s t e r o i d s 7 3 ( 2 0 0 8 ) 417–423

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Human �4-3-oxosteroid 5�-reductase (AKR1D1) deficiencyand steroid metabolism�

Mario Palermoa, Maria Grazia Marazzib, Beverly A. Hughesc, Paul M. Stewart c,Peter T. Claytond, Cedric H.L. Shackletonc,∗

a Department of Endocrinology, Azienda Sanitaria Locale (ASL) 1, Sassari, Italyb Department of Paediatric Science, Gaslini Institute, University of Genoa, Italyc Division of Medical Sciences University of Birmingham, Birmingham, UKd UCL Institute of Child Health, Guilford Street, London, UK

a r t i c l e i n f o

Article history:

Received 27 July 2007

Received in revised form

14 November 2007

Accepted 3 December 2007

Published on line 14 December 2007

Keywords:

Cortisol metabolism

Steroid metabolism

5�-reductase

Urine steroids

GC/MS

a b s t r a c t

Conclusive in vivo evidence regarding the enzyme responsible for steroid hormone 5�-

reduction has not been obtained, although studies have suggested it may be the same

enzyme as that utilized for cholic acid and chenodeoxycholic bile-acid synthesis. We have

recorded the steroid metabolome of a patient with a defect in the “bile-acid” 5�-reductase

(AKR1D1) and from this confirm that this enzyme is additionally responsible for steroid hor-

mone metabolism. The 13-year old patient has been investigated since infancy because of a

cholestasis phenotype caused by bile-acid insufficiency. Several years ago it was shown that

she had a 662C > T missense mutation in AKR1D1 causing a Pro198Leu substitution. It was

found that the patient had an almost total absence of 5�-reduced metabolites of corticos-

teroids and severely reduced production of 5�-reduced metabolites of other steroids. The

patient is healthy in spite of her earlier hepatic failure and is on no treatment. All her vital

signs were normal, as were results of many biochemical analyses. She had normal pubertal

changes and experiences regular menstrual cycles. There was no evidence for any clinical

condition that could be attributed to attenuated ability to metabolize steroids in normal

fashion. Both parents were heterozygous for the mutation but the steroid excretion was

entirely normal, although an older female sibling showed definitive evidence for attenuated

5�-reduction of cortisol. A younger brother had a normal steroid metabolome. The sibling

genotypes were not available.

© 2008 Elsevier Inc. All rights reserved.

� Steroid metabolites without specified 5-hydrogen orientation are 3�-hydroxy-5�-steroids.∗ Corresponding author at: Division of Medical Sciences University of Birmingham, Institute of Biomedical Research, Birmingham, B15

2TT, UK.E-mail address: cshackleton@chori.org (C.H.L. Shackleton).Abbreviations: And, androsterone; Etio, etiocholanolone; 11OxoAnd, 11-oxoandrosterone; 11�OHAnd, 11�-hydroxyandrosterone;

11�OHEtio, 11�-hydroxyetiocholanolone; THE, tetrahydrocortisone; 5�THE, 5�-tetrahydrocortisone; THF, tetrahydrocortisol; 5�THF, 5�-tetrahydrocortisol; �C′one, �-cortolone; �C′one5�, (5�)-�-cortolone (equivalent for �-cortolones); �C′ol, �-cortol; �C′ol5�, (5�)-�-cortol(equivalent for �-cortols); THA, tetrahydro-11-dehydrocorticosterone; 5�THA, 5�-tetrahydro-11-dehydrocorticosterone; THB, tetrahy-drocorticosterone; 5�THB, 5�-tetrahydrocorticosterone; P′diol, pregnanediol; 5�P′diol, 5�-pregnanediol; P′triol, pregnanetriol; 5�P′triol,5�-pregnanetriol; 17HP, 17-hydroxypregnanolone; 17HP5�, (5�)-17-hydroxypregnanolone; 6�OHF, 6�-hydroxycortisol; 20�DHE, 20�-dihydrocortisone; 20�DHE, 20�-dihydrocortisone; 20�-DHF, 20�-dihydrocortisol; 20�DHF, 20�-dihydrocortisol.0039-128X/$ – see front matter © 2008 Elsevier Inc. All rights reserved.doi:10.1016/j.steroids.2007.12.001

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1. Introduction

Reduction of sterols and steroids with a 3-oxo-4-ene structureis a common transformation. This is a two-step procedure,5�- or 5�-reduction occurring first followed by the actionof 3�-hydroxysteroid dehydrogenase. Reduction at 5�- hasbeen most extensively studied following the finding thatthis reaction was involved in generation of the hormone5�-dihydrotestosterone (5�-DHT), while comparatively littleattention has been paid to 5�-reduction. However, this reac-tion has vital synthetic roles as an integral part of bile-acidsynthesis, and a catabolic role in the degradative metabolismof active steroid hormones, about 2/3 of the mass of which aredeactivated by this mechanism prior to excretion.

The human bile-acid biosynthetic enzyme has been clonedand is now known as AKR1D1. It has been expressed in COScells and shows high 5�-reductase activity to bile-acid inter-mediates but low or absent activity to steroid hormones [1].This suggests that may be more than one enzyme is involvedin human 5�-reduction, a question that could be resolved bystudying the urinary steroid metabolome of a human “knock-out” of the enzyme, and over a period of 20 years there havebeen several reports of patients with putative 5�-reductasedeficiency [2–6]. This disorder (congenital bile-acid synthesisdefect type 2 (CBAS2), OMIM:235555) is known for a cholesta-sis and liver failure phenotype, which can be fatal in infancy.Lemonde et al. found inactivating AKR1D1 mutations in threepatients with the disorder [7]. Typically the treatment for thesepatients is administration of exogenous bile-acids or livertransplantation. However, 11 years after publication of a casereport on one of these individuals [3,7], the female patientis thriving and has apparently no current need for bile-acidsupplementation, possibly as a result of active formation of“replacement” 5�- (or “allo”) bile-acids. Daugherty et al. [8]have also studied patients with the disorder and have notednear-normalization of liver function with age and/or oral bile-acid administration.

We have revisited this case to determine if steroid (as dis-tinct from sterol) 5�-reduction was affected, and evaluate theimpact of the disorder on steroid hormone homeostasis, par-ticularly related to clinical problems of an endocrine nature.

2. Materials and methods

2.1. Patient

The patient is from the Italian province of Sardinia, the secondchild of parents not known to be consanguineous. Sardiniais a Mediterranean island with much of the population rel-atively isolated from mainland European communities forcenturies. The patient’s ancestors are autochthonous and theparents are both from the north-west region of the island. Thepatient presented at 3 weeks of age with cholestasis associ-ated with steatorrhoea, failure to thrive and rickets. A liver

biopsy at 3 months showed lobular disarray, and hepatocytescontained fat and bile pigments. Failure to thrive, malabsorb-tion of fat soluble vitamins continued despite ursodeoxycholicacid treatment. At 8 months the liver still showed giant cell

0 8 ) 417–423

hepatitis with steatosis. Treatment with cholic acid and chen-odeoxycholic acid led to normalization of liver function within3 months. She remained well on this treatment for severalyears. Lemonde et al. [7] published biochemical and clinicaldata at the age of 9 years. Her bile-acid synthesis had beenevaluated in detail when she was 8 months old using liq-uid ion mass spectrometry (LSIMS) of a urine extract, whichshowed a complete absence of chenodeoxycholic acid andcholic acid, strong evidence for 5�-reductase deficiency. TheAKR1D1 gene was sequenced and it was determined thatshe had a 662C > T missense mutation causing a Pro198Leusubstitution. Currently she is 13 years old and even after termi-nation of treatment (by personal decision, not medical advice)at the age of 10 years she has apparently remained healthy.She has shown normal growth (height 156, weight 53, 50thcentile for Sardinian population); regular menses started atage of 11, and secondary sexual characteristics are normal.Neither acne nor hirsutism was evident. Blood pressure wasrecently determined to be normal at 110/70 mmHg and clinicalsigns of cholestasis or jaundice were no longer evident. Liveranalytes (transaminase, bilirubin, gGT, cholinesterase) werenormal. Blood glucose, uric acid, cholesterol, electrophore-sis of proteins, plasma electrolytes were all in the normalrange. Plasma creatinine was 0.45 mg/dl (normal 0.50–1.4),urea 19.5 mg/dl (normal 15–50) and urine creatinine 75.8 mg/dl.Serum levels of the lipophilic vitamins A, D, E and K wereall normal, thus showing no indication of malabsorbtion.Liver sonography showed no abnormalities. Serum cortisol,17-hydroxyprogesterone and ACTH were also all in the nor-mal range. The patient appears well and takes no medication.We suggest that she is compensating for her conventional bile-acid deficiency through increased synthesis of 5�- (or “allo”)bile-acids which could be hepatoprotective through their abil-ity, shared with conventional bile-acids, to activate bile flow.Serum bile-acids were measured at the age of 11 years [3]1 year after cessation of supplementary bile-acid therapy,results are given in Table 1. It was found that 62% of her circu-lating acids were of 5�-configuration.

2.2. Family members

The mother and father were heterozygous for the 662C > Tmutation. Genotypes of the brother and sister (7 and 16 years,respectively) were not determined. They are asymptomatic.The patient’s grandfathers died of bowel cancer and stroke,respectively, but the grandmothers are alive and healthy.

2.3. Methods

Informed consent for these studies was obtained from all fam-ily members and the study was approved by the institutionalreview board of ASL, Sassari. Urine samples were collectedfrom the patient and immediate family members for measure-ment of urinary steroids. In view of the family being rural andnot under current formal investigation only random sampleswere collected during a single visit by one of us (MCP) to their

home. This was not considered to be a disadvantage sincethe interrelationships between individual steroid metaboliteexcretions would be sufficient to establish the activity of 5�-reductase to hormonal steroids.

s t e r o i d s 7 3 ( 2 0 0 8 ) 417–423 419

Table 1 – Serum bile-acid concentrations in patient with 5�-reductase deficiency

Bile-acid (C = cholanoate) Trivial name Plasma concentration (mM) Normal range (mM)

5�-Bile-acids5�C-3�,7�-ol Chenodeoxycholic 0.05 0.22–12.45�C-3�,7�-ol Isochenodeoxycholic 0.06 <0.055�C-3�,7�-ol Ursodeoxycholic ND 0–2.15�C-3�,12�-ol Deoxycholic 0.60 0–4.45�C-3�,7�,12�-ol Cholic ND 0.05–4.6

5�-Bile-acids (“allo”)5�C-3�,7�-ol Allochenodeoxycholic 0.26 ND5�C-3�,7�-ol 0.32 ND5�C-3�,12�-ol Allodeoxycholic 0.09 ND5�C-3�,12�-ol 0.14 ND5�C-3�,12�-ol 0.31 ND

0.05 <0.05

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Fig. 1 – Comparison of SIM chromatograms of urinesteroids from proband and normal individuals. The figureillustrates the virtual absence of the classical major cortisolmetabolites, THE and THF from the urine of the patient

5�C-3�,7�,12�-ol Allocholic

ND = not detectable.

Steroids were measured according to a method previouslyescribed [9]. In brief, free steroids and steroid conjugates werextracted by solid phase extraction (SPE, C-18 phase), thenydrolyzed with �-glucuronidase and sulfatase prior to re-xtraction by SPE and conversion to methyloxime trimethylsi-yl ethers suitable for GC/MS analysis. Steroid quantitationas carried out by selected-ion-monitoring (SIM) on an Agi-

ent 5975 MSD with separation on a non-polar (DB1) capillaryolumn. The compounds measured (the “steroid panel”), andelected ions utilized for quantification were detailed in ourrevious communication. As previously described, quantita-ion was achieved by running a callibrant mixture containingnown amounts of the steroids of interest and relating the

on-intensity values obtained from the sample analyses tohis. In this study we quantified some normally minor “5�”

etabolites not normally part of our panel. These had theame mass spectra as the corresponding 5�-metabolites, butifferent retention times. We utilized the same selected-ionsor both 5�- and 5�-metabolites. The 5�- and 5�-epimers of �-nd �-cortol were not resolved so these compounds could note separately quantified. While quantification was achieved byIM, structures of almost all steroids were confirmed by scan-ing GC/MS. Individual compounds had identical retentionimes and mass spectra to authentic compounds.

. Results

he excretion of corticosteroids (cortisol and corticosteroneetabolites) by the patient and family are given in Table 2.

he absolute amounts (�g/24 h) are approximations sincehey were calculated by multiplying quantitative values webtained (�g/ml) by average volume excretions of the age-atched controls utilized in our laboratory. The relative

xcretions of individual compounds are authentic. Whilealues listed this way are not accurate they permit easy com-arison of the excretions of the proband, family and normal

ndividuals. The results illustrate that for the corticosteroids,

�-reduction is negligible in the proband since many 5�-teroids were below the level of detection. The virtual absencef 5�-reduction of cortisone and cortisol is represented visu-lly in the SIM chromatograms illustrated in Fig. 1.

with AKR1D1 deficiency. The normally insignificantmetabolite 5�THE is a major cortisone metabolite in5�-reductase deficiency. The monitored ions for THE andTHF are m/z 531 and m/z 562, respectively.

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Table 2 – Individual corticosteroid metabolite excretion (�g/24) in proband and family with values for age-matched controls

Steroid P 13 yearsa B 7 years S 16 years F 38 years M 36 years NC 5–8 years(n = 13)

NF 11–15 (n = 6) NFA (n = 34)b

mean, rangeNMA (n = 26)b

mean, range

THA <1c 14 66 182 67 72 20–111 112 27–209 144, 38–299 177, 48–3505�THA 73 19 116 207 38 58 29–106 69 26–113 137, 38–270 181, 36–383THB <1 6.0 47 284 59 38 14–69 62 23–108 141, 35–353 170, 23–3005�THB 193 26 386 643 80 166 87–297 188 51–334 264, 50–577 379, 125–90011OxoAnd 61 8.3 586 204 353 126 36–363 303 30–707 334, 71–946 482, 148–92211�OHAnd 281 12 487 1049 302 135 60–241 558 147–913 555, 191–979 1019, 345–174611�OHEtio 12 2.2 158 93 305 96 16–223 204 21–635 265, 16–859 346, 74–748THE 1.9 298 1586 4921 1162 1124 402–1612 2053 573–3449 2004, 775–4174 3193, 1037–63675�THE 149 23 170 325 46 NA NA 129d 205d

THF <2.0 64 467 2603 484 472 153–915 748 347–1202 1140, 384–3190 1786, 615–29975�THF 748 168 1352 1742 228 513 215–838 594 151–964 767, 141–1865 1510, 414–2934�C′one 6.9 115 1036 2776 969 316 94–468 953 242–1385 1080, 422–2499 1143, 450–2128�C′one 13 97 306 1367 230 268 98–490 578 84–1073 445, 171–1291 656, 231–1834�C′one 5� 293 7.4 61 80 12 NA 45 8.8–81 35d 54d

�C′ol(5� + 5�)e 244 23 242 729 197 56 25–92 133 54–210 315, 83–873 284, 96–704�C′ol(5� + 5�)e 71 68 222 1464 160 166 58–386 337 54–785 359, 72–2143 386, 139–862F 31 9.2 35 195 43 58 29–106 55 13–106 85, 26–145 87, 25–145E 82 10 55 178 56 72 20–111 67 31–121 150, 48–300 193, 76–3266�OHF 92 53 54 240 223 61 13–99 39 17–72 181, 53–416 255, 122–48620�DHE 85 9.0 24 46 36 7.0 1.7–14 13 4–25 31, 14–120 83, 27–19520�DHE 291 18 38 77 56 16 3.3–25 32 9.6–56 63, 28–120 89, 17–15020�DHF 295 12 22 88 27 13 4.1–29 17 7.5–34 53, 20–99 88, 25–25820�DHF 280 18 38 141 36 24 4.8–90 39 9.5–82 56, 22–111 88, 29–158Total �4 F metabolites 1176 129 266 965 477 215 54–466 262 94–496 619 872Total F metabolites 3059 1014 6939 17318 4925 3449 1438–5667 6771 1932–10719 8047 11840

The 24 h values given are estimated and approximately based on average volume excretion values obtained for our normal controls. These are 1.7 l for normal males; 1.4 l for normal females; 1 l fornormal 11–15 years old children;1.2 l for 15–16 years old children and 0.46 l for 6–8 years old children.a P, proband; B, brother; S, sister; M, mother; F, fatherb NMA and NFA are normal male and female adults, respectively, age range 18–55.c Figures in bold are 5�-metabolites with markedly attenuated excretions.d Figures for individual normal controls were not measured when normative ranges were established some years ago so excretion range is not available. The listed excretions were obtained from

analysis of urine pooled from an equal proportion of daily excretion of an equal number (n = 17) of male and female adults.e The epimers were not chromatographically separated.

s t e r o i d s 7 3 ( 2 0 0 8 ) 417–423 421

Table 3 – Steroid excretion ratios comparing activities of 5�-reductase, 5�-reductase, overall A-ring reduction and20-reduction

Ratios Proband Brother Sister Mother Father Normaladultsa

5�/5� ratios, cortisol (F) and corticosterone (B) metabolitesTHE/5�THE 0.013 12.9 9.3 25.3 15.1 15.6THF/5�THF <0.003 0.38 0.34 2.12 1.49 1.33�C′one/�C′one(5�) 0.046 13.1 5.0 19.2 17.1 12.4THB/5�THB <0.01 0.22 0.14 0.74 0.43 0.49THA/5�THA <0.01 0.74 0.57 1.77 0.88 1.0211�OHEtio/11�OHAnd 0.04 0.18 0.32 1.0 0.09 0.41

5�/5� ratios, not corticosteroidsEtio/And 0.15 1.71 1.44 0.79 0.87 0.97P′diol/5�P′diol 1.13 6.63 ≥20 ≥2.0 4.0 6.817HP/17HP5� 0.59 18 40 2.5 5.5 10Ptriol/5�P′triol 6.7 40 20 8.0 20 23

5� metabolites as % of total F metabolites5�THF, % total 24 17 19 4.6 9.4 115�THE, % total 4.9 2.3 2.4 0.9 1.7 1.7�Cone5�/ % total 9.6 0.7 0.9 0.2 0.4 0.53-Oxo-�4 metabolites as % total F metabolites 38 8.9 5.4 9.8 6.4 7.6Saturated 20�-, 20�-reduced/total saturated metabolites 33 35 28 33 35 26

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Unsaturated 20�-, 20�-reduced/total unsaturated metabolites 8

a These values were calculated by combining the normal female and

The overall alteration in corticosteroid metabolism ismphasized by the low 5�/5� ratio values given in Table 3. Inddition, ratios have been included which attempt to definehe affect of 5�-reductase deficiency on other metabolic pro-esses. These include whether 5�-reductase and 20-reductions utilized to greater extent to counteract loss of 5�-reductasectivity. Three parameters were evaluated regarding increased�-reductase activity; these were the excretion of 5�THE,�THF and �-cortolone-5� relative to the total of all mea-ured cortisol metabolites. All of these values were greatlyncreased suggesting an increased utilization of 5�-reductasen the patient. However, this apparent increased activity doesot compensate fully for inactive 5�-reductase as a muchreater proportion of cortisol is excreted with intact 3-oxo-�4

onfiguration in the proband (approximately 40%) comparedo controls (<10%).

Measurement of 20�- and 20�-reduced cortisol metabo-ites to evaluate possible accentuated activity showed mixedesults. There was no apparent increased excretion ofaturated 20-reduced cortisol metabolites such as the cor-ols and cortolones relative to total saturated metabolitesTable 3). However, for 3-oxo-�4 steroids there seemedo be an increase in excretion of 20-reduction products.eduction of non-corticosteroid metabolites at 5� wasvaluated and 5�/5� ratios of otherwise identical com-ounds are given in Table 3. These include androgenetabolites, progesterone metabolites (pregnanediols) andetabolites of 17-hydroxyprogesterone (pregnanetriols and

7-hydroxypregnanolones).While all measurements and ratio calculations showed

hat the proband had severely attenuated 5�-reductase activ-ty, the family members had results generally in the normalange. The one exception was the proband’s sister who had a

arkedly reduced ratio of 5�/5�THF.

44 46 36 32 37

values given in Table 2.

4. Discussion

The presence in urine of steroid metabolites reduced at 5� hasbeen known since 1930s but almost all studies on the enzyme5�-reductase have concerned its important role in bile-acidsynthesis. The “bile-acid synthesis enzyme” has been shownto be cytosolic and utilizes NADPH as electron donor. Berseusin 1967 partially purified the enzyme from rat liver and deter-mined that it was effective in the reduction of bile-acidprecursors and all forms of steroid hormones containing the3-oxo-4-ene group [10]. Okuda and Okuda [11] succeeded inobtaining a highly purified enzyme in 1984, and Onishi et al.utilizing an enzyme isolated and purified accordingly, pre-pared a monoclonal antibody to the enzyme allowing them todetermine the amino acid sequence from a rat cDNA library[12,13]. Kondo et al. reported the cDNA structure of the corre-sponding human enzyme and were successful in expressingit in COS cells [1]. The substrate specificity was evaluatedshowing that cholestenones involved in bile-acid synthe-sis were preferred substrates, and cortisol and testosteronewere 1/5 efficiently reduced. Reduction of androstenedioneand progesterone was not observed. Charbonneau and The[14] studied the activity of the enzyme expressed in HEK-293 cells and, in contrast to the Kondo et al. [1] study,found that the highest activity was towards androstenedione,testosterone, progesterone and 17-hydroxyprogesterone. Theyfound that corticosteroids and mineralocorticoids were infe-rior substrates. It is possible that this discrepancy was due todifferent experimental conditions utilized. Kondo et al. used

cell homogenates while Charbonneau and The used intactcells in culture. The latter authors also suggest that theremay be more than one human 5�-reductase. Clearly, the con-fusion resulting from the contrasting results from in vitro

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experiments could only be clarified by study of a patient withproven 5�-reductase deficiency. Although the mass spectro-metric profile of bile-acids from AKR1D1 deficient patientsclearly showed the absence of 5�-reduced compounds, theprofile of steroid hormone metabolites has not been previouslystudied.

Our results clearly demonstrate that AKR1D1 is the steroid5�-reductase entirely responsible for the 5�-reduction of cor-ticosterone and cortisol; only trace amounts of 5�-metaboliteswere detected. The enzyme is also largely responsible forthe reduction of androstenedione, progesterone and 17-hydroxyprogesterone, although some 5�-steroid was stillproduced. Either the mutant enzyme has low residual activ-ity to these precursors or else a secondary enzyme hassome 5�-reductase activity. It is interesting that AKR1D1 hasan extremely broad precursor tolerance, reducing moleculesat opposite ends of the sterol/steroid spectrum such ascholestenones and androstenedione, with one or two oxy-gen functionalities, to cortisol metabolites with five. In 1994Kondo et al. compared the predicted secondary structureof 5�-reductase with another NADPH binding protein, P450oxidoreductase and suggested that the mutated Pro198 wasin the NADPH binding domain of the 5�-reductase protein[1,7]. However, in recent years several crystal structures ofsteroid transforming aldoketoreductases have been depositedin the protein database (PDB) and inspection of these struc-tures shows that Pro198 is not in the cofactor-binding siteso its mutations do not provide a specific reason for reducedenzyme activity. However, a mutation involving substitutionof a proline residue in any position is likely to have a sig-nificant effect on secondary structure, which could lead toreduced enzyme activity. The high urinary levels of steroidswith the hormonally active intact A-ring opens up the pos-sibility of clinical problems related to excess availability ofactive hormones. Both aldosterone, cortisol and active andro-gen depend highly on 5�-reduction for catabolism and theabsence of this mechanism could effectively render moreactive hormone available. Neither the patient nor heterozy-gous parents showed evidence of hypercortisolemia, or highBP secondary to cortisol or aldosterone excess, and thepatient had no apparent disorder of androgen or estrogenorigin. However, it must be emphasized that we studied asingle affected individual, and other 5�-reductase deficientpatients may experience a more severe clinical phenotype.Several articles have described altered 5�-reductase activityassociated with hypertensive conditions. In apparent min-eralocorticoid excess syndrome an increase in the excretionof 5�THF has been reported, but this probably representsdecreased 5�-reductase since activity values of the enzymesinvolved are usually determined by ratios of 5�- to 5�-metabolites [15,16]. Soro et al. [17] have suggested that5�-reduction of cortisol in patients with untreated hyper-tension is reduced. However, it is unlikely that these minorchanges in metabolism, if real, contribute to disorder phe-notype. In addition, Westerbacka et al. [18] have shownincreased 5�-reduction of cortisol in obesity, specifically asso-

ciated with fatty accumulation in the liver rather than adiposetissue.

A significant role for 5�-reduced steroids has been pro-posed by Bertillson et al. [19]. Their studies provided strong

0 8 ) 417–423

evidence that 5�-pregnanes (but not equivalent 5�-reducedsteroids) were important ligands for the hPAR receptorinvolved in modulating cytochrome CYP3A induction. Theimpact of 5�-reductase deficiency on this mechanism couldnot be evaluated in our study.

Acknowledgements

This work was supported by the Wellcome Trust program grant066357 to PMS. Thanks to Catherine Chong for expert technicalassistance.

e f e r e n c e s

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