall of blood vessels transversing the supepithelial and stromal space. Immunosignals specific for PDE2 were also identified in interstitial-like cells located in the basal epithelial layer. Staining for PDE11A was observed in single nerve trunks located in the clitoral stroma. The results are in favor of a role of the cyclic GMP signaling in the control of clitoral blood flow. It seems likely that PDE2 and PDE11 are also involved in the mechanism of local (neuro)transmission in the clitoris.

Biomechanical characterization of double-bundle femoral press-fit fixation techniques.

PURPOSE: Press-fit fixation of patellar tendon bone anterior cruciate ligament autografts is an interesting technique because no hardware is necessary. To date, no biomechanical data exist describing an implant-free double-bundle press-fit procedure. The purpose of this study was to characterize the biomechanical properties of three double-bundle press-fit fixations. METHODS: In a controlled laboratory study, the patellar-, quadriceps- and hamstring tendons of 10 human cadavers (age: 49.2 ± 18.5 years) were used. An inside out press-fit fixation with a knot in the semitendinosus and gracilis tendons (SG) combined with an additional bone block, with two quadriceps tendon bone block grafts (QU) was compared with press-fit fixation of two bone patellar tendon bone block (PT) grafts in 30 porcine femora. Constructs were cyclically stretched and then loaded until failure. Maximum load to failure, stiffness and elongation during failure testing and cyclical loading were investigated. RESULTS: The maximum load to failure was 703 ± 136 N for SG fixation, 632 ± 130 N for QU and 656 ± 127 N for PT fixation. Stiffness of the constructs averaged 138 ± 26 N/mm for SG, 159 ± 74 N/mm for QU, and 154 ± 50 N/mm for PT fixation. Elongation during initial cyclical loading was 1.2 ± 1.4 mm for SG, 2.0 ± 1.4 mm for QU, and 1.0 ± 0.6 mm for PT (significantly larger for PT and QU between the first 5 cycles compared with cycles 15-20th, P < 0.01). CONCLUSION: All investigated double-bundle fixation techniques were equal in terms of maximum load to failure, stiffness, and elongation. Unlike with single-bundle press-fit fixation techniques that have been published, no difference was observed between pure tendon combined with an additional bone block and tendon bone grafts. All techniques exhibited larger elongation during initial cyclical loading. All three press-fit fixation techniques that were investigated exhibit comparable biomechanical properties. Preconditioning of the constructs is critical.

[Biomechanical analysis of press-fit fixation of anterior cruciate ligament transplants]. / Biomechanische Analyse der Press-fit-Fixierung von Kreuzbandtransplantaten.

Press-fit fixation of anterior cruciate ligament autografts is an interesting technique because no hardware is necessary. A total of 168 cadaveric human patellar, quadriceps and hamstring tendons (average age 49.2 +/- 18.5 years) were used and 15 different fixation methods were tested. Constructs were cyclically stretched and then loaded until failure. Maximum load to failure, stiffness and elongation during failure testing and cyclical loading were compared. Some techniques showed comparable biomechanical qualities to interference screw fixation. All investigated double bundle fixation techniques were equal in terms of maximum load to failure, stiffness and elongation. All techniques exhibited greater elongation during initial cyclical loading. Some of the press-fit fixation techniques investigated exhibited comparable biomechanical properties and preconditioning of the constructs is critical. Press-fit fixation enhances tendon to bone contact at the entry of the bone tunnel to the joint.

[Evaluation of blood and breath alcohol measurement in patients with hepatocellular carcinoma during procedures in intubated patients with percutaneous ethanol instillation]. / Vergleich von Blut- und Atemalkoholkonzentrationen während der perkutanen Athanolinstillationstherapie (PEIT) bei intubierten Patienten mit hepatozellulärem Karzinom.

The accuracy of breath alcohol measurements in intubated patients with assisted ventilation was evaluated. The breath alcohol concentration was measured in 24 patients undergoing percutaneous alcohol injection therapy for hepatocellular carcinoma with Alcotest 7410 med, from Dräger, Germany. The blood ethanol concentrations (BACs) were determined in each blood serum applying the German forensic criteria standard, namely, two alcohol dehydrogenase (ADH) and two gas chromatography (GC) measurements. The blood alcohol concentrations were between 0.07 and 1.51 per thousand in the central venous samples and 0.02-1.66 per thousand in the arterial samples. An excellent correlation between both the venous and arterial blood alcohol concentrations (r2=0.94), as well as between the breath alcohol concentrations and the venous (r2=0.84) or arterial alcohol concentration (r2=0.89), p<0.01 for both parameters was revealed. Determination of breath alcohol concentration using the Alcotest in intubated patients is reliable and reflects the blood alcohol values.

Postmortem determination of concentrations of stress hormones in various body fluids--is there a dependency between adrenaline/noradrenaline quotient, cause of death and agony time?

To find out whether a certain cause of death or a certain length of an agonal period shows specific adrenaline or noradrenaline profiles, heart blood, femoral vein blood, liquor, urine and vitreous humour were taken from corpses (n = 98) at the Medical School Hannover, and noradrenaline and adrenaline were determined using high-performance liquid chromatography (HPLC). Corpses were classified according to the following five categories: short agony, long agony, state after hanging, state after asphyxiation and state after CPR with documented administration of epinephrine. Once results were collected the adrenaline/noradrenaline quotient was determined. It became clear that there were no significant differences regarding the concentration of adrenaline and noradrenaline in the various body fluids in relation to the above-mentioned categories. The means adrenaline/noradrenaline quotients in femoral vein blood were 0.21 +/- 0.29 for hanged persons, 0.38 +/- 0.47 for asphyxiated persons, 0.17 +/- 0.19 for those with short agony and 0.42 +/- 0.43 for those with long agony, significantly below 1 (p < 0.001; p = 0.001; p = 0.003). For condition after CPR we found an adrenaline/noradrenaline quotient of 2.81 +/- 5.8. In liquor the adrenaline/noradrenaline quotients for short agony was 0.17 +/- 0.17, for hanged persons 0.18 +/- 0.19 and for asphyxiated ones 0.30 +/- 0.38, significantly lower than 1 (p < 0.001). In urine the adrenaline/noradrenaline quotients for all categories are lower than 1 (p < 0.001); short agony (0.13 +/- 0.09), long agony (0.21 +/- 0.16), hanged (0.15 +/- 0.16), asphyxiated (0.14 +/- 0.08) and CPR (0.14 +/- 0.06). In vitreous humour the quotients for short agony (0.14 +/- 0.28), long agony (0.13 +/- 0.12), hanged (0.07 +/- 0.09) and asphyxiated (0.09 +/- 0.11) are lower than 1 (p < 0.001). The spread of data for the adrenaline/noradrenaline quotient did not allow for any conclusions about cause of death and length of agony in individual cases.

Effect of ethanol on cardiac single sodium channel gating.

Alcohol in modest and higher doses has the potential to induce cardiac arrhythmias. The most famous alcohol-related arrhythmia is the "holiday heart syndrome". Furthermore, there is a clear association between excessive alcohol consumption and the risk of sudden cardiac death. However, the acute effects of ethanol on arrhythmia induction are not well understood. The effect of ethanol on single cardiac sodium channels has not been studied yet. To elucidate the effect of ethanol on human cardiac sodium channels we performed a patch clamp study in HEK-293 cells overexpressing the human cardiac sodium channel. We used HEK-293 cells overexpressing the human cardiac sodium channel (Na(1.5)). Single channel gating was investigated by the cell-attached patch clamp technique. Sodium channel currents were elicited by depolarizing pulses from -120 to -20mV for a duration of 150ms. Single channel availability, open probability and peak average current were assessed baseline and after addition of ethanol in increasing concentrations (0.50 per thousand (10.9mM), 1.00 per thousand (21.7mM), 2.00 per thousand (43.5mM) and 4.00 per thousand (87.0mM)). We found a concentration-dependent reduction of open probability which was statistically significant at 2.00 per thousand ethanol (66.5+/-14% of control). At higher concentrations (4.00 per thousand) also availability decreased to 66.5+/-11.0% of control. This resulted in a significant decrease of peak average current at 2.00 per thousand and at 4.00 per thousand ethanol (61.8+/-7.4 and 53.0+/-8.2% of control). For the first time the present study demonstrates acute inhibitory effects of ethanol on single cardiac sodium channel gating and provides one potential mechanism for the well known clinical observation that ethanol triggers supraventricular and ventricular arrhythmias.

[Spontaneous ruptures of the urinary bladder in the routine forensic examination]. / Spontane Harnblasenrupturen im Rechtsmedizinischen Untersuchungsgut.

This article analyses three cases of death following a spontaneous rupture of the urinary bladder. One case is based on an extensive tamponade of the bladder eight days after a transurethral resection of the prostate gland. Two other cases of death by spontaneous rupture resulted from increased alcohol consumption. The paper presents an overview of pathomorphological findings such as the typical intraperitoneal rupture localisation at the posterior wall of the urinary bladder or bladder dome and the subsequent diagnosed causes of death. In addition predisposing (anamnestic) influencing factors such as chronic alcoholism are highlighted and their relevance for the clinical urologist and the forensic pathologist are discussed.

Evaluation of the correlation between time corpses spent in in-ground graves and findings at exhumation.

To determine how long pathological findings persist after burial and which factors play a role in decomposition of a corpse, we evaluated all bodies exhumed under the auspices of the Institute of Legal Medicine at the Hannover Medical School between 1978 and 1997. A total of 87 exhumations (54 men, 33 women) were performed in this period. The time bodies remained buried varied between 5 days and 16.8 years (mean 1.5 years, median 2.3 months). Fifty-six percent of the bodies were exhumed after at most 3 months, 10% remained buried for greater than 3 years. Pathomorphological changes of the soft tissues and the internal organs remained evident after several months, in some cases after several years of burial. Overall, it was possible to evaluate internal organs after 5 years of burial. Bodies became mostly decomposed after approximately 8 years at the earliest, although it was still possible to evaluate some soft tissue remnants after 16.8 years. In stepwise logistic regression, both the length of time the body was buried (p < 0.00005) and the time of year (p < 0.0019) clearly affected the rate of physical change. The variables of sex (p = 0.33), age (p = 0.61) and changes in the integrity of the body before burial (trauma, autopsy before burial; p = 0.15) did not influence the physical state of the body after exhumation. Our data show that much information may be gained from an exhumation even after significant time has passed since burial.

[Cricothyroidotomy training on cadavers - experiences in the education of medical students, anaesthetists, and emergency physicians]. / Koniotomieübungen an der Leiche - Erfahrungen in der Ausbildung mit Medizinstudenten, Anästhesisten und Notärzten.

OBJECTIVE: Should the technique of surgical cricothyroidotomy be practiced on cadavers and should it be a compulsory part of the teaching curriculum? Is it wise to use a speculum for the insertion of the endotracheal tube? What is the optimum size of the tube? METHODS: A surgical cricothyroidotomy with a speculum was carried out on 30 cadavers from the Institute of Legal Medicine, Medical School Hannover. This took place as part of a official and voluntary course for students of advanced semesters, anaesthetists and emergency doctors with the subjects "cricothyroidotomy, chest drainage and venous cut-down". The surgical cricothyroidotomy without the use of a speculum was carried out on 5 cadavers by two clinicians well practiced in this technique. The elapsed time between skin incision and the insertion of the endotracheal tube was measured on all five subjects. After the course the participants were asked if they were able to carry out a cricothyroidotomy in an emergency. They were also asked whether this course should be a compulsory part of their curriculum and whether practical sessions should take place. During autopsies at the Institute of Legal Medicine the length of the ligamentum conicum was measured on 40 corpses with reclined and non-reclined heads. RESULTS: The average time of storage of the cadavers was 4.2 days +/- 1.9 days. The cricothyroidotomy was possible on all 35 cadavers. In one case (3,3 %) the result was a complete rupture of the cricoid cartilage. In 5 cases (16.7 %) the horizontal incision was torn due to prising with the speculum. Difficult situations always occured when the skin incision was not exactly in the midline. The average time to place the endotracheal tube into the trachea by the surgical procedure of cricothyroidotomy was 22.4 seconds +/- 3.1 seconds (minimum 18 seconds, maximum 26 seconds). 10 % of the medical students and 50 % of the anaesthetists and emergency doctors felt they would be prepared to carry out a cricothyroidotomy in an emergency. 90 % of the students and respectively 80 % of the anaesthetists and emergency doctors stated that they would like to practice the technique on a cadaver again. Almost all participants were of the opinion that the course should be integrated as a compulsory course in a future educational curriculum. The average distance between the thyroid cartilage and the cricoid cartilage was 9.5 mm +/- 1.9 mm with non-reclined head (minimum 6 mm, maximum 14 mm) and 11.9 mm +/- 2.5 mm with reclined head (minimum 7 mm, maximum 18 mm). The average difference of distances was 2.4 mm +/- 1.2 mm (minimum 1 mm, maximum 6 mm) in reclined and non-reclined heads. CONCLUSIONS: In our opinion it is highly recommended that the technique of cricothyroidotomy should be practiced on cadavers and that the course should become a compulsory part in a future educational curriculum. In addition the incision of the ligamentum conicum using dilators or a speculum is not to be recommended from the point of view of this study. The tracheal tube used in this study (reinforced wire tube, ID 6.0) was best suited for surgical cricothyroidotomy.

Applied anatomy of the superior vena cava-the carina as a landmark to guide central venous catheter placement.

BACKGROUND: Cardiac tamponade is a serious complication of central venous catheter (CVC) insertion. Current guidelines strongly advise that the CVC tip should be located in the superior vena cava (SVC) and outside the pericardial sac. This may be difficult to verify as the exact location of the pericardium cannot be seen on a normal chest x-ray. The carina is an alternative radiographic marker for correct CVC placement, suggested on the basis of studies of embalmed cadavers. METHODS: We set out to confirm this radiographic landmark in 39 fresh cadavers (age 58.4 (3.4) (mean and SE) yr) and to compare the results with those from ethanol-formalin-fixed cadavers. RESULTS: We found that the carina was 0.8 (0.05) cm above the pericardial sac as it transverses the SVC. In no case was the carina inferior to the pericardial reflection and our study confirmed the previous findings. All the measured distances were significantly greater in fresh cadavers. CONCLUSIONS: We confirm that the carina is a reliable, simple anatomical landmark that can be identified in vivo for the correct placement of CVCs outside the boundaries of the pericardial sac.

Accidental autoerotic deaths between 1978 and 1997. Institute of Legal Medicine, Medical School Hannover.

Between 1978 and 1997 the Institute of Legal Medicine of the Hannover Medical School examined 17 fatal autoerotic deaths. The incidence for the Hannover region was 0.49 cases per million inhabitants per year. The victims included 17 men with an average age of 36.8 years; a peak in the age distribution was seen between 20 and 29 years. Twelve of the men were found by friends or family in a domestic environment, while other situations in which the victims were found included the victim's own car, a hotel room, a canal embankment, a public parking lot as well as the holding cell of the youth detention center. The men were of varying socioeconomic status and held a number of different types of jobs or still attended school. Five of the men were found completely nude, while five were only undressed below the waist. Four men wore women's clothes and two were fully clothed with exposed genitals. Besides women's clothes, other objects found at the scene included various types of sexual aids, including ropes, chains, metal bars, locks, sex magazines, condoms, plastic bags, rubber items, etc. In four cases blood alcohol levels between 0.1 and 2.5 per thousand (urine alcohol levels between 0.2 and 2.5 per thousand ) were found. Toxicologic examination revealed chloroform, ketamine, a propane-butane gas mixture in one case each, and in two cases cocaine and morphine. Causes of death included central paralysis after strangulation (seven cases), asphyxiation (4), subarachnoid hemorrhage (2), intoxication (1), hypothermia (1), left heart failure (1), and drowning (1). The history, findings at scene, and autopsy findings and, in individual cases, other investigations are of utmost importance to accurately reconstruct a fatal autoerotic accident.

Autoerotic accident associated with self-applied ketamine.

We present a rare case of an autoerotic accident involving a fatal combination of asphyxia by suffocation and intoxication with self-administered intravenous ketamine. Of note in this case is the fact that the victim was an emergency medical technician. Ketamine causes complete analgesia with superficial unconciousness and amnesia called "dissociative anasthesia". Futhermore low anaesthetic doses of ketamine induce alterations in mood, cognition and body image and the substance is an emerging drug of abuse. We discuss the death scene investigation, findings at autopsy and the toxicological report.

Acarbose and 1-deoxynojirimycin inhibit maltose and maltooligosaccharide hydrolysis of human small intestinal glucoamylase-maltase in two different substrate-induced modes.

The inhibition of the glucoamylase-maltase-catalyzed maltose and maltooligosaccharide hydrolysis by acarbose and 1-deoxynojirimycin has been demonstrated. Acarbose and 1-deoxynojirimycin act as potent competitive inhibitors with Ki = 0.8 microM for the hydrolysis of maltose and with Ki values of 0.4 and 0.3 microM, respectively, for the hydrolysis of maltooligosaccharides. In a previous work (Günther et al., Arch. Biochem. Biophys. 327, 295-302, 1996) using maltitol and maltobionate as inhibitors we were able to discriminate two different binding modes for glucoamylase-maltase: a maltose and an oligosaccharide binding mode. Here we found that structurally quite different substances, namely, the pseudotetrasaccharide acarbose and the monomeric glucose analog 1-deoxynojirimycin, act as competitive inhibitors for maltose and maltooligosaccharide hydrolysis. The Ki values for all used maltooligosaccharides are nearly equal, but for maltose hydrolysis the Ki values are significantly higher by a magnitude factor of two. The differences concerning Ki values can be explained by means of the two-binding-mode model.

Human small intestinal sucrase-isomaltase: different binding patterns for malto- and isomaltooligosaccharides.

The hydrolysis of maltose and isomaltose and of sucrose and isomaltose at two different catalytic sites of sucrase-isomaltase has been demonstrated. Maltose and sucrose are competing for the same catalytic center. This competing can be described by alternative substrate kinetics. Steady-state kinetic parameters Km and k0 (maximal reaction velocity per mol enzyme) for linear alpha-1,4 and alpha-1,6 glucosyloligosaccharides has been determined. Using these parameters subsite affinities for the catalytic sites of sucrase and isomaltase were computed. The different numbers of subsites for sucrase (2 subsites) and isomaltase (4 subsites) indicate, that the binding patterns for maltooligosaccharides and isomaltooligosaccharides are different. That means that for sucrase unproductive enzyme-maltooligosaccharide complexes are definitely less probable than the productive one. As in human small intestinal glucoamylase-maltase in the isomaltase moiety four subsites can be evaluated with affinity values (Ai): A1 = 2.6 (+/- 0.91), A2 = 13.8 (+/- 0.70), A3 = 1.1 (+/- 0.13) and A4 = 1.5 (+/- 0.13) kJ/mol using isomaltooligosaccharides. The two subsites of sucrase are evaluated to be A1 = 4.9 (+/- 0.70) and A2 = 16.7 (+/- 0.51) kJ/mol using maltooligosaccharides. The four subsite model for isomaltase and glucoamylase-maltase is an indication that these two enzymes are mechanistically homologous in binding linear glucosyl-oligosaccharides.