Transcript
Page 1: 3β-Hydroxy-Δ5 -C27-steroid dehydrogenase deficiency: Diagnosis and treatment

This report describes a Japanese male infant with 3β-hydroxy-∆5 -C27-steroid dehydrogenase/isomerase (3β-HSD) deficiencywho received ursodeoxycholic acid (UDCA) and chenodeoxy-cholic acid (CDCA) treatment. To evaluate the effects of thetreatment and to obtain further information about the pathwayof bile acid biosynthesis in this patient, samples of urine andserum were analyzed qualitatively and quantitatively by gas-chromatography-mass spectrometry (GC-MS) using selectedion monitoring.

MATERIALS AND METHODS

Patient

The family history and the initial presentation of this patienthave been described previously.1 Our patient was a Japanese2-month-old male infant, born after 41 weeks gestation withoutcomplications. His birthweight was 3910 g. At 1 month of age,he developed jaundice and showed elevated serum levels oftotal bilirubin and alanine aminotransferase.

The laboratory data on admission were as follows: totalbilirubin 9.3 mg/dL, conjugated bilirubin 5.9 mg/dL, aspartateaminotransferase 587 IU/L (13–33 IU/L), alanine aminotrans-ferase 596 IU/L (6–27 IU/L), alkaline phosphatase 1555 IU/L,γ-GTP 23 IU/L (10–47 IU/L) and total bile acids 2.5 µmol/L

by an enzymatic method for 3α-hydroxy bile acids. The prothrombin time was 12.2 s (control 10.8 s). The anti-thrombin III (80–120%) was 48%. Fibrinogen degradationproducts (< 100 ng/mL) were 556 ng/mL. Antibody titresshowed no evidence of infection with hepatitis B or C virus,herpes simplex virus, Epstein–Barr virus, cytomegalovirus or toxoplasma. The histological findings in the liver showedcholestasis, giant cell transformation in the lobular architectureand mild fibrosis around the portal area.

The patient underwent medical therapy for cholestasis,consisting of UDCA (50 mg/day), prednisolone (6 mg/day)and vitamins A, E and K (Fig. 1). Beginning at the age of14 months, while he was receiving this medication, hischolestasis and liver dysfunction improved. At the age of18 months, his liver function test results were as follows: totalbilirubin 0.9 mg/dL, conjugated bilirubin 0.5 mg/dL, aspartateaminotransferase 53 IU/L, alanine aminotransferase 66 IU/L,and total bile acids 2.2 µmol/L. A second biopsy was not ableto be performed.

Sample collection

Urine and serum samples were collected from the patient andstored at – 25°C until analysis. The concentrations of individual

J. Paediatr. Child Health (2001) 37, 516–519

3�-Hydroxy-�5 -C27-steroid dehydrogenase deficiency:Diagnosis and treatment

Y YAMATO,1 A KIMURA,1 T MURAI,2 T YOSHIMURA,2 T KUROSAWA,2 S TERAZAWA,3 A TAKAO,4 K MAEDA,1

E NAKASHIMA,1 Y YAMASHITA1 and H KATO1

1Department of Pediatrics and Child Health, Kurume University School of Medicine, Kurume, Fukuoka, 2Faculty ofPharmaceutical Sciences, Health Sciences University of Hokkaido, Ishikari-Tobetsu, Hokkaido, 3Division of Pediatrics,

Gifu Kouseiren Gihoku General Hospital, Yamagata-Takatomi, Gifu, Japan, and 4Division of Pediatrics,Gifu Municipal Hospital, Kashima, Gifu, Japan

Abstract: The aim of this study was to evaluate the effects of bile acid treatment and to obtain further information aboutthe pathway of bile acid biosynthesis in a patient with 3β-hydroxy-∆5-C27-steroid dehydrogenase/isomerase (3β-HSD) defi-ciency by gas chromatography-mass spectrometry. Results showed that at 2 months of age, 3β-hydroxy-5-cholen-24-oic acid (3.0 µmol/mmol Cr, 7.9%) was detected in the urine in essentially the same relative amount as 3β,7α-dihydroxy- and3β,7α,12α-trihydroxy-5-cholen-24-oic acids (3.7 µmol/mmol Cr, 9.8%) during ursodeoxycholic acid treatment combinedwith prednisolone. As a result, diagnosis was delayed until 18 months of age. One month later with substitution of cheno-deoxycholic acid treatment, urinary 3β,7α-dihydroxy- and 3β,7α,12α-trihydroxy-5-cholen-24-oic acids decreased signifi-cantly, and subsequent improvement of liver dysfunction was accelerated. Chenodeoxycholic acid treatment is useful in3β-HSD deficiency. However, in the diagnosis of this disease in early life, it should be noted that the acidic pathway may bethe major route for bile acid biosynthesis in the neonatal period. Diagnosis of 3β-HSD deficiency may have been delayed byadministration of ursodeoxycholic acid, resulting in prolonged diagnostic investigation in this child with cholestasis. Further,use of prednisolone may have been contraindicated.

Key words: 3β-hydroxy-∆5-C27-steroid dehydrogenase/isomerase deficiency; acidic pathway; bile acid therapy;chenodeoxycholic acid; ursodeoxycholic acid.

Correspondence: A Kimura, MD, Department of Pediatrics and Child Health, Kurume University School of Medicine, 67 Asahi-machi, Kurume,Fukuoka 830–0011, Japan. Fax: +81 942 38 1792; email: [email protected]

Accepted for publication 8 March 2001.

Page 2: 3β-Hydroxy-Δ5 -C27-steroid dehydrogenase deficiency: Diagnosis and treatment

bile acids in the urine were corrected according to the creati-nine (Cr) concentration and expressed as µmol/mmol of Cr.Urine samples were obtained from the patient at 2 and18 months of age during the period of UDCA (12.5 mg/kg perday and 4.2 mg/kg per day, respectively) treatment combinedwith prednisolone (1.5 mg/kg per day and 0.5 mg/kg everyother day, respectively) and vitamins A, E and K, at 18 monthsof age during a period with no treatment, and at 19 months ofage before and after CDCA treatment (8.3 mg/kg per day). Wealso obtained serum samples at 19 months of age before andafter CDCA treatment.

Analysis of bile acids

In the standard procedure, samples of human biological fluidswere routinely prepared for GC-MS analysis as describedpreviously.2

RESULTS

Analysis of bile acids during UDCA treatment

Concentrations of urinary 3β,7α-dihydroxy- and 3β,7α,12α-trihydroxy-5-cholen-24-oic acids (54.0 µmol/mmol Cr, 79.6%of total bile acids) were relatively high at 18 months of ageduring UDCA treatment, together with prednisolone andvitamins A, E and K. However, the concentrations of theseunusual urinary bile acids at that time were less than thelevels observed at 18 months of age (56.8 µmol/mmol Cr,93.2%) during a period when no treatment was received. At2 months of age, we detected trace amounts of urinary 3β,7α-dihydroxy- and 3β,7α,12α-trihydroxy-5-cholen-24-oicacids (3.7 µmol/mmol Cr, 9.8%) during the period of UDCAtreatment, with prednisolone and vitamins A, E and K. In-terestingly, the levels of 3β-hydroxy-5-cholen-24-oic acid(3.0 µmol/mmol Cr, 7.9%), an intermediate in the acidicpathway, were essentially the same as those of 3β,7α-dihydroxy- and 3β,7α,12α-trihydroxy-5-cholen-24-oicacids in the urine at the same time. The percentages of 3β,7α-dihydroxy- and 3β,7α,12α-trihydroxy-5-cholen-24-oicacids, and 3β-hydroxy-5-cholen-24-oic acid as a proportion oftotal urinary bile acids were 39.7% and 32.2%, respectively,when UDCA was excluded from total urinary bile acids. Thedaily dose of UDCA administered was the same at 2 or18 months of age (50 mg/day), at which times the concentra-tions of urinary UDCA were 20.6 µmol/L and 21.3 µmol/L,respectively (not corrected for Cr) (Table 1).

Analysis of bile acids before and after CDCA treatment

Before CDCA treatment, at 19 months of age, large amounts of3β,7α-dihydroxy- and 3β,7α,12α-trihydroxy-5-cholen-24-oicacids were detected in urine and serum, 63.2 µmol/mmol Cr(94.7%) and 7.7 µmol/L (87.1%), respectively.

Two weeks after CDCA treatment, the concentrations of3β,7α-dihydroxy- and 3β,7α,12α-trihydroxy-5-cholen-24-oic acids significantly decreased from 63.2 µmol/mmolCr (94.7%) to 16.5 µmol/mmol Cr (73.8%) in urine and from7.7 µmol/L (87.1%) to 2.0 µmol/L (13.5%) in serum(Tables 2, 3).

517Diagnosis and oral bile acid treatment

Table 1 Analysis of bile acids in urine during periods of ursodeoxycholic treatment and no treatment

UDCA treatment* No treatment2 months 18 months

Bile acid (µmol/mmol Cr) (12.5 mg/kg/day) (4.2 mg/kg/day) 18 months

Cholic acid 0.239 0.779 0.480Chenodeoxycholic acid 0.569 (1.5)** 1.494 (2.2) 1.610 (2.7)1β,3α,6α,12α-Tetrahydroxy-5β-cholan-24-oic acid Trace 0.444 Trace3α,6α,12α-Trihydroxy-5β-cholan-24-oic acid 1.414 0.484 0.384Hyocholic acid 0.410 0.472 0.464Ursodeoxycholic acid 28.816 (75.4) 8.944 (13.2) Trace3β,7α,12α-Trihydroxy-5-cholen-24-oic acid 2.439 (6.4) 36.635 (54.0) 38.888 (64.4)3β,7α-Dihydroxy-5-cholen-24-oic acid 1.237 (3.4) 17.346 (25.6) 17.895 (28.8)3β-Hydroxy-5-cholen-24-oic acid 3.020 (7.9) 1.219 (1.8) 1.128 (1.9)Total bile acids 38.184 67.817 60.349Cr (µmol/L) 716.04 2386.8 3005.6

UDCA, Ursodeoxycholic acid; Cr, creatinine; *combined with prednisolone; **% of total bile acids.

Fig. 1 Clinical course of the patient before chenodeoxycholictreatment. Interval A indicates treatment with ursodeoxycholic acid(50 mg/day), prednisolone (6 mg/day), and vitamins A, E, and K.Interval B indicates treatment with ursodeoxycholic acid (50 mg/day),prednisolone (0.5 mg/kg every other day), and vitamins A, E, and K.(�) GOT, aspartate aminotransferase; (�) GPT, alanine aminotrans-ferase; T. Bil, total bilirubin; D. Bil, conjugated bilirubin.

Page 3: 3β-Hydroxy-Δ5 -C27-steroid dehydrogenase deficiency: Diagnosis and treatment

DISCUSSION

In this study, during UDCA treatment, small amounts ofendogenous bile acids, such as cholic and chenodeoxycholicacids and 3β-hydroxy-, 3β,7α-dihydroxy-, and 3β,7α,12α-trihydroxy-5-cholen-24-oic acids were detected in the urine, aswell as large mounts of the exogenous bile acid, UDCA, at2 months of age (Table 1). At 18 months of age, we found thatthe levels of urinary 3β,7α-dihydroxy- and 3β,7α,12α-trihydroxy-5-cholen-24-oic acids had increased, despite UDCAtreatment (Table 1). However, serum transaminase andbilirubin levels gradually decreased between 2 and 18 monthsof age under UDCA treatment. Hepatocyte functions areknown to be improved by administration of a hydrophilic bileacid (such as UDCA) which may protect against liver injury,3

reducing the risk of bile acid-induced damage to liver cellmembranes.4 Therefore, long-term oral administration ofUDCA to a patient with 3β-HSD deficiency may not beeffective treatment for accumulation of 3β,7α-dihydroxy- and3β,7α,12α-trihydroxy-5-cholen-24-oic acids in hepatocytes,because the catabolism of cholesterol through its conversion tobile acids is certainly not reduced by UDCA treatment and maywell be an enhanced by it.5 This is compatible with failure ofUDCA to inhibit the rate-limiting step of bile acid synthesis –cholesterol 7α-hydroxylase. The levels of 3β,7α-dihydroxy-

and 3β,7α,12α-trihydroxy-5-cholen-24-oic acids may beslightly decreased by UDCA treatment, since the rate of biliaryexcretion of endogenous bile acids is increased. Actually,greater amounts of urinary 3β,7α-dihydroxy- and 3β,7α,12α-trihydroxy-5-cholen-24-oic acids were detected during theperiod in which no treatment was received at 18 months of agethan during UDCA treatment at the same age (Table 1).

The benefits of prednisolone in the treatment of cholestasisis unproved. However, according to one report, high-dose pred-nisolone was effective in stimulating bile flow.6

Interestingly, 3β-hydroxy-5-cholen-24-oic acid was detectedat essentially the same levels as 3β,7α-dihydroxy- and3β,7α,12α-trihydroxy-5-cholen-24-oic acids in the urine at2 months of age (Table 1) during UDCA treatment with pred-nisolone. On the other hand, according to the report of firstpatient with 3β-HSD deficiency, large amounts of 3β,7α-dihydroxy- and 3β,7α,12α-trihydroxy-5-cholen-24-oic acids(90–100%) were detected in the urine at 3 months of age.7

Trace amounts of 3β-hydroxy-5-cholen-24-oic acid (< 8.5%)were detected in this first patient. Therefore, we suggest thatthe acidic pathway8 is unquestionably the most important onefor bile acid biosynthesis in humans in the perinatal andneonatal periods of early life.

Setchell et al. reported a patient with cholestatic jaundice inwhose case no 7α-hydroxylated bile acids were synthesized

518 Y Yamato et al.

Table 3 Analysis of bile acids in serum before and after chenodeoxycholic acid treatment*

Before After treatmentBile acid (µmol/L) treatment 7 days 14 days

Cholic acid 0.734 0.734 0.294Chenodeoxycholic acid 0.408 (4.6)** 27.927 (84.1) 11.208 (76.5)Hyocholic acid Trace 0.162 TraceUrsodeoxycholic acid n.d. Trace 1.1723β,7α,12α-Trihydroxy-5-cholen-24-oic acid 5.915 (67.0) 2.893 (8.7) 1.408 (9.6)3β,7α-Dihydroxy-5-cholen-24-oic acid 1.771 (20.1) 1.033 (3.1) 0.566 (3.9)Total bile acids 8.828 33.199 14.648

n.d., Not detected; *8.3 mg/kg per day; **% of total bile acids.

Table 2 Analysis of bile acids in urine before and after chenodeoxycholic acid treatment*

Before After treatmentBile acid (µmol/mmol Cr) treatment 1 3 7 14 days

Cholic acid 0.826 0.614 0.287 0.121 0.067Chenodeoxycholic acid 1.436 (2.1)** 1.090 (2.0) 1.814 (5.8) 1.901 (6.0) 1.964 (8.9)Deoxycholic acid 0.025 0.032 Trace Trace Trace1β,3α,7α,12α-Tetrahydroxy-5β-cholan-24-oic acid 0.126 0.032 Trace Trace Trace2β,3α,7α,12α-Tetrahydroxy-5β-cholan-24-oic acid 0.037 0.033 n.d. n.d. n.d.3α,4β,7α,12α-Tetrahydroxy-5β-cholan-24-oic acid 0.033 Trace n.d. n.d. n.d.3α,6α,12α-Trihydroxy-5β-cholan-24-oic acid 0.262 0.234 0.632 Trace n.d.1β,3α,7α,-Trihydroxy-5β-cholan-24-oic acid Trace Trace Trace Trace 0.037Hyocholic acid 0.481 0.372 1.292 1.350 1.976Ursodeoxycholic acid 0.071 (0.1) 0.059 (0.1) Trace 0.106 (0.3) 1.643 (7.4)3β,7α,12α-Trihydroxy-5-cholen-24-oic acid 46.218 (68.5) 36.854 (68.8) 17.612 (56.1) 23.365 (73.7) 14.153 (63.5)3β,7α-Dihydroxy-5-cholen-24-oic acid 17.023 (25.2) 13.486 (25.2) 9.384 (29.9) 4.414 (14.6) 2.305 (10.3)3β-Hydroxy-5-cholen-24-oic acid 0.519 (0.8) 0.331 (0.6) 0.201 (0.6) 0.074 (0.2) 0.078 (0.3)12α-Hydroxy-3-oxo-4,6-cholen-24-oic acid 0.288 0.277 0.163 0.154 0.055Total bile acids 67.452 53.575 31.385 31.685 22.288Cr (µmol/L) 5038.8 6453.2 2386.8 5038.8 6541.6

n.d., Not detected; Cr, creatinine; *8.3 mg/kg per day; **% of total bile acids.

Page 4: 3β-Hydroxy-Δ5 -C27-steroid dehydrogenase deficiency: Diagnosis and treatment

519Diagnosis and oral bile acid treatment

and acidic intermediates in the bile acid pathway accumulatedin the serum.9 In this patient, the cholesterol 7α-hydroxylasegene was normal. However, there was a mutation in theoxysterol 7α-hydroxylase gene which codes for an enzymewhich is important in the acidic pathway but not in the classicalpathway. They therefore suggested that the acidic pathway maybe the major route for 7α-hydroxylated bile acid biosynthesisin the human neonate.9 Moreover, we have speculated that theenzyme activities in the developmental stage may be differentfrom those of cholesterol 7α-hydroxylase and 27 hydroxylasein humans in early life, especially in patients with neonatalcholestasis. Actually, in our patient, trace amounts of 3β-hydroxy-5-cholen-24-oic acid were detected during lateinfancy (Tables 1–3). Large amounts of 3β-hydroxy-5-cholen-24-oic acid, an intermediate in the acidic pathway in early life,and large amounts of 3β,7α-dihydroxy- and 3β,7α,12α-trihydroxy-5-cholen-24-oic acids, an intermediate in theclassical pathway in late infancy, were detected in our patientduring UDCA treatment with prednisolone (Table 1). Asalready mentioned, it may be the UDCA treatment with pred-nisolone that reduced the level metabolism in this patient,leading to difficulty with diagnostic monitoring.

Primary bile acids administered as replacement therapy enterthe enterohepatic circulation and down regulate the activity ofcholesterol 7α-hydroxylase, thereby reducing the production of hepatotoxic 3β-hydroxy-∆5 -bile acids.10 In our patient, thelevels of 3β,7α-dihydroxy- and 3β,7α,12α-trihydroxy-5-cholen-24-oic acids in urine and serum gradually decreasedafter CDCA treatment (Tables 2, 3). Primary bile acid therapywas more effective than UDCA treatment. Considering thisexperience, primary bile acid therapy with CDCA should beinitiated immediately after diagnosis of 3β-HSD deficiency.We recommend a CDCA dose of 5–10 mg/kg per day duringinfancy.

Finally, hepatotoxic 3β-hydroxy-∆5 -bile acids accumulatedin hepatocytes even during ursodeoxycholic acid treatment inthis patient with 3β-HSD deficiency, because ursodeoxycholicacid does not inhibit cholesterol 7α-hydroxylase activity.However, chenodeoxycholic acid inhibits this key enzyme inthe metabolism of cholesterol. Therefore, chenodeoxycholicacid treatment is useful in the treatment this disease to preventliver cirrhosis. However, in the diagnosis of 3β-HSD deficiencyin early life it should be noted that the acidic pathway may be

the major route for bile acid biosynthesis in the neonatalperiod. Moreover, 3β-HSD deficiency may have been maskedand the diagnosis delayed by treatment with ursodeoxycholicacid, regiving further diagnostic investigations in the presentlydescribed child with cholestasis. Also, in retrospect, empiricuse of prednisolone in cholestasis may have been contra-indicated.

REFERENCES

1 Terazawa S, Kimura A, Inoue T, Murai T, Kurosawa T, Takao A.An infant with 3β-hydroxy-∆5-C27-steroid dehydrogenase/isomerase deficiency presenting with typical neonatal hepatitissyndrome: the first Japanese case. Acta Paediatr. Jpn 1998; 40:638–40.

2 Kimura A, Suzuki M, Murai T et al. Perinatal bile acid metabo-lism analysis of urinary bile acids in pregnant women andnewborns. J. Lipid Res. 1997; 38: 1954–62.

3 Attili AF, Angelico M, Cantrafora A, Alvaro D, Capocaccia L.Bile acid-induced liver toxicity relation to the hydrophobic-hydrophilic balance of bile acids. Med. Hypotheses 1986; 19:57–68.

4 Batta AK, Salen G, Arora R et al. Effect of ursodeoxycholic acidon bile acid metabolism in primary biliary cirrhosis. Hepatology1989; 10: 414–9.

5 Tint GS, Salen G, Shefer S. Effect of ursodeoxycholic acid andchenodeoxycholic acid on cholesterol and bile acid metabolism.Gastroenterology 1986; 91: 1007–18.

6 Karrer FM, Lilly JR. Corticosteroid therapy in biliary atresia.J. Pediatr. Surg. 1985; 20: 693–5.

7 Clayton PT, Leonard JV, Setchell KDR, Andersson S, Egestad B,Sjövall J. Familial giant cell hepatitis associated with synthesis of3β,7α-dihydroxy- and 3β,7α,12α-trihydroxy-5-cholenoic acids. J. Clin. Invest. 1987; 79: 1031–8.

8 Javitt NB, Kok E, Carubbi F, Blizzard T, Gut M, Byon CY. Bileacid biosynthesis: metabolism of 3β-hydroxy-5-cholenoic acid tochenodeoxycholic acid. J. Biol. Chem. 1986; 261: 12486–9.

9 Setchell KDR, Schwarz M, O’Connell NC et al. Identification of anew inborn error in bile acid synthesis: mutation of the oxysterol7α-hydroxylation gene causes neonatal liver disease. J. Clin.Invest. 1998; 102: 1690–703.

10 Ichimiya H, Egestad B, Nazer H, Baginski ES, Clayton PT,Sjövall J. Bile acids and bile alcohols in a child with hepatic 3β-hydroxy-∆5-C27-steroid dehydrogenase deficiency: effects ofchenodeoxycholic acid treatment. J. Lipid Res. 1991; 32: 829–41.


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