A comparison of4b-hydroxycholesterol :cholesterol and6b-hydroxycortisol :cortisol as markers ofCYP3A4 inductionYvonne Mårde Arrhén,1 Hanna Nylén,2 Anita Lövgren-Sandblom,2

Kajsa P. Kanebratt,3 Katarina Wide4 & Ulf Diczfalusy2

1Clinical Chemistry, Karolinska University Hospital, Huddinge, Sweden, 2Department of Laboratory

Medicine, Karolinska Institutet, Huddinge, Sweden, 3CVGI iMed DMPK, AstraZeneca R&D, Mölndal,

Sweden and 4Department of CLINTEC, Karolinska Institutet, Huddinge, Sweden

CorrespondenceDr Ulf Diczfalusy, Karolinska Institutet,Department of Laboratory Medicine,Division of Clinical Chemistry, KarolinskaUniversity Hospital, Huddinge C1.74,SE-14186 Stockholm, Sweden.Tel.: +46 8 5858 1253Fax: +46 8 5858 1260E-mail: ulf.diczfalusy@karolinska.se-----------------------------------------------------------------------

KeywordsCYP3A4, endogenous markers, induction,LC-MS/MS, rifampicin-----------------------------------------------------------------------

Received6 October 2011

Accepted29 October 2012

Accepted ArticlePublished Online1 November 2012

AIMTo compare plasma 4b-hydroxycholesterol : cholesterol with urinary6b-hydroxycortisol : cortisol as markers of cytochrome P4503A4 activitybefore and after treatment with rifampicin for 2 weeks.

METHOD6b-hydroxycortisol and cortisol were determined by liquidchromatography tandem mass spectrometry and4b-hydroxycholesterol was determined by gas chromatography–massspectrometry in three groups of healthy volunteers.

RESULTSInduction ratios for 6b-hydroxycortisol : cortisol were 1.8, 3.9 and 4.5 for20 mg day-1, 100 mg day-1 or 500 mg day-1 of rifampicin, respectively.The corresponding ratios for 4b-hydroxycholesterol : cholesterol were1.5, 2.4 and 3.8.

CONCLUSIONSPlasma 4b-hydroxycholesterol : cholesterol gave similar induction ratiosto urinary 6b-hydroxycortisol : cortisol.

WHAT IS ALREADY KNOWN ABOUTTHIS SUBJECT• We have suggested that plasma

4b-hydroxycholesterol may be used as anendogenous marker for cytochrome P4503A4/5 (CYP3A)4/5 activity. We have shownthat it can be used to monitor bothinduction and inhibition of CYP3A activity.

WHAT THIS STUDY ADDS• Plasma 4b-hydroxycholesterol : cholesterol

was compared with another marker ofCYP3A activity, 6b-hydroxycortisol : cortisolin urine. Urine collections from volunteerstreated with rifampicin for 2 weeks wereanalyzed for this ratio and compared toearlier determined plasma4b-hydroxycholesterol : cholesterol. The twomarkers gave similar induction ratios.

Introduction

Cytochrome P4503A (CYP3A) is an important enzymefamily metabolizing both drugs and endogenous sub-stances such as steroids. We have shown previously that

CYP3A4 converts cholesterol into 4b-hydroxycholesterol[1] and suggested that plasma 4b-hydroxycholesterol maybe used as a phenotypic marker of CYP3A activity [2].CYP3A4 also metabolizes cortisol to 6b-hydroxycortisol,which has been used as an endogenous marker for CYP3A

British Journal of ClinicalPharmacology

DOI:10.1111/bcp.12016

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activity. Since cortisol shows a pronounced diurnal varia-tion, the ratio of 6b-hydroxycortisol : cortisol in urine isused rather than 6b-hydroxycortisol itself [3, 4]. We havepreviously studied 4b-hydroxycholesterol as a marker ofCYP3A-activity in healthy volunteers treated withrifampicin [5]. There is a weak but significant correlationbetween 4b-hydroxycholesterol and cholesterol. There-fore, the 4b-hydroxycholesterol to cholesterol ratioshould be used rather than 4b-hydroxycholesterol itself tocorrect for variations in cholesterol concentration duringthe study period [6]. In the present investigation wecompared urinary 6b-hydroxycortisol : cortisol to plasma4b-hydroxycholesterol : cholesterol determined previously[5].

Methods

Chemicals6b-hydroxycortisol, cortisol and cortisone were fromSigma-Aldrich Sweden AB, Stockholm, Sweden. 2H4-6b-hydroxycortisol was from Toronto Research Chemicals Inc.,North York, Canada and 2H4-cortisol was purchased fromLarodan Fine Chemicals AB, Malmö, Sweden.

Calibration standardsWorking solutions of calibrators were prepared from stocksolutions (300 mmol l-1 in methanol) by dilution with waterto 700 nmol l-1.

Quality control (QC) samplesQuality control samples were prepared from spikedhuman urine at two concentrations. The concentrations of6b-hydroxycortisol, cortisol and cortisone were 255, 89 and127 nmol l-1, respectively (concentration 1) and 933, 235and 482 nmol l-1 (concentration 2).

Healthy volunteersTwenty-four healthy Swedish volunteers were recruited tothe study, 12 males and 12 females, as described previously[5]. All subjects possessed the CYP3A5*3/*3 genotype [5].Urine was collected from 20.00 h to 08.00 h (12 h) beforetreatment and after 2 weeks of treatment with rifampicin.All volunteers gave their written informed consent to par-ticipate in the study. The study was approved by theHuman Ethics Committee at Karolinska Institutet, Stock-holm, Sweden.

Determination of 6b-hydroxycortisol andcortisol in urine6b-hydroxycortisol, cortisol and cortisone in urine weredetermined by liquid chromatography tandem massspectrometry (LC-MS/MS) using deuterium labelled6b-hydroxycortisol (2H4-6b-hydroxycortisol) and deute-rium labelled cortisol (2H4-cortisol) as internal standards.

Sample preparationUrine (200 ml), QC samples or calibrators were dilutedwith an equal volume of water containing the internal

standards 2H4-6b-hydroxycortisol (1000 nmol l-1) and 2H4-cortisol (400 nmol l-1).The diluted sample was applied to a96-well solid phase extraction plate (Oasis HLB mElutionPlate, Waters). After washing with 200 ml 5% methanol inwater, analytes were eluted with 100 ml acetonitrile :isopropanol 2:3 (v/v) and diluted with 400 ml water.The samples were prepared on a Tecan Genesis RSP 150/TeVacS robot.

Analysis by LC-MS/MSThe samples (5 ml) were analyzed on a Waters Acquity-Quattro Premier LC-MS/MS system with a Waters UPLCBEH ShieldRP18 column (1.7 mm, 2.1 ¥ 150 mm) at 55°C.The mobile phase was a 7 min linear gradient of 5–50%acetonitrile in water with formic acid (0.1%), flow rate0.400 ml min-1 and total run time 12 min. Retention timesfor 6b-hydroxycortisol, cortisol and cortisone were 4.27,6.27 and 6.30 min, respectively.

The mass spectrometer was operated in negative elec-trospray mode for 6b-hydroxycortisol and positive modefor cortisol and cortisone. The transitions used were347→313 and 347→125 (6b-hydroxycortisol), 363→121and 363→97 (cortisol) and 361→163 and 361→121 (corti-sone).Transitions for the internal standards were 351→128(2H4-6b-hydroxycortisol) and 367 →121 (2H4-cortisol).

Method validationThe linear range was 10–14 000 nmol l-1 for 6b-hydroxycortisol and 5–14 000 nmol l-1 for cortisol andcortisone. Urine samples spiked with 0.8–7 times theoriginal concentration (255, 89 and 127 nmol l-1, respec-tively) gave recoveries of 90–97%. Urine samples spikedwith 30–150 times the original concentration (933, 235and 482 nmol l-1, respectively) gave recoveries of 105–123%. The total relative standard deviations (CV) werebetween 5.0 and 9.2% (n = 80, 117 and 115, respectively).The cortisol analysis is accredited according to SS-ENISO 15189. Our laboratory participates in an externalquality control programme for cortisol (UKNEQAS) on aregular basis.

Determination of 4b-hydroxycholesterol inblood plasmaThe results from the determination of 4b-hydroxycholesterol in plasma were from a previous inves-tigation [5]. 4b-hydroxycholesterol was determined bycombined gas chromatography-mass spectrometry asdescribed in [1].

Determination of cholesterol in blood plasmaCholesterol was determined by an enzymatic method,CHOD-PAPP, utilizing a Roche/Hitachi Modular instrument.The CV was 4% at 3 mmol l-1.

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Results

Determination of 6b-hydroxycortisol : cortisolin urine from rifampicin treated volunteersThe urinary concentrations of 6b-hydroxycortisol and cor-tisol were determined in three groups of volunteers (eightin each group) treated with rifampicin (20 mg day-1,100 mg day-1 or 500 mg day-1) for 14 days and the6b-hydroxycortisol : cortisol ratios were calculated. Asshown in Figure 1, the ratios before treatment were similar,6.3, 5.4 and 7.3 and increased after 14 days treatment to10.5, 16.2 and 27.3, respectively.Three subjects, two receiv-ing 20 mg day-1 and one receiving 100 mg day-1, showed areduced 6b-hydroxycortisol : cortisol ratio after 14 days ofrifampicin treatment.

Plasma 4b-hydroxycholesterol concentrationsin rifampicin treated volunteersThe plasma 4b-hydroxycholesterol concentrations in thevolunteers were published previously [5]. In contrast to6b-hydroxycortisol : cortisol, the plasma concentration of4b-hydroxycholesterol increased in all volunteers afterrifampicin treatment.

Calculation of induction ratiosThe induction ratio was defined as 6b-hydroxycortisol : cortisol after treatment divided bythe 6b-hydroxycortisol : cortisol before treatment. Theseinduction ratios and the corresponding ratios for4b-hydroxycholesterol and quinine metabolic ratio pub-lished previously [5] are shown in Table 1. The ratio of4b-hydroxycholesterol : cholesterol should be used whenthe cholesterol concentration changes during a treatmentperiod. There was a large interindividual variation in6b-hydroxycortisol : cortisol. The ratios determined beforerifampicin treatment showed a 10-fold variation and theratios after treatment showed a 15-fold variation. The cor-responding variations for 4b-hydroxycholesterol werethree-fold and six-fold, respectively.

Discussion

The induction of CYP3A4 could be monitored by 6b-hydroxycortisol : cortisol at all three doses of rifampicin.

Compared with 4b-hydroxycholesterol : cholesterol, theP values for the differences in the 6b-hydroxycortisol :cortisol before and after treatment were higher [5] andin contrast to 4b-hydroxycholesterol : cholesterol someindividuals had lower ratios after treatment than beforetreatment (Figure 1). However, the induction ratios for6b-hydroxycortisol : cortisol and 4b-hydroxycholesterol :cholesterol are similar and also similar to the metabolicratio of the exogenous CYP3A marker quinine (Table 1,data on 4b-hydroxycholesterol and quinine are from [5]).An advantage with 4b-hydroxycholesterol is that only asingle blood sample is required for the determinationwhile a urine collection over at least 12 h is necessary whenusing 6b-hydroxycortisol : cortisol. The long half-life of4b-hydroxycholesterol [7] results in small variations in con-centration, but excludes this marker in short term studies.

In conclusion, similar estimates for the induction ofCYP3A4 following rifampicin treatment were obtainedusing either urinary 6b-hydroxycortisol : cortisol or plasma4b-hydroxycholesterol : cholesterol.

Competing Interests

K.P. Kanebratt is an employee of AstraZeneca. Unrestrictedfinancial support was provided by AstraZeneca. This hasnot influenced the design or the interpretation of results ofthis study.All authors have completed the Unified Compet-ing Interest form at http://www.icmje.org/coi_disclosure.pdf (available on request from the corresponding author)and declare no support from any organization for the sub-mitted work (YMA, HN, AL-S, KPK, KW, UD had support(grant) from AstraZeneca for the submitted work and nofinancial relationships with any organizations that mighthave an interest in the submitted work in the previous 3years (YMA,HN,AL-S,KPK,KW).UD received support (grant,consultancy) from AstraZeneca in the previous 3 years andno other relationships or activities that could appear tohave influenced the submitted work (YMA, HN, AL-S, KPK,KW, UD).

This work was supported by grants from AstraZeneca, theSwedish Research Council, Medicine (3902), Torsten andRagnar Söderbergs Foundation and through the regionalagreement on medical training and clinical research (ALF)

Table 1Induction ratios for 6b-hydroxycortisol : cortisol, 4b-hydroxycholesterol, 4b-hydroxycholesterol : cholesterol and quinine metabolic ratio (MR) in volunteerstreated for 2 weeks with 20, 100 or 500 mg rifampicin day-1 (mean � SD, n = 8). Data on 4b-hydroxycholesterol and quinine metabolic ratio (MR) are froma previous publication [5]

Rifampicin (mg day-1) 6b-OH-cortisol : cortisol 4b-OH-cholesterol 4b-OH-cholesterol : cholesterol Quinine MR

20 1.77 � 0.82 1.46 � 0.24 1.53 � 0.20 1.57 � 0.33100 3.92 � 3.28 2.49 � 0.66 2.40 � 0.75 2.95 � 1.10

500 4.46 � 1.81 4.06 � 0.85 3.76 � 0.67 4.25 � 2.07

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Figure 1Urinary 6b-hydroxycortisol : cortisol and plasma 4b-hydroxycholesterol : cholesterol (¥ 104) in adult healthy volunteers before and after 2 weeks ofrifampicin treatment (500 mg day-1, upper panels; 100 mg day-1, middle panels; 20 mg day-1, lower panels). The 6b-hydroxycortisol : cortisol ratios weredetermined in 12 h urine collections. P values from analysis using Wilcoxon matched pairs test. 6b-OH-F: 6b-hydroxycortisol; F: cortisol; 4b-OH-chol:4b-hydroxycholesterol; chol: cholesterol

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between Stockholm County Council and Karolinska Institutet(SLL 582107).

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