Simultaneous LC-MS/MS quantification of P-glycoprotein and cytochrome P450 probe substrates and their metabolites in DBS and plasma.

BACKGROUND: An LC-MS/MS method has been developed for the simultaneous quantification of P-glycoprotein (P-gp) and cytochrome P450 (CYP) probe substrates and their Phase I metabolites in DBS and plasma. P-gp (fexofenadine) and CYP-specific substrates (caffeine for CYP1A2, bupropion for CYP2B6, flurbiprofen for CYP2C9, omeprazole for CYP2C19, dextromethorphan for CYP2D6 and midazolam for CYP3A4) and their metabolites were extracted from DBS (10 µl) using methanol. Analytes were separated on a reversed-phase LC column followed by SRM detection within a 6 min run time. RESULTS: The method was fully validated over the expected clinical concentration range for all substances tested, in both DBS and plasma. The method has been successfully applied to a PK study where healthy male volunteers received a low dose cocktail of the here described P-gp and CYP probes. Good correlation was observed between capillary DBS and venous plasma drug concentrations. CONCLUSION: Due to its low-invasiveness, simple sample collection and minimal sample preparation, DBS represents a suitable method to simultaneously monitor in vivo activities of P-gp and CYP.

Spin-labeling coronary MR angiography with steady-state free precession and radial k-space sampling: initial results in healthy volunteers.

The purpose of this study was to prospectively compare free-breathing navigator-gated cardiac-triggered three-dimensional steady-state free precession (SSFP) spin-labeling coronary magnetic resonance (MR) angiography performed by using Cartesian k-space sampling with that performed by using radial k-space sampling. A new dedicated placement of the two-dimensional selective labeling pulse and an individually adjusted labeling delay time approved by the institutional review board were used. In 14 volunteers (eight men, six women; mean age, 28.8 years) who gave informed consent, signal-to-noise ratio (SNR), contrast-to-noise ratio (CNR), vessel sharpness, vessel length, and subjective image quality were investigated. Differences between groups were analyzed with nonparametric tests (Wilcoxon, Pearson chi2). Radial imaging, as compared with Cartesian imaging, resulted in a significant reduction in the severity of motion artifacts, as well as an increase in SNR (26.9 vs 12.0, P < .05) in the coronary arteries and CNR (23.1 vs 8.8, P < .05) between the coronary arteries and the myocardium. A tendency toward improved vessel sharpness and vessel length was also found with radial imaging. Radial SSFP imaging is a promising technique for spin-labeling coronary MR angiography.

Identification of early metabolic markers of various degrees of ischemia in the mouse brain by localized H-1 magnetic resonance spectroscopy

Objectives: Magnetic resonance (MR) imaging and spectroscopy (MRS) allow the establishment of the anatomical evolution and neurochemical profiles of ischemic lesions. The aim of the present study was to identify markers of reversible and irreversible damage by comparing the effects of 10-mins middle cerebral artery occlusion (MCAO), mimicking a transient ischemic attack, with the effects of 30-mins MCAO, inducing a striatal lesion. Methods: ICR-CD1 mice were subjected to 10-mins (n = 11) or 30-mins (n = 9) endoluminal MCAO by filament technique at 0 h. The regional cerebral blood flow (CBF) was monitored in all animals by laser- Doppler flowmetry with a flexible probe fixed on the skull with < 20% of baseline CBF during ischemia and > 70% during reperfusion. All MR studies were carried out in a horizontal 14.1T magnet. Fast spin echo images with T2-weighted parameters were acquired to localize the volume of interest and evaluate the lesion size. Immediately after adjustment of field inhomogeneities, localized 1H MRS was applied to obtain the neurochemical profile from the striatum (6 to 8 microliters). Six animals (sham group) underwent nearly identical procedures without MCAO. Results: The 10-mins MCAO induced no MR- or histologically detectable lesion in most of the mice and a small lesion in some of them. We thus had two groups with the same duration of ischemia but a different outcome, which could be compared to sham-operated mice and more severe ischemic mice (30-mins MCAO). Lactate increase, a hallmark of ischemic insult, was only detected significantly after 30-mins MCAO, whereas at 3 h post ischemia, glutamine was increased in all ischemic mice independently of duration and outcome. In contrast, glutamate, and even more so, N-acetyl-aspartate, decreased only in those mice exhibiting visible lesions on T2-weighted images at 24 h. Conclusions: These results suggest that an increased glutamine/glutamate ratio is a sensitive marker indicating the presence of an excitotoxic insult. Glutamate and NAA, on the other hand, appear to predict permanent neuronal damage. In conclusion, as early as 3 h post ischemia, it is possible to identify early metabolic markers manifesting the presence of a mild ischemic insult as well as the lesion outcome at 24 h.

Selective three-dimensional visualization of the coronary arterial lumen using arterial spin tagging.

Conventional coronary magnetic resonance angiography (MRA) techniques display the coronary blood-pool along with the surrounding structures, including the myocardium, the ventricular and atrial blood-pool, and the great vessels. This representation of the coronary lumen is not directly analogous to the information provided by x-ray coronary angiography, in which the coronary lumen displayed by iodinated contrast agent is seen. Analogous "luminographic" data may be obtained using MR arterial spin tagging (projection coronary MRA) techniques. Such an approach was implemented using a 2D selective "pencil" excitation for aortic spin tagging in concert with a 3D interleaved segmented spiral imaging sequence with free-breathing, and real-time navigator technology. This technique allows for selective 3D visualization of the coronary lumen blood-pool, while signal from the surrounding structures is suppressed.

Three-dimensional high-resolution fast spin-echo coronary magnetic resonance angiography.

Due to SNR constraints, current "bright-blood" 3D coronary MRA approaches still suffer from limited spatial resolution when compared to conventional x-ray coronary angiography. Recent 2D fast spin-echo black-blood techniques maximize signal for coronary MRA at no loss in image spatial resolution. This suggests that the extension of black-blood coronary MRA with a 3D imaging technique would allow for a further signal increase, which may be traded for an improved spatial resolution. Therefore, a dual-inversion 3D fast spin-echo imaging sequence and real-time navigator technology were combined for high-resolution free-breathing black-blood coronary MRA. In-plane image resolution below 400 microm was obtained. Magn Reson Med 45:206-211, 2001.

Free-breathing renal magnetic resonance angiography with steady-state free-precession and slab-selective spin inversion combined with radial k-space sampling and water-selective excitation.

The impact of radial k-space sampling and water-selective excitation on a novel navigator-gated cardiac-triggered slab-selective inversion prepared 3D steady-state free-precession (SSFP) renal MR angiography (MRA) sequence was investigated. Renal MRA was performed on a 1.5-T MR system using three inversion prepared SSFP approaches: Cartesian (TR/TE: 5.7/2.8 ms, FA: 85 degrees), radial (TR/TE: 5.5/2.7 ms, FA: 85 degrees) SSFP, and radial SSFP combined with water-selective excitation (TR/TE: 9.9/4.9 ms, FA: 85 degrees). Radial data acquisition lead to significantly reduced motion artifacts (P < 0.05). SNR and CNR were best using Cartesian SSFP (P < 0.05). Vessel sharpness and vessel length were comparable in all sequences. The addition of a water-selective excitation could not improve image quality. In conclusion, radial k-space sampling reduces motion artifacts significantly in slab-selective inversion prepared renal MRA, while SNR and CNR are decreased. The addition of water-selective excitation could not improve the lower CNR in radial scanning.

A novel interferon-gamma regulated human melanoma-associated antigen, gp33-38, defined by monoclonal antibody Me14-D12. II. Molecular cloning of a genomic probe.

The human Me14-D12 antigen is a cell surface glycoprotein regulated by interferon-gamma (IFN-gamma) on tumor cell lines of neuroectodermal origin. It consists of two non-convalently linked subunits with apparent mol. wt sizes of 33,000 and 38,000. Here we describe the molecular cloning of a genomic probe for the Me14-D12 gene using the gene transfer approach. Mouse Ltk- cells were stably cotransfected with human genomic DNA and the Herpes Simplex virus thymidine kinase (TK) gene. Primary and secondary transfectants expressing the Me14-D12 antigen were isolated after selection in HAT medium by repeated sorting on a fluorescence activated cell sorter (FACS). A recombinant phage harboring a 14.3 kb insert of human DNA was isolated from a genomic library made from a positive secondary transfectant cell line. A specific probe derived from the phage DNA insert allowed the identification of two mRNAs of 3.5 kb and 2.2 kb in primary and secondary L cell transfectants, as well as in human melanoma cell lines expressing the Me14-D12 antigen. The regulation of Me14-D12 antigen by INF-gamma was retained in the L cell transfectants and could be detected both at the level of protein and mRNA expression.

An improved synthesis of dansylated α-galactosylceramide and its use as a fluorescent probe for the monitoring of glycolipid uptake by cells.

A highly efficient synthesis of the biologically important fluorescent probe dansyl α-GalCer is presented. Key in our strategy is the incorporation of the fluorescent dansyl group at an early stage in the synthesis to facilitate in the monitoring and purification of intermediates via TLC and flash column chromatography, respectively, and the use of a high yielding α-selective glycosylation reaction between the phytosphingosine lipid and a galactosyl iodide donor. The ability of dansyl α-GalCer to activate iNKT cells and to serve as a fluorescent marker for the uptake of glycolipid by dendritic cells is also presented.

Continuous arterial spin labeling of mouse cerebral blood flow using an actively-detuned two-coil system at 9.4T.

Among numerous magnetic resonance imaging (MRI) techniques, perfusion MRI provides insight into the passage of blood through the brain's vascular network non-invasively. Studying disease models and transgenic mice would intrinsically help understanding the underlying brain functions, cerebrovascular disease and brain disorders. This study evaluates the feasibility of performing continuous arterial spin labeling (CASL) on all cranial arteries for mapping murine cerebral blood flow at 9.4 T. We showed that with an active-detuned two-coil system, a labeling efficiency of 0.82 ± 0.03 was achieved with minimal magnetization transfer residuals in brain. The resulting cerebral blood flow of healthy mouse was 99 ± 26 mL/100g/min, in excellent agreement with other techniques. In conclusion, high magnetic fields deliver high sensitivity and allowing not only CASL but also other MR techniques, i.e. (1)H MRS and diffusion MRI etc, in studying murine brains.

Oral administration of a low dose of midazolam (75 microg) as an in vivo probe for CYP3A activity.

OBJECTIVE: We investigated whether the oral administration of a low dose (75 micro g) of midazolam, a CYP3A probe, can be used to measure the in vivo CYP3A activity. METHODS: Plasma concentrations of midazolam, 1'OH-midazolam and 4'OH-midazolam were measured after the oral administration of 7.5 mg and 75 micro g midazolam in 13 healthy subjects without medication, in four subjects pretreated for 2 days with ketoconazole (200 mg b.i.d.), a CYP3A inhibitor, and in four subjects pretreated for 4 days with rifampicin (450 mg q.d.), a CYP3A inducer. RESULTS: After oral administration of 75 micro g midazolam, the 30-min total (unconjugated + conjugated) 1'OH-midazolam/midazolam ratios measured in the groups without co-medication, with ketoconazole and with rifampicin were (mean+/-SD): 6.23+/-2.61, 0.79+/-0.39 and 56.1+/-12.4, respectively. No side effects were reported by the subjects taking this low dose of midazolam. Good correlations were observed between the 30-min total 1'OH-midazolam/midazolam ratio and midazolam clearance in the group without co-medication (r(2)=0.64, P<0.001) and in the three groups taken together (r(2)=0.91, P<0.0001). Good correlations were also observed between midazolam plasma levels and midazolam clearance, measured between 1.5 h and 4 h. CONCLUSION: A low oral dose of midazolam can be used to phenotype CYP3A, either by the determination of total 1'OH-midazolam/midazolam ratios at 30 min or by the determination of midazolam plasma levels between 1.5 h and 4 h after its administration.

Renal arteries: navigator-gated balanced fast field-echo projection MR angiography with aortic spin labeling: initial experience.

A cardiac-triggered free-breathing three-dimensional balanced fast field-echo projection magnetic resonance (MR) angiographic sequence with a two-dimensional pencil-beam aortic labeling pulse was developed for the renal arteries. For data acquisition during free breathing in eight healthy adults and seven consecutive patients with renal artery disease, real-time navigator technology was implemented. This technique allows high-spatial-resolution and high-contrast renal MR angiography and visualization of renal artery stenosis without exogenous contrast agent or breath hold. Initial promising results warrant larger clinical studies.

Free-breathing renal MR angiography with steady-state free-precession (SSFP) and slab-selective spin inversion: initial results.

BACKGROUND: The aim of our study was the investigation of a novel navigator-gated three-dimensional (3D) steady-state free-precession (SSFP) sequence for free-breathing renal magnetic resonance angiography (MRA) without contrast medium, and to examine the advantage of an additional inversion prepulse for improved contrast. METHODS: Eight healthy volunteers (mean age 29 years) and eight patients (mean age 53 years) were investigated on a 1.5 Tesla MR system (ACS-NT, Philips, Best, The Netherlands). Renal MRA was performed using three navigator-gated free-breathing cardiac-triggered 3D SSFP sequences [repetition time (TR) = 4.4 ms, echo time (TE) = 2.2 ms, flip angle 85 degrees, spatial resolution 1.25 x 1.25 x 4.0 mm(3), scanning time approximately 1 minute 30 seconds]. The same sequence was performed without magnetization preparation, with a non-slab selective and a slab-selective inversion prepulse. Signal-to-noise ratio (SNR), contrast-to-noise (CNR) vessel length, and subjective image quality were compared. RESULTS: Three-dimensional SSFP imaging combined with a slab-selective inversion prepulse enabled selective and high contrast visualization of the renal arteries, including the more distal branches. Standard SSFP imaging without magnetization preparation demonstrated overlay by veins and renal parenchyma. A non-slab-selective prepulse abolished vessel visualization. CNR in SSFP with slab-selective inversion was 43.6 versus 10.6 (SSFP without magnetization preparation) and 0.4 (SSFP with non-slab-selective inversion), P < 0.008. CONCLUSION: Navigator-gated free-breathing cardiac-triggered 3D SSFP imaging combined with a slab-selective inversion prepulse is a novel, fast renal MRA technique without the need for contrast media.

Specific delineation of BK polyomavirus in kidney tissue with a digoxigenin-labeled DNA probe.

The detection of BK polyomavirus (BK virus, BKV) in kidney tissue is hampered by nonspecificity of antibodies suited to immunohistochemistry, and nonspecific background with in situ hybridization. The biotin-labeled DNA probe that is commercially available (Enzo Life Sciences, Inc.) shows good signal, but the intrinsic background in kidney tissue is high. We determined that the intrinsic background is due to endogenous biotin or biotin-binding activity in the renal tubular epithelium. Neither antibody blocking procedures nor an avidin/biotin block were entirely satisfactory for eliminating this background staining. We developed a digoxigenin-labeled DNA probe, and protocol, for detecting BK virus in formalin-fixed, paraffin embedded, kidney tissue obtained at autopsy. The hybridization signal is strong and there is no perceptible background staining. Eleven negative control kidneys all failed to hybridize. Conditions for low stringency hybridization may be employed, detecting both the related JC polyomavirus and BKV. Alternatively, high stringency hybridization conditions may be utilized, detecting BKV only. BK associated tubular necrosis is clearly demonstrated in two cases of BK nephritis.