Unconjugated Fraction Research Articles

Reference data on estrogen metabolome in healthy pregnancy

IntroductionEstrogen hormones and their metabolites are implicated in the maintenance of healthy pregnancy and adequate fetal development. Abnormal levels were related to increased risk of pregnancy complications, particularly preeclampsia. Our aims were (1) to develop a methodological platform for the comprehensive assessment of estrogen metabolome in pregnancy; (2) to collect healthy reference data for relevant elements of estrogen metabolome in each trimester; (3) to assess unconjugated fractions of the estrogen metabolome, (4) to assess the dominant metabolic pathways of estrogen compounds. MethodsWe enrolled healthy pregnant mothers between gestational week 5–15 (on the confirmation of pregnancy; 79 samples), gestational weeks 19–27 (70 samples), and gestational week 34–39 (54 samples). A method employing liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed to assess estrone, 17-beta-estradiol, estriol levels, and their metabolites as conjugated and unconjugated forms. Descriptive statistics were used to characterize the level of compounds in each trimester. ResultsEstrone, 17-beta-estradiol and estriol levels are dramatically increasing with the advancement of pregnancy. Measured levels were in a very wide range. 17-beta-estradiol is neither glucuronated nor sulphated. To the contrary, estriol and estrone are significantly conjugated; unconjugated fraction is <15% of total hormone levels in any trimester. Regarding metabolism, 4-methoxy-estradiol and 17-epiestriol were not detected. ConclusionWe concluded that (1) the levels of estrogen compounds and metabolites increase with advancing gestational age; (2) the wide ranges of levels challenge the establishment of a healthy reference range for clinical purposes; (3) 17-beta-estradiol is not conjugated significantly; (4) 4-methylation and 17-epimerization pathways of estrogens are negligible with our LC-MS/MS method.

Measurement and Clinical Usefulness of Delta Bilirubin in Liver Disease

Delta bilirubin is albumin bound conjugated bilirubin that can be calculated by formula [Total bilirubin - (conjugated + unconjugated bilirubin)]. Newer technology like dry chemistry provide conjugated and unconjugated bilirubin fraction which is more specific and informative than direct and indirect bilirubin by conventional method in which only approximate values of conjugated bilirubin can be measured. In cholestasis conjugated bilirubin combines with albumin non enzymatically and form delta bilirubin in circulation and remains for long duration. The present review describes the usefulness of delta bilirubin in cholestasis, chronicity of liver diseases, in pediatric jaundice, transplant rejection and other jaundice condition.clinical correlation of delta bilirubin in all conditions have been summarized and their future perspectives are discussed.

Circulating bile acid profile characteristics in PCOS patients and the role of bile acids in predicting the pathogenesis of PCOS.

The metabolic profile of bile acids and their potential role as biomarkers in the pathogenesis of polycystic ovary syndrome (PCOS) have not been thoroughly characterized. Assessing their predictive value for PCOS is of significant importance. In this study, we enrolled 408 women with PCOS and 204 non-PCOS controls. The serum bile acid profile was measured using high-performance liquid chromatography-tandem mass spectrometry (LC/MS). We analyzed the differences in serum bile acid profiles between PCOS patients using the OPLS-DA model. Additionally, we examined the relationship between bile acid profiles and parameters related to glucose metabolism and hyperandrogenism. ROC analysis was employed to identify potential biomarkers for PCOS pathogenesis. XGboost was utilized for cross-validation. The bile acid profile was found to be altered in PCOS patients. Specifically, the primary and secondary unconjugated bile acid fractions were significantly higher in the PCOS population. We identified five bile acid metabolite candidates that exhibited the most significant differences between PCOS and non-PCOS controls. DCA was associated with deposition index, fasting and postprandial insulin but was influenced by testosterone. CDCA and LCA combined with testosterone showed potential as biomarkers for the pathogenesis of PCOS. The circulating bile acid profile undergoes changes in PCOS. DCA is associated with deposition index, fasting and postprandial insulin and its level is influenced by testosterone. CDCA and LCA combined with testosterone have the potential to serve as biomarkers for the pathogenesis of PCOS.

Derivatized versus non-derivatized LC-MS/MS techniques for the analysis of estrogens and estrogen-like endocrine disruptors in human plasma

Bisphenols, parabens, alkylphenols and triclosan are anthropogenic substances with a phenolic group that have been introduced to the environment in recent decades. As they possess hormone-like effects, they have been termed endocrine disruptors (EDs), and can interfere with steroid pathways in organisms. To evaluate the potential impact of EDs on steroid biosynthesis and metabolism, sensitive and robust methods enabling the concurrent measurement of EDs and steroids in plasma are needed. Of crucial importance is the analysis of unconjugated EDs, which possess biological activity. The aim of the study was to develop and validate LC-MS/MS methods with and without a derivatization step for the analysis of unconjugated steroids (estrone-E1, estradiol-E2, estriol-E3, aldosterone-ALDO) and different groups of EDs (bisphenols, parabens, nonylphenol-NP and triclosan-TCS), and compare these methods on a set of 24 human plasma samples using Passing-Bablok regression analysis. Both methods were validated according to FDA and EMA guidelines. The method with dansyl chloride derivatization allowed 17 compounds to be measured: estrogens (E1, E2, E3), bisphenols (bisphenol A-BPA, BPS, BPF, BPAF, BPAP, BPZ, BPP), parabens (methylparaben-MP, ethylparaben-EP, propylparaben-PP, butylparaben-BP, benzylparaben-BenzylP), TCS and NP, with lower limits of quantification (LLOQs) between 4 and 125 pg/mL. The method without derivatization enabled 15 compounds to be analyzed: estrogens (E1, E2, E3), ALDO, bisphenols (BPA, BPS, BPF, BPAF, BPAP, BPZ), parabens (MP, EP, PP, BP, BenzylP) with LLOQs between 2 and 63 pg/mL, and NP and BPP in semiquantitative mode. Adding 6 mM ammonium fluoride post column into mobile phases in the method without derivatization achieved similar or even better LLOQs than the method with the derivatization step. The uniqueness of the methods lies in the simultaneous determination of different classes of unconjugated (bioactive) fraction of EDs together with selected steroids (estrogens + ALDO in the method without derivatization), which provides a useful tool for evaluating the relationships between EDs and steroid metabolism.

Gilbert syndrome as a risk factor for the development of cholelithiasis in children

Introduction/Objective. Gilbert syndrome (GS) is the most common hereditary hyperbilirubinemia. As well as mild unconjugated hyperbilirubinemia, it is characterized by the excess of bilirubin monoglucuronide over diglucuronide in the bile and thus increases the risk of biliary calculosis. The aim of the study was to determine the importance of GS as a risk factor in the development of cholelithiasis in children. Methods. The study included a sample of 31 children (14 male and 17 female, mean age 12.16 ? 4.11 years, range 3?16.75 years) with symptomatic cholelithiasis. The diagnosis of cholelithiasis was based on an ultrasonographic finding, and for GS the diagnosis was based on at least a double increase of unconjugated bilirubin fraction after a three-day hypocaloric diet (400 kcal per day). Results. GS was confirmed in five or 16.13% of patients (three male and two female, mean age 14.71 ? 0.55 years, range 14?15.3 years). In addition to GS, in the history of the disease they all had some of the additional risk factors for the development of cholelithiasis. One of them had an identical problem as its mother, one had hereditary elliptocytosis, one had sudden weight loss, one was overweight, and one had premature birth and sepsis. Conclusion. GS registers in one-sixth of children with cholelithiasis, but in none of them as the only risk factor for developing this disease. This finding suggests that GS is a risk factor for the development of cholelithiasis, but not sufficient in itself in that respect.

Informativeness of biochemical markers of connective tissue metabolism in canine babesiosis

The aim: to analyze the diagnostic informativeness of biochemical markers of connective tissue in the blood of dogs with babesiosis.&#x0D; Materials and methods. German Shepherd (n=7), Labrador Retriever (n=3) and Rottweiler (n=2) dogs aged 1 to 5 years and diagnosed with babesiosis were studied. As a control group, clinically healthy dogs were used, which came to the veterinary clinic for a preventive examination, the age of the animals was from 1 to 5 years (n=10). The animals were examined according to the following scheme: collection of anamnestic data, clinical examination according to generally accepted methods, general and biochemical blood analysis, urine examination. Glycoproteins in blood serum were determined according to Shteinberg – Dotsenko, sialic acids – according to the Hess method, chondroitin sulfates – Nemeth–Csoka in the modification of L.I. Slutsky.&#x0D; Results. The biochemical examination of the blood revealed the presence of acute cytolytic syndrome and cholestasis in the animal's body. Cholestasis in sick animals was characterized by an increase in the content of bilirubin and the activity of alkaline phosphatase, as well as an increase in the content of β-lipoproteins. The increase in the content of total bilirubin in the blood was obviously due mainly to its unconjugated fraction, since intravascular hemolysis took place. In the blood serum of patients with babesiosis in dogs, there was an increase in the content of markers of connective tissue metabolism – glycoproteins by 1.63, sialic acids – by 1.36, and chondroitin sulfates by 1.8 times, respectively.&#x0D; Conclusions. Glycoproteins have "acute phase proteins" in their composition, which are indicators for assessing the degree of the inflammatory process in the body of dogs with babesiosis. Sialic acids are components of sialoproteins, which are also markers of the inflammatory process and destructive changes in the body. An increase in the content of chondroitin sulfates in the blood during babesiosis indicates the development of a compensatory reaction, associated with the action of toxic hemolysis products on the endothelium of blood vessels. Thus, the increased content of biochemical markers of connective tissue in the blood of dogs with babesiosis indicates the presence of a systemic inflammatory-protective reaction in animals, which makes it possible to recommend them for assessing the acute phase of the inflammatory process in the body and protecting blood vessels from damage due to toxic hemolysis

Circulating Conjugated and Unconjugated Vitamin D Metabolite Measurements by Liquid Chromatography Mass Spectrometry.

ContextVitamin D status is conventionally defined by measurement of unconjugated circulating 25-hydroxyvitamin D (25OHD), but it remains uncertain whether this isolated analysis gives sufficient weight to vitamin D’s diverse metabolic pathways and bioactivity. Emerging evidence has shown that phase II endocrine metabolites are important excretory or storage forms; however, the clinical significance of circulating phase II vitamin D metabolites remains uncertain.ObjectiveIn this study we analyzed the contribution of sulfate and glucuronide vitamin D metabolites relative to unconjugated levels in human serum.MethodsAn optimized enzyme hydrolysis method using recombinant arylsulfatase (Pseudomonas aeruginosa) and beta-glucuronidase (Escherichia coli) was combined with liquid chromatography mass spectrometry (LC-MS/MS) analysis to measure conjugated and unconjugated vitamin D metabolites 25OHD3, 25OHD2, 3-epi-25OHD3, and 24,25(OH)2D3. The method was applied to the analysis of 170 human serum samples from community-dwelling men aged over 70 years, categorized by vitamin D supplementation status, to evaluate the proportions of each conjugated and unconjugated fraction.ResultsAs a proportion of total circulating vitamin D metabolites, sulfate conjugates (ranging between 18% and 53%) were a higher proportion than glucuronide conjugates (ranging between 2.7% and 11%). The proportion of conjugated 25OHD3 (48 ± 9%) was higher than 25OHD2 conjugates (29.1 ± 10%) across all supplementation groups. Conjugated metabolites correlated with their unconjugated forms for all 4 vitamin D metabolites (r = 0.85 to 0.97).ConclusionSulfated conjugates form a high proportion of circulating vitamin D metabolites, whereas glucuronide conjugates constitute a smaller fraction. Our findings principally in older men highlight the differences in abundance between metabolites and suggest a combination of both conjugated and unconjugated measurements may provide a more accurate assessment of vitamin D status.

Проблема диагностики приобретенных гемолитических анемий в детском возрасте

Аутоиммунная гемолитическая анемия (АИГА) — это группа заболеваний, в основе которых лежит декомпенсированный приобретенный гемолиз. АИГА у детей чаще всего наблюдается после вирусного заболевания. Это достаточно редкое состояние с частотой менее 0,2 случая на 100 000 педиатрического населения. Целью статьи было представить клинический случай больного АИГА. Представлен случай гемолитической анемии у ребенка 4 лет на фоне острой вирусной инфекции с пневмонией и диспептическим синдромом. Во время пребывания в больнице состояние мальчика резко ухудшилось за счет быстрого нарастания слабости и бледности кожных покровов. Общий анализ крови выявил прогрессирующую гемолитическую анемию (Hb 57 г/л, эритроциты 2,58 × 1012/л, Ht 0,16, лейкоциты 13,88 × 109/л, СОЭ 69 мм/ч, тромбоциты 400 × 109/л). Наблюдалось умеренное повышение билирубина за счет неконъюгированной фракции (31,3 и 21,2 мкмоль/л соответственно). В связи с критическим нарастанием анемического синдрома встал вопрос о необходимости гемотрансфузии, но в результате теста на индивидуальную совместимость 20 пакетов крови одной группы было отклонено из-за несовместимости. Интенсивная иммуносупрессивная терапия (комплексное введение высоких доз кортикостероидов в сочетании с внутривенными иммуноглобулинами в дозе 2 г/кг/день) позволила остановить гемолиз и улучшить состояние пациента, вопрос о переливании крови был отклонен. Особенностью представленного случая было критическое нарастание анемии при отсутствии классических признаков гемолиза, невозможность проведения гемотрансфузии и положительная динамика при назначении высоких доз кортикостероидов и иммуноглобулина. Осведомленность об АИГА должна присутствовать у педиатров, и важно расширить их знания о правильном алгоритме диагностики в таких случаях. Препаратами выбора для лечения АИГА являются внутривенные иммуноглобулины в иммуносупрессивных дозах и глюкокортикоиды коротким курсом. В случаях резистентности к такому лечению показано переливание перфторана.

In vitro and in vivo metabolism of the anabolic-androgenic steroid oxandrolone in the horse.

Oxandrolone is an anabolic-androgenic steroid with favourable anabolic to androgenic ratio, making it an effective anabolic agent with less androgenic side effects. Although its metabolism has been studied in humans, its phase I and II metabolism has not been previously reported in the horse. The purpose of this study was to investigate the in vitro metabolism of oxandrolone (using both equine liver microsomes and S9) and in vivo metabolism following oral administration (three daily doses of 50 mg of oxandrolone to a single Thoroughbred horse), using both gas and liquid chromatography-mass spectrometry techniques. The in vitro phase I transformations observed included 16-hydroxylated (two epimers), 17-methyl-hydroxylated and 16-keto metabolites. In addition to parent oxandrolone and these hydroxylated metabolites, the 17-epimer and a 17,17-dimethyl-18-norandrost-13-ene analogue were detected in biological samples following the administration. 16-keto-oxandrolone was only observed in urine. The 16- and 17-methyl-hydroxylated oxandrolone metabolites were predominantly excreted as sulfate conjugates in urine, whereas parent oxandrolone, its epimer and 17,17-dimethyl-18-norandrost-13-ene derivative were found predominantly in the unconjugated urine fraction. The most abundant analyte detected in both plasma and urine was parent oxandrolone. However, the longest detection period using the developed analytical method was provided by 17-hydroxymethyl-oxandrolone in both matrices. The results of this study provided knowledge of how best to detect the use of oxandrolone in regulatory samples.

Detection of urinary arimistane metabolites in humans using liquid chromatography-mass spectrometry: Complementary results to gas chromatography mass spectrometric data and its application to antidoping analyses.

The metabolism of arimistane (Arim) was first described in 2015, and androst-3,5-diene-7β-ol-17-one was proposed as the main metabolite excreted in urine. Recently, a more detailed study describing the findings in urine after the administration of Arim has been published. This study corroborated the previously described metabolite but also described several phase I and II metabolites, analyzing trimethylsilylated urinary extracts using accurate mass spectrometry coupled to gas chromatography (GC/qTOF). The present communication is an extension of this late investigation aiming to implement the results of Arim metabolism using either accurate mass spectrometry and/or triple quadrupole tandem mass spectrometry, both coupled to liquid chromatography (LC/qTOF and LC/QqQ). The samples used in this study were the same as previously studied using GC/qTOF. One single oral dose of Arim was administered to three volunteers, and samples collected before and up to 10 h after the Arim administration were analyzed. The unconjugated fraction of urine was removed, and the hydrolysis was performed with β-glucuronidase from Escherichia coli. The extracts were reconstituted in water:acetonitrile before the LC/qTOF and LC/QqQ analysis. The presence of the proposed metabolites studied using GC was verified by accurate mass measurements. Twelve metabolites not found in the blank urine samples were identified by the accurate mass spectra with acceptable errors between -7.5 and 8.1 ppm: 4 reduced metabolites, 4 monohydroxylated metabolites, and 4 with an additional hydroxylation (bis-hydroxylated metabolites). Unlike in the study carried out using GC/qTOF, Arim itself was found in the samples of the three volunteers. Twelve metabolites were identified, and specific transitions were proposed. Despite the good results, some limitations remain. As for GC/qTOF, the α- or β configuration of hydroxy groups, as well as the exact position for some unsaturation, cannot be assigned with certainty. Because certified reference materials of these metabolites are not yet available, the molecular structures were hypothesized considering the previous study using GC.

Unconjugated Hyperbilirubinemia after Open Heart Surgery

The occurrence of hyperbilirubinaemia after heart surgery using cardiopulmonary bypass or post-operative heart failure is fairly common. Mechanism of hyperbilirubinemiais still not completely clarified, and there are so few specific therapies available for acute hepatobiliary injury. Post-operative mortality well correlates with increasing total bilirubin values, particularly for bilirubin-associate acute kidney tubular necrosis. The difficulty to reduce mortality is partially a consequence of not completely understood physiopathology. It is obvious that long-lasting CPB plays an important role, in association with hemodilution, hypotension, ischemia-reperfusion, and increasing hematic level of endogenous catecholamine with reduction of hepatic blood flow. Case report. A 68 years old man with severe mitral valve regurgitation and pulmonary hypertension and low EF 30%. Mitral valve replacement and tricuspid anuloplastic was performed. Due to low cardiac output syndrome severe hyperbilirubiemeia was seen (24 mg/dl. and unconjugated fraction 16mg/dl) days after. Phenobarbital (luminal) was started 15 mg/kg daily. Two days later the level decreased until 8 mg/dl with normalization of conjugation/unconjugation ratio.Postoperative hyperbilirubinemia is a multifactorial process caused by both impaired liver function of bilirubin transport. In case of elevated level of unconjugated fraction we suggest to use Luminal as alternative for decreasing unconjugated fraction.

Functional activity of the liver under the conditions of immersion and effects of countermeasures

Ultrasound investigations (USI) of the liver, organs and vessels of the gastroduodenal area, as well as blood biochemistry, were performed in two groups of male volunteers on the 4th day of their stay in the conditions of “dry” immersion with and without the application of countermeasures, including the support load imitator (SLI) or high-frequency electromyostimulation. Using 13С-methacetin breath test (13C-MBT), two other groups were investigated for the effect of immersion on the detoxification activity and metabolic capacity of the liver and the efficacy of SLI. The performed USIs have identified deceleration in the hepatic venous flow and the signs of plethora in the abdominal venous system. Elevated blood levels were detected in pepsinogen, pancreatic amylase, bilirubin total, due to its unconjugated fraction, insulin, and C-peptide. The 13C-MBT has shown a slowdown in the rate of 13C-methacetin inactivation and a reduction in the hepatic metabolic capacity. The application of countermeasures during the immersion has not affected the ultrasound patterns of the hemodynamic rearrangement in both the liver and the abdomen. High frequency electromyostimulation during the immersion has neutralized the changes in all biochemical indicators except C-peptide, while the application of SLI has led to the restoration of only pepsinogen and amylase to the initial values. In addition, the use of SLI during the immersion counteracted the reduction in the 13C-methacetin inactivation rate and did not substantially affect the reduction in the metabolic capacity of the liver.

Genotype-dependent metabolism of exogenous testosterone - new biomarkers result in prolonged detectability.

Testosterone (T) misuse still represents a major problem in sports drug testing. Many strategies have been developed and applied to routine doping controls in recent years to enable both to identify suspicious samples in initial testing procedures and to confirm the exogenous origin of urinary T by means of carbon isotope ratio (CIR) determinations. Depending on the tested individual's genotype of UGT2B17, significantly different amounts of T are glucuronidated and excreted, which results in unaffected T/epitestosterone ratios after T misuse in those subjects with the deletion/deletion polymorphism (del/del). The aim of this study was to investigate differences in metabolic pathways of orally administered T between persons of del/del and insertion/insertion (ins/ins) genetic polymorphism. Therefore, a recently established method using hydrogen isotope ratios together with high-resolution and high-accuracy mass spectrometry was applied after administration of deuterated T to n = 4 subjects including both genotypes. Participants collected urine specimens directly before and for up to 8 days after the application. Urine aliquots were prepared to yield unconjugated, glucuronidated, and sulphoconjugated fractions of urinary steroids. Besides the significant difference in the excretion of T-glucuronide, all measured metabolites varied rather on an individual basis than due to a genotype difference. New T metabolites (both methylated and de-methylated) were detected and investigated regarding their potential to enhance the screening for T misuse. Sulphoconjugated epiandrosterone was further identified as the biomarker allowing for a prolonged retrospective detection of T misuse by means of CIR determinations for up to 5 days compared to 1 day if currently applied sports drug testing procedures were used. Copyright © 2016 John Wiley & Sons, Ltd.

Urine and serum biomonitoring of exposure to environmental estrogens I: Bisphenol A in pregnant women

Despite its very low oral bioavailability and rapid elimination, multiple reports of unexpectedly high bisphenol A (BPA) concentrations in the serum of pregnant mothers or cord blood have raised questions about BPA exposures during pregnancy. Thirty healthy pregnant women recruited to the study were evaluated for total BPA exposure over a 30-h period comprising one-half day in the field and one day in a clinical setting. BPA and its metabolites were measured in serum and total BPA was measured in matching urine samples. The mean total exposure was similar to the 50th percentile of exposure for U.S. women and pregnant women in a large North American cohort. Twenty volunteers had total daily exposures equal to or exceeding the U.S. mean, and six volunteers had exposures exceeding the 75th percentile. Women working as cashiers did not have higher total BPA exposure. BPA was detected in some serum samples (0.25–0.51 ng/ml), but showed no relationship to total BPA in corresponding urine samples, no relationship to total BPA exposure, and had unconjugated BPA fractions of 60–80%, consistent with established criteria for sample contamination. We conclude that typical exposures of North American pregnant women produce internal exposures to BPA in the picomolar range.

Galactosemia como causa de ictericia neonatal

Jaundice is a commonly occurring problem in hospitals' neonatal units. This report describes a newborn with hypefbilirrubinaemia; the clinical picture was initially dominated by the unconjugated bilirubin fraction and later by anaemia, hepatomegaly, increased conjugated bilirubin fraction, impaired liver function, renal tubular dysfunction, cataracts and injury to CNS white matter. Various causes of direct hyperbilirrubinaemia in the newborn were ruled out. Thin-layer chromatography revealed urine-reducing substances (i.e. galactose). A marked reduction in galactose uridyl transferaseactivity was documented, thereby confirming a diagnosis of galactosaemia. Breastfeedingwas discontinued and soy milk started which led to the symptoms becoming resolved.

The effect of zinc sulphate supplementation on atazanavir/ritonavir‐associated hyperbilirubinemia

BackgroundAtazanavir (ATV) causes an exposure dependent elevation of unconjugated hyperbilirubinemia (HBR) as a result of UGT1A1 inhibition. Zinc sulphate reduces unconjugated hyperbilirubinemia in individuals with Gilbert's syndrome. We assessed the changes in total and conjugated bilirubin following single dose and 14 daily doses of zinc sulphate (ZnSO4) and its impact on ATV pharmacokinetics (PK).MethodsHIV‐infected individuals stable on ATV/ritonavir (r) containing regimens with a total bilirubin level &gt; 25 mmol/L were administered 125 mg once daily of as solvazinc effervescent tablets for 14 days. ATV and bilirubin concentrations were measured pre‐ATV‐dose, 2, 4, 6, 8 and 24 hours post‐ATV‐dose before ZnSO4 intake initiation (phase 1), after a single dose of ZnSO4 (phase 2), and following 14 days of ZnSO4 intake (phase 3). Changes in bilirubin and ATV concentrations in the absence and presence of ZnSO4 were evaluated by geometric mean ratios (GMR) and 90% confidence intervals (CI, phase 1 as reference).ResultsAll 16 male patients completed the study maintaining virologic suppression throughout. ZnSO4 was well tolerated. We observed statistically significant declines in total bilirubin Cmax and AUC0–24 of −12 and −13% after single dose ZnSO4 and −19 and −20% after steady state, compared to reference phase; GM Cmax decreasing from 57 nmol/L before zinc intake to 50 and 46 nmol/L after ZnSO4 single dose and steady state, respectively. No significant changes in conjugated bilirubin were observed, indicating that the changes were secondary to declines in the unconjugated fraction (data pending). ATV GMR (90% CI) for Ctrough, Cmax and AUC were −16% (−33 to +6), −8% (−20 to +8) and −12% (−23 to +0.1) after single dose ZnSO4, but changed by −26% (−38 to −11) −18% (−30 to −3) and −22% (31 to −12) after multiple dose ZnSO4 compared to reference. All individuals with the exception of one (whose levels were low throughout the study) maintained ATV concentration above the suggested MEC of 150 ng/mL.ConclusionsThe intake of ZnSO4 led to a moderate decrease in total bilirubin maximum concentration and overall exposure. However, a decrease in ATV concentrations was also observed. ZnSO4 supplementation may represent a useful tool in the management of ATV‐related HBR.

Postprandial plasma bile acid responses in normal weight and obese subjects

Bile acids can act as signalling molecules via various receptors including the nuclear farnesoid X receptor (FXR) and pregnane X receptor (PXR), and the cell surface G-protein-coupled receptor TGR5. The signalling has been implicated in the release of peptide YY (PYY) and glucagon-like peptide 1 (GLP-1), which improves glycaemic control and energy expenditure. We investigated whether morbidly obese subjects have altered postprandial bile acid responses in comparison to normal weight subjects. Blood samples were taken every 30 min from 0 to 180 min following a 400 kcal test meal. Samples were taken from 12 normal weight subjects with a body mass index (BMI) of 23.2 (2.8) kg/m(2) (median [interquartile range (IQR)]) and seven obese patients with a BMI of 47.2 (7.2) kg/m(2). Fractionated bile acids were measured on these samples using high-performance liquid chromatography tandem mass spectrometry. The obese subjects showed a lower postprandial response in total bile acids compared with the normal weight subjects. An increase of 6.4 (5.0) and 2.6 (3.3) micromol/L (median [IQR]) in normal weight and obese subjects was observed, respectively (P = 0.02). The difference was predominantly due to the glycine-conjugated fraction (P = 0.03). There was no difference in the increase of the unconjugated or taurine-conjugated fractions. The decreased postprandial bile acid response in obese subjects compared with normal weight subjects may partly explain the suboptimal GLP-1 and PYY responses and could affect appetite, glycaemic control and energy expenditure.

Synthesis and Metabolism of 16α-Hydroxy-Steroids and Corticosteroids in Fetuses and Neonates

This study involved the biosynthesis and metabolism of a series of 16α-hydroxy-steroids and gluco- and mineralocorticoids in the fetus and their concentrations in materno-fetal plasma and amniotic fluid. In the biosynthesis of 16α-hydroxy-steroids, 17α-hydroxylase and C17–20-lyase in the adrenals and 16α-hydroxylase in the liver which are distributed in the microsomal fractions, extremely high activities are observed in 16–20 week fetuses. High sulfokinase activity is also found in the 105,000xg supernatant fraction and the activity in decreasing order is adrenal, liver and small intestine. These enzyme activities are affected by some steroids present in the umbilical plasma. In the biosynthesis of gluco- and mineralocorticoids, 17α-, 21-hydroxylase are distributed in the extramitochondrial fraction of the definitive adrenal cortex while 11β-, 18-hydroxylase and 18-dehydrogenase are in the mitochondrial fraction. All of these enzymes show high activities relatively early in gestation and angiotensin-II shows a selective stimulation for 11β-, 18-hydroxylase and potassium together with ACTH for 18-dehydrogenase. All of the 16α-hydroxy-steroids are consistently higher in the fetal plasma than in maternal plasma and show a 2 to 4 fold increase during the 3rd trimester of gestation. All of corticosteroids assayed are also higher in the fetal plasma and increase during this period. It is well established that a portion of cortisol undergoes sulfate conjugation or conversion to tetrahydro-derivative and that aldosterone is largely converted to 3α' 5β-tetrahydro-aldosterone and a portion undergoes glucosiduronation. Thus, in the fetus, some trophic hormones and endogenous steroids become active in the control of 16α-hydroxy-steroid and corticosteroid biosynthesis and metabolism. In conjunction, the presence of protein bound, unbound fractions as well as sulfate or glucuronide conjugated, unconjugated fractions in the blood tends to maintain an equilibrium and control over these systems.

Arias icterus: Prolonged unconjugated hyperbilirubinemia caused by breast milk

Breast milk jaundice occurs in 1-2% of healthy breast-fed newborns and young infants. It develops as the result of liver immaturity and the inhibitory effect of mother's milk to the clearance of unconjugated bilirubin. The paper analyzes variations in the level and length of unconjugated hyperbilirubinemia in breast-fed infants. The study was conducted on a sample of 29 young infants (19 male) with breast milk jaundice. All infants were born on time, by natural delivery and without complications. All were on breast-feeding only and developed optimally. None of the infants had either haemolysis or any other disease associated with unconjugated hyperbilirubinemia. All infants had physiological jaundice in the first week after birth, with unconjugated bilirubin level of 166-260 micromol (201.50 +/- 36.37 micromol). In the postneonatal period the highest bilirubin level was recorded in the fifth week of life and was 87-273 micromol (166.82 +/- 45.06 micromol), which then spontaneously, without interruption of breast-feeding, gradually declined. The decrease of the unconjugated fraction of serum bilirubin between the fourth and fifth week was significant, and after that highly significant. The normalization of serum bilirubin occurred in the seventh and thirteenth week (10.41 +/- 1.68 micromol). Negative consequences of hyperbilirubinemia were not noted in any of the infants. Breast milk jaundice presents a harmless and transitory disorder of bilirubin metabolism. It occurs in healthy breast-fed neonates and young infants. Jaundice is most marked in early neonatal period, and then it gradually declines and disappears between the seventh and thirteenth week.

Gilbert’s syndrome in children: Our experience

Gilbert's syndrome represents the most frequent hereditary disorder of bilirubin metabolism. It occurs in 2-7% of subjects in general population, and is manifested by mild unconjugated hyperbilirubinaemia of benign nature. The study was conducted in order to analyse our experience related to the circumstances of disclosure, age at onset and sex distribution of Gilbert's syndrome in children. The diagnosis of Gilbert's syndrome was based on the findings of mild unconjugated hyperbilirubinaemia in the absence of haemolysis and organic liver disease, as well as significant increase of bilirubin unconjugated fraction in serum after a 3-day hypocaloric diet (400 kcal/daily). Of 58 subjects with Gilbert's syndrome, there were 40 (68.97%) boys and 18 (31.03%) girls, indicating a 2.22 fold higher incidence of boys than girls. The age at diagnosis of the disorder was similar in boys and girls, and was 12.2-18 (X =14.71 +/- 1.55) years for boys and 10.5-16.4 (X = 14.38 +/- 2.10) years for girls (p > 0.05). Except for 3 girls aged below 12 years, in all other patients the diagnosis was made after that age; in 20, between age 12 and 14 years and in 14, between age 14 and 16 years, while in 10 patents, it was made at age between 16 and 18 years. In 20 (34.48%) subjects, hyperbilirubinaemia was disclosed on routine paediatric examination, in 19 (32.76%) when differentiating the cause of recurrent or acute abdominal pain, in 17 (29.3%) during febrile condition and in 2 during insufficient caloric intake. The values of unconjugated bilirubinaemia, both before a hypocaloric test and after a 3-day energy deprivation, were higher in boys than girls, but this difference was statistically significant only after the 3-day hypocaloric diet in age group between 16 and 18 years (p = 0.038). Also, no significant difference was found in bilirubinaemia level between the different age groups either in boys or girls. According to our findings, in children, Gilbert's syndrome is manifested only in puberty, and 2.22 times more often in boys than girls. It is most frequently revealed during a routine paediatric examination, as well as when investigating the cause of abdominal pain and fever. A significantly higher level of serum bilirubin in boys as related to girls is registered only after a hypocaloric diet in age group between 16 and 18 years.