Objectives: Cerebrotendinous xanthomatosis (CTX) is caused by defects in sterol 27-hydroxylase (CYP27A1, encoded by CYP27A1), a key enzyme in the bile acid synthesis pathway. CTX usually presents as neurologic disease in adults or older children. The rare reports of CTX manifest as neonatal cholestasis assess the cholestasis as transient, with patient survival. Our experience differs. Methods: Homozygous or compound heterozygous CYP27A1 mutations were detected in 8 neonatal cholestasis patients by whole exome sequencing, panel sequencing, or Sanger sequencing. Their clinical and biochemical data were retrospectively reviewed. Immunostaining for CYP27A1 was conducted in liver of 4 patients. Mass spectrometry was used to analyze patients' urine samples. Results: All 8 infants had severe cholestasis. Five died from, or were transplanted for, liver failure; 3 cleared their jaundice eventually. Marking for CYP27A1 was weak or absent in 3 of the 4 patient specimens. Mass spectrometry of urine revealed a predominance of sulfated and doubly conjugated (sulfated-glucuronidated) bile alcohols. No patient harbored a putatively pathogenic mutation in genes other than CYP27A1 that have been implicated in cholestatic liver disease. Conclusions: CTX manifest as neonatal cholestasis has a bile acid profile different from CTX manifest in later life, and thus may be overlooked. Immunostaining, mass spectrometry of urine, and genetic studies can support one another in making the diagnosis. A substantial proportion of CTX patients with severe neonatal cholestasis may die or need liver transplantation. CTX manifest in infancy as severe cholestasis warrants further investigation of biochemical diagnostic criteria and best management.

BACKGROUND 3β-Hydroxy-Δ(5)-C27-steroid oxidoreductase (HSD3B7) deficiency, a progressive cholestatic liver disease, is the most common genetic defect in bile acid synthesis. Early diagnosis is important because patients respond to oral primary bile acid therapy, which targets the negative feedback regulation for bile acid synthesis to reduce the production of hepatotoxic 3β-hydroxy-Δ(5)-bile acids. These atypical bile acids are highly labile and difficult to accurately measure, yet a method for accurate determination of 3β-hydroxy-Δ(5)-bile acid sulfates is critical for dose titration and monitoring response to therapy. METHODS We describe a electrospray ionization LC-MS/MS method for the direct measurement of atypical 3β-hydroxy-Δ(5)-bile acid sulfates in urine from patients with HSD3B7 deficiency that overcomes the deficiencies of previously used GC-MS methods. RESULTS Separation of sulfated 3β-hydroxy-Δ(5)-bile acids was achieved by reversed-phase HPLC in a 12-min analytical run. The mean (SE) urinary concentration of the total 3β-sulfated-Δ(5)-cholenoic acids in patients with HSD3B7 deficiency was 4650 (1711) μmol/L, approximately 1000-fold higher than in noncholestatic and cholestatic patients with intact primary bile acid synthesis. GC-MS was not reliable for measuring 3β-hydroxy-Δ(5)-bile acid sulfates; however, direct analysis of urine by fast atom bombardment mass spectrometry yielded meaningful semiquantitative assessment of urinary excretion. CONCLUSIONS The tandem mass spectrometry method described here for the measurement of 3β-hydroxy-Δ(5)-bile acid sulfates in urine can be applied to the diagnosis and accurate monitoring of responses to primary bile acid therapy in HSD3B7 patients.

BACKGROUND: 3 beta-Hydroxy-Delta(5)-C-27-steroid oxidoreductase (HSD3B7) deficiency, a progressive cholestatic liver disease, is the most common genetic defect in bile acid synthesis. Early diagnosis is important because patients respond to oral primary bile acid therapy, which targets the negative feedback regulation for bile acid synthesis to reduce the production of hepatotoxic 3 beta-hydroxy-Delta(5)-bile acids. These atypical bile acids are highly labile and difficult to accurately measure, yet a method for accurate determination of 3 beta-hydroxy-Delta(5)-bile acid sulfates is critical for dose titration and monitoring response to therapy.METHODS: We describe a electrospray ionization LC-MS/MS method for the direct measurement of atypical 3 beta-hydroxy-Delta(5)-bile acid sulfates in urine from patients with HSD3B7 deficiency that overcomes the deficiencies of previously used GC-MS methods.RESULTS: Separation of sulfated 3 beta-hydroxy-Delta(5)-bile acids was achieved by reversed-phase HPLC in a 12-min analytical run. The mean (SE) urinary concentration of the total 3 beta-sulfated-Delta(5)-cholenoic acids in patients with HSD3B7 deficiency was 4650 (1711) mu mol/L, approximately 1000-fold higher than in noncholestatic and cholestatic patients with intact primary bile acid synthesis. GC-MS was not reliable for measuring 3 beta-hydroxy-Delta(5)-bile acid sulfates; however, direct analysis of urine by fast atom bombardment mass spectrometry yielded meaningful semiquantitative assessment of urinary excretion.CONCLUSIONS: The tandem mass spectrometry method described here for the measurement of 3 beta-hydroxy-Delta(5)-bile acid sulfates in urine can be applied to the diagnosis and accurate monitoring of responses to primary bile acid therapy in HSD3B7 patients. (C) 2015 American Association for Clinical Chemistry

Secoisolariciresinol-diglycoside (SDG), a natural dietary lignan of flaxseeds now available in dietary supplements, is converted by intestinal bacteria to the mammalian lignans enterodiol and enterolactone. High levels of these lignans in blood and urine are associated with reduced risk of many chronic diseases. Our objective was to determine the bioavailability and pharmacokinetics of SDG in purified flaxseed extracts under dose-ranging and steady-state conditions, and to examine whether differences in secoisolariciresinol-diglycoside purity influence bioavailability. Pharmacokinetic studies were performed on healthy postmenopausal women after oral intake of 25, 50, 75, 86 and 172 mg of secoisolariciresinol-diglycoside. Extracts differing in secoisolariciresinol-diglycoside purity were compared, and steady-state lignan concentrations measured after daily intake for one week. Blood and urine samples were collected at timed intervals and secoisolariciresinol, enterodiol and enterolactone concentrations measured by mass spectrometry. Secoisolariciresinol-diglycoside was efficiently hydrolyzed and converted to secoisolariciresinol. Serum concentrations increased rapidly after oral intake, peaking after 5-7 h and disappearing with a plasma elimination half-life of 4.8 h. Maximum serum concentrations of the biologically active metabolites, enterodiol and enterolactone were attained after 12-24 h and 24-36 h, respectively, and the half-lives were 9.4 h and 13.2 h. Linear dose-responses were observed and secoisolariciresinol bioavailability correlated (r(2) = 0.835) with cumulative lignan excretion. There were no significant differences in the pharmacokinetics of extracts differing in purity, and steady-state serum lignan concentrations were obtained after one-week of daily dosing. In conclusion, this study defines the pharmacokinetics of secoisolariciresinol-diglycoside and shows it is first hydrolyzed and then metabolized in a time-dependent sequence to secoisolariciresinol, enterodiol and ultimately enterolactone, and these metabolites are efficiently absorbed.

BACKGROUND & AIMS:The final step in bile acid synthesis involves conjugation with glycine and taurine, which promotes a high intraluminal micellar concentration to facilitate lipid absorption. We investigated the clinical, biochemical, molecular, and morphologic features of a genetic defect in bile acid conjugation in 10 pediatric patients with fat-soluble vitamin deficiency, some with growth failure or transient neonatal cholestatic hepatitis.METHODS:We identified the genetic defect that causes this disorder using mass spectrometry analysis of urine, bile, and serum samples and sequence analysis of the genes encoding bile acid-CoA:amino acid N-acyltransferase (BAAT) and bile acid-CoA ligase (SLC27A5).RESULTS:Levels of urinary bile acids were increased (432 ± 248 μmol/L) and predominantly excreted in unconjugated forms (79.4% ± 3.9%) and as sulfates and glucuronides. Glycine or taurine conjugates were absent in the urine, bile, and serum. Unconjugated bile acids accounted for 95.7% ± 5.8% of the bile acids in duodenal bile, with cholic acid accounting for 82.4% ± 5.5% of the total. Duodenal bile acid concentrations were 12.1 ± 5.9 mmol/L, which is too low for efficient lipid absorption. The biochemical profile was consistent with defective bile acid amidation. Molecular analysis of BAAT confirmed 4 different homozygous mutations in 8 patients tested.CONCLUSIONS:Based on a study of 10 pediatric patients, genetic defects that disrupt bile acid amidation cause fat-soluble vitamin deficiency and growth failure, indicating the importance of bile acid conjugation in lipid absorption. Some patients developed liver disease with features of a cholangiopathy. These findings indicate that patients with idiopathic neonatal cholestasis or later onset of unexplained fat-soluble vitamin deficiency should be screened for defects in bile acid conjugation.

The precise role that isoflavones play in the health-related effects of soy foods, and their potential for adverse effects are controversial. This may be due in part to a lack of basic knowledge regarding their bioavailability and metabolism, particularly as it relates to the soy source. To date, there is little information concerning possible differences in the bioavailability of isoflavones derived from natural soy foods consumed at physiologically relevant intakes and whether age- or gender-related differences influence that bioavailability. In the current study of healthy adults [premenopausal (n = 21) and postmenopausal (n = 17) women and a group of men (n = 21)], we examined the effect of age, gender, and the food matrix on the bioavailability of isoflavones for both the aglycon and glucoside forms that are naturally present in 3 different soy foods, soy milk, textured vegetable protein, and tempeh. The study was designed as a random crossover trial so that all individuals received each of the 3 foods. The dose of isoflavones administered to each individual as a single bolus dose was 0.44 mg/kg body weight. Pharmacokinetic parameters were normalized to mg of each isoflavone ingested per kilogram body weight to account for differences in daidzein and genistein content between the diets. Serum isoflavone concentrations in all individuals and groups increased rapidly after the ingestion of each soy food; as expected, genistein concentrations exceeded daidzein concentrations in serum. In this small study, gender differences in peak concentrations of daidzein were observed, with higher levels attained in women. Consumption of tempeh (mainly isoflavone aglycon) resulted in higher serum peak levels of both daidzein (P < 0.001) and genistein (P < 0.01) and the associated area under the curve (P < 0.001 and P < 0.03, respectively) compared with textured vegetable protein (predominantly isoflavone glucosides). However, soy milk was absorbed faster and peak levels of isoflavones were attained earlier than with the other soy foods. Only 30% of the subjects were equol producers and no differences in equol production with age or gender were observed.

BACKGROUND:The discovery of equol in human urine more than 2 decades ago and the finding that it is bacterially derived from daidzin, an isoflavone abundant in soy foods, led to the current nutritional interest in soy foods. Equol, unlike the soy isoflavones daidzein or genistein, has a chiral center and therefore can occur as 2 distinct diastereoisomers.OBJECTIVE:Because it was unclear which enantiomer was present in humans, our objectives were to characterize the exact structure of equol, to examine whether the S- and R-equol enantiomers are bioavailable, and to ascertain whether the differences in their conformational structure translate to significant differences in affinity for estrogen receptors.DESIGN:With the use of chiral-phase HPLC and mass spectrometry, equol was isolated from human urine and plasma, and its enantiomeric structure was defined. Human fecal flora were cultured in vitro and incubated with daidzein to ascertain the stereospecificity of the bacterial production of equol. The pharmacokinetics of S- and R- equol were determined in 3 healthy adults after single-bolus oral administration of both enantiomers, and the affinity of each equol enantiomer for estrogen receptors was measured.RESULTS:Our studies definitively establish S-equol as the exclusive product of human intestinal bacterial synthesis from soy isoflavones and also show that both enantiomers are bioavailable. S-equol has a high affinity for estrogen receptor beta (K(i) = 0.73 nmol/L), whereas R-equol is relatively inactive.CONCLUSIONS:Humans have acquired an ability to exclusively synthesize S-equol from the precursor soy isoflavone daidzein, and it is significant that, unlike R-equol, this enantiomer has a relatively high affinity for estrogen receptor beta.

Pharmacokinetic studies of soybean isoflavones have shown that following oral ingestion, the two major isoflavones, daidzin and genistin, are hydrolyzed in the intestine, rapidly absorbed into the peripheral circulation, and eliminated from the body with a terminal half-life of 7-8 h. These characteristics make maintenance of steady-state plasma isoflavone concentrations difficult to attain unless there is repeated daily ingestion of foods or supplements containing isoflavones. In an attempt to sustain more constant plasma isoflavone concentrations, a new slow-release formulation of a soybean isoflavone extract was prepared by microencapsulation with a mixture of hydroxypropylcellulose and ethylcellulose to alter its dissolution characteristics. In vitro experiments confirmed slow aqueous dissolution of isoflavones from this formulation when compared with the conventional isoflavone extract. The pharmacokinetics of this slow-release isoflavone extract was studied in 10 healthy postmenopausal women after oral administration of a single capsule containing the equivalent of 22.3 mg of genistein and 7.47 mg of daidzein expressed as aglycons. A comparison of the key pharmacokinetic parameters obtained in this study with those established in extensive studies performed previously in this laboratory indicated that the mean residence time of genistein and daidzein increased 2-fold with microencapsulation. These findings are indicative of a decreased rate of absorption, consistent with the observed slow in vitro dissolution rate. These findings show that it is feasible to employ polymer matrices that slow the aqueous dissolution for preparing sustained-release formulations of soy isoflavones. Further studies to optimize such formulations are warranted.

The pharmacokinetics of isoflavones in 10 healthy women were determined from serum appearance/disappearance concentration profiles and urinary excretions after single-bolus ingestion of 10, 20 or 40 g of soy nuts delivering increasing amounts of the conjugated forms of daidzein (6.6, 13.2 and 26.4 mg) and genistein (9.8, 19.6 and 39.2 mg). Peak serum daidzein and genistein concentrations were attained after 4-8 h, and elimination half-lives were 8.0 and 10.1 h, respectively. There were no differences in the pharmacokinetics of daidzein and genistein between pre- and postmenopausal women, indicating absorption and disposition of isoflavones to be independent of age or menopausal status. A curvilinear relationship was observed between the bioavailability of daidzein and genistein, apparent from the area under the curve to infinity (AUC(inf)) of the serum concentration-time profiles and the amount of isoflavones ingested. The mean fraction of the isoflavones excreted in urine decreased with increasing intake when expressed as a percentage of the administered dose (63.2 +/- 8.0, 54.4 +/- 8.1 and 44.0 +/- 4.3%, respectively, for daidzein, and correspondingly, 25.2 +/- 5.3, 13.4 +/- 2.1 and 15.8 +/- 2.7% for genistein), underscoring the trend toward nonlinear pharmacokinetics. Equol was identified as a metabolite in 30% of women; it was present consistently in urine and blood from the same subjects. Its delayed appearance was consistent with colonic synthesis. On the basis of the pharmacokinetics, optimum steady-state serum isoflavone concentrations would be expected from modest intakes of soy foods consumed regularly throughout the day rather than from a single highly enriched product.

Campesterol is present in all the phytosterol-containing dietary hypocholesterolemic agents in current use. Campesterol is absorbed more efficiently than sitosterol, and the question of its possible atherogenicity has been raised. To test this possibility, rabbits were fed either a semipurified, cholesterol-free diet that has been shown to be atherogenic for this species or the same diet augmented with 0.5 g of phytosterol-rich diet preparations (spreads) containing either sitosterol or sitostanol. The diets contained 295 mg phytosterol per 100 g. After 60 d, serum cholesterol levels in the two phytosterol groups were 78±4 mg/dL (sitosterol) and 76±4 mg/dL (sitostanol), respectively. The serum cholesterol level of rabbits fed the control diet was 105±8 mg/dL. Serum campesterol (μg/mL) levels were higher than sitosterol or sitostanol levels in all groups. Aortic phytosterols were present in nanogram quantities compared to cholesterol, which was present in microgram quantities. The ratio of campesterol/sitosterol/sitostanol in the aortas was: control, 1.00∶0.43∶0.02; sitosterol, 1∶00∶0.32∶0.01; sitostanol, 1∶00∶0.34∶0.11. Aortic campesterol was present at 4% the concentration of aortic cholesterol, sitosterol at 1.4%, and sitostanol at 0.14%. Aortic lesions were not present in any of the animals.

BACKGROUND:The isoflavones daidzein and genistein occur naturally in most soyfoods, conjugated almost exclusively to sugars. Controversy exists regarding the extent of bioavailability of isoflavone glycosides, and the mechanism of intestinal absorption of isoflavones in humans is unclear. Evidence from intestinal perfusion and in vitro cell culture studies indicates that isoflavone glycosides are poorly absorbed, yet isoflavones are bioavailable and appear in high concentrations in plasma, irrespective of whether they are ingested as aglycones or glycoside conjugates.OBJECTIVE:The objective was to determine whether isoflavone glycosides are absorbed from the intestine intact and reach the peripheral circulation unchanged.DESIGN:Plasma was collected at timed intervals before and after healthy adults ingested 50 mg of one of the isoflavone beta-glycosides (daidzin or genistin) or 250 mL soymilk containing mainly isoflavone glycosides. Electrospray ionization mass spectrometry was used to detect daidzin and genistin after solid-phase extraction of these conjugates from plasma. Bioavailability of isoflavones was confirmed by gas chromatography-mass spectrometry analysis.RESULTS:Specific and sensitive electrospray mass spectrometry failed to detect even traces of daidzin or genistin in plasma collected 1, 2, and 8 h after their ingestion as pure compounds or in a soyfood matrix. However, plasma was enriched in isoflavones that were hydrolyzable with a combined beta-glucuronidase and sulfatase enzyme preparation.CONCLUSION:Isoflavone glycosides are not absorbed intact across the enterocyte of healthy adults, and their bioavailability requires initial hydrolysis of the sugar moiety by intestinal beta-glucosidases for uptake to the peripheral circulation.

The pharmacokinetic behavior of naturally occurring isoflavones has been determined for the first time in healthy adults. We compared plasma kinetics of pure daidzein, genistein and their beta-glycosides administered as a single-bolus dose to 19 healthy women. This study demonstrates differences in the pharmacokinetics of isoflavone glycosides compared with their respective beta-glycosides. Although all isoflavones are efficiently absorbed from the intestinal tract, there are striking differences in the fate of aglycones and beta-glycosides. Mean time to attain peak plasma concentrations (t(max)) for the aglycones genistein and daidzein was 5.2 and 6.6 h, respectively, whereas for the corresponding beta-glycosides, the t(max) was delayed to 9.3 and 9.0 h, respectively, consistent with the residence time needed for hydrolytic cleavage of the glycoside moiety for bioavailability. The apparent volume of distribution of isoflavones confirms extensive tissue distribution after absorption. Plasma genistein concentrations are consistently higher than daidzein when equal amounts of the two isoflavones are administered, and this is accounted for by the more extensive distribution of daidzein (236 L) compared with genistein (161 L). The systemic bioavailability of genistein [mean AUC = 4.54 microg/(mL x h)] is much greater than that of daidzein [mean AUC = 2.94 microg/(mL x h)], and bioavailability of these isoflavones is greater when ingested as beta-glycosides rather than aglycones as measured from the area under the curve of the plasma appearance and disappearance concentrations. The pharmacokinetics of methoxylated isoflavones show distinct differences depending on the position of the methoxyl group in the molecule. Glycitin, found in two phytoestrogen supplements, underwent hydrolysis of the beta-glycoside moiety and little further biotransformation, leading to high plasma glycitein concentrations. Biochanin A and formononetin, two isoflavones found in one phytoestrogen supplement, were rapidly and efficiently demethylated, resulting in high plasma genistein and daidzein concentrations typically observed after the ingestion of soy-containing foods. These differences in pharmacokinetics and metabolism have implications for clinical studies because it cannot be assumed that all isoflavones are comparable in their pharmacokinetics and bioavailability. An analysis of 33 phytoestrogen supplements and extracts revealed considerable differences in the isoflavone content from that claimed by the manufacturers. Plasma concentrations of isoflavones show marked qualitative and quantitative differences depending on the type of supplement ingested. These studies indicate a need for improvement in quality assurance and standardization of such products.