bufuralol , 
  1-hydroxybufuralol , 7-benzyloxy-4-trifluoromethylcoumarin ( bfc ) , 
  7-methoxy-4-trifluoromethylcoumarin ( mfc ) , 
  7-hydroxy-4-trifluoromethylcoumarin , and hydroxytolbutamide were purchased 
  from bd gentest ( cowley , uk ) .
midazolam , 1-hydroxymidazolam , and 
  4-hydroxymidazolam were kind gifts from roche applied science , and 
  1-hydroxymetoprolol and o - demethylmetoprolol were generous gifts from 
  astra hsle ( mlndal , sweden ) .
a targeting vector was constructed from an 18-kb dna fragment , 
  produced by fusing overlapping pcr fragments generated from mouse 129/ola 
  genomic dna ( supplemental fig .
a cassette , flanked by same 
  orientation loxp sites and containing a selectable marker ( neomycin ) , 
  driven by the herpes simplex thymidine kinase promoter , was cloned into a bcli 
  site in intron 1 , and a third loxp site was cloned into a kpni site 
  in intron 5 .
the construct was checked by pcr and sequencing and transfected 
  into gk129/1 embryonic stem cells by electroporation ; the embryonic stem cells 
  were subsequently cultured in 96-well plates under g418 selection . 
 
g418-resistant clones were screened for specific homologous recombination by 
  southern blot analysis , using bglii and an 800-bp pcr fragment generated using 
  5-ggcacaacaccaattatttgtc-3 and 
  5-gacagtccttaacacaagctc-3 as forward and reverse primers , 
  respectively .
two correctly targeted embryonic stem cell clones ( cytb 
 5 ) were expanded , injected into 
  c57bl/6 blastocysts , and transferred into pseudopregnant mice .
male chimeric 
  mice were bred to c57bl/6 mice , and heterozygous offspring were screened by 
  southern blot and multiplex pcr to confirm germ line transmission of the 
  cytb 5 genotype 
  ( supplemental fig .
1b ) using the following primer set : 1 ) forward 
  primer , 5-ccaatggtctctccttggtc-3;2 ) lox / neomycin 
  reverse primer , 5-caatagcagccagtcccttc-3 ; 3 ) wild - type reverse 
  primer , 5-gatggagttccccgatgat-3. mouse breeding and 
  maintenance 
cytb5 
  mice were crossed to produce homozygous 
  cytb5 mice and maintained by 
  random breeding on a 129p2  c57bl/6 genetic background . 
 
cytb5 mice were crossed with a 
  transgenic mouse line expressing cre recombinase under the control of the 
  hepatocyte - specific rat albumin promoter ( cre ) 
  ( 22 ) on a c57bl/6 background , 
  and 
  cytb5::cre 
  offspring were backcrossed with 
  cytb5 mice to generate 
  liver - specific microsomal cytochrome b5 conditional 
  knock - out mice ( hbn ; 
  cytb5::cre ) and 
  control ( wild - type , cytb5 ) 
  mice .
the presence of the cre transgene was determined as 
  previously described ( 23 ) .
all mice were maintained under standard animal house conditions , with free 
  access to food and water , and a 12-h light/12-h dark cycle .
all animal work 
  was carried out on male 10-week - old mice in accordance with the animal 
  scientific procedures act ( 1986 ) and after local ethical review . in vivo drug treatments
 hbn and wild - type mice were 
  administered the following drugs either concomitantly as a mixture or 
  individually : chlorzoxazone ( 5 mg / kg ) , metoprolol ( 2 mg / kg ) , midazolam ( 5 
  mg / kg ) , phenacetin ( 5 mg / kg ) , and tolbutamide ( 5 mg / kg ) , dissolved in mixture 
  buffer ( 5% ethanol , 5% dmso , 35% polyethylene glycol 200 , 40% phosphate 
  buffered saline , and 15% water ) , by intravenous injection or orally by 
  gavage .
preparation of microsomes  microsomes were prepared from 
  wild - type and hbn mouse tissues , using 0.30.5 g of tissue , by a 
  modified method of meehan et al . 
 
( 24 ) , using sonication instead 
  of mechanical homogenization 
  ( 25 ) .
por 
  activity was estimated by nadph - dependent cytochrome c reduction 
  ( 26 ) .
western blot analysis was carried out as 
  previously described using polyclonal antisera raised against human por 
  ( 27 ) ; rat cytochrome 
  b5 ; rat cyp2a1 , cyp2b1 , cyp2c6 , cyp3a1 , and cyp4a1 
  ( 28 ) ; or human full - length 
  cyp2a4 , cyp2d6 , and cyp2e1 
  ( 25 , 
  29 ) .
polyclonal antiserum to 
  rat cytochrome b5 reductase and a monoclonal antibody 
  raised against rat cyp1a1 were also 
  used.3 the polyclonal 
  antiserum to cytochrome b5 oxidoreductase was a kind gift 
  from dr .
immunoreactive proteins were detected using polyclonal goat anti - rabbit , 
  anti - mouse , or anti - sheep horseradish peroxidase immunoglobulins as secondary 
  antibodies ( dako , ely , uk ) and visualized using immobilon 
  chemiluminescent horseradish peroxidase substrate ( millipore , watford , uk ) and 
  a fujifilm las-3000 mini - imaging system ( fujifilm uk ltd . ) .
densitometric 
  analysis was performed using multi gauge version 2.2 software ( fujifilm uk 
  ltd . ) .
microsomal incubations were 
  carried out in triplicate in 50 mm hepes , ph 7.4 , 30 mm 
  mgcl2 containing mouse liver microsomes and substrate prewarmed to 
  37 c before initiation of the reaction by the addition of either nadph or 
  nadh to a final concentration of 0.5 or 1 mm , respectively .
assays were performed in a 
  final volume of 150 l using white 96-well plates and the following 
  substrate and microsome concentrations : bfc , 50 m substrate , 20 
  g of mouse liver microsomes ; efc , 40 m substrate , 15 g 
  of mouse liver microsomes ; mfc , 180 m of substrate , 15 g of 
  mouse liver microsomes ; ethoxyresorufin ( er ) and benzoxyresorufin ( br ) , 1 
  m substrate , 11.25 g of mouse liver microsomes .
reactions 
  were measured in real time for 3 min , using the recommended excitation and 
  emission wavelengths for each probe using a fluroskan ascent fl plate reading 
  fluorimeter ( labsystems , uk ) .
turnover rates were calculated using authentic 
  metabolite standards ( 7-hydroxy-4-trifluoromethylcoumarin for bfc , efc , and 
  mfc assays and resorufin for er and br assays ) .
nadh - mediated incubations for hplc or liquid chromatography - tandem mass 
  spectrometry ( lc - ms / ms ) analysis  nadh - mediated reactions were 
  performed in triplicate using the following conditions : bufuralol , 300 
  m ( final concentration ) , 20 g of mouse liver microsomes in 
  a final volume of 150 l for 6 min ; chlorzoxazone , 1 mm ( final 
  concentration ) and 20 g of mouse liver microsomes in a final volume of 150 
  l for 15 min ; midazolam , 50 m ( final concentration ) and 25 
  gof mouse liver microsomes in a final volume of 150 l for 9 min ; 
  metoprolol , 800 m ( final concentration ) and 30 g of mouse 
  liver microsomes in a final volume of 150 l for 60 min ; phenacetin , 200 
  m ( final concentration ) and 20 g of mouse liver microsomes 
  in a final volume of 100 l for 9 min ; tolbutamide , 800 m 
  ( final concentration ) and 30 g of mouse liver microsomes in a final volume 
  of 150 l for 60 min .
assays were stopped by the addition of either 0.5 
  assay volumes ( for bufuralol , chlorzoxazone , and tolbutamide assays ) or 1 
  assay volume ( for midazolam and phenacetin assays ) of ice - cold methanol and 
  incubated on ice for 10 min .
assays to determine the apparent 
  kinetic parameters were performed in triplicate with wild - type and hbn liver 
  microsomes under conditions of linearity for time and protein ( data not shown ) 
  using the same buffer / nadph conditions as described above , with the following 
  concentrations of substrates : chlorzoxazone , 101000 m ; 
  phenacetin , 1.7150 m ; midazolam , 0.975 
  m ; metoprolol , 101000 m ; tolbutamide , 
  101000 m ( 12 concentration points / determination ) . 
 
metabolites were detected by lc - ms / ms as described in the supplemental 
  materials .
the data generated were analyzed by nonlinear regression using the 
  michaelis - menten equation ( chlorzoxazone , phenacetin , midazolam , and 
  metoprolol ) , whereas the hill equation ( tolbutamide ) 
  ( equation 1 ) was used to 
  determine the kinetic parameters vmax , s50 , and 
  the hill coefficient ( n ) . 
 
( eq .  1)\documentclass[10pt]{article } 
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 \begin{document } 
 \begin{equation*}\;\;v=\frac{v_{{\mathrm{max}}}{\times}s^{n}}{s_{50}^{n}+s^{n}}\end{equation*}\end{document } 
  in vivo pharmacokinetics  whole blood ( 10 l ) was taken 
  from the tail vein at intervals after drug administration and transferred into 
  a tube containing heparin ( 10 l , 15 iu / ml ) .
internal standard solution ( 10 
  l ; 500 ng of caffeine and 500 ng of resorpine ) was added to each tube . 
 
protein precipitation was carried out by adding methanol ( 75 l ) , followed 
  by 8% sulfosalicylic acid ( 55 l ) .
samples were mixed for 1 min and 
  centrifuged at 13,000 rpm for 5 min , and the supernatant was analyzed by 
  hplc .
the range of concentrations for the standard curves was constructed for 
  quantifying blood levels by spiking blank blood samples with known amounts of 
  chlorzoxazone , metoprolol , midazolam , phenacetin , and tolbutamide .
extraction 
  and protein precipitation were carried out as outlined above for the test 
  samples .
analysis of in vitro and in vivo data  average rates of 
  metabolism were calculated for each triplicate incubation of mouse liver 
  microsomes from each genotype ( n = 6 ) , and these data were then used 
  to calculate p values using an unpaired t test ( available on 
  the world wide web ) .
a simple noncompartmental model was used to 
  calculate area under the curve ( auc ) , terminal half - life , maximum plasma 
  concentration ( cmax ) , and clearance .
details of assays and 
  separation conditions for hplc and lc - ms / ms are given in the supplemental 
  materials .
hepatic microsomal cytochrome b5 null mice  mice 
  nulled for hepatic microsomal cytochrome b5 
  ( cytb5::cre ) 
  were born at the expected mendelian ratio , developed normally , and displayed 
  no overt phenotype when compared with their wild - type littermates 
  ( cytb5 ) .
livers from adult hbn 
  mice looked similar to those of wild - type animals , with no gross changes in 
  general morphology or pathology noted . to establish whether hepatic microsomal 
  cytochrome b5 protein had been deleted specifically , 
 
microsomes from liver , lung , kidney , and small and large intestine were 
  immunoblotted for cytochrome b5 expression 
  ( fig .
cytochrome 
  b5 protein was virtually undetectable in microsomes from 
  the livers of hbn mice ; however , occasionally , a very faint band was observed 
  in some samples , possibly originating from liver cells other than hepatocytes . 
 
there were no discernible differences in cytochrome b5 
  expression between the hbn and wild - type mice in the other organs examined .
no 
  cytochrome b5 protein could be detected in microsomes from 
  the large intestine of either genotype ( data not shown ) . in order to investigate whether concomitant changes in p450 expression had 
  occurred ,
three representative liver microsomal samples of each genotype were 
  analyzed for p450 enzyme profiles , por , and cytochrome b5 
  reductase levels ( fig . 
 
although the expression of some p450s was slightly 
  elevated in the hbn mice , these changes were not statistically significant . 
 
cytochrome b5 reductase level was slightly increased when 
  the immunoreactive bands were quantified by densitometry 
  ( fig .
a slight 
  reduction in cyp2d expression was also observed . as can be seen from 
  supplemental fig .
2a , there was no difference observed in the amount 
  of spectrally active p450 in the hbn as compared with wild - type liver 
  microsomes , with both samples containing 0.15 nmol of p450/mg of 
  microsomal protein .
por protein levels were also essentially unchanged in hbn 
  compared with wild - type mice , and although a slight increase in por activity 
  in hbn mice was measured using cytochrome c as substrate ( 228 
   65 versus 180  26 nmol cytochrome c 
  reduced / min / mg ) , this change was not significant .
a , immunoblot analysis 
  for cytochrome b5 expression in liver , kidney , lung , and 
  small intestine microsomes ( 5 g of protein / lane ) from 
  cytb5::cre 
  ( hbn ) and cytb5 ( wild type ) , as detailed 
  under  experimental procedures .
 b , immunoblot analysis 
  for expression of cytochrome p450-dependent monooxygenase components in liver 
  microsomes ( 5 g of protein / lane ) from 
  cytb5::cre 
  ( hbn ) and cytb5 ( wild type ) , as 
  detailed under  experimental procedures .
wild - type expression was 
  fixed at 100% , and percentage difference was calculated for cytochrome 
  b5 liver null ( hbn ) samples .
a , immunoblot analysis 
  for cytochrome b5 expression in liver , kidney , lung , and 
  small intestine microsomes ( 5 g of protein / lane ) from 
  cytb5::cre 
  ( hbn ) and cytb5 ( wild type ) , as detailed 
  under  experimental procedures .
 b , immunoblot analysis 
  for expression of cytochrome p450-dependent monooxygenase components in liver 
  microsomes ( 5 g of protein / lane ) from 
  cytb5::cre 
  ( hbn ) and cytb5 ( wild type ) , as 
  detailed under  experimental procedures .
wild - type expression was 
  fixed at 100% , and percentage difference was calculated for cytochrome 
  b5 liver null ( hbn ) samples .
figure 2.nadph- and nadh - dependent cytochrome p450 activities in hepatic 
  microsomes from wild - type and microsomal cytochrome b5 
  hepatic null mice .
black bars 
  represent nadph - mediated , and white bars nadh - mediated activities , 
  respectively .
* , p  0.05 ; * * , p  0.005 ; * * * , 
  p  0.001 .
nadph- and nadh - dependent cytochrome p450 activities in hepatic 
  microsomes from wild - type and microsomal cytochrome b5 
  hepatic null mice .
black bars 
  represent nadph - mediated , and white bars nadh - mediated activities , 
  respectively . * ,
p  0.05 ; * * , p  0.005 ; * * * , 
  p  0.001 . in vitro cytochrome p450 activities
 six commonly used p450 
  model substrates were used to establish if there were any in vitro 
  differences in metabolism between liver microsomes from hbn and wild - type 
  mice : bfc ( cyp3a ) , mfc ( cyp2c ) , efc ( cyp1a/2b ) , er ( cyp1a1/2 , cyp1b1 ) , br 
  ( cyp2b/3a ) , and bufuralol ( cyp2d ) 
  ( 3036 ) . 
 
assays were performed at substrate concentrations of 5 times 
  km ( mouse ) , as defined by the literature , or data were 
  generated with recombinant mouse p450s ( data not shown ) . with nadph as 
  electron donor ,
significantly lower turnover rates were measured in hbn mice 
  relative to wild - type samples for all substrates tested .
the nadph - mediated 
  rates in hbn liver microsomes , expressed relative to wild - type samples , were 
  as follows : er ( 73% ) , bufuralol ( 44% ) , br ( 34% ) , bfc ( 19% ) , mfc ( 11% ) , and efc 
  ( 10% ) ( fig .
the addition of recombinant cytochrome b5 to hbn 
  microsomes led to a concentration - dependent increase in bfc or chlorzoxazone 
  metabolism , restoring activities to wild - type levels , demonstrating that the 
  change in metabolism was due to the absence of cytochrome 
  b5 .
nadh could support monooxygenase activity for all model substrates in the 
  wild - type samples , although the activity relative to nadph was highly 
  substrate - dependent , ranging from 4% ( br ) to 74% ( er ) 
  ( fig . 2 and supplemental table 
  3 ) .
all nadh - catalyzed reaction rates with the exception of br were 
  significantly lower in the hbn samples compared with wild type , with barely 
  detectable activities using bfc ( 0.66% ) , efc ( 0.79% ) , and mfc ( 0.28% ) as 
  substrates ( fig . 2 and 
  supplemental table 3 ) .
the metabolism of er and bufuralol was also reduced , to 
  52 and 28% of wild - type values , respectively .
the nadh - mediated in vitro turnovers of the probe drugs 
  chlorzoxazone ( cyp2e1 ) , metoprolol ( cyp2d ) , midazolam ( cyp2c , cyp3a ) , 
  tolbutamide ( cyp2c ) , and phenacetin ( cyp1a ) 
  ( 3741 ) 
  were also examined with liver microsomes from both hbn and wild - type mice . 
 
nadh supported the metabolism of all substrates examined in wild - type liver 
  microsomes , with the exception of tolbutamide .
rates observed were 601 
   152 ( chlorzoxazone 6-hydroxylation ) , 128  60 
  ( metoprolol o - demethylation ) , 17.6  0.2 ( metoprolol 
  -hydroxylation ) , 17.1  8.0 ( midazolam 1-hydroxylation ) , 
  9.1  3.8 ( midazolam 4-hydroxylation ) , and 244  63 pmol / min / mg 
  ( phenacetin hydroxylation ) .
these rates observed in wild - type liver microsomes 
  were significantly lower than those observed using nadph as co - factor , ranging 
  from 7 to 29% ( o - demethylmetoprolol and acetaminophen production , 
  respectively ) of the nadph rate .
furthermore , the nadh - dependent turnover 
  rates observed in hbn liver microsomes ( 177  64 ( chlorzoxazone 
  6-hydroxylation ) , 90  21 ( metoprolol o - demethylation ) , 
  7.6  2.2 ( metoprolol -hydroxylation ) , 4.3  2.0 
  ( midazolam 1-hydroxylation ) , 2.1  2.7 ( midazolam 
  4-hydroxylation ) , and 67  18 pmol / min / mg ( phenacetin hydroxylation ) ) 
  were significantly lower than the nadh - dependent wild - type rates 
  ( chlorzoxazone ( 29% reduction ) , metoprolol ( o - demethyl 71% , 
  -hydroxy 44% , acid 83% reduction ) , midazolam ( 1- and 4-hydroxy 
  25% reduction ) , and phenacetin ( 29% reduction ) ) .
figure 3.pharmacokinetic disposition of an intravenously administered p450 drug 
  mixture in wild - type and hepatic microsomal cytochrome b5 
  null mice .
a p450 drug mixture containing chlorzoxazone ( 5 mg / kg ) , 
  metoprolol ( 2 mg / kg ) , midazolam ( 5 mg / kg ) , phenacetin ( 5 mg / kg ) , and 
  tolbutamide ( 5 mg / kg ) was administered intravenously at the indicated doses to 
  wild - type ( black circles ) or hbn ( open circles ) mice , and 
  blood samples were taken at timed intervals to determine the in vivo 
  pharmacokinetic parameters of the parent compounds . * , p  0.05 ; 
  * * , p  0.005 ; * * * , p  0.001 , where
n = 
  5 . pharmacokinetic disposition of an intravenously administered p450 drug 
  mixture in wild - type and hepatic microsomal cytochrome b5 
  null mice .
a p450 drug mixture containing chlorzoxazone ( 5 mg / kg ) , 
  metoprolol ( 2 mg / kg ) , midazolam ( 5 mg / kg ) , phenacetin ( 5 mg / kg ) , and 
  tolbutamide ( 5 mg / kg ) was administered intravenously at the indicated doses to 
  wild - type ( black circles ) or hbn ( open circles ) mice , and 
  blood samples were taken at timed intervals to determine the in vivo 
  pharmacokinetic parameters of the parent compounds . * , p  0.05 ; 
  * * , p  0.005 ; * * * , p  0.001 , where n = 
  5 . in vitro kinetics  the apparent kinetic parameters of the 
  probe drugs using nadph as co - factor were determined in wild - type and hbn 
  liver microsomes , and metabolism of chlorzoxazone , phenacetin , midazolam , and 
  metoprolol followed standard michaelis - menten kinetics 
  ( table 1 ) . the michaelis 
  constant for chlorzoxazone 6-hydroxylation ,
midazolam 1-hydroxylation , 
  phenacetin oxidation , and metoprolol -hydroxylation were unchanged by 
  the removal of microsomal cytochrome b5 .
conversely , for 
  those reactions , the vmax was markedly reduced in the hbn 
  samples , ranging from a 2-fold ( phenacetin oxidation ) to a 6-fold ( metoprolol 
  -hydroxylation ) decrease . midazolam
4-hydroxylation and metoprolol 
  o - demethylation reactions again showed decreased 
  vmax values for the hbn samples ( 20- and 3-fold , 
  respectively ) ; however , the km values were marginally 
  affected , with the hbn samples exhibiting a 1.7-fold reduction in 
  km for midazolam 4-hydroxylation and a 1.8-fold increase 
  in km for metoprolol o - demethylation as compared 
  with wild type .
the data obtained for tolbutamide hydroxylation could not be 
  fitted using the michaelis - menten equation but did conform to the hill 
  equation .
interestingly , the hbn sample exhibited 68-fold higher 
  km for tolbutamide as well as a 5-fold reduction in 
  vmax as compared with wild type .
the hill coefficient for 
  both wild type and hbn were similar ( 0.38 versus 0.29 ) and indicative 
  of tolbutamide binding in a negatively cooperative manner .
the alternative of 
  a 2 km,2 vmax fit was considered for 
  these data ; however , when plotted in such a manner , the 
  km1 parameter for both wild type and hbn was subject to 
  such a large error as to render it meaningless . table 1kinetic analyses of the metabolism of probe drugs by hepatic microsomes 
  from hbn mice and wild - type controlsassays were performed in triplicate for each concentration of substrate . 
 
values given are from the fit of the curve as calculated using the 
  michaelis - menten equation ( grafit version 5 ( erithacus software , horley , uk ) ) . 
  for tolbutamide , the hill equation ( grafit version 5 ) was used to fit the 
  data , giving hill coefficients ( n ) of 0.38 and 0.29 for wild type and 
  hbn , respectively.substrate 
 metabolite 
 wild type 
 hbn 
 kmvmaxkmvmax mpmol / min / mg mpmol / min / mg chlorzoxazone 
   6-hydroxychlorzoxazone 
   47  9 
   1930  100 
   53  6 
   387  11 
   midazolam 
   1-hydroxymidazolam 
   2.1  0.3 
   389  15 
   2.0  0.3 
   116  4 
   midazolam 
   4-hydroxymidazolam 
   13.3  0.4 
   146  2 
   7.7  0.9 
   7.3  0.3 
   phenacetin 
   acetaminophen 
   5.6  1.0 
   133  6 
   4.1  0.7 
   77  3 
   metoprolol 
   -hydroxymetoprolol 
   84  7 
   483  14 
   99  9 
   76  3 
   metoprolol 
  o - desmethylmetoprolol 
   86  9 
   1650  2 
   157  14 
   531  21 
 
  tolbutamide 
 
  hydroxytolbutamide 
 
  11200  3400 
 
  211
 17 
 
  165  48 
 
  44  165 
   kinetic analyses of the metabolism of probe drugs by hepatic microsomes 
  from hbn mice and wild - type controls assays were performed in triplicate for each concentration of substrate . 
 
values given are from the fit of the curve as calculated using the 
  michaelis - menten equation ( grafit version 5 ( erithacus software , horley , uk ) ) . 
  for tolbutamide , the hill equation ( grafit version 5 )
was used to fit the 
  data , giving hill coefficients ( n ) of 0.38 and 0.29 for wild type and 
  hbn , respectively . to ensure that the observed changes in p450 activity were not a consequence 
  of perturbations of the phospholipid bilayer ,
fatty acid profile analysis was 
  carried out on endoplasmic reticulum membrane fractions from the livers of 
  both wild type and hbn mice .
in addition , hepatic lipid levels were unchanged , as 
  determined by oil red o staining ( supplemental fig .
3 ) . in vivo pharmacokinetics of probe drugs in wild - type and hbn 
  mice  we next investigated the effect of the absence of hepatic 
  microsomal cytochrome b5 on the in vivo 
  pharmacokinetics of the probe drugs in wild - type and hbn mice .
initially , mice 
  were administered chlorzoxazone , metoprolol , midazolam , tolbutamide , and 
  phenacetin either as an intravenous or oral mixture . previous work with these 
  drugs in wild - type mice , individually and as a mixture , had demonstrated that 
  simultaneous delivery did not result in altered pharmacokinetics as compared 
  with single administration ( not shown ) . following dosing of the drug cassette 
  to wild - type and hbn mice , quantification of individual drugs and their 
  metabolites was carried out by lc - ms / ms .
the complete set of pharmacokinetic 
  parameters for each drug is summarized in supplemental tables 4 ( intravenous ) 
  and 5 ( oral ) , and the elimination profiles of the drugs are shown in 
  fig .
following intravenous 
  administration of the drug mixture , wild - type mice showed a higher 
  concentration of parent drug in the blood at the early time points ( 060 
  min ) compared with the hbn mice for all drugs in the mixture .
in contrast , 
  from 2 h , the average concentration of parent drug was statistically much 
  lower in the wild - type than in hbn mice for most of the compounds and remained 
  so for the duration of the experiment .
pharmacokinetic analysis for each drug 
  showed that the clearance was consistently faster , and the auc was 
  consistently lower ( except in the case of midazolam ) in wild - type compared 
  with hbn mice .
furthermore , the terminal half - life for phenacetin , 
  tolbutamide , chlorzoxazone , and midazolam was statistically different between 
  the two lines , being significantly longer in hbn mice , increasing from 53 to 
  168 min , 197 to 321 min , 92 to 257 min , and 83 to 329 min , respectively 
  ( table 2 , 
  fig . 3 , and supplemental table 
  4 ) .
for metoprolol , although a 2.7-fold increase of the terminal half - life was 
  found in hbn mice , this did not reach statistical significance ( p = 
  0.1 ) .
table 2 in vivo pharmacokinetic data from hbn mice and wild - type 
  controls dosed with a drug mixtureadult male hbn and wt mice were administered a drug mixture intravenously ; 
  serial blood samples were collected over an 8-h period and analyzed for plasma 
  drug concentrations by lc - ms / ms and key pharmacokinetic parameters calculated . 
 
absolute oral bioavailability was calculated by dividing auc ( 0  
   , oral ) by auc ( 0   , intravenous ) ( supplemental tables 4 
  and 5).drug 
 terminal half - life ( intravenous ) 
 bioavailability ( % ) 
 wild typehbnwild typehbn midazolam 
   83  15 
   329  132 
   14 
   55 
   phenacetin 
   53  11 
   168  56 
   0.6 
   0.8 
   metoprolol 
   96  13 
   260  65 
   0.3 
   0.3 
   chlorzoxazone 
   92  19 
   257  76 
   3.7 
   1.9 
 
  tolbutamide 
 
  197  37 
 
  321  25 
 
  95 
  ( 154)a 
  47 
  ( 135)aacomparative data
where tolbutamide was dosed as a single agent . in vivo pharmacokinetic data from hbn mice and wild - type 
  controls
dosed with a drug mixture adult male hbn and wt mice were administered a drug mixture intravenously ; 
  serial blood samples were collected over an 8-h period and analyzed for plasma 
  drug concentrations by lc - ms / ms and key pharmacokinetic parameters calculated . 
 
absolute oral bioavailability was calculated by dividing auc ( 0  
   , oral ) by auc ( 0   , intravenous ) ( supplemental tables 4 
  and 5 ) . comparative data where tolbutamide was dosed as a single agent .
the major metabolites of metoprolol ( o - demethylmetoprolol ) , 
  phenacetin ( acetaminophen ) , and midazolam ( 1-oh midazolam ) were also measured 
  in the serum of hbn and wild - type mice after intravenous administration ; the 
  results are presented in supplemental fig .
interestingly , the 
  overall profiles of metabolite production in hbn mice were shifted to the 
  right , consistent with a delayed metabolism of the parent compound 
  ( fig . 3 and supplemental fig . 
 
tolbutamide and chlorzoxazone metabolites were below the limit 
  of detection at all time points .
oral pharmacokinetics  following oral administration of the 
  drug mixture , differences in the elimination profiles of all of the drugs were 
  observed between wild - type and hbn mice . in the case of midazolam ,
phenacetin , 
  and metoprolol , auc and cmax were consistently higher in hbn mice , 
  whereas the clearance was slower ( supplemental fig .
were significantly increased from 34 to 
  124 ming / ml and from 0.4 to 1.0 g / ml , respectively , whereas 
  clearance was decreased from 149 to 41 ml / min / kg ( supplemental table 5 ) .
the 
  phenacetin elimination profile also showed significant changes , with 
  cmax increasing from 0.06 to 0.09 g / ml in hbn and clearance 
  decreasing from 688 to 550 ml / min / kg .
although the phenacetin auc increased 
  from 1.5 to 1.8 ming / ml , this change was not significant . in the 
  case of metoprolol , similar trends were observed in cmax and auc ; 
  however , these did not reach statistical significance .
in contrast to the 
  observations above , the alterations in the pharmacokinetic parameters for 
  tolbutamide gave significantly lower auc and cmax values and a 
  ( nonsignificant ) higher clearance rate , relative to wild - type animals 
  ( supplemental fig . 4 and supplemental table 5 ) .
this unexpected effect 
  appeared to be a consequence of using the drug mixture ( see below ) . 
 
interestingly , although the terminal half - life for chlorzoxazone was 
  significantly decreased in hbn mice from 194.0 to 46 min , the decreased auc 
  and cmax and increased clearance in hbn mice were not significantly 
  different ( supplemental table 5 ) .
additionally , there were no significant 
  differences between hbn and wild - type mice in the plasma concentration of 
  chlorzoxazone at any of the time points analyzed ( supplemental fig .
administration of the probe drugs individually to hbn and wild - type mice 
  yielded essentially the same elimination profiles for phenacetin , metoprolol , 
  midazolam , and chlorzoxazone , compared with those obtained from use of the 
  drug mixture ( data not shown ) .
4a , inset ) , the elimination profiles were 
  found to be reversed in comparison with that obtained from the drug mixture 
  ( i.e. significantly increased auc and cmax and 
  significantly decreased clearance in hbn mice ) .
similar to data obtained with 
  the drug mixture administered intravenously , the pattern of metabolite 
  production in the hbn animals showed a delay in maximal levels of circulating 
  metabolites ( supplemental fig .
hbn 
  animals showed a 4-fold increase in midazolam bioavailability , whereas for 
  phenacetin , metoprolol , and chlorzoxazone , bioavailability was very low in the 
  wild - type animals ( < 4% ) and unaltered in the nulls . when dosed as part of 
  the drug mixture , tolbutamide bioavailability in the hbn mice
was decreased by 
  50% as compared with wild type ; however , when orally dosed as a single 
  agent and recalculated , both wild - type and hbn animals showed > 100% 
  bioavailability .
all previously published work on the effects of cytochrome 
  b5 on nadph - dependent p450 activity has been carried out 
  in vitro . in order to establish the in vivo role of this 
  hemoprotein in drug disposition ,
we have generated a mouse where this enzyme 
  has been conditionally inactivated in the liver . to exclude the possibility that a global deletion of microsomal cytochrome 
  b5 would be lethal ,
mice deleted for hepatic microsomal cytochrome b5 
  developed normally , with the 
  cytb5::cre 
  genotypes being found in mendelian proportions .
no alterations in liver 
  morphology , hepatic lipid content , or hepatic serum markers were observed .
the hepatic deletion of microsomal cytochrome b5 was 
  shown to be both specific and essentially complete by western blotting 
  ( fig .
1a ) and , 
  importantly , unlike the hepatic deletion of cytochrome p450 reductase 
  ( 23 , 
  42 ) , did not result in 
  significantly increased expression of hepatic p450 proteins 
  ( fig . 1 , b and 
  c ) .
these data indicate that at the time points studied , 
  a reduction in p450 activity , as a consequence of microsomal cytochrome 
  b5 deletion , is not compensated by an increase in p450 
  expression .
the use of a range of both model substrates and probe drugs 
  clearly demonstrated that the absence of microsomal cytochrome 
  b5 significantly attenuated p450-mediated metabolism 
  in vitro ( fig . 2 and 
  table 1 ) .
the extent of 
  suppression of enzyme activity varied markedly between substrates , as has been 
  previously reported ( 2 , 
  6 ) .
for all fluorocoumarin 
  substrates employed , there was a highly significant decrease in nadph - mediated 
  p450 metabolism in liver microsomes from the hbn mice ( as high as 90% for 
  efc ) , whereas for the two resorufin analogues and bufuralol , the change was 
  still significant ( at least 50% decrease for each compound ) but less marked . 
 
this variability in cytochrome b5 effects was further 
  illustrated by the use of a series of probe drugs , where the kinetic 
  parameters defining the in vitro metabolism were markedly changed 
  ( table 1 ) .
for all substrates , 
  with the exception of tolbutamide , the data followed michaelis - menten 
  kinetics , and km values were essentially unchanged in the 
  hbn samples .
this suggests that cytochrome b5 binding does 
  not cause a conformational change in the structure of the p450s responsible 
  for this metabolism .
in contrast , in both wild type and hbn samples , the data 
  for tolbutamide oxidation did not fit michaelis - menten kinetics , exhibiting 
  negatively cooperative binding .
furthermore , the removal of cytochrome 
  b5 had a pronounced effect on tolbutamide binding , 
  decreasing the km by 68-fold , indicating that the presence 
  of cytochrome b5 does affect tolbutamide binding .
in all cases , vmax values were substantially reduced by 
  the removal of cytochrome b5 ( range 1.720-fold 
  lower for acetaminophen and 4-hydroxymidazolam production , respectively ) , 
  implying that the presence of cytochrome b5 is critical 
  for optimal turnover in the p450s investigated .
interestingly , the effects of 
  cytochrome b5 were different dependent on the site of 
  metabolism ( e.g. with midazolam , the vmax for 
  oxidation at the 1- versus 4-hydroxy sites was 29 versus 5% 
  of the control , respectively ) .
a similar effect was also seen for metoprolol 
  oxidation ( table 1 ) , indicating 
  that cytochrome b5 effects show specificity for different 
  sites of oxidation .
these findings provide strong genetic evidence that the modulatory effects 
  of cytochrome b5 are both substrate- and p450-dependent 
  and that the activities of many p450 enzymes are affected by this protein . 
 
similar reductions in p450 activities ( 6070% ) have also been 
  demonstrated using antibodies to cytochrome b5 , both in 
  microsomal fractions and with purified protein 
  ( 43 , 
  44 ) .
furthermore , the addition 
  of recombinant membrane - bound cytochrome b5 to hbn liver 
  microsomes led to a concentration - dependent increase of catalytic activities 
  toward bfc and chlorzoxazone , indicating that the loss of activity in hbn 
  samples was indeed due to the absence of cytochrome b5 
  ( supplemental fig .
the in vitro data 
  generated from the probe drugs suggests that in general , the absence of 
  cytochrome b5 results in reduced rates of p450-mediated 
  metabolism .
it is interesting to note that in some cases the fluorocoumarin substrates 
  and tolbutamide nadh - mediated metabolism were essentially abolished in hbn 
  mice , whereas for the other compounds , residual activity remained 
  ( e.g. in the case of er or br ) , indicating the existence of an 
  alternative metabolic pathway involving nadh but independent of cytochrome 
  b5 ( fig . 
  2 ) .
one possible explanation for the residual activity could be 
  the presence of an alternate electron donor , such as the recently described 
  outer mitochondrial cytochrome b5 , which is derived from a 
  gene locus on chromosome 8 
  ( 45 , 
  46 ) and encodes for a protein 
  of 16.3 kda , slightly larger than microsomal cytochrome b5 
  at 15.2 kda and 45% identical at the amino acid level .
however , to date , 
  there is no evidence for the interaction of outer mitochondrial cytochrome 
  b5 with the microsomal p450 system .
recently , a novel , soluble , nad(p)h reductase ( nad(p)h cytochrome 
  b5 oxidoreductase ) , located in the lumen of the 
  endoplasmic reticulum , was reported 
  ( 47 ) , which contains both 
  cytochrome b5 and cytochrome b5 
  reductase domains . in order to confirm that this enzyme was not induced in the 
  hbn mice , and potentially compensating for the loss of microsomal cytochrome 
  b5
, we obtained antiserum to nad(p)h cytochrome 
  b5 oxidoreductase ; immunoblotting liver samples from 
  wild - type and hbn mice found no difference in expression levels ( data not 
  shown ) .
li and porter ( 48 ) 
  have also described a second microsomal reductase , which is active with 
  squalene monooxygenase and apparently capable of supporting up to 40% of the 
  activity of this enzyme , although the identity of this reductase and its 
  interactions with other enzymes remain unclear .
in addition , due to its role in fatty acid desaturation , it is possible 
  that changes in the phospholipid bilayer , as observed in 
  stearoyl - coa - desaturase null mice 
  ( 49 ) , are responsible for the 
  observed effects of cytochrome b5 on p450 metabolism . 
 
however , no changes in the fatty acid composition of endoplasmic reticulum 
  membranes from wild - type and hbn mice were found ( supplemental fig .
3 ) . in order to establish the in vivo effects of the hepatic 
  cytochrome b5 deletion , we administered a series of probe 
  drugs by both intravenous and oral routes to control and hbn mice .
these drugs 
  were chosen because they have been shown to be metabolized in a number of 
  species by different cytochrome p450s .
the interpretation of such experiments 
  is inevitably complex , particularly because in the mouse there is little 
  background historical information on the key factors that define their 
  pharmacokinetic parameters .
however , consistent with the in vitro 
  studies , in general , it was evident that the deletion of hepatic cytochrome 
  b5 caused marked changes in drug pharmacokinetics 
  independent of the route of administration . when administered intravenously ,
the terminal half - life was increased in 
  hbn mice for all drugs used ( table 
  2 ) .
this increase is consistent with decreased liver metabolism as 
  a consequence of hepatic microsomal cytochrome b5 
  deficiency and provides the first evidence that strongly supports the 
  conclusion that cytochrome b5 plays a role in in 
  vivo p450-mediated drug metabolism .
in addition , the profiles of 
  metabolite production for midazolam , phenacetin , and metoprolol were different 
  between hbn and wild - type mice ; for example , maximal metabolite levels of 
  1-hydroxymidazolam occurred 3 times more slowly in hbn mice than in 
  wild type ( 120 versus 40 min ) when dosed intravenously ( supplemental 
  fig .
these differences in metabolite production are consistent with the 
  in vitro kinetic data shown in 
  table 1 . for example , the 
  vmax values for 1-hydroxymidazolam were 3 
  times higher in wild type as compared with hbn .
no major differences in the 
  formation of the demethyl metabolite of metoprolol were observed at the early 
  time points between hbn and wild - type mice , despite major in vitro 
  differences ( supplemental fig .
the interpretation of 
  pharmacokinetic parameters is more complex due to factors such as the 
  absorption across gastrointestinal tract and hepatic blood flow .
this added 
  complexity was manifest in the fact that for some drugs , such as midazolam , 
  microsomal cytochrome b5 deficiency and the consequent 
  reduction in hepatic metabolism can lead to increased bioavailability 
  ( table 2 ) as well as a 
  decreased rate of elimination .
these alterations in pharmacokinetics could 
  therefore potentially lead to an exaggerated response to the drug .
however , 
  for the other drugs , first pass liver metabolism does not appear to be 
  important in determining bioavailability , which is presumably dependent 
  primarily on efficiency of gastrointestinal absorption or metabolism .
when 
  dosed orally , the cmax appears to be the most reliable indicator of 
  a change in metabolizing capacity and acts as a valuable corollary to the 
  increased half - life observed following intravenous administration . in summary , mice with a hepatic deletion of microsomal cytochrome 
  b5 display a significant change in the metabolism of a 
  range of substrates by a number of cytochrome p450 enzymes in vitro 
  and , more importantly , in vivo .
the data presented in this report 
  suggest that the level and/or activity of hepatic microsomal cytochrome 
  b5 can play a significant role in the metabolism , 
  disposition , and therapeutic effectiveness of clinically used drugs .
the 
  extrapolation of in vitro data to in vivo and the relative 
  importance to drug pharmacokinetics and disposition are very complex and 
  involve a wide range of parameters that could include the cytochrome 
  p450/cytochrome b5 ratio , the cytochrome p450 enzyme 
  involved , the substrate concentration , the p450 turnover number , and the 
  overall role of cytochrome p450 metabolism as a rate - limiting factor in drug 
  disposition .
 in vitro , cytochrome b5 modulates the rate of 
  cytochrome p450-dependent mono - oxygenation reactions . 
 however , the role of 
  this enzyme in determining drug pharmacokinetics in vivo and the 
  consequential effects on drug absorption distribution , metabolism , excretion , 
  and toxicity are unclear . in order to resolve this issue , we have carried out 
  the conditional deletion of microsomal cytochrome b5 in 
  the liver to create the hepatic microsomal cytochrome b5 
  null mouse . 
 these mice develop and breed normally and have no overt phenotype . 
  in vitro studies using a range of substrates for different p450 
  enzymes 
 showed that in hepatic microsomal cytochrome b5 
  null nadh - mediated metabolism was essentially abolished for most substrates , 
  and the nadph - dependent metabolism of many substrates was reduced by 
  5090% . 
 this reduction in metabolism was also reflected in the in 
  vivo elimination profiles of several drugs , including midazolam , 
  metoprolol , and tolbutamide . in the case of chlorzoxazone , 
 elimination was 
  essentially unchanged . 
 for some drugs , the pharmacokinetics were also markedly 
  altered ; for example , when administered orally , the maximum plasma 
  concentration for midazolam was increased by 2.5-fold , and the clearance 
  decreased by 3.6-fold in hepatic microsomal cytochrome b5 
  null mice . 
 these data indicate that microsomal cytochrome 
  b5 can play a major role in the in vivo 
  metabolism of certain drugs and chemicals but in a p450- and 
  substrate - dependent manner . 
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
Supplementary Material