Prenatal exposure to valproic acid during pregnancy and limb deficiencies: A case‐control study

We conducted a case‐control study using data from the Spanish Collaborative Study of Congenital Malformations (ECEMC) on the relationship between prenatal exposure to valproic acid (VPA) and the presence of limb deficiencies in newborn infants. Among a total of 22,294 consecutive malformed infants (once we excluded genetic syndromes) and 21,937 control infants with specified data on antiepileptic drugs during gestation, 57 malformed infants and 10 control infants were exposed to VPA during the first trimester of pregnancy. Of the total of malformed infants exposed to VPA, 36.8% (21/57) presented with congenital limb defects of different types (including overlapping digits, talipes, clubfoot, clinodactyly, arachnodactyly, hip dislocation, pre‐ and postaxial polydactyly, etc.), three of them having limb deficiencies. The result of the case‐control analysis shows a risk for limb deficiencies of odds ratio = 6.17 [confidence interval (CI) 1.28–29.66, P = 0.023], after controlling for potential confounder factors. If we consider that in our population the prevalence at birth of this type of defect is 6.88 per 10,000 livebirths (95% CI 6.43–7.36) we can estimate that the risk for women treated with VPA of having a baby with limb deficiencies would be around 0.42%. The limb deficiencies in the three patients exposed to VPA were the following: the first case was a newborn infant with hypoplasia of the left hand, the second patient was a newborn infant with unilateral forearm defect and hypoplastic first metacarpal bone in the left hand, and the third patient presented with short hands with hypoplastic first metacarpal bone, absent and hypoplastic phalanges, retrognathia, facial asymmetry, hypospadias, teleangiectatic angioma in skull, and hypotonia. Am. J. Med. Genet. 90:376–381, 2000. © 2000 Wiley‐Liss, Inc.     

1H NMR and hyperpolarized 13C NMR assays of pyruvate–lactate: a comparative study

Pyruvate–lactate exchange is mediated by the enzyme lactate dehydrogenase (LDH) and is central to the altered energy metabolism in cancer cells. The measurement of exchange kinetics using hyperpolarized ¹³C NMR has provided a biomarker of response to novel therapeutics. However, the observable signal is restricted to the exchanging hyperpolarized ¹³C pools and the endogenous pools of ¹²C‐labelled metabolites are invisible in these measurements. In this study, we investigated an alternative in vitro ¹H NMR assay, using [3‐¹³C]pyruvate, and compared the measured kinetics with a hyperpolarized ¹³C NMR assay, using [1‐¹³C]pyruvate, under the same conditions in human colorectal carcinoma SW1222 cells. The apparent forward reaction rate constants (k_PL) derived from the two assays showed no significant difference, and both assays had similar reproducibility (k_PL = 0.506 ± 0.054 and k_PL = 0.441 ± 0.090 nmol/s/10⁶ cells; mean ± standard deviation; n = 3); ¹H, ¹³C assays, respectively). The apparent backward reaction rate constant (k_LP) could only be measured with good reproducibility using the ¹H NMR assay (k_LP = 0.376 ± 0.091 nmol/s/10⁶ cells; mean ± standard deviation; n = 3). The ¹H NMR assay has adequate sensitivity to measure real‐time pyruvate–lactate exchange kinetics in vitro, offering a complementary and accessible assay of apparent LDH activity. Copyright © 2013 John Wiley & Sons, Ltd.     

Ursodeoxycholic acid treatment of hepatic steatosis: a (13)C NMR metabolic study

Ursodeoxycholic acid (UDCA) is commonly used for the treatment of hepatobiliary disorders. In this study, we tested whether a 4‐week treatment with this bile acid (12–15 mg/kg/day) could improve hepatic fatty acid oxidation in obese Zucker rats – a model for nonalcoholic fatty liver disease and steatosis. After 24 h of fasting, livers were perfused with physiological concentrations of [U‐¹³C]nonesterified fatty acids and [3‐¹³C]lactate/[3‐¹³C]pyruvate. Steatosis was associated with abundant intracellular glucose, lactate, alanine and methionine, and low concentrations of choline and betaine. Steatotic livers also showed the highest output of glucose and lactate. Glucose and glycolytic products were mostly unlabeled, indicating active glycogenolysis and glycolysis after 24 h of fasting. UDCA treatment resulted in a general amelioration of liver metabolic abnormalities with a decrease in intracellular glucose and lactate, as well as their output. Hepatic betaine and methionine were also normalized after UDCA treatment, suggesting the amelioration of anti‐oxidative defenses. Choline levels were not affected by the bile acid, which may indicate a deficient synthesis of very‐low‐density lipoproteins. The percentage contribution of [U‐¹³C]nonesterified fatty acids to acetyl‐coenzyme A entering the tricarboxylic acid (TCA) cycle was significantly lower in livers from Zucker obese rats relative to control rats: 23.1 ± 4.9% versus 44.1 ± 2.7% (p < 0.01). UDCA treatment did not alter significantly fatty acid oxidation in control rats, but improved significantly oxidation in Zucker obese rats to 46.0 ± 6.1% (p > 0.05), comparable with control group values. The TCA cycle activity subsequent to fatty acid oxidation was reduced in steatotic livers and improved when UDCA was administered (0.24 ± 0.04 versus 0.37 ± 0.05, p = 0.05). We further suggest that the mechanism of action of UDCA is either related to the activity of the farnesoid receptor, or to the amelioration of the anti‐oxidative defenses and cell nicotinamide adenine dinucleotide (NAD⁺/NADH) ratio, favoring TCA cycle activity and β‐oxidation. Copyright © 2011 John Wiley & Sons, Ltd.     

Ultrashort‐TE MRI longitudinal study and characterization of a chronic model of asthma in mice: inflammation and bronchial remodeling assessment

Asthma is a chronic disease characterized by bronchial hyperresponsiveness (BHR), bronchial inflammation and remodeling. The great improvements in ¹H MRI ultrashort‐TE (UTE) sequences in the last decade have allowed lung images with high‐resolution and good signal‐to‐noise ratio to be obtained in parenchymal tissues. In this article, we present a UTE ¹H MRI high‐resolution study of a chronic model of asthma in mice with the aim to longitudinally assess the main features of asthma using a fully noninvasive approach. Balb/c mice (n = 6) were sensitized with ovalbumin over a period of 75 days. The control group (n = 3) received normal saline on the same days. MRI acquisitions were performed on days 0, 38 and 78 to study the inflammatory volumes and bronchial remodeling (peribronchial signal intensity index, PBSI). Plethysmographic studies were performed on days 0, 39 and 79 to assess BHR to methacholine using the enhanced pause (Penh) ratio. The average inflammatory volume measured by MRI in the ovalbumin group (15.6 ± 2.4 μL) was increased significantly relative to control mice (–0.3 ± 0.7 μL) on day 38. The inflammatory volume was larger (34.2 ± 3.1 μL) on day 78 in the ovalbumin group. PBSI was significantly higher in the ovalbumin group on day 78 (1.53 ± 0.08) relative to the control group (1.16 ± 0.10), but not on day 38. After sensitization, asthmatic mice presented BHR to methacholine on days 39 and 79. Penh ratios correlated significantly with the inflammatory volume on day 39 and with the PBSI on day 79. This study shows, for the first time, that high‐resolution UTE ¹H MRI of the lungs may allow the noninvasive quantification of peribronchial eosinophilic inflammation with airways occlusion by mucus and of bronchial remodeling in a murine asthma model that correlates with functional parameters. Copyright © 2013 John Wiley & Sons, Ltd.     

Valproic acid therapy decreases serum 25-hydroxyvitamin D level in female infants and toddlers with epilepsy-a pilot longitudinal study

To evaluate if valproic acid(VPA)therapy is associated with vitamin D deficiency among infants and toddlers with epilepsy,a cross-sectional clinical study was conducted in 25 children with epilepsy taking VPA.Blood levels of calcium,phosphorus,alkaline phosphatase,and 25-hydroxy vitamin D[25(OH)D]and plasma VPA level were measured at 1-to 3-month intervals.At the initial and final measurements,vitamin D deficiency or insufficiency was recognized in 8(32%)and 12(42%),respectively.In girls,a decreasing trend in serum25(OH)D levels(P<0.05)was observed.Polytherapy had a significant negative effect on the longitudinal change of 25(OH)D(P<0.05)in girls.In conclusion,our study indicates that a high proportion of girls after VPA therapy had hypovitaminosis D.     

1H NMR spectroscopic method for diagnosis of malabsorption syndrome: a pilot study

Despite its well-documented limitations, colorimetry has been commonly used for the d-xylose test in the diagnosis of malabsorption syndrome (MAS). With a possibility of overcoming its limitations, the use of (1)H NMR spectroscopy for D-xylose test is explored herein. Urine samples from 35 adults with suspected MAS were obtained before and after oral ingestion of D-xylose. The diagnosis of MAS was based on fecal fat (72 h excretion using Van de Kamer's technique, normal < 7 g/24 h and/or Sudan III stain of spot stool specimen, normalor=1 g/5 g/5 h). In vitro experiments on the standard specimens of D-xylose were also performed independently using both methods. Colorimetry showed a lower value for the quantity of D-xylose excreted in urine than NMR [median 0.73 (0.17-1.89 g) vs 1.37 (0.17-3.23 g), respectively; p<0.0001, Wilcoxon's signed ranks test]. Colorimetry and NMR correctly diagnosed 11/12 and 10/12 (p=N.S.) patients with MAS and 14/23 and 20/23 (p<0.05) without MAS, respectively. Sensitivity and specificity of colorimetry and NMR were 91.6 and 60.7% vs 83.3 and 86.9%, respectively. In in vitro experiments, the values obtained for standard xylose using NMR showed a maximum error of 7%, whereas the colorimetric method showed 20%. The NMR method is simple and may be more accurate for the D-xylose absorption test. Colorimetry was found to be inferior as compared with NMR due to its low specificity.     

Stereochemical Pseudohexad 13C NMR Resonances and Regioregular Propylene/Ethylene‐1‐13C Copolymers

This paper reports the assignment of the ¹³C NMR resonances of the S_ββ and S_αγ methylenes observed in the spectra of copolymers of propylene with trace amounts of enriched ethylene‐1‐¹³C. The assignment is achieved by comparing the chemical shifts calculated by an empirical method (at the CH₂ stereochemical pseudohexad level), with the experimental spectra of regioregular copolymers with different stereochemical structure. The implications of the observed S_ββ and S_αγ resonances on the stereochemical polymerization mechanisms are also discussed.

     

Hyperpolarized [1,4‐13C]‐diethylsuccinate: a potential DNP substrate for in vivo metabolic imaging

The tricarboxylic acid (TCA) cycle performs an essential role in the regulation of energy and metabolism, and deficiencies in this pathway are commonly correlated with various diseases. However, the development of non‐invasive techniques for the assessment of the cycle in vivo has remained challenging. In this work, the applicability of a novel imaging agent, [1,4‐¹³C]‐diethylsuccinate, for hyperpolarized ¹³C metabolic imaging of the TCA cycle was explored. In vivo spectroscopic studies were conducted in conjunction with in vitro analyses to determine the metabolic fate of the imaging agent. Contrary to previous reports (Zacharias NM et al. J. Am. Chem. Soc. 2012; 134: 934–943), [¹³C]‐labeled diethylsuccinate was primarily metabolized to succinate‐derived products not originating from TCA cycle metabolism. These results illustrate potential issues of utilizing dialkyl ester analogs of TCA cycle intermediates as molecular probes for hyperpolarized ¹³C metabolic imaging. Copyright © 2014 John Wiley & Sons, Ltd.     

Increased interhemispheric resting‐state functional connectivity in functional dyspepsia: a pilot study

Recent brain imaging studies have emphasized the role of regional brain activity abnormalities in the pathophysiology of functional dyspepsia (FD). However, whether the functional connectivity between brain regions is changed, especially between the cerebral hemispheres, in patients with FD remains unknown. Thus, the present study aimed to examine the interhemispheric resting‐state functional connectivity (RSFC) changes in patients with FD. Resting‐state functional MRI (fMRI) was performed in 26 patients with FD and in 20 matched healthy controls. An interhemispheric RSFC map was obtained by calculating the Pearson correlation (Fisher Z transformed) between each pair of homotopic voxel time series for each subject. The between‐group difference in interhemispheric RSFC was then examined at global and voxelwise levels separately. The global difference in interhemispheric RSFC between groups was tested using the independent two‐sample t‐test. Voxelwise comparisons were carried out using a permutation‐based nonparametric test, and multiple comparisons across space were corrected using the threshold‐free cluster enhancement (TFCE) method. The results showed that patients with FD had higher global interhemispheric RSFC than healthy controls (p < 0.01). Furthermore, voxelwise analysis revealed that patients with FD had increased interhemispheric RSFC in brain regions including the anterior cingulate cortex, insula and thalamus (p < 0.01, TFCE corrected). Our findings provide preliminary evidence of interhemispheric correlation abnormalities in patients with FD and contribute to a better understanding of the pathophysiology of the disease. Copyright © 2012 John Wiley & Sons, Ltd.     

Complement C5a potentiates uric acid crystal‐induced IL‐1β production

Anaphylatoxin C5a released upon complement activation is associated with both acute and chronic inflammations such as gout. The pathogenesis of gout was identified as uric acid crystal deposition in the joints that activates inflammasome, leading to IL‐1β release. However, little is known about the interaction between complement activation and monosodium urate/uric acid (MSU) crystal‐induced inflammasome activation or IL‐1β production. Here, we report that MSU crystal‐induced proinflammatory cytokines/chemokines in human whole blood is predominantly regulated by C5a through its interaction with C5a receptor. C5a induces pro‐IL‐1β and IL‐1β production in human primary monocytes, and potentiates MSU or cholesterol crystals in IL‐1β production. This potentiation is caspase‐1 dependent and requires intracellular Ca²⁺ mobilization, K⁺ efflux, and cathepsin B activity. Our results provide insight into the role of C5a as an endogenous priming signal that is required for the initiation of uric acid crystal‐induced IL‐1β production. C5a could potentially be a therapeutic target together with IL‐1β antagonists for the treatment of complement‐dependent and inflammasome‐associated diseases.     

A calibration‐based approach to real‐time in vivo monitoring of pyruvate C1 and C2 polarization using the JCC spectral asymmetry

A calibration‐based technique for real‐time measurement of pyruvate polarization by partial integral analysis of the doublet from the neighbouring J‐coupled carbon is presented. In vitro calibration data relating the C₂ and C₁ asymmetries to the instantaneous C₁ and C₂ polarizations, respectively, were acquired in blood. The feasibility of using the in vitro calibration data to determine the instantaneous in vivo C₁ and C₂ polarizations was demonstrated in the analysis of rat kidney and pig heart spectral data. An approach for incorporating this technique into in vivo protocols is proposed. Copyright © 2013 John Wiley & Sons, Ltd.     

Radical Copolymerization of Vinylidene Fluoride with 8‐Bromo‐1H,1H,2H‐perfluorooct‐1‐ene: Microstructure,Crosslinking and Thermal Properties

An original synthesis of 8‐bromo‐1H,1H,2H‐perfluorooct‐1‐ene (BDFO) and its radical copolymerization with vinylidene fluoride (VDF), initiated by 2,5‐bis(tert‐butylperoxy)‐2,5‐dimethylhexane at 134 °C, are presented. The fluorinated bromoalkene was obtained by dehydrobromination of 1,8‐dibromo‐1H,1H,2H,2H‐perfluorooctane in a satisfactory yield. Although BDFO did not homopolymerize under radical initiation, it did copolymerize with VDF. The compositions of the resulting random type copolymers were calculated by means of ¹⁹F NMR spectroscopy and allowed the quantification of the respective amounts of both comonomers in the copolymers, showing good incorporation of the brominated monomer. Nevertheless, obtaining PVDF copolymers containing a high molar percentage of BDFO in good yields was difficult to achieve from initial molar ratios of BDFO higher than 9.2 mol‐%. Radical terpolymerization of VDF, BDFO and hexafluoropropene (HFP) was also successfully achieved. BDFO contents in these co‐ or terpolymers ranged from 3.6 to 12.2 mol‐%. The bromoalkene acted as a cure site monomer and the resulting poly(VDF‐co‐BDFO) copolymers were crosslinked via the bromine atom in the presence of a triallyl isocyanurate/peroxide system. The materials obtained led to more thermally stable copolymers than the uncured ones and their thermostabilities were compared to those of commercially available poly(VDF‐co‐HFP) copolymers crosslinked using diamines.

     

Combined treatment of pediatric high-grade glioma with the oncolytic viral strain MTH-68/H and oral valproic acid

The case of a 12-year-old boy with anaplastic astrocytoma of the left thalamus is reported. Postoperative irradiation and chemotherapy could not repress tumor progression; therefore, treatment was undertaken with an oncolytic virus, MTH-68/H, an attenuated strain of Newcastle disease virus (NDV), and valproic acid (VPA), an antiepileptic drug, which also has antineoplastic properties. This treatment resulted in a far-reaching regression of the thalamic glioma, but 4 months later a new tumor manifestation, an extension of the thalamic tumor, appeared in the wall of the IVth ventricle, which required a second neurosurgical intervention. Under continuous MTH-68/H - VPA administration the thalamic tumor remained under control, but the rhombencephalic one progressed relentlessly and led to the fatal outcome. In the final stage, a third tumor manifestation appeared in the left temporal lobe. The possible reasons for the antagonistic behavior of the three manifestations of the same type of glioma to the initially most successful therapy are discussed. The comparative histological study of the thalamic and rhombencephalic tumor manifestations revealed that MTH-68/H treatment induces, similar to in vitro observations, a massive apoptotic tumor cell decline. In the rhombencephalic tumor, in and around the declining tumor cells, NDV antigen could be demonstrated immunohistochemically, and virus particles have been found in the cytoplasm of tumor cells at electron microscopic investigation. These findings document that the oncolytic effect of MTH-68/H treatment is the direct consequence of virus presence and replication in the neoplastic cells. This is the first demonstration of NDV constituents in an MTH-68/H -treated glioma.     

Effect of Mixed Sol‐Gel Precursors on the Metal‐Oxo Phase Within a UV‐Curable Silicone Hybrid Material

Summary: An inorganic hybrid material was produced using a polysiloxane binder and metal‐oxo‐clusters which were derived from sol‐gel precursors. The continuous phase is composed of an elastomeric polysiloxane functionalized with cycloaliphatic epoxide groups. Pendant alkoxy silane groups serve as a coupling agent to form a network between the metal‐oxo‐clusters and the crosslinked polysiloxane. Methyl, cyclopentyl, and cyclohexyl substituted polysiloxanes were formulated with a variety of sol‐gel precursors (tetraethylorthosilicate oligomers, titanium(IV) iso‐propoxide, zirconium(IV) propoxide, and zinc acetate). Phase‐modulated FT‐IR, X‐ray photoelectron spectroscopy, solid state ²⁹Si NMR, and solid state ¹³C NMR were used to investigate the internal structure of the mixed metal‐oxo/silicon‐oxo colloids prepared within the cured polysiloxane matrix. Results indicate that the cycloaliphatic groups inhibit the complete hydrolysis and condensation of the pendant triethoxysilane group and reaction of the sol‐gel precursors. Analysis also indicated that the metal‐oxo‐clusters were comprised of mixed species of sol‐gel precursors resulting in hetero‐bonded (Si? O? Metal) colloids.

Possible network compositions of the cured material (a) specific metal‐oxo‐clusters, (b) hetero‐bonded metal‐oxo‐clusters, and (c) core/shell type metal‐oxo‐cluster configuration.     

1H and 13C NMR Study of the Intermediates Formed by (Cp‐R)2ZrCl2 Activation with MAO and AlMe3/[CPh3][B(C6F5)4]. Correlation of Spectroscopic and Ethene Polymerization Data

Using ¹H and ¹³C NMR spectroscopy, cationic intermediates formed by activation of (Cp‐R)₂ZrCl₂ (R = nBu, tBu and 1,2,3‐Me₃) with MAO in toluene were monitored at Al/Zr ratios from 50 to 1 000. The catalysts (Cp‐R)₂ZrCl₂/AlMe₃/CPh₃⁺B(C₆F₅)₄^? (nBu, tBu and 1,2,3‐Me₃) were also studied for comparison of spectroscopic and polymerization data with MAO based systems. Complexes of type (Cp‐R)₂ZrMe⁺ ← Me^?‐Al?MAO ( IV ) with different Me‐MAO^? counter anions have been identified in the (Cp‐R)₂ZrCl₂/MAO systems at low Al/Zr ratios. At Al/Zr ratios of 200–1 000, the complex [(Cp‐R)₂Zr(μ‐Me)₂AlMe₂]⁺ Me‐MAO^? ( III ) dominates in all MAO‐based reaction systems. Ethene polymerization activity strongly depends on the Al/Zr ratio (Al/Zr = 200–1 000) for the systems (Cp‐nBu)₂ZrCl₂/MAO and (Cp‐tBu)₂ZrCl₂/MAO, while it is virtually constant in the same range of Al/Zr ratios for the catalytic system (Cp‐1,2,3‐Me₃)₂ZrCl₂/MAO. The data obtained are interpreted on the assumption that complex III is the actual precursor of active centers of polymerization in MAO based systems.

Formation of cationic intermediates by activation with MAO.     

Tract‐specific and age‐related variations of the spinal cord microstructure: a multi‐parametric MRI study using diffusion tensor imaging (DTI) and inhomogeneous magnetization transfer (ihMT)

Being able to finely characterize the spinal cord (SC) microstructure and its alterations is a key point when investigating neural damage mechanisms encountered in different central nervous system (CNS) pathologies, such as multiple sclerosis, amyotrophic lateral sclerosis or myelopathy. Based on novel methods, including inhomogeneous magnetization transfer (ihMT) and dedicated SC probabilistic atlas post‐processing, the present study focuses on the in vivo characterization of the healthy SC tissue in terms of regional microstructure differences between (i) upper and lower cervical vertebral levels and (ii) sensory and motor tracts, as well as differences attributed to normal aging. Forty‐eight healthy volunteers aged from 20 to 70 years old were included in the study and scanned at 3 T using axial high‐resolution T₂*‐w imaging, diffusion tensor imaging (DTI) and ihMT, at two vertebral levels (C2 and C5). A processing pipeline with minimal user intervention, SC segmentation and spatial normalization into a reference space was implemented in order to assess quantitative morphological and structural parameters (cross‐sectional areas, scalar DTI and MT/ihMT metrics) in specific white and gray matter regions of interest. The multi‐parametric MRI metrics collected allowed upper and lower cervical levels to be distinguished, with higher ihMT ratio (ihMTR), higher axial diffusivity (λ_∥) and lower radial diffusivity (λ_⊥) at C2 compared with C5. Significant differences were also observed between white matter fascicles, with higher ihMTR and lower λ_∥ in motor tracts compared with posterior sensory tracts. Finally, aging was found to be associated with significant metric alterations (decreased ihMTR and λ_∥). The methodology proposed here, which can be easily transferred to the clinic, provides new insights for SC characterization. It bears great potential to study focal and diffuse SC damage in neurodegenerative and demyelinating diseases. Copyright © 2016 John Wiley & Sons, Ltd.     

Nanocomposites Based on Layered Silicates and Epoxy Resins: Measurement of Clay Dispersion and Exfoliation using TEM,Solid‐State NMR,and X‐ray Diffraction Methods

In an epoxy/layered silicate nanocomposite containing different organically modified layered silicats, the effect of various organic modifications and the layered silicate content on the layer distances are investigated by transmission electron microscopy (TEM), X‐ray diffraction (XRD) measurements, and ¹H solid‐state NMR. TEM micrographs were evaluated using a semi‐quantitative method. The effect of paramagnetic impurities present in the clay is used to obtain overall sample information related to the degree of exfoliation by ¹H solid‐state NMR. A new model for determination of the degree of exfoliation by proton longitudinal relaxation rate measurements is developed.     

Combined use of filtered and edited 1H NMR spectroscopy to detect 13C‐enriched compounds in complex mixtures

In conventional metabolism and pharmacokinetic studies, radioactive isotopes are used to identify and quantify the breakdown products of xenobiotics. However, the stable isotope ¹³C provides a cheaper and less hazardous alternative. Metabolites of ¹³C‐enriched xenobiotics can be detected, quantified and identified by ¹³C‐filtered NMR spectroscopy. However, one obstacle to using ¹³C is its 1.1% natural abundance that produces a background signal in ¹³C‐filtered NMR spectra of crude biological extracts. The signal makes it difficult to distinguish between ¹³C‐enriched xenobiotics resonances from endogenous metabolites unrelated to the xenobiotic. This study proposes that the ¹³C background signal can be distinguished from resonances of ¹³C‐enriched xenobiotics by the absence of a ¹²C component in the xenobiotic. This is detected by combined analysis of ¹³C‐filtered and ‐edited NMR spectra. The theory underlying the approach is described and the method is demonstrated by the detection of sub‐microgram amounts of ¹³C‐enriched phenacetin in crude extracts of hepatocyte microsomes. Copyright © 2012 John Wiley & Sons, Ltd.