Driving under the influence of cocaine: Quantitative determination of basic drugs in oral fluid obtained during roadside controls and a controlled study with cocaine users.

Using the Belgian Drugs and Driving procedure, 36% of the cocaine-positive oral fluid (OF) screening results were not confirmed in plasma. This study investigates the impact of the choice of screening devices and confirmation matrix on the detection of cocaine use. An ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method quantifying cocaine, benzoylecgonine (BZE), and other basic drugs in OF was developed and validated. This method monitored OF samples obtained either from a roadside (n = 12) or a double-blind controlled study with cocaine users (n = 10) who were given either a capsule containing 300 mg of cocaine-HCl or a placebo. The OF data were compared to plasma concentrations to obtain concentration-time profiles. In addition, the sensitivity and accuracy of the Drugwipe5S® was assessed. A significant difference between the OF volume collected at baseline/placebo (median 0.93 mL [range 0.43-1.92 mL]) or after cocaine-HCL intake (0.79 mL [0.30-1.21 mL]) was observed. The median OF/Plasma at the 3 collection time points were 10.7, 13.8, 6.7 for cocaine and 0.8, 1.7, 0.8 for BZE, respectively. The Drugwipe5S® detected cocaine use until at least 4 hours after intake. When applying the Belgian legal confirmation decision limit of 10 ng/mL in OF, an accuracy of 75%-98% was observed, depending on the study setting. Cocaine concentrations in OF were much higher and were detected longer as compared to plasma, when applying the same decision limit. From a toxicological viewpoint, the longer detection window with the higher sensitivity of Cocaine and BZE is beneficial to detect drivers in the crash/fatigue phase.

Evaluation of Δ(9) -tetrahydrocannabinol detection using DrugWipe5S(®) screening and oral fluid quantification after Quantisal™ collection for roadside drug detection via a controlled study with chronic cannabis users.

Oral fluid (OF) is potentially useful to detect driving under the influence of drugs because of its ease of sampling. While cannabis is the most prevalent drug in Europe, sensitivity issues for Δ(9) -tetrahydrocannabinol (THC) screening and problems during OF collection are observed. The ability of a recently improved OF screening device - the DrugWipe5S(®) , to detect recent THC use in chronic cannabis smokers, was studied. Ten subjects participated in a double-blind placebo-controlled study. The subjects smoked two subsequent doses of THC; 300 µg/kg and 150 µg/kg with a pause of 75 min using a Volcano vapourizer. DrugWipe5S(®) screening and OF collection using the Quantisal™ device were performed at baseline, 5 min after each administration and 80 min after the last inhalation. Blood samples were drawn simultaneously. The screening devices (n = 80) were evaluated visually after 8 min, while the corresponding OF and serum samples were analyzed respectively with ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) or gas chromatography-mass spectrometry (GC-MS). Neat OF THC concentrations ranged from 12 361 ng/g 5 min after smoking down to 34 ng/g 80 min later. Under placebo conditions, a median THC concentration of 8 ng/g OF (0-746 ng/g) and < 1 ng/ mL serum (0-7.8 ng/mL) was observed. The DrugWipe5S(®) was positive just after smoking (90%); however, sensitivity rapidly decreased within 1.5 h (50%). Sensitivity of DrugWipe5S(®) should be improved. As chronic cannabis users have high residual THC concentrations in their serum and OF, confirmation cut-offs should be set according to the aim of detecting recent drug use or establishing zero tolerance.

Extracranial sources of S100B do not affect serum levels.

S100B, established as prevalent protein of the central nervous system, is a peripheral biomarker for blood-brain barrier disruption and often also a marker of brain injury. However, reports of extracranial sources of S100B, especially from adipose tissue, may confound its interpretation in the clinical setting. The objective of this study was to characterize the tissue specificity of S100B and assess how extracranial sources of S100B affect serum levels. The extracranial sources of S100B were determined by analyzing nine different types of human tissues by ELISA and Western blot. In addition, brain and adipose tissue were further analyzed by mass spectrometry. A study of 200 subjects was undertaken to determine the relationship between body mass index (BMI) and S100B serum levels. We also measured the levels of S100B homo- and heterodimers in serum quantitatively after blood-brain barrier disruption. Analysis of human tissues by ELISA and Western blot revealed variable levels of S100B expression. By ELISA, brain tissue expressed the highest S100B levels. Similarly, Western blot measurements revealed that brain tissue expressed high levels of S100B but comparable levels were found in skeletal muscle. Mass spectrometry of brain and adipose tissue confirmed the presence of S100B but also revealed the presence of S100A1. The analysis of 200 subjects revealed no statistically significant relationship between BMI and S100B levels. The main species of S100B released from the brain was the B-B homodimer. Our results show that extracranial sources of S100B do not affect serum levels. Thus, the diagnostic value of S100B and its negative predictive value in neurological diseases in intact subjects (without traumatic brain or bodily injury from accident or surgery) are not compromised in the clinical setting.

A fast ultra-performance liquid chromatography method for simultaneous quantification of mycophenolic acid and its phenol- and acyl-glucuronides in human plasma.

Several studies have demonstrated a close relationship between mycophenolic acid (MPA) exposure and the risk for graft rejection or side effects. Measurements of MPA and its metabolites plasma levels are therefore recommended. A new chromatographic method has been developed using ultra-performance liquid chromatography (UPLC) to improve both analytical throughput and sensitivity. MPA and its phenol-glucuronide and acyl-glucuronide were extracted from plasma using Isolute C2 solid phase extraction (SPE) cartridges (100 mg, 3 mL). UPLC separations were performed with a Waters BEH C18 column (50 x 2.1 mm, 1.7 microm) maintained at 65 degrees C on a Waters Acquity instrument equipped with a photodiode array detector. The total UPLC run time was 3.5 minutes. The method was linear in the range of 0.1-40 microg/mL for MPA and acyl-glucuronide, and 1-400 microg/mL for phenol-glucuronide. Relative standard error and mean relative prediction error were <15% for all tested quality controls (in-house and external proficiency panels). UPLC performances are characterized by a dramatic reduction in retention times together with an improvement of the sensitivity without affecting peak resolution. Further validations have been obtained by analyzing routine and clinical trial patients' samples. Significant improvement of the analytical throughput (reduction of run time from >10 to 3.5 minutes) was obtained using UPLC for MPA analyses. This retention time reduction was accompanied by an improvement of other analytical performances such as sensitivity.

Combined effects of prenatal inhibition of vasculogenesis and neurogenesis on rat brain development.

Malformations of cortical development (MCD) are one of the most common causes of neurological disabilities including autism and epilepsy. To disrupt cortical formation, methylazoxymethanol (MAM) or thalidomide (THAL) has been used to affect neurogenesis or vasculogenesis. Although previous models of MCD have been useful, these models primarily attack a single aspect of cortical development. We hypothesized that simultaneous prenatal exposure to MAM or THAL will lead to the development of a novel and specific type of brain maldevelopment. Rats were prenatally exposed to MAM and THAL. At early postnatal days, brains displayed abnormal ventricular size and hemispheric asymmetry due to altered brain water homeostasis. The postnatal brain was also characterized by gliosis in regions of focal leakage of the blood brain barrier. These morphological abnormalities gradually disappeared at adult stages. Although the adult MAM-THAL rats showed normal cortical morphology, abnormal hippocampal connectivity and mossy fiber sprouting persisted well into adulthood.

ProApolipoprotein A1: a serum marker of brain metastases in lung cancer patients.

BACKGROUND: Central nervous system (CNS) diagnostics is a promising tool for detection of neurological disorders, including brain metastases. One of the earliest applications of CNS diagnostics was based on serum markers of blood-brain barrier (BBB) dysfunction, which often correlates with acute, chronic, or incipient brain disease. In the case of brain metastases, serum levels of S100beta demonstrated a good negative predictive value comparable to radiologic investigations. However, a confounding factor was the presence of BBB changes due to cerebrovascular disease. METHODS: Of 103 patients enrolled in a lung cancer study, greater than 50% presented with magnetic resonance imaging (MRI) changes consistent with chronic cerebrovascular disease and reflected by elevated serum S100beta. To unveil serum protein, the authors used proteomic techniques that allow discrimination between patients with brain metastases and lung cancer patients affected by cerebrovascular ischemic changes without infiltrating tumor. RESULTS: ProApolipoprotein A1, transferrin, haptoglobin, and transthyretin were upregulated in patients affected by chronic cerebrovascular disease and brain metastases compared with those affected only by vascular diseases. ProApolipoprotein A1 was significantly increased (p<.05) in patients with CNS disease. CONCLUSIONS: In conclusion, these data support the use of serum markers for the early detection of brain metastases. ProApolipoprotein A1 may be used in conjunction with S100beta for serum-based, MRI-independent diagnosis of metastatic brain tumors.

Seizure-promoting effect of blood-brain barrier disruption.

PURPOSE: It is generally accepted that blood-brain barrier (BBB) failure occurs as a result of CNS diseases, including epilepsy. However, evidences also suggest that BBB failure may be an etiological factor contributing to the development of seizures. METHODS: We monitored the onset of seizures in patients undergoing osmotic disruption of BBB (BBBD) followed by intraarterial chemotherapy (IAC) to treat primary brain lymphomas. Procedures were performed under barbiturate anesthesia. The effect of osmotic BBBD was also evaluated in naive pigs. RESULTS: Focal motor seizures occurred immediately after BBBD in 25% of procedures and originated contralateral to the hemisphere of BBBD. No seizures were observed when BBB was not breached and only IAC was administered. The only predictors of seizures were positive indices of BBBD, namely elevation of serum S100beta levels and computed tomography (CT) scans. In a porcine model of BBBD, identical procedures generated an identical result, and sudden behavioral and electrographic (EEG) seizures correlated with successful BBB disruption. The contribution of tumor or chemotherapy to acute seizures was therefore excluded. CONCLUSION: This is the first study to correlate extent of acute BBB openings and development of seizures in humans and in a large animal model of BBB opening. Acute vascular failure is sufficient to cause seizures in the absence of CNS pathologies or chemotherapy.

Alternating current electrical stimulation enhanced chemotherapy: a novel strategy to bypass multidrug resistance in tumor cells.

BACKGROUND: Tumor burden can be pharmacologically controlled by inhibiting cell division and by direct, specific toxicity to the cancerous tissue. Unfortunately, tumors often develop intrinsic pharmacoresistance mediated by specialized drug extrusion mechanisms such as P-glycoprotein. As a consequence, malignant cells may become insensitive to various anti-cancer drugs. Recent studies have shown that low intensity very low frequency electrical stimulation by alternating current (AC) reduces the proliferation of different tumor cell lines by a mechanism affecting potassium channels while at intermediate frequencies interfere with cytoskeletal mechanisms of cell division. The aim of the present study is to test the hypothesis that permeability of several MDR1 over-expressing tumor cell lines to the chemotherapic agent doxorubicin is enhanced by low frequency, low intensity AC stimulation. METHODS: We grew human and rodent cells (C6, HT-1080, H-1299, SKOV-3 and PC-3) which over-expressed MDR1 in 24-well Petri dishes equipped with an array of stainless steel electrodes connected to a computer via a programmable I/O board. We used a dedicated program to generate and monitor the electrical stimulation protocol. Parallel cultures were exposed for 3 hours to increasing concentrations (1, 2, 4, and 8 microM) of doxorubicin following stimulation to 50 Hz AC (7.5 microA) or MDR1 inhibitor XR9576. Cell viability was assessed by determination of adenylate kinase (AK) release. The relationship between MDR1 expression and the intracellular accumulation of doxorubicin as well as the cellular distribution of MDR1 was investigated by computerized image analysis immunohistochemistry and Western blot techniques. RESULTS: By the use of a variety of tumor cell lines, we show that low frequency, low intensity AC stimulation enhances chemotherapeutic efficacy. This effect was due to an altered expression of intrinsic cellular drug resistance mechanisms. Immunohistochemical, Western blot and fluorescence analysis revealed that AC not only decreases MDR1 expression but also changes its cellular distribution from the plasma membrane to the cytosol. These effects synergistically contributed to the loss of drug extrusion ability and increased chemo-sensitivity. CONCLUSION: In the present study, we demonstrate that low frequency, low intensity alternating current electrical stimulation drastically enhances chemotherapeutic efficacy in MDR1 drug resistant malignant tumors. This effect is due to an altered expression of intrinsic cellular drug resistance mechanisms. Our data strongly support a potential clinical application of electrical stimulation to enhance the efficacy of currently available chemotherapeutic protocols.

Potentiation of cyclophosphamide chemotherapy using the anti-angiogenic drug thalidomide: importance of optimal scheduling to exploit the 'normalization' window of the tumor vasculature.

The aim of this work was to study how administration schedule affects potentiation of cyclophosphamide, an alkylating agent, by thalidomide, an anti-angiogenic agent. Tumor oxygenation after thalidomide administration was determined over time by EPR oximetry. Such measurements provide a surrogate marker for determining the timing of 'normalization' of tumor vasculature. Re-growth delays were measured using different combinations and schedules of treatments. Additionally, the uptake of the metabolite of cyclophosphamide (hydroxycyclophosphamide or OH-CP) into tumors was determined by high performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS). A significant increase in pO(2) was observed after 2 and 3 days of treatment before eventually declining on day 4. Thalidomide potentiated the effect of cyclophosphamide only when cyclophosphamide was administered after 2 days of treatment with thalidomide (no significant benefit using other schedules). In this time frame, the HPLC/MS/MS measurements showed that the quantity of OH-CP penetrating into the tumor was about twice in mice treated by thalidomide compared to controls. In conclusion, the present study demonstrates that the benefit of combined therapy using an anti-angiogenic agent with a cytotoxic agent requires knowledge of the time window during which the vessels initially become normalized.

S100beta as a predictor of brain metastases: brain versus cerebrovascular damage.

BACKGROUND: The identification of brain metastases in patients with malignant disease has important implications for determining their treatment and prognosis. Asymptomatic metastatic brain tumors may be detected by surveillance imaging techniques, but longitudinal follow-up of patients who are at risk is sporadic primarily due to cost. Because the development of brain metastases is accompanied and detected by extravasation of contrast agents across the blood-brain barrier (BBB), the authors hypothesized that peripheral analysis of the BBB indicator S100beta may be useful as a screening tool for brain metastases in patients who have no neurologic symptoms. METHODS: Thirty-eight patients were enrolled for the current study. All patients had newly diagnosed lung carcinoma and had no neurologic symptoms or known history of brain metastasis. Patients underwent an initial magnetic resonance imaging (MRI) scans and S100beta blood tests. S100beta tests were repeated in a subset of patients at the time of routine follow-up MRI scans. RESULTS: Based on imaging studies and on serum S100beta analyses, the patients were divided in 3 categories: 1) patients with normal S100beta levels (0.08 +/- 0.02 microg/L; n = 22 patients) and normal MRI scans; 2) patients with elevated S100beta levels (0.5 +/- 0.28 microg/L; n = 8 patients) and pronounced microvascular changes on MRI scans but with no metastases; and 3) patients with elevated S100beta levels (0.28 +/- 0.19 microg/L; n = 7 patients) and metastatic brain tumor(s) on MRI scans. CONCLUSIONS: Because of the significant overlap in S100beta levels between patients with cerebral microvascular diseases and patients with brain metastases, the authors concluded that the serum S100beta level may be used as a surveillance tool to predict or detect brain metastases if appropriate prescreening radiologic tests are obtained and if patients who are candidates for false-positive results are identified and excluded.

Very low intensity alternating current decreases cell proliferation.

Electric fields impact cellular functions by activation of ion channels or by interfering with cell membrane integrity. Ion channels can regulate cell cycle and play a role in tumorigenesis. While the cell cycle may be directly altered by ion fluxes, exposure to direct electric current of sufficient intensity may decrease tumor burden by generating chemical products, including cytotoxic molecules or heat. We report that in the absence of thermal influences, low-frequency, low-intensity, alternating current (AC) directly affects cell proliferation without a significant deleterious contribution to cell survival. These effects were observed in normal human cells and in brain and prostate neoplasms, but not in lung cancer. The effects of AC stimulation required a permissive role for GIRK2 (or K(IR)3.2) potassium channels and were mimicked by raising extracellular potassium concentrations. Cell death could be achieved at higher AC frequencies (>75 Hz) or intensities (>8.5 microA); at lower frequencies/intensities, AC stimulation did not cause apoptotic cellular changes. Our findings implicate a role for transmembrane potassium fluxes via inward rectifier channels in the regulation of cell cycle. Brain stimulators currently used for the treatment of neurological disorders may thus also be used for the treatment of brain (or other) tumors.

Peripheral detection of S100beta during cardiothoracic surgery: what are we really measuring?

BACKGROUND: S100beta has been used in cardiac surgery to identify patients with postoperative neurologic complications. However, extracranial proteins may falsely elevate measurements of serum S100beta;. Objectives of this study were (1) to quantify S100 beta levels in serum and pericardial cavity during coronary artery bypass grafting (CABG), and (2) to identify proteins recognized by standard immunodetection as S100beta. METHODS: Systemic and pericardial cavity blood from 5 patients undergoing CABG were sampled before, during, and after cardiopulmonary bypass (CPB). A commercially available enzyme-linked immunosorbent assay (ELISA) kit was used to quantify S100beta. Two-dimensional gel electrophoresis, Western blot, and mass spectroscopy were also performed to identify S100a and other proteins. RESULTS: Mean S100beta levels measured by ELISA, systemic and pericardial cavity blood were (in ng x mL(-1)) 1.0 +/- 0.46 and 111 +/- 71 before CPB, 0.6 +/- 0.11 and 113 +/- 54 during CPB, and 1.7 +/- 0.64 and 101 +/- 42 after CPB, respectively. However, gel electrophoresis and Western blot analysis revealed proteins other than S100beta to be present in the pericardial cavity giving a falsely elevated serum S100a levels measured by immunoassay. Mass spectroscopy of identified potential candidates revealed contaminants including haptoglobin I precursor, apolipoprotein A-1 precursor, complement factor B precursor, and complement C3 precursor. CONCLUSIONS: S100beta immunoassays are not specific for S100a and give a falsely elevated reading due to contaminants from the surgical field that cross react with the assay's antibody. This does not appear to be an issue in nonsurgical patients. Caution must be exerted when evaluating immunodetection results for low-abundance proteins under conditions where contamination of the sample is likely.

Plasma and tissue determination of 4-methylpyrazole for pharmacokinetic analysis in acute adult and pediatric methanol/ethylene glycol poisoning.

Methanol and ethylene glycol poisoning may result in severe intoxication. The inhibition of alcohol dehydrogenase by ethanol or 4-methylpyrazole (4-MP, fomepizole) is fundamental to their treatment. 4-MP presents several advantages over ethanol therapy and has been recently approved as a specific antidote for both intoxications. The authors have developed a simple gas chromatographic method to determine blood and tissue 4-MP concentrations. This method has been validated for its reproducibility (between-day CV < 6.3%), sensitivity (LOD 0.2 microg/mL), and linearity. It has been used in 4 adult patients intoxicated by methanol and 1 child accidentally intoxicated by ethylene glycol. 4-MP was used for each patient, and its blood levels were monitored every 4 hours over 2-3 days for pharmacokinetics purposes. In the population studied, after repeated administration of 10 mg/kg fomepizole, plasma 4-MP concentrations ranged from 1.4 to 21.6 microg/mL, always above the active level of 0.8 microg/mL. The mean peak concentration observed in the 4 adult patients was 18.5 +/- 2.6 microg/mL and in the child was 18.9 +/- 2.2 microg/mL. Even though 4-MP is characterized by a dose-dependent kinetic profile, under our conditions of dosage and blood sampling, its elimination better fitted a first-order kinetic model. At steady state and without any concomitant therapies, the mean apparent elimination half-life was 14.5 +/- 3 hours. Elimination seemed faster in the child. A trend toward a progessive enhancement of the 4-MP elimination rate is suggested in the pediatric case, with the duration of the treatment resulting in a t(1/2) below 5 hours after 48 hours. One patient died, and samples of blood and hepatic tissue were removed simultaneously during autopsy for 4-MP analysis. Interestingly, when the plasma concentration was subtherapeutic (<1 microg/mL) the tissue concentration observed was still significant with 12 microg/g, supporting an intermittent scheme of administration.

Serum S100beta: a noninvasive marker of blood-brain barrier function and brain lesions.

BACKGROUND: S100beta protein is expressed constitutively by brain astrocytes. Elevated S100beta levels in cerebrospinal fluid and serum reported after head trauma, subarachnoid hemorrhage, and stroke were correlated with the extent of brain damage. Because elevated serum S100beta also was shown to indicate blood-brain barrier (BBB) dysfunction in the absence of apparent brain injury, it remains unclear whether elevation of serum levels of S100beta reflect BBB dysfunction, parenchymal damage, or both. METHODS: The authors conducted a prospective study of serum S100beta levels in six patients who underwent hyperosmotic BBB disruption (BBBD) with intraarterial chemotherapy for primary central nervous system lymphoma. In addition, 53 serum S100beta samples were measured in 51 patients who had a variety of primary or metastatic brain lesions at the time of neuroimaging. RESULTS: S100beta was correlated directly with the degree of clinical and radiologic signs of BBBD in patients who were enrolled in the hyperosmotic study. In patients with neoplastic brain lesions, gadolinium enhancement on a magnetic resonance image was correlated with elevated S100beta levels (n = 45 patients; 0.16 +/- 0.1 microg/L; mean +/- standard error of the mean) versus nonenhancing scans (n = 8 patients; 0.069 +/- 0.04 microg/L). Primary brain tumors (n = 8 patients; 0.12 +/- 0.08) or central nervous system metastases also presented with elevated serum S100beta levels (n = 27 patients; 0.14 +/- 0.34). Tumor volume was correlated with serum S100beta levels only in patients with vestibular schwannoma (n = 6 patients; 0.13 +/- 0.10 microg/L) but not in patients with other brain lesions. CONCLUSIONS: S100beta was correlated directly with the extent and temporal sequence of hyperosmotic BBBD, further suggesting that S100beta is a marker of BBB function. Elevated S100beta levels may indicate the presence of radiologically detectable BBB leakage. Larger prospective studies may better determine the true specificity of S100beta as a marker for BBB function and as an early detection or follow-up marker of brain tumors.

Blood-brain barrier damage induces release of alpha2-macroglobulin.

Blood-brain barrier (BBB) failure occurs in many neurological diseases and is caused in part by activation of proinflammatory factors including matrix metalloproteinases. Counterbalancing, "BBB protective" cascades have recently been described, including NO-mediated interleukin 6 release by glia. Interleukin 6 has been shown to trigger production of matrix metalloproteinase inhibitors such as alpha2-macroglobulin (alpha2M). We hypothesized that BBB failure may result in increased alpha(2)M release by perivascular astrocytes. This was initially tested in patients undergoing iatrogenic BBB disruption by hyperosmotic mannitol for intra-arterial chemotherapy of brain tumors. Serum samples revealed significantly increased levels of alpha2M at 4 h after BBB disruption by hyperosmotic mannitol. In parallel in vitro experiments, we observed a similar increase of alpha2M release by astrocytes under conditions mimicking BBB failure and perivascular edema. For both experiments, protein analysis was initially performed by bidimensional gel electrophoresis and mass spectrometry followed by Western blotting immunodetection. We conclude that, in addition to proinflammatory changes, BBB failure may also trigger protective release of alpha2M by perivascular astrocytes as well as peripheral source.