A Japanese prospective multicenter study of urinary oxysterols in biliary atresia.

Diagnosis of biliary atresia (BA) can involve uncertainties. In the present prospective multicenter study, we considered whether urinary oxysterols represent a useful marker for diagnosis of BA in Japanese children. Subjects under 6 months old at 7 pediatric centers in Japan were prospectively enrolled, including patients with cholestasis and healthy controls (HC) without liver disease. Patients with cholestasis constituted 2 groups representing BA patients and others with cholestasis from other causes (non-BA). We quantitatively analyzed 7 oxysterols including 4ß-, 20(S)-, 22(S)-, 22(R)-, 24(S)-, 25-, and 27-hydroxycholesterol by liquid chromatography/electrospray ionization-tandem mass spectrometry. Enrolled subjects included 14 with BA (median age 68 days; range 26-170) and 10 non-BA cholestatic controls (59; 14-162), as well as 10 HC (57; 25-120). Total urinary oxysterols were significantly greater in BA (median, 153.0 µmol/mol creatinine; range 24.1-486.7; P < 0.001) and non-BA (36.2; 5.8-411.3; P < 0.05) than in HC (2.7; 0.8-7.6). In patients with BA, urinary 27-hydroxycholesterol (3.61; 0.42-11.09; P < 0.01) was significantly greater than in non-BA (0.71; 0-5.62). In receiver operating characteristic (ROC) curve analysis for distinguishing BA from non-BA, the area under the ROC curve for urinary 27-hydroxycholesterol was 0.83. In conclusion, this first report of urinary oxysterol analysis in patients with BA indicated that 27-hydroxycholesterol may be a useful marker for distinguishing BA from other causes of neonatal cholestasis.

Assessment of induced CYP3A activity in pregnant women using 4ß-hydroxycholesterol: Cholesterol ratio as an appropriate metabolic marker.

AIMS: This study was aimed at evaluating changes in CYP3A activity following and during pregnancy by analyzing metabolic markers for CYP3A activity, which can help avoid unnecessary drug exposure and invasive sampling. METHODS: Forty-eight pregnant women and 25 non-pregnant women were enrolled in this study. Plasma and urine samples were collected from the pregnant women during each trimester and from the non-pregnant women for evaluation of metabolic markers for CYP3A activity. Metabolic markers for CYP3A activity were measured using GC-MS. RESULTS: An increased 4ß-hydroxycholesterol/cholesterol ratio, consistent with high CYP3A activity, was observed in pregnant women compared with that in non-pregnant women; however, no differences were observed among trimesters. No significant differences were observed in urinary markers. CONCLUSIONS: We observed an increase in the activity of CYP3A following but not during pregnancy when measured using the 4ß-hydroxycholesterol/cholesterol ratio. In addition, based on our results, we suggest that the plasma 4ß-hydroxycholesterol/cholesterol ratio be used to measure CYP3A activity in pregnant women.

LC/MS/MS of steroids having vicinal diol as electrospray-active boronates.

A derivatization procedure with (3-dimethylaminophenyl)dihydroxyborane (DAPB) was introduced to enhance the detectability of steroids having a vicinal diol in LC/electrospray ionization (ESI)-MS/MS. DAPB reacted with the vicinal diol on the steroids [4ß-hydroxycholesterol (4-HCh), pregnanetriol (PT) and 20R,22R-dihydroxycholesterol] in pyridine at 50°C within 1 h. The resulting DAPB-derivatives were highly responsive in ESI-MS operating in the positive-ion mode and gave characteristic product ions during MS/MS, which enabled sensitive detection using a selected reaction monitoring mode; the detection responses of the DAPB-derivatives were increased by 20-160-fold over those of the intact steroids and the limits of detection were in the low femtomole or attomole range. The derivatization procedure was successfully applied to biological sample analysis; the derivatization followed by LC/ESI-MS/MS enabled the specific detection of trace amounts of 4-HCh in human plasma and PT in human urine with a small sample volume, simple pretreatment and short chromatographic run time.

Neonatal cholestasis with increased 3ß-monohydroxy-Δ5 bile acids in serum and urine: not necessarily primary oxysterol 7α hydroxylase deficiency.

BACKGROUND: Inborn errors of bile acid synthesis are rare genetic disorders that can present with cholestatic liver disease. Recently we encountered 3 infants with neonatal cholestasis and excessive 3ß-monohydroxy-Δ5-C24 bile acids in serum and urine. We investigated whether identification of 3ß-hydroxy-5-cholestenoic acid and 27-hydroxycholesterol in serum and urine of cholestatic patients is necessary for diagnosis of primary oxysterol 7α-hydroxylase deficiency. METHODS: These 3 patients initially led us to suspected oxysterol 7α-hydroxylase deficiency. However, sequence analysis of genomic DNA resulted in diagnosis of 2 patients with oxysterol 7α-hydroxylase deficiency and 1 patient with 3ß-hydroxy-Δ5-C27-steroid dehydrogenase/isomerase deficiency. We examined identification of 3ß-hydroxy-5-cholestenoic acid and 27-hydroxycholesterol by gas chromatography-mass spectrometry after diagnosis. RESULTS: Interestingly, we detected a peak for 3ß-hydroxy-5-cholestenoic acid in serum and 27-hydroxycholesterol of the neutral sterol in urine from 2 patients who were diagnosed with primary oxysterol 7α-hydroxylase deficiency. CONCLUSION: In evaluating infants with cholestasis and excessive 3ß-monohydroxy-Δ5-C24 bile acids in infancy, one needs to conduct C24 bile acid analysis serially. Results can guide performance and interpretation of genomic DNA analysis. Moreover, identification of 3ß-hydroxy-5-cholestenoic acid in serum and 27-hydroxycholesterol in urine is highly important for diagnosis of oxysterol 7α-hydroxylase deficiency as is genomic DNA analysis.

Role of 8-iso-prostaglandin F2alpha and 25-hydroxycholesterol in the pathophysiology of endometriosis.

OBJECTIVE: To investigate the involvement of 8-iso-PGF(2alpha) and 25-hydroxycholesterol (25-OH-Chol) in the pathophysiology of endometriosis. DESIGN: Observational case-control study using enzyme immunoassay and high-performance liquid chromatography (HPLC). SETTING: Postgraduate Institute of Medical Education and Research. PATIENT(S): Forty-five women undergoing laparoscopy (n = 25), laparotomy (n = 19), or tubal ligation (n =1). INTERVENTION(S): Venipuncture and laparoscopic peritoneal fluid (PF) collection. MAIN OUTCOME MEASURE(S): The levels of 8-iso-PGF(2alpha) were determined both in urine and PF of all the patients using enzyme immunoassay. The levels of 25-OH-Chol were determined by using reversed phase HPLC both in the plasma and PF samples. Oxidative damage to DNA was assessed by agarose gel electrophoresis. RESULT(S): Significantly increased levels of 8-iso-PGF(2alpha) were observed both in urine and PF of women with endometriosis compared with control women. Similarly, higher levels of 25-OH-Chol were observed both in plasma and PF of patients compared with controls and the difference was statistically significant. A clear-cut tailing pattern was observed in DNA of patients with endometriosis, indicating significant DNA damage. CONCLUSION(S): Our observations implicate oxidative stress in the pathophysiology of endometriosis. For the first time, we demonstrate that 8-iso-PGF(2alpha) and oxysterols (the known promoters of steroidogenesis) might be the culprits in this disease.

Acceleration of lipid peroxidation in alpha-tocopherol transfer protein-knockout mice following the consumption of drinking water containing a radical initiator.

To assess the antioxidative role of vitamin E (VE) in a mouse model of severe VE deficiency by using biomarkers, alpha-tocopherol transfer protein (alpha-TTP(-/-))-knockout mice were maintained on a VE-deficient diet for 28 weeks [KO group, n = 6]. Wild-type C57BL/6 mice were maintained on a diet containing 0.002% alpha-tocopherol [WT group, n = 6]. The animals were housed individually in a metabolic cage from the age of 9 weeks (Week 0) to 27 weeks. Urine was collected every week, and the levels of total hydroxyoctadecadienoic acid (tHODE), 7-hydroxycholesterol (t7-OHCh), and 8-iso-prostaglandin F(2alpha)(t8-isoPGF(2alpha)), which are biomarkers for lipid peroxidation, were measured by gas chromatography (GC)-mass spectrometry. From the age of 21 weeks (Week 12), three mice in each group were provided drinking water containing the water-soluble radical initiator 2,2'-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride (AIPH) until the end of the study (Week 19). Blood and tissue samples were collected, and the levels of the abovementioned biomarkers therein were assessed. AIPH consumption clearly elevated the plasma and erythrocyte levels of tHODE and t8-isoPGF(2alpha) in both the WT and KO groups except for the erythrocyte level of tHODE in the WT group. Furthermore, this elevation was more prominent in the KO group than in the WT group. Interestingly, AIPH consumption reduced the stereoisomer ratio of HODE (ZE/EE), which is reflective of the efficacy of a compound as an antioxidant in vivo; this suggests that free radical-mediated oxidation reduces the antioxidant capacity in vivo. The urine levels of tHODE, t7-OHCh, and t8-isoPGF(2alpha) tended to increase with AIPH consumption, but these individual levels fluctuated. It was clearly demonstrated by the proposed biomarkers that maintaining alpha-TTP(-/-) mice on a VE-deficient diet results in a severe VE deficiency and promotes lipid peroxidation.

High levels of (24S)-24-hydroxycholesterol 3-sulfate, 24-glucuronide in the serum and urine of children with severe cholestatic liver disease.

Extracts of urine and serum from children with cholestatic liver disease were analyzed by fast atom bombardment (FAB) mass spectrometry. About half of all spectra showed a peak at m/z 657, compatible with the presence of a glucuronidated cholestenediol sulfate. Separation by ion exchange chromatography before and after solvolysis and treatment with beta-glucuronidase, combined with analyses by gas chromatography-mass spectrometry and FAB mass spectrometry with collision-induced dissociation, showed that the major compound responsible for the peak at m/z 657 was (24S)-24-hydroxycholesterol 3-sulfate, 24-glucuronide. The double conjugate of 27-hydroxycholesterol was also identified and double conjugates of cholestene- and cholestanetriols were also present. Semiquantitative analyses of the double conjugate of 24-hydroxycholesterol in patients whose FAB spectra showed a peak at m/z 657 indicated serum levels of 2-18 microM and a daily urinary excretion of 0.1-2.7 mumol/24 h. Eleven of 13 studied patients with a prominent peak at m/z 657 in the FAB spectra of their serum or urine either underwent liver transplantation or died. It is concluded that double conjugation of hydroxysterols with sulfuric and glucuronic acids can be an important metabolic pathway, particularly for (24S)-24-hydroxycholesterol. It is speculated that serious cholestatic liver disease may induce an increased formation and release of (24S)-24-hydroxycholesterol from brain (Lütjohann et al. 1996, Proc. Nutl. Acad. Sci. USA. 93: 9799-9804) with subsequent extracerebral conjugation with sulfuric and glucuronic acids.

Two cases of adrenal myelolipoma.

We report myelolipoma found in two patients, of whom one had hormonal abnormalities related to adrenal function. The first patient was a 36-year-old woman, who was found incidentally to have a left adrenal tumor by CT scan during admission for treatment of Guillain-Barré syndrome. Obesity, hirsutism and osteoporosis were also evident, and the patient was forwarded for additional endocrine function analysis, which revealed elevation of serum cortisol, urine 17-OHCS and 17-KS, and a decreased level of ACTH. These abnormalities returned to normal after excision of the tumor. Pathologically, the tumor was composed of mature fat cells and hematopoietic components, and was diagnosed as myelolipoma. The second patient was a 63-year-old woman, who was receiving follow-up care for hyperthyroidism. A right adrenal tumor was noted incidentally in a routine examination by CT scan. Endocrinologically, she was found to have no abnormalities of adrenal function. The tumor was excised, and diagnosed pathologically as myelolipoma, being composed of mature fat cells and hematopoietic components. Generally, although most myelolipomas have no endocrine function, our first patient showed features of Cushing's syndrome. Thus it is suggested that an interrelationship may exist between myelolipoma and endocrinological alteration.

26-Hydroxycholesterol disulfate: metabolism and excretion in the normal neonate.

Deuterated 26-hydroxycholesterol disulfate has been given in a tracer amount to neonates to evaluate the pool size, metabolism and excretion of the endogenously occurring compound in meconium. In a group of 5 normal neonates excretion of endogenous 26-hydroxycholesterol during the initial 72 h of life ranged from 327 to 1096 micrograms. Recovery of administered isotope during the same period was 66-98%. Only trace amounts of 26-hydroxycholesterol were recovered in urine. The findings indicate that relative to bile acid pool size, the normal neonate has a small intestinal pool of 26-hydroxycholesterol which, for the most part, is rapidly excreted and does not contribute significantly to bile acid synthesis. The techniques developed during the course of the study provide an approach to non invasive metabolic studies that give insights on the normal transition from fetal to neonatal life.