R. Aderjan

Ethyl Glucuronide Concentration in Serum of Human Volunteers, Teetotalers, and Suspected Drinking Drivers

The kinetic profile of ethanol and ethyl glucuronide (EtG) in serum was investigated in three subject groups: 1) Healthy, moderately drinking volunteers (daily intake less than 30 g ethanol) who ingested a single dose of ethanol. In this group the maximum of serum ethyl glucuronide concentration (SEtGC) and of serum ethanol concentration (SEC) did not exceed 3.7 mg/L and 1.5 g/L respectively. EtG peaked 2 to 3.5 h later than ethanol. EtG was eliminated with a terminal half-life of 2 to 3 h. EtG decreased slower than ethanol--the metabolite could still be determined in serum up to 8 h after complete ethanol elimination. 2) In serum samples of teetotalers neither ethanol nor EtG could be found. 3) In 37 of 50 serum samples of drivers suspected of driving under the influence of ethanol, SEtGC was found between the limit of detection (0.1 mg/L) and 20 mg/L. If the SEC is less than 1 g/L and the SEtGC is significantly higher than 5 mg/L, we assume alcohol misuse.

Postmortem distribution pattern of morphine and morphine glucuronides in heroin overdose

The postmortem distribution of morphine and its metabolites was investigated in four cases of heroin overdose to evaluate some of the factors that influence intravasal blood concentrations. Variables included were the chemical stability of morphine conjugates, hemoconcentration, incomplete distribution of the drug and diffusion processes. Blood samples from different sampling sites including the aorta, the infra- and suprarenal portion of the inferior vena cava, the superior vena cava, the femoral and subclavian veins, and the right and left ventricles were examined for morphine, morphine-3-glucuronide and morphine-6-glucuronide, hematocrit and water content. Drug concentrations were determined by HPLC based on the native fluorescence of the analytes. Morphine glucuronides proved to be stable for a time period of 72 h. The water content ranged from 65 to 83% and hematocrit values from 25 to 75%, and were seen as contributory factors to the dramatic differences observed for drug concentrations from different sampling sites. The differences could neither be attributed to incomplete distribution during life-time nor to a diffusion process following the different distribution volumes of morphine and its conjugates. A definite relationship between the ratio of the molar concentrations of morphine and its glucuronides, as assessed in pharmacokinetical studies after morphine dosing, could not be established. For a better understanding more cases and changes over time and tissue concentrations should be analysed.

Formation and clearance of active and inactive metabolites of opiates in humans.

The results of recent investigations of the analgesic and the nonanalgesic effects of opioid glucuronides are relevant to the research on drug abuse in forensic toxicology. As has been shown for heroin, knowledge of the state of distribution and elimination of active and inactive metabolites and glucuronides offers new possibilities in forensic interpretation of analytic results. Because of similar metabolic degradation, calculation of the time-dependent ratio of the concentration of morphine and its glucuronide metabolites in blood or serum allows a rough estimation of increased dosage and of time elapsed since the last application. Drug effects can be examined with respect to individual case histories, including overdose and survival time if the patient died. However, different methods of administration and the strong influence of different volumes or compartments of distribution of parent compounds and metabolites on concentrations in human body tissues require careful use of glucuronide concentration data. In Germany, dihydrocodeine (DHC) is prescribed as a heroin substitute, and relative overdoses are needed to be effective. DHC metabolism was studied in three patients who died from overdoses. All metabolites (dihydrocodeine-6-glucuronide [DHC6], nor-DHC [NDHC], dihydromorphine [DHM], nor-DHM [NDHM], and DHM-3- and 6-glucuronide [DHM3G, DHM6G]) were determined using HPLC and fluorescence detection. Concentrations of DHM (0.16 mg/L to 0.22 mg/L serum) were found. The DHM glucuronide ratios were similar to those of morphine. Receptor binding studies showed that the binding affinity of DHM to porcine mu-receptor was higher than that of morphine, and DHM6Gs binding affinity was as high as that of morphine-6-glucuronide (M6G). Metabolites may play an important role in the effectiveness of DHC in substitution and toxicity. Because of enzyme polymorphism, the formation of DHC poses a risk for proper dosage in patients who are either poor or extensive metabolizers. The distribution of opioid glucuronides in cerebral spinal fluid in relation to transcellular transport in central nervous tissue is discussed with respect to the receptor binding of opiates and drug effect.

Postmortem distribution of dihydrocodeine and metabolites in a fatal case of dihydrocodeine intoxication

A report of a fatal dihydrocodeine ingestion under substitution therapy is given. Quantitation of dihydrocodeine, dihydromorphine, N-nordihydrocodeine, dihydrocodeine-6-, dihydromorphine-6- and dihydromorphine-3-glucuronide was performed simultaneously after solid-phase extraction prior to HPLC analysis, and the analytes were detected using their native fluorescence. Postmortem concentrations of blood samples from different sampling sites as well as from liver, kidney and cerebrum are reported. A hair sample was investigated to prove long-term use of the substitute drug. Site-to-site differences of the analytes from blood samples were very small. The partition behavior of the opioid glucuronides depended on the hematocrit value of the particular blood sample. Most important findings seemed that dihydromorphine and dihydromorphine-6-glucuronide concentrations decisively contributed to the toxicity of dihydrocodeine. This case report outlines that in dihydrocodeine related deaths the concentrations of the pharmacologically active metabolites should additionally be determined for reliable interpretation.

Helicobacter pylori infection decreases gastric alcohol dehydrogenase activity and first-pass metabolism of ethanol in man.

Background/Aims: Ethanol is metabolized by alcohol dehydrogenase in the human stomach. This metabolism contributes to the so-called first-pass metabolism of ethanol which is affected by gender, medication, and morphological alterations of the gastric mucosa. Recently, it has been shown that Helicobacter pylori is capable to oxidize ethanol to acetaldehyde in vitro. Since H. pylori also injures gastric mucosa, the present study examines the effect of this bacterium on gastric alcohol dehydrogenase activity and systemic availability of ethanol in vivo. Methods: Thirteen volunteers (7 men and 6 women, aged 18–52 years) with gastric H. pylori infection diagnosed by a positive CLO test and positive gastric histology received ethanol (0.225 g/kg) either orally or intravenously before and after H. pylori elimination to determine systemic availability of ethanol. In addition, gastric biopsy specimens were taken from all subjects before and after H. pylori elimination for histological assessment of mucosal alterations and determinations of gastric alcohol dehydrogenase activity and phenotype of the enzyme. Results: In the presence of H. pylori the first-pass metabolism of ethanol was found to be significantly reduced (625 ± 234 vs. 1,155 ± 114 mg/dl/min, p = 0.046). This reduction of first-pass metabolism of ethanol was associated with a significant decrease in alcohol dehydrogenase activity (4.8 ± 1.5 vs. 12.1 ± 2.3 nmol/mg protein × min, p < 0.05) and an increase in the severity of mucosal damage as determined by a histological score (p < 0.05). Conclusions: H. pylori infection leads to gastric mucosal injury which is associated with a decrease in gastric alcohol dehydrogenase activity and first-pass metabolism of ethanol. Ethanol metabolism by H. pylori does not play an important role in vivo. However, gastric morphology is one important factor determining systemic availability of ethanol in man.

Fatal overdose of 2,4-dichlorophenoxyacetic acid (2,4-D)

An ingestion of an unknown quantity of U 46 D-Fluid (500 g dichlorophenoxyacetic acid/l) in a suicide is described. Although 2,4-dichlorophenoxyacetic acid (2,4 D) is widely used as a herbicide, intoxications are relatively rare. Quantitation of 2,4-D was performed by diethyl ether extraction from acidified samples (viscera) or by deproteinization (blood, plasma) with methanol before HPLC analysis. Postmortem concentrations of 2,4-D in body fluids and tissues are given. The proposed method resulted in a rapid procedure most useful in cases of deliberate poisoning with phenoxyacetic herbicides.

Freie und glucuronidierte Cannabinoide im Urin - Untersuchungen zur Einschätzung des Konsumverhaltens

Abstract Urine specimens were collected from 49 persons up to 10 days after abstaining from cannabis use. Urine samples (n = 135) were investigated for free and conjugated 9-carboxy-11-nor tetrahydrocannabinol (THCCOOH) by LC/MS/MS and for free and glucuronidated tetrahydrocannabinol (THC) and 11-hydroxy-THC (11-OH-THC) by GC/MS prior to and after enzyme hydrolysis. Concentrations for free and conjugated THCCOOH were measured with reference to 100 mg creatinine/dL urine. Cannabis use was classified as light, moderate and heavy according to the current literature. In all groups chosen free and bound THCCOOH was present in urine samples on the day of the last use and on the next day. Urine samples from occasional and frequent users generally differed in the amount of THCCOOH and THCCOOH glucuronide. Within 4–6 days the concentrations of free and glucuronidated THCCOOH in urine samples of heavy users decreased to those ranges present in urine samples of moderate and light users. In frequent users, THC glucuronide and 11-OH-THC glucuronide could be found up to 1 and 3 days after abstinence, respectively. In light users, neither THC glucuronide nor 11-OH-THC glucuronide was detectable. The determination of conjugated THC and 11-OH-THC in addition to free and bound ¶THCCOOH is suggested as an aid in assessing the frequency of cannabis use, but is limited to samples obtained within a few days after the last consumption.Zusammenfassung Spontanurinproben (n = 135) von ¶49 Cannabiskonsumenten wurden auf freie und glucuronidierte Tetrahydrocannabinolcarbonsäure (THCCOOH) mittels LC/MS/MS sowie auf freies und glucuronidiertes Tetrahydrocannabinol (THC) und 11-Hydroxytetrahydrocannabinol (11-OH-THC) mittels GC/MS direkt sowie nach enzymatischer Hydrolyse bis zu 10 Tagen nach dem letztmaligen Konsum untersucht. Die Einteilung des Konsumverhaltens erfolgte in schwer, moderat und leicht und orientierte sich an den Zuordnungskriterien neuerer, einschlägiger Publikationen. Die Konzentrationsangaben für freie und glucuronidierte THCCOOH wurden auf 100 mg Kreatinin/dL Urin bezogen. In den Konsumentengruppen schwer, moderat und leicht war THCCOOH in konjugierter und freier Form am Tag des Konsums sowie am nachfolgenden Tag in unterschiedlichen Konzentrationsbereichen nachweisbar. Die Höhe der Konzentrationen ließ prinzipiell die Abgrenzung eines sporadischen von einem gewohnheitsmäßigen Konsum zu. Nach 4–6 Tagen ergab sich jedoch ein Konzentrationsangleich für beide Analyte. Bei regelmäßigen Rauchern konnte THCglucuronid teilweise bis zum ersten Tag und 11-OH-THCglucuronid bis zum dritten Tag nach Abstinenz bestimmt werden. Bei ¶gelegentlichen Rauchern waren konjugiertes THC und ¶11-OH-THC im Urin nicht faßbar. Die Ergebnisse zeigten, daß eine zusätzliche Bestimmung der Glucuronidkonjugate von THC und 11-OH-THC in konsumnah abgegebenen Urinproben die Kategorisierung des Konsumverhaltens erleichtern kann.

Pharmacokinetics of 11-nor-9-Carboxy-Δ9-Tetrahydrocannabinol (CTHC) After Intravenous Administration of CTHC in Healthy Human Subjects

After cannabis consumption there is only limited knowledge about the pharmacokinetic (PK) and metabolic properties of 11‐nor‐9‐carboxy‐Δ9‐tetrahydrocannabinol (CTHC), which is formed by oxidative breakdown from Δ9‐tetrahydrocannabinol (THC). Despite widely‐varying concentrations observed in smoking studies, attempts have been made to interpret consumption behavior with special regard to a cumulated or decreasing concentration of CTHC in serum. Ten healthy nonsmoking white male individuals received 5 mg CTHC intravenously over 10 min. Highest serum concentrations of CTHC were observed at the end of the infusion (336.8±61.7 μg/l) followed by a quick decline. CTHC concentration could be quantified up to 96 h after administration, with a terminal elimination half‐life of 17.6±5.5 h. Total clearance was low (91.2±24.0 ml/min), with renal clearance having only a minor contribution (0.136±0.094 ml/min). This first metabolite‐based kinetic approach will allow an advanced understanding of CTHC PKs data obtained in previous studies with THC.

Saliva testing after single and chronic administration of dihydrocodeine

Abstract In the present study, concentrations of dihydrocodeine and its metabolites in saliva and serum were compared after single low-dose and chronic high-dosage administration of the drug. In the first investigation, blood and saliva were collected periodically from six subjects after oral administration of 60 mg dihydrocodeine. In the second study, 20 subjects on oral dihydrocodeine maintenance provided single samples of blood and saliva simultaneously. Serum protein binding of salivary analytes and their recovery from the adsorbing material of the collection device as well as pH values of saliva samples were determined. The fluids were analyzed for dihydrocodeine and the major metabolites by high-performance liquid chromatography. In the single dose study dihydrocodeine was the only analyte found in saliva for up to 12–24 h post-dose. The half-life of dihydrocodeine in saliva was about twice that found in blood. The ratios of saliva/ serum concentrations ranged from 1.2 to 17.0. After chronic high-dosage use, dihydrocodeine was the main salivary analyte and N-nordihydrocodeine was present in a few samples. Saliva/serum concentration ratios of dihydrocodeine were strongly dependent on the pH value of saliva and, to a lesser extent, on serum-protein binding. The saliva/ serum ratios were more similar after chronic administration. The data suggest a passive diffusion process as the underlying mechanism for the transport of dihydrocodeine into saliva. After both single and chronic use, the presence of the drug in saliva can be used as evidence of recent substance administration.

Zur Wertigkeit postmortaler Digoxin-Konzentrationen im Blut

SummaryThe digoxin concentration in the blood of ten patients with terminal illness were determined by radioimmunoassay sampling the specimens 10–30 min before death, 30 min after death (subclavial catheter), and at autopsy (femoral vein and heart). At autopsy increased levels were found: In heart blood 6.64±4.45 ng/ml, in femoral vein blood 4.42±2.45 ng/ml, in ante mortem vein blood 3.36±1.75 ng/ml, mean difference in vein blood: 1.06±0.79 ng/ml (mean value and standard deviation). Only the corresponding concentration values in vein blood are closely correlated. The increase is determined statistically significant by the initial concentration found before death. A gradient dependent diffusion back from the tissues surrounding the vessels is to be considered. The walls of the vessels, however, do not contain higher digoxin concentration than the blood. Six of the ten cases showed levels of digoxin in the ante mortem blood clearly above the therapeutic range of 0.5–2.0 ng/ml. Reasons for this observation are discussed.ZusammenfassungBei 10 digitalisierten Patienten wurden präfinal, ca. 10–30 min vor Todeseintritt, Blutproben entnommen und deren Digoxin-Konzentration mit Werten verglichen, die in ca. 30 min postmortal entnommenem Leichenblut festzustellen sind. Nach der Sektion wurden die Digoxin-Konzentrationen in Schenkelvenenblut und Herzblut bestimmt. Gegenüber den präfinalen Konzentrationen (3,36±1,75 ng/ml) fand sich bis zur Sektion im Venenblut ein Anstieg der Digoxin-Konzentration um 1,06±0,79 auf 4,42±2,45 ng/ml, im Herzblut durchschnittlich auf 6,64±4,45 ng/ml. Nur im venösen Blut sind präfinale und postmortale Konzentrationen eng miteinander korreliert. Der Konzentrations-Anstieg im Schenkelvenenblut wird durch die präfinale Ausgangs-Konzentration statistisch signifikant bestimmt. Es ist eine Gradienten-abhängige Rückdiffusion aus den gegenüber den Gefäßen höher konzentrierten umliegenden Geweben anzunehmen. Die Gefäßwände jedoch speichern kaum Digoxin. Die präfinalen Digoxin-Konzentrationen im Blut lagen in 6 von 10 Fällen deutlich über dem therapeutischen Bereich von 0,5–2 ng/ml. Gründe für diese Beobachtung werden diskutiert.