Matthew H. Slawson

Plasma and liver acetaminophen-protein adduct levels in mice after acetaminophen treatment: dose-response, mechanisms, and clinical implications.

At therapeutic doses, acetaminophen (APAP) is a safe and effective analgesic. However, overdose of APAP is the principal cause of acute liver failure in the West. Binding of the reactive metabolite of APAP (NAPQI) to proteins is thought to be the initiating event in the mechanism of hepatotoxicity. Early work suggested that APAP-protein binding could not occur without glutathione (GSH) depletion, and likely only at toxic doses. Moreover, it was found that protein-derived APAP-cysteine could only be detected in serum after the onset of liver injury. On this basis, it was recently proposed that serum APAP-cysteine could be used as diagnostic marker of APAP overdose. However, comprehensive dose-response and time course studies have not yet been done. Furthermore, the effects of co-morbidities on this parameter have not been investigated. We treated groups of mice with APAP at multiple doses and measured liver GSH and both liver and plasma APAP-protein adducts at various timepoints. Our results show that protein binding can occur without much loss of GSH. Importantly, the data confirm earlier work that showed that protein-derived APAP-cysteine can appear in plasma without liver injury. Experiments performed in vitro suggest that this may involve multiple mechanisms, including secretion of adducted proteins and diffusion of NAPQI directly into plasma. Induction of liver necrosis through ischemia-reperfusion significantly increased the plasma concentration of protein-derived APAP-cysteine after a subtoxic dose of APAP. While our data generally support the measurement of serum APAP-protein adducts in the clinic, caution is suggested in the interpretation of this parameter.

Effects of intrathecal morphine on the ventilatory response to hypoxia.

BACKGROUND Intrathecal administration of morphine produces intense analgesia, but it depresses respiration, an effect that can be life-threatening. Whether intrathecal morphine affects the ventilatory response to hypoxia, however, is not known. METHODS We randomly assigned 30 men to receive one of three study treatments in a double-blind fashion: intravenous morphine (0.14 mg per kilogram of body weight) with intrathecal placebo; intrathecal morphine (0.3 mg) with intravenous placebo; or intravenous and intrathecal placebo. The selected doses of intravenous and intrathecal morphine produce similar degrees of analgesia. The ventilatory response to hypercapnia, the subsequent response to acute hypoxia during hypercapnic breathing (targeted end-tidal partial pressures of expired oxygen and carbon dioxide, 45 mm Hg), and the plasma levels of morphine and morphine metabolites were measured at base line (before drug administration) and 1, 2, 4, 6, 8, 10, and 12 hours after drug administration. RESULTS At base line, the mean (+/-SD) values for the ventilatory response to hypoxia (calculated as the difference between the minute ventilation during the second full minute of hypoxia and the fifth minute of hypercapnic ventilation) were similar in the three groups: 38.3+/-23.2 liters per minute in the placebo group, 33.5+/-16.4 liters per minute in the intravenous-morphine group, and 30.2+/-11.6 liters per minute in the intrathecal-morphine group (P=0.61). The overall ventilatory response to hypoxia (the area under the curve) was significantly lower after either intravenous morphine (20.2+/-10.8 liters per minute) or intrathecal morphine (14.5+/-6.4 liters per minute) than after placebo (36.8+/-19.2 liters per minute) (P=O.003). Twelve hours after treatment, the ventilatory response to hypoxia in the intrathecal-morphine group (19.9+/-8.9 liters per minute), but not in the intravenous-morphine group (30+/-15.8 liters per minute), remained significantly depressed as compared with the response in the placebo group (40.9+/-19.0 liters per minute) (P= 0.02 for intrathecal morphine vs. placebo). Plasma concentrations of morphine and morphine metabolites either were not detectable after intrathecal morphine or were much lower after intrathecal morphine than after intravenous morphine. CONCLUSIONS Depression of the ventilatory response to hypoxia after the administration of intrathecal morphine is similar in magnitude to, but longer-lasting than, that after the administration of an equianalgesic dose of intravenous morphine.

Quantitative confirmation of testosterone and epitestosterone in human urine by LC/Q-ToF mass spectrometry for doping control.

Testosterone (T) is the primary male sex hormone. In addition to the development of secondary sex characteristics, testosterone has anabolic effects including increases in muscle size and strength and increases in lean body mass, making it an attractive candidate to enhance athletic performance. In the case of exogenous administration of testosterone, the ratio of testosterone to its isomer, epitestosterone (E), is elevated. WADA has set a standard for T/E ratios of 4.0 as indicative of possible exogenous testosterone administration. Typically, a sample that screens for a T/E ratio above that threshold is then subjected to quantitative confirmation by GC/MS. This methodology, however, can limited due to sensitivity issues as well as a limited number of qualifying ions that can be used for unambiguous identification. We have developed a confirmation method which uses liquid/liquid extraction, followed by room temperature Girard P derivatization, and analysis using LC/MS-ToF. We observe a number of advantages over conventional GC/MS analysis. Analysis time is decreased. Sensitivity is increased, resulting in limits of detection of 2 and 0.5 ng/ml for testosterone and epitestosterone, respectively. The number of diagnostic qualifier ions is also increased allowing more confident identification of the analytes. Finally, while this method has been developed on a QToF instrument, it should be easily transferable to any tandem LC/MS/MS system.

Correlations of the induction of microsomal epoxide hydrolase activity with phase II drug conjugating enzyme activities in rat liver

Within the selective induction of phase II enzymes following treatment with dipyridyls or N-heterocyclic analogs of phenanthrene, strong correlations (r > or = 0.70) are observed between the increase of microsomal epoxide hydrolase (mEH) activity and UDP-glucuronosyltransferase (UGT) activities towards 4-nitrophenol, 1-naphthol and morphine. The present study investigates whether this correlation is maintained with inducing agents known to also increase phase I enzyme activities. Rats were treated with beta-naphthoflavone, isosafrole, phenobarbital, ethanol, dexamethasone and clofibric acid regimens in which P450 isozyme induction could be confirmed. Comparisons between the responses of mEH, UGT and glutathione S-transferase (GST) activities were made. mEH activity was increased by beta-naphthoflavone, isosafrole, phenobarbital and clofibric acid. The elevation in mEH activity by these agents showed modest but significant correlations with GST activities toward all the substrates monitored (r values range between 0.49 and 0.65) and a strong correlation with UGT activity towards only one substrate, morphine (r = 0.70). This study suggests that induction of mEH activity correlates with the increases in select phase II enzyme activities whether it is accompanied by P450 induction or not.

Selective induction of rat liver phase II enzymes by N-heterocycle analogues of phenanthrene: a response exhibiting high correlation between UDP-glucuronosyltransferase and microsomal epoxide hydrolase activities

1. Among nitrogen heterocycles based on the planar phenanthrene structure are three (1,7- and 4,7-phenanthroline and phenanthridine) which selectively increase rat hepatic phase II drug metabolizing enzyme activities without increasing cytochrome P450 concentration. Of six monooxygenase activities investigated, only ethoxyresorufin dealkylase was raised but this was only minor. 2. The detergent-activated UDP-glucuronosyltransferase activities towards morphine, 4-nitrophenol, and 1-naphthol were increased up to five-, three- and two-fold of control respectively. Microsomal epoxide hydrolase activity towards cis-stilbene oxide was increased up to three-fold and cytosolic glutathione S-transferase activity towards 1-chloro-2, 4-dinitrobenzene reached twice the control value. 3. Cytosolic 4-nitrophenol sulphotransferase activity was not increased by any compound and like some monooxygenase reactions, was decreased by 4,7- and 1,7-phenanthrolines. 4. 1,10-Phenanthroline and two compounds which lack a heterocyclic nitrogen atom, (phenanthrene and 9-phenanthrol), failed to elicit any induction of enzyme activities. 5. Changes in microsomal epoxide hydrolase activity showed high correlation (r = 0.97) with changes in UDP-glucuronosyltransferase (4-nitrophenol) activity.

Demystifying Analytical Approaches for Urine Drug Testing to Evaluate Medication Adherence in Chronic Pain Management

ABSTRACT This comprehensive review of analytical methods used for urine drug testing for the support of pain management describes the methods, their strengths and limitations, and types of analyses used in clinical laboratories today. Specific applications to analysis of opioid levels are addressed. Qualitative versus quantitative testing, immunoassays, chromatographic methods, and spectrometry are discussed. The importance of proper urine sample collection and processing is addressed. Analytical explanations for unexpected results are described. This article describes the scientific basis for urine drug testing providing information which will allow clinicians to differentiate between valid and questionable claims for urine drug testing to monitor medication adherence among chronic pain patients.

Assessing orally bioavailable commercial silver nanoparticle product on human cytochrome P450 enzyme activity

Abstract Nanotechnology produces a wide range of medicinal compounds, including nanoparticulate silver, which are increasingly introduced in various forms for consumer use. As with all medicinal compounds, potential drug interactions are an important consideration for ingested silver nanoparticles. Nanoparticulate silver–drug interactions may be mediated through induced oxidative stress in liver tissue where the majority of systemically bioavailable silver nanoparticles is found. To investigate whether an orally ingested commercially available colloidal silver nanoproduct produces pharmacokinetic interference on select cytochrome P450 enzymes, a prospective, single-blind, controlled in vivo human study using simultaneous administration of standardized probes for P450 enzyme classes CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 was conducted. Oral ingestion of a commercial colloidal silver nanoproduct produces detectable silver in human serum after 14 days of dosing. This silver, however, elicits no demonstrable clinically significant changes in metabolic, hematologic, urinary, physical findings or cytochrome P450 enzyme inhibition or induction activity. Given their increasingly broad, diverse human exposures, future characterization of human cytochrome P450 enzyme activity for other systemically bioavailable nanotechnology products are warranted.

Analysis of Selected Low-Dose Benzodiazepines by Mass Spectrometry

The benzodiazepines discussed in this chapter are alprazolam, lorazepam, midazolam, and triazolam. They are all prescription medications that are used as anti-anxiety agents, preoperative medications, or as sedative—hypnotics (1). All are prescribed in very low doses (because of their potency), rapidly metabolized, and have short plasma half-lives (Table 1). Generally, they are biotransformed to hydroxylated metabolites and are excreted in the urine as glucuronide conjugates. Because the recommended therapeutic doses of alprazolam, lorazepam, midazolam, and triazolam may be 1 mg or less and the drugs are rapidly metabolized, plasma concentrations of the parent drugs and their metabolites are in nanogram per milliliter concentrations. Therefore, the detection and quantification of alprazolam, lorazepam, midazolam, triazolam, and their respective metabolites present a significant challenge to the analytical laboratory.