This report describes a Japanese male infant with 3-hydroxy-5 -C27-steroid dehydrogenase/isomerase (3-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
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 (1333 IU/L), alanine aminotrans-ferase 596 IU/L (627 IU/L), alkaline phosphatase 1555 IU/L,-GTP 23 IU/L (1047 IU/L) and total bile acids 2.5 mol/L
by an enzymatic method for 3-hydroxy bile acids. The prothrombin time was 12.2 s (control 10.8 s). The anti-thrombin III (80120%) 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, EpsteinBarr 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 mol/L. A second biopsy was not ableto be performed.
Urine and serum samples were collected from the patient andstored at 25C until analysis. The concentrations of individual
J. Paediatr. Child Health (2001) 37, 516519
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,1E 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 3-hydroxy-5-C27-steroid dehydrogenase/isomerase (3-HSD) defi-ciency by gas chromatography-mass spectrometry. Results showed that at 2 months of age, 3-hydroxy-5-cholen-24-oic acid (3.0 mol/mmol Cr, 7.9%) was detected in the urine in essentially the same relative amount as 3,7-dihydroxy- and3,7,12-trihydroxy-5-cholen-24-oic acids (3.7 mol/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 3,7-dihydroxy- and 3,7,12-trihydroxy-5-cholen-24-oic acids decreased signifi-cantly, and subsequent improvement of liver dysfunction was accelerated. Chenodeoxycholic acid treatment is useful in3-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 3-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: 3-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 8300011, Japan. Fax: +81 942 38 1792; email: email@example.com
Accepted for publication 8 March 2001.
bile acids in the urine were corrected according to the creati-nine (Cr) concentration and expressed as mol/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
Analysis of bile acids during UDCA treatment
Concentrations of urinary 3,7-dihydroxy- and 3,7,12-trihydroxy-5-cholen-24-oic acids (54.0 mol/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 mol/mmol Cr,93.2%) during a period when no treatment was received. At2 months of age, we detected trace amounts of urinary 3,7-dihydroxy- and 3,7,12-trihydroxy-5-cholen-24-oicacids (3.7 mol/mmol Cr, 9.8%) during the period of UDCAtreatment, with prednisolone and vitamins A, E and K. In-terestingly, the levels of 3-hydroxy-5-cholen-24-oic acid(3.0 mol/mmol Cr, 7.9%), an intermediate in the acidicpathway, were essentially the same as those of 3,7-dihydroxy- and 3,7,12-trihydroxy-5-cholen-24-oicacids in the urine at the same time. The percentages of 3,7-dihydroxy- and 3,7,12-trihydroxy-5-cholen-24-oicacids, and 3-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 mol/L and 21.3 mol/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 of3,7-dihydroxy- and 3,7,12-trihydroxy-5-cholen-24-oicacids were detected in urine and serum, 63.2 mol/mmol Cr(94.7%) and 7.7 mol/L (87.1%), respectively.
Two weeks after CDCA treatment, the concentrations of3,7-dihydroxy- and 3,7,12-trihydroxy-5-cholen-24-oic acids significantly decreased from 63.2 mol/mmolCr (94.7%) to 16.5 mol/mmol Cr (73.8%) in urine and from7.7 mol/L (87.1%) to 2.0 mol/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 (mol/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)1,3,6,12-Tetrahydroxy-5-cholan-24-oic acid Trace 0.444 Trace3,6,12-Trihydroxy-5-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) Trace3,7,12-Trihydroxy-5-cholen-24-oic acid 2.439 (6.4) 36.635 (54.0) 38.888 (64.4)3,7-Dihydroxy-5-cholen-24-oic acid 1.237 (3.4) 17.346 (25.6) 17.895 (28.8)3-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 (mol/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.
In this study, during UDCA treatment, small amounts ofendogenous bile acids, such as cholic and chenodeoxycholicacids and 3-hydroxy-, 3,7-dihydroxy-, and 3,7,12-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 3,7-dihydroxy- and 3,7,12-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,3reducing the risk of bile acid-induced damage to liver cellmembranes.4 Therefore, long-term oral administration ofUDCA to a patient with 3-HSD deficiency may not beeffective treatment for accumulation of 3,7-dihydroxy- and3,7,12-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 7-hydroxylase. The levels of 3,7-dihydroxy-
and 3,7,12-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 3,7-dihydroxy- and 3,7,12-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, 3-hydroxy-5-cholen-24-oic acid was detectedat essentially the same levels as 3,7-dihydroxy- and3,7,12-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 3-HSD deficiency, large amounts of 3,7-dihydroxy- and 3,7,12-trihydroxy-5-cholen-24-oic acids(90100%) were detected in the urine at 3 months of age.7Trace amounts of 3-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 7-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 (mol/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 1.1723,7,12-Trihydroxy-5-cholen-24-oic acid 5.915 (67.0) 2.893 (8.7) 1.408 (9.6)3,7-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 (mol/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 Trace1,3,7,12-Tetrahydroxy-5-cholan-24-oic acid 0.126 0.032 Trace Trace Trace2,3,7,12-Tetrahydroxy-5-cholan-24-oic acid 0.037 0.033 n.d. n.d. n.d.3,4,7,12-Tetrahydroxy-5-cholan-24-oic acid 0.033 Trace n.d. n.d. n.d.3,6,12-Trihydroxy-5-cholan-24-oic acid 0.262 0.234 0.632 Trace n.d.1,3,7,-Trihydroxy-5-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)3,7,12-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)3,7-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)3-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)12-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 (mol/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.
519Diagnosis and oral bile acid treatment
and acidic intermediates in the bile acid pathway accumulatedin the serum.9 In this patient, the cholesterol 7-hydroxylasegene was normal. However, there was a mutation in theoxysterol 7-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 7-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 7-hydroxylase and 27 hydroxylasein humans in early life, especially in patients with neonatalcholestasis. Actually, in our patient, trace amounts of 3-hydroxy-5-cholen-24-oic acid were detected during lateinfancy (Tables 13). Large amounts of 3-hydroxy-5-cholen-24-oic acid, an intermediate in the acidic pathway in early life,and large amounts of 3,7-dihydroxy- and 3,7,12-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 7-hydroxylase, thereby reducing the production of hepatotoxic 3-hydrox...