Probiotics improve the neurometabolic profile of rats with chronic cholestatic liver disease.

Chronic liver disease leads to neuropsychiatric complications called hepatic encephalopathy (HE). Current treatments have some limitations in their efficacy and tolerability, emphasizing the need for alternative therapies. Modulation of gut bacterial flora using probiotics is emerging as a therapeutic alternative. However, knowledge about how probiotics influence brain metabolite changes during HE is missing. In the present study, we combined the advantages of ultra-high field in vivo 1H MRS with behavioural tests to analyse whether a long-term treatment with a multistrain probiotic mixture (VIVOMIXX) in a rat model of type C HE had a positive effect on behaviour and neurometabolic changes. We showed that the prophylactic administration of this probiotic formulation led to an increase in gut Bifidobacteria and attenuated changes in locomotor activity and neurometabolic profile in a rat model of type C HE. Both the performance in behavioural tests and the neurometabolic profile of BDL + probiotic rats were improved compared to the BDL group at week 8 post-BDL. They displayed a significantly lesser increase in brain Gln, a milder decrease in brain mIns and a smaller decrease in neurotransmitter Glu than untreated animals. The clinical implications of these findings are potentially far-reaching given that probiotics are generally safe and well-tolerated by patients.

A Comparison of Smoflipid® and Intralipid® in the Early Management of Infants with Intestinal Failure.

PURPOSE: Cholestasis is problematic for infants with intestinal failure (IF). The soy-based lipid Intralipid® (IL) has been implicated. An alternative, Smoflipid® (SMOF), is increasingly used. However, its role in cholestasis prevention is unclear. This study compares the incidence and degree of cholestasis between infants with IF receiving SMOF or IL. METHODS: Infants with IF receiving SMOF or IL during the first 8 weeks of parenteral nutrition (PN) support between 2014 and 2017 were reviewed. Clinical characteristics, cholestasis incidence (conjugated bilirubin (Cbili) >2 mg/dL for >2 weeks), and nutritional parameters were compared using Welch's t-test. RESULTS: 91% (21/23) of IL and 76% (16/21) of SMOF babies became cholestatic (p = 0.18). There was no significant difference in median peak Cbili, but SMOF babies normalized more quickly (p = 0.04). Median z-scores for weight were similar throughout the study. SMOF patients getting full PN had a lower incidence of cholestasis compared to IL patients (78% vs. 92%, p = 0.057), but those with cholestasis had similar peak Cbili, time to resolution, and growth. CONCLUSION: Early use of Smoflipid® did not reduce the incidence of cholestasis compared to Intralipid® in infants with IF, but hyperbilirubinemia did resolve more quickly. SMOF may be most beneficial for infants tolerating no enteral nutrition. LEVEL OF EVIDENCE: Level III Retrospective Comparative Treatment Study. TYPE OF STUDY: Retrospective Review.

Andrographolide impairs alpha-naphthylisothiocyanate-induced cholestatic liver injury in vivo.

To investigate if andrographolide impairs cholestatic liver injury. All rats were randomly divided into six groups-(1) control (n = 6), (2) control + 200 mg/kg andrographolide (n = 6), (3) alpha-naphthylisothiocyanate (ANIT)-control (n = 6), (4) ANIT + 50 mg/kg andrographolide (n = 6), (5) ANIT + 100 mg/kg andrographolide (n = 6), and (6) ANIT + 200 mg/kg andrographolide (n = 6). We gavaged 50 mg/kg ANIT to mimic cholestatic liver injury in rats. Seven days after treatment, all the rats were killed. Serum biochemistry and hepatic histopathological assays were performed to evaluate liver injury. We observed that 200 mg/kg andrographolide significantly decreased the level of alanine transaminase, aspartate aminotransferase, alkaline phosphatase, γ-glutamyltranspeptidase, total bilirubin, and total bile acid in the blood. It also markedly decreased hepatic interleukin-6 and tumor necrosis factor α. Furthermore, 200 mg/kg andrographolide significantly decreased malondialdehyde but increased superoxide dismutase, glutathione, and erythrocyte glutathione peroxidase. Moreover, 200 mg/kg andrographolide effectively increased the accumulation of sirtuin 1 and nuclear erythroid 2-related factor-2. It also attenuated the level of nuclear factor kappa-light-chain-enhancer of activated B and cyclooxygenase-2. These data suggest that andrographolide may impair cholestatic liver injury via anti-inflammatory and anti-oxidative stress.

Long-Term Outcomes in Children With Intestinal Failure-Associated Liver Disease Treated With 6 Months of Intravenous Fish Oil Followed by Resumption of Intravenous Soybean Oil.

BACKGROUND: Intravenous soybean oil (SO) is a commonly used lipid emulsion for children with intestinal failure (IF); however, it is associated with IF-associated liver disease (IFALD). Studies have demonstrated that intravenous fish oil (FO) is an effective treatment for IFALD. However, there is a lack of long-term data on children who stop FO and resume SO. This study's objective was to investigate our institution's outcomes for children with IFALD treated with 6 months of FO and who then restarted SO. METHODS: Inclusion criteria for FO included children with IFALD. Parenteral nutrition (PN)-dependent children resumed SO after FO and were prospectively followed for 4.5 years or until death, transplant, or PN discontinuation. The primary outcome was the cumulative incidence rate (CIR) for cholestasis after FO. RESULTS: Forty-eight subjects received FO, and conjugated bilirubin decreased over time (-0.22 mg/dL/week; 95% confidence interval [CI]: -0.25, -0.19; P < .001). The CIR for cholestasis resolution after 6 months of FO was 71% (95% CI: 54%, 82%). Twenty-seven subjects resumed SO and were followed for a median of 16 months (range 3-51 months). While the CIR for enteral autonomy after 3 years of follow-up was 40% (95% CI: 17%, 26%), the CIR for cholestasis and transplant was 26% (95% CI: 8%, 47%) and 6% (95% CI: 0.3%, 25%), respectively. CONCLUSION: In this study, FO effectively treated cholestasis, and SO resumption was associated with cholestasis redevelopment in nearly one-fourth of subjects. Long-term FO may be warranted to prevent end-stage liver disease.

Association of serum bilirubin in newborns affected by jaundice with gut microbiota dysbiosis.

BACKGROUND AND AIMS: Breast milk jaundice (BMJ) is common and benign, but neonatal cholestasis (NC) is rare and not benign, so early differentiation between NC and non-NC jaundice is important and may facilitate diagnosis and treatment. Gut microbiota plays an important role in enterohepatic circulation, which in turn plays an important role in the secretion of bilirubin. We aimed to determine the composition of gut microbiota in patients with NC and BMJ, and to identify the gut microbiota composition associated with NC and BMJ. METHODS: Data on age, gender, delivery, feeding mode, serum total bilirubin, direct bilirubin, and liver function were collected for NC patients, BMJ patients and healthy controls, respectively. Shotgun metagenomic sequencing and metagenome-wide association were performed. RESULTS: Forty NC patients, 16 patients affected by BMJ, and 14 healthy controls (CON) without jaundice were enrolled. A significant increase in species richness, especially Bacteroides, was found in NC patients. The abundances of potentially pathogenic species and KEGG orthologies (KOs) of virulence factor genes were positively correlated with serum bilirubin level. The abundances of nine species of Bifidobacterium and three KOs of galactose metabolism were significantly decreased in the jaundice group (NC and BMJ) and were negatively correlated with serum bilirubin level. CONCLUSIONS: The gut microbiota in NC patients is characterized by a significant increase in species richness, possibly due to the proliferation of potentially pathogenic species. Additionally, the gut microbiota in jaundice patients is characterized by a decreased abundance of Bifidobacterium. Decreased Bifidobacterium has been associated with elevated bilirubin and abnormal gut microbiota galactose metabolic pathway. Further, ten bacteria species were identified as potential biomarker of jaundice. KEY POINTS: Question Is there any alteration of gut microbiotain neonatal cholestasis patients? Does gut microbiota have any involvement in the occurrence of neonatal cholestasis or breast milk jaundice? Findings The alteration of gut microbiota in neonatal cholestasis patients mainly manifested as a significant increase in species richness and an increased abundance of potentially pathogenic species, while the main manifestation in jaundice patients was a significant decrease in Bifidobacterium which may be involved in the metabolism of bilirubin through the galactose metabolic pathway. Meaning The results suggest that an imbalance of gut microbiota exist in neonatal cholestasis and breast milk jaundice patients, primarily in the form of a substantial reduction in the abundance of Bifidobacterium, suggesting the possibility of intervention treatment for neonatal cholestasis and breast milk jaundice by supplementing probiotics.

Prevalence and Prognostic Value of Abnormal Liver Test Results in Critically Ill Children and the Impact of Delaying Parenteral Nutrition.

OBJECTIVES: In the Early versus Late Parenteral Nutrition in the Pediatric ICU randomized controlled trial, delaying parenteral nutrition to beyond day 7 (late parenteral nutrition) was clinically superior to supplemental parenteral nutrition initiated within 24 hours (early parenteral nutrition), but resulted in a higher rise in bilirubin. We aimed to document prevalence and prognostic value of abnormal liver tests in the PICU and the impact hereon of withholding early parenteral nutrition. DESIGN: Preplanned secondary analysis of the Early versus Late Parenteral Nutrition in the Pediatric ICU randomized controlled trial. Total bilirubin, alanine aminotransferase, aspartate aminotransferase, gamma-glutamyl transpeptidase, alkaline phosphatase plasma concentrations were measured systematically in PICU. Liver test analyses were adjusted for baseline characteristics including severity of illness. SETTING: Three PICUs in Belgium, the Netherlands, and Canada. PATIENTS: As neonatal jaundice was considered a confounder, only the 1,231 of the 1,440 Early versus Late Parenteral Nutrition in the Pediatric ICU-patients 28 days to 17 years old were included. INTERVENTIONS: Late parenteral nutrition as compared with early parenteral nutrition. MEASUREMENTS AND MAIN RESULTS: During the first seven PICU days, the prevalence of cholestasis (> 2 mg/dL [34.2 µmol/L] bilirubin) ranged between 3.8% and 4.9% and of hypoxic hepatitis (≥ 20-fold upper limit of normality for alanine aminotransferase and aspartate aminotransferase) between 0.8% and 2.2%, both unaffected by the use of parenteral nutrition. Throughout the first week in PICU plasma bilirubin concentrations were higher in late parenteral nutrition patients (p < 0.05), but became comparable to early parenteral nutrition patients as soon as parenteral nutrition was started on day 8. Plasma concentrations of gamma-glutamyl transpeptidase, alkaline phosphatase, alanine aminotransferase, and aspartate aminotransferase were unaffected by parenteral nutrition. High day 1 plasma concentrations of gamma-glutamyl transpeptidase, alanine aminotransferase, and aspartate aminotransferase (p ≤ 0.01), but not alkaline phosphatase, were independent risk factors for PICU mortality. Day 1 plasma bilirubin concentrations displayed a U-shaped association with PICU mortality, with higher mortality associated with bilirubin less than 0.20 mg/dL and greater than 0.76 mg/dL (< 3.42 µmol/L and > 13 µmol/L) (p ≤ 0.01). CONCLUSIONS: Overt cholestasis and hypoxic hepatitis were rare and unrelated to the nutritional strategy. However, withholding parenteral nutrition up to 1 week in PICU increased plasma bilirubin. A mild elevation of bilirubin on the first PICU day was associated with lower risk of death and may reflect a stress response, rather than true cholestasis.

Na+ -taurocholate cotransporting polypeptide inhibition has hepatoprotective effects in cholestasis in mice.

Accumulation of bile salts (BSs) during cholestasis leads to hepatic and biliary injury, driving inflammatory and fibrotic processes. The Na+ -Taurocholate Cotransporting Polypeptide (NTCP) is the major hepatic uptake transporter of BSs, and can be specifically inhibited by myrcludex B. We hypothesized that inhibition of NTCP dampens cholestatic liver injury. Acute cholestasis was induced in mice by a 3.5-diethoxycarbonyl-1.4-dihydrocollidine (DDC) diet or by bile duct ligation (BDL). Chronic cholestasis was investigated in Atp8b1-G308V and Abcb4/Mdr2 deficient mice. Mice were injected daily with myrcludex B or vehicle. Myrcludex B reduced plasma alkaline phosphatase (ALP) levels in DDC-fed, Atp8b1-G308V and BDL mice by 39%, 27% and 48% respectively. Expression of genes involved in fibrosis, proliferation and inflammation was reduced by myrcludex B treatment in DDC-fed and Atp8b1-G308V mice. NTCP-inhibition increased plasma BS levels from 604±277 to 1746±719 µm in DDC-fed mice, 432±280 to 762±288 µm in Atp8b1-G308V mice and from 522±130 to 3625±378 µm in BDL mice. NTCP-inhibition strongly aggravated weight loss in BDL mice, but not in other cholestatic models studied. NTCP-inhibition reduced biliary BS output in DDC-fed and Atp8b1-G308V mice by ∼50% while phospholipid (PL) output was maintained, resulting in a higher PL/BS ratio. Conversely, liver injury in Abcb4 deficient mice, lacking biliary phospholipid output, was aggravated after myrcludex B treatment. Conclusion: NTCP-inhibition by myrcludex B has hepatoprotective effects, by reducing BS load in hepatocytes and increasing the biliary PL/BS ratio. High micromolar plasma BS levels after NTCP-inhibition were well tolerated. NTCP-inhibition may be beneficial in selected forms of cholestasis. (Hepatology 2018).

Evaluation of vitamin D status bone mineral density and dental health in children with cholestasis.

BACKGROUND: Hepatic osteodystrophy caused by vitamin D and calcium malabsorption is thought to develop in children with cholestatic liver disease leading to secondary hyperparathyroidism and rickets or osteomalacia. The aim of this study was to evaluate the dental and bone mineral densities and the serum level of vitamin D in cholestatic infants and children and to correlate this process with clinical and laboratory parameters. METHODS: This is a cross-sectional study that include 50 patients presenting with cholestasis. Thirty age and sex matched controls recruited not complaining of liver disease. All cases were subjected to full history taking, clinical and dental examination, 25(OH)D level, ALT, AST, bilirubin, albumin, GGT, alkaline phosphatase, PT, INR, calcium, corrected calcium, phosphorus and DXA scan to those above 5 years old. Controls were subjected to measuring the serum levels of 25(OH)D, total bilirubin, direct bilirubin, ALT, GGT, AST, PT, INR, alkaline phosphatase, albumin, calcium and phosphorus. RESULTS: Out of the 50 cases; 23 were females (46%), with a mean age of 6.17±3.9 years ranging from 1.1 to 17 years. Twenty-eight of the cases had signs of rickets (56%), 6 of them had bone fracture (12%) and 42.8% had milky teeth caries. The level of 25(OH) vitamin D was below normal range in around half of the patients. There was significant difference between cases and controls in calcium and phosphorus levels, ALT and alkaline phosphatase. Low bone mineral density (BMD) was present in 50% and 5 cases (17.9%) were diagnosed as having osteoporosis. There was a negative correlation between the Z-score, BMD of total body, BMD and bone mineral content (BMC) of spine and total and direct bilirubin. There was a positive correlation between (BMD of total body, spine and BMC of spine) and serum phosphorus, alkaline phosphatase and albumin. There was a positive correlation between the Z-score of total body and serum calcium. CONCLUSION: Decreased level of 25-OH vitamin D is present in more than half of cholestatic patients, and is correlated positively to serum calcium. Decreased BMD was present in more than half of studied cholestatic patients correlated to the low serum calcium rather than the vitamin D level. The decreased BMD and the dental affection in cholestatic children is related to the level of hyperbilirubinemia.

Dithiothreitol supplementation mitigates hepatic and renal injury in bile duct ligated mice: Potential application in the treatment of cholestasis-associated complications.

Cholestasis is a disorder characterized by impaired bile flow and accumulation of cytotoxic bile acids in the liver. On the other hand, oxidative stress and its deleterious consequences seem to have a significant role in cholestasis-induced organ injury. Hence, antioxidants and thiol-reducing agents could have potential protective effect against this complication. The current investigation was designed to evaluate the effect of dithiothreitol (DTT) as a safe and clinically applicable thiol-reductant in cholestatic animals. DTT is a dithiol compound which effectively reduces disulfide bonds in glutathione molecule or different proteins and preserves cellular redox environment. Bile duct ligated (BDL) mice were supplemented with DTT-containing drinking water (0.25% and 1% w: v) for 14 days. Blood, liver, kidney, and spleen samples were collected at scheduled time intervals (3, 7, and 14 days after BDL operation). Significant elevation in plasma biomarkers of liver and kidney injury was detected in BDL animals. Liver and kidney injury was also histopathologically evident by necrosis, inflammation, and fibrosis. Furthermore, high levels of reactive oxygen species in addition to lipid peroxidation, depleted glutathione reservoirs, and impaired tissue antioxidant capacity was detected in the liver and kidney of cholestatic animals. It was found that DTT supplementation (0.25% and 1% w:v) alleviated markers of oxidative stress in the liver and kidney. Moreover, liver and kidney histopathological changes and collagen deposition were markedly attenuated by DTT treatment. The beneficial effects of DTT administration in cholestasis and its associated complications might be linked to its ability for preserving cellular redox environment and preventing oxidative stress.

Herbal medicine Yinchenhaotang protects against α-naphthylisothiocyanate-induced cholestasis in rats.

Cholestasis is a clinical disorder defined as an impairment of bile flow, and that leads to toxic bile acid (BA) accumulation in hepatocytes. Here, we investigated the hepatoprotective effect of Yinchenhaotang (YCHT), a well-known formulae for the treatment of jaundice and liver disorders, against the cholestasis using the α-naphthylisothiocyanate (ANIT)-induced cholestasis in male Wistar rats. ANIT feeding induced significant cholestasis with substantially increased intrahepatic retention of hydrophobic BAs. The dynamic changes of serum and liver BAs indicated that YCHT was able to attenuate ANIT-induced BA perturbation, which is consistent with the histopathological findings that YCHT significantly decreased the liver damage. YCHT treatment substantially reduced serum alanine aminotransferase (ALT), alkaline phosphatase (AST), total bilirubin (TBIL) and direct bilirubin (DBIL) with minimal bile duct damage in the ANIT treated rats. Elevated mRNA expression of liver IL-6, IL-17A, IL-17F, TGF-ß1, α-SMA, TGR5, NTCP, OATP1a1, and ileum ASBT and decreased liver IL-10, FXR, CAR, VDR, BSEP, MRP2, MRP3, MRP4 was also observed in ANIT-induced cholestasis but were attenuated or normalized by YCHT. Our results demonstrated that the BA profiles were significantly altered with ANIT intervention and YCHT possesses the hepatoprotective potential against cholestatic liver injury induced by hepatotoxin such as ANIT.

Low-Dose Parenteral Soybean Oil for the Prevention of Parenteral Nutrition-Associated Liver Disease in Neonates With Gastrointestinal Disorders.

BACKGROUND: Neonates with gastrointestinal disorders (GDs) are at high risk for parenteral nutrition-associated liver disease (PNALD). Soybean-based intravenous lipid emulsions (S-ILE) have been associated with PNALD. This study's objective was to determine if a lower dose compared with a higher dose of S-ILE prevents cholestasis without compromising growth. MATERIALS AND METHODS: This multicenter randomized controlled pilot study enrolled patients with GDs who were ≤5 days of age to a low dose (~1 g/kg/d) (LOW) or control dose of S-ILE (~3 g/kg/d) (CON). The primary outcome was cholestasis (direct bilirubin [DB] >2 mg/dL) after the first 7 days of age. Secondary outcomes included growth, PN duration, and late-onset sepsis. RESULTS: Baseline characteristics were similar between the LOW (n = 20) and CON groups (n = 16). When the LOW group was compared with the CON group, there was no difference in cholestasis (30% vs 38%, P = .7) or secondary outcomes. However, mean ± SE DB rate of change over the first 8 weeks (0.07 ± 0.04 vs 0.3 ± 0.09 mg/dL/wk, P = .01) and entire study (0.008 ± 0.03 vs 0.2 ± 0.07 mg/dL/wk, P = .02) was lower in the LOW group compared with the CON group. CONCLUSION: In neonates with GDs who received a lower dose of S-ILE, DB increased at a slower rate in comparison to neonates who received a higher dose of S-ILE. Growth was comparable between the groups. This study demonstrates a need for a larger, randomized controlled trial comparing 2 different S-ILE doses for cholestasis prevention in neonates at risk for PNALD.

Parenteral nutrition-associated cholestasis and triglyceridemia in surgical term and near-term neonates: A pilot randomized controlled trial of two mixed intravenous lipid emulsions.

BACKGROUND: Cholestasis is a common complication in infants receiving prolonged parenteral nutrition (PN). We studied the effects of two intravenous lipid emulsions composed with either 30% soybean oil, 30% medium-chain triglycerides (MCT), 25% olive oil, and 15% fish oil (SMOF) or with 50% MCT and 50% soybean oil n-6 (MCT/SOY) on the incidence of cholestasis in surgical term and near-term neonates. METHODS: A single-center, double-blinded, randomized controlled trial compared the incidence of cholestasis using either SMOF or MCT/SOY in neonates born at gestational age ≥34 weeks undergoing major surgery. The primary outcome was the incidence of conjugated serum bilirubin >1 mg/dL. Other liver enzymes were assessed as secondary outcomes. A post-hoc analysis assessed serum triglycerides levels. Odds ratios were estimated by mixed-effects regression models. RESULTS: Enrollment was prematurely interrupted because the MCT/SOY became unavailable, thus 49 infants (SMOF 22, MCT/SOY 27) completed the study. The exposure (time on PN, cumulative dose of lipids) was similar in both groups. Similar cumulative incidence rates were found for elevated conjugated bilirubinemia and other liver enzymes. Hypertriglyceridemia >250 mg/dL (12/49) was more frequent in MCT/SOY (37.0%, 95% CI 21.53-55.77) than in SMOF (9.1%, 95% CI 2.53-27.81, p = 0.024). Triglyceridemia at the first assessment (median 8 postnatal days) was significantly higher with MCT/SOY than with SMOF (181 vs. 134 mg/dL, p = 0.006). Over the whole study period, mean triglyceride concentration was 36.5 mg/dL higher with MCT/SOY compared with SMOF (p = 0.013). CONCLUSION: Both emulsions had similar effects on the incidence of cholestasis and markers of liver integrity, but MCT/SOY induced higher serum triglyceride concentrations. TRIAL REGISTRATION: ClinicalTrials.gov, NCT02633384.

Predictors of failure of fish-oil therapy for intestinal failure-associated liver disease in children.

BACKGROUND: Parenteral fish-oil (FO) therapy is a safe and effective treatment for intestinal failure-associated liver disease (IFALD). Patients whose cholestasis does not resolve with FO may progress to end-stage liver disease. OBJECTIVE: We sought to identify factors associated with the failure of FO therapy in treating IFALD to guide prognostication and referral guidelines. DESIGN: Prospectively collected data for patients treated with FO at Boston Children's Hospital from 2004 to 2014 were retrospectively reviewed. Resolution of cholestasis was defined as sustained direct bilirubin (DB) <2 mg/dL, and treatment failure as liver transplantation or death while DB was >2 mg/dL as of July 2015. Demographics, laboratory values, and medical history at FO therapy initiation were compared between patients who achieved resolution of cholestasis and those who failed therapy. RESULTS: Among 182 patients treated with FO, 86% achieved resolution of cholestasis and 14% failed therapy. Patients who failed therapy had median (IQR) lower birth weight [1020 g (737, 1776 g) compared with 1608 g (815, 2438 g); P = 0.03] and were older at FO initiation [20.4 wk (9.9, 38.6 wk) compared with 11.7 wk (7.3, 21.4 wk); P = 0.02] than patients whose cholestasis resolved. Patients who failed therapy had more advanced liver disease at therapy initiation than patients whose cholestasis resolved, as evidenced by lower median (IQR) γ-glutamyltransferase [54 U/L (41, 103 U/L) compared with 112 U/L (76, 168 U/L); P < 0.001], higher DB [10.4 mg/dL (7.5, 14.1 mg/dL) compared with 4.4 mg/dL (3.1, 6.6 mg/dL); P < 0.001], and a higher pediatric end-stage liver disease (PELD) score [22 (14, 25) compared with 12 (7, 15); P < 0.001]. A PELD score of ≥15, history of gastrointestinal bleeding, age at FO initiation ≥16 wk, presence of nongastrointestinal comorbidities, and mechanical ventilation at FO initiation were independent predictors of treatment failure. CONCLUSIONS: Most infants with IFALD responded to FO therapy with resolution of cholestasis, and liver transplantation was rarely required. Early FO initiation once biochemical cholestasis is detected in parenteral nutrition-dependent patients is recommended. This trial was registered at clinicaltrials.gov as NCT00910104.

Promising toxicological biomarkers for the diagnosis of liver injury types: Bile acid metabolic profiles and oxidative stress marker as screening tools in drug development.

Promising biomarkers were identified in adult male Crl:CD (SD) rats for the screening of new chemical entities for their potential to cause liver injury. We examined the serum biochemistry, liver histopathology, and bile acid profiles by LC-MS/MS, and the mRNA expression of transporters and CYPs by an RT-PCR after the following treatments to male Crl:CD (SD) rats: (a) bile duct ligation (BDL); (b) a single oral dose of 150 mg/kg α-naphthylisothiocyanate (ANIT); and (c) repeated oral doses of a novel pyrrolidinecarboxylic acid derivative (abbreviated as PCA) at 30, 300, and 1000 mg/kg. The serum total bile acid levels and bilirubin concentrations were found to be elevated in all of the groups. However, the bile acid component profiles of the PCA group differed significantly from BDL and ANIT models: deoxycholic acid, lithocholic acid, and sulfated bile acids were upregulated in a dose-dependent manner only in the PCA group. In addition, the PCA group demonstrated high levels of hepatic heme oxygenase-1 expression, whereas the profiles of the mRNA levels of the hepatic transporters and CYPs of all groups were found to be similar. The histopathological findings, for both the BDL and ANIT groups, were of bile duct hyperplasia, hepatocyte degeneration and necrosis. In contrast, only bile duct hyperplasia and hepatocyte degeneration were observed in the PCA group, even at a lethal dose. These results indicated that PCA induced a cholestatic condition and the increase of oxidative stress markers implies that this will also lead hepatocellular injury. In conclusion, the serum bile acid components and sulfated bile acid levels, and the expression of oxidative stress markers could provide information that aids in the diagnosis of liver injury type and helps to elucidate the mechanisms of hepatotoxicity. These findings can be extrapolated into our clinical investigation. The analysis of these crucial biomarkers is likely to be a useful screening tool in the lead optimization phase of drug discovery.

Paeonia lactiflora Pall. protects against ANIT-induced cholestasis by activating Nrf2 via PI3K/Akt signaling pathway.

BACKGROUND: Paeonia lactiflora Pall. (PLP), a traditional Chinese herbal medicine, has been used for hepatic disease treatment over thousands of years. In our previous study, PLP was shown to demonstrate therapeutic effect on hepatitis with severe cholestasis. The aim of this study was to evaluate the antioxidative effect of PLP on alpha-naphthylisothiocyanate (ANIT)-induced cholestasis by activating NF-E2-related factor 2 (Nrf2) via phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway. MATERIALS AND METHODS: Liquid chromatography-mass spectrometry (LC-MS) was performed to identify the main compounds present in PLP. The mechanism of action of PLP and its therapeutic effect on cholestasis, induced by ANIT, were further investigated. Serum indices such as total bilirubin (TBIL), direct bilirubin (DBIL), aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), γ-glutamyl transpeptidase (γ-GT), and total bile acid (TBA) were measured, and histopathology of liver was also performed to determine the efficacy of treatment with PLP. Moreover, in order to illustrate the underlying signaling pathway, liver glutathione (GSH) content and mRNA or protein levels of glutamate-cysteine ligase catalytic subunit (GCLc), glutamate-cysteine ligase modulatory subunit (GCLm), Akt, heme oxygenase-1 (HO-1), NAD(P)H/quinone oxidoreductase 1 (Nqo1), and Nrf2 were further analyzed. In addition, validation of PLP putative target network was also performed in silico. RESULTS: Four major compounds including paeoniflorin, albiflorin, oxypaeoniflorin, and benzoylpaeoniflorin were identified by LC-MS analysis in water extract of PLP. Moreover, PLP could remarkably downregulate serum levels of TBIL, DBIL, AST, ALT, ALP, γ-GT, and TBA, and alleviate the histological damage of liver tissue caused by ANIT. It enhanced antioxidative system by activating PI3K/Akt/Nrf2 pathway through increasing Akt, Nrf2, HO-1, Nqo1, GCLc, and GCLm expression. The putative targets network validation also confirmed the important role of PLP in activating Akt expression. CONCLUSION: The potential mechanism of PLP in alleviating ANIT-induced cholestasis could to be related to the induction of GSH synthesis by activating Nrf2 through PI3K/Akt-dependent pathway. This indicates that PLP might be a potential therapeutic agent for cholestasis.

Large dose means significant effect--dose and effect relationship of Chi-Dan-Tui-Huang decoction on alpha-naphthylisothiocyanate-induced cholestatic hepatitis in rats.

BACKGROUND: Large dose application of traditional Chinese medicines has attracted more and more attentions in recent years. However, the scientific connotation of large dose application has not been clarified so far. The present study was designed to investigate the protective effects of Chi-Dan-Tui-Huang decoction (CDTHD) against Alpha-naphthylisothiocyanate (ANIT) induced acute cholestatic hepatitis in rats and explore the dose-effect relationship of CDTHD as a reference for clinical application. METHODS: The administration of CDTHD at a series of doses was performed twice each day for 5 days. The acute cholestasic hepatitis models were induced by intragastric administration of ANIT on the third day of CDTHD administration. Then, the protective effects on cholestatic hepatitis were investigated by examining the following parameters: body weights of rats, morphological and histopathological liver changes, the levels of serum biomarkers including alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, total bilirubin, direct bilirubin and γ-glutamyltranspeptidase. Furthermore, the dose-effect relationship was investigated with the application of correspondence analysis. RESULT: In the tested range of doses, CDTHD at the maximum tolerance dose did not show any toxicity as time went on. The efficacy result showed that CDTHD from 21.6 g/kg⋅d to 86.4 g/kg⋅d exhibited significant hepatoprotective effect against ANIT-induced acute cholestatic hepatitis. It alleviated liver injury and reversed adverse biochemical and histopathological changes in a dose-dependent manner. Correspondence analysis showed that Radix Paeoniae Rubra in CDTHD was the main effective component and CDTHD could enhance the integrated efficacy in dose-dependent manner. CONCLUSIONS: CDTHD is beneficial to liver protection in a dose-dependent manner. Especially large dose demonstrates potent efficacy and Radix Paeoniae Rubra in the formula contributes the main effect on ANIT-induced acute cholestatic hepatitis without toxicity.

A double-blind randomised controlled trial of fish oil-based versus soy-based lipid preparations in the treatment of infants with parenteral nutrition-associated cholestasis.

BACKGROUND: Infants receiving prolonged parenteral nutrition (PN) are at risk of PN-associated cholestasis (PNAC). This can progress to hepatic failure and death if PN cannot be discontinued. Fish oil-based parenteral lipid preparation (FOLP) has been shown to be beneficial in case studies. OBJECTIVES: (1) To evaluate whether FOLP could halt or reverse the progression of PNAC compared with soy-based parenteral lipid preparation (SLP) and (2) to assess the effects of FOLP on liver function and physical growth. DESIGN: double-blind randomised controlled trial. SETTING: level III neonatal intensive care unit. PARTICIPANTS: infants with PNAC (plasma-conjugated bilirubin concentration ≥ 34 µmol/l or 2 mg/dl) expected to be PN-dependent for >2 weeks. INTERVENTION: to receive either FOLP or SLP at 1.5 g/kg/day. PRIMARY OUTCOME MEASURE: reversal of PNAC within 4 months after commencement of lipid treatment; secondary outcomes: rate of change of weekly liver function tests, infant growth parameters, blood lipid profile and episodes of late-onset sepsis. RESULTS: A total of 9 infants were randomised to the FOLP group and 7 to the SLP group. There was no significant difference in reversal of PNAC at 4 months between groups. Rates of increase of plasma-conjugated bilirubin and alanine aminotransferase in the SLP group were significantly greater than the FOLP group (13.5 vs. 0.6 µmol/l per week and 9.1 vs. 1.1 IU/l per week, respectively, p = 0.03). Increased enteral nutrition was associated with significant improvement of PNAC in infants receiving FOLP compared with SLP (-8.5 vs. -1.6 µmol/l per 10% increase in enteral nutrition, respectively). The study was terminated prematurely. CONCLUSIONS: progression of PNAC in PN-dependent infants can be halted by replacing SLP with FOLP and reversed by increasing the proportion of enteral nutrition in infants receiving FOLP. Replacement of SLP with FOLP in PN-dependent infants who develop PNAC may be considered.

Pediatric intestinal failure-associated liver disease is reversed with 6 months of intravenous fish oil.

BACKGROUND: Studies have suggested that when intravenous (IV) soybean oil (SO) is replaced with fish oil (FO), direct hyperbilirubinemia is more likely to resolve. The necessary duration of FO has not been established. This study seeks to determine if 24 weeks of FO is an effective and safe therapy for intestinal failure-associated liver disease (IFALD). MATERIALS AND METHODS: This is a clinical trial using patients with IFALD between the ages of 2 weeks and 18 years. SO was replaced with FO (1 g/kg/d) in 10 patients who were receiving most of their calories from parenteral nutrition (PN). Patients were compared with 20 historic controls receiving SO. SO for both groups was prescribed by the primary medical team at variable doses. The primary outcome was time to reversal of cholestasis. Secondary outcomes were death, transplant, and full enteral feeds. Safety measurements included growth, essential fatty acid deficiency, and laboratory markers to assess bleeding risk. RESULTS: The Kaplan-Meier method estimated that 75% in the FO group would experience resolution of cholestasis by 17 weeks vs 6% in the SO group (P < .0001). When compared with the SO group, the FO group had decreased serum direct bilirubin concentrations at weeks 8 (P = .03) and 12, 16, 20, and 24 weeks (P < .0001). Although length z score at the end of the study increased in the FO group compared with baseline (P = .03), there were no significant differences in other outcomes. CONCLUSIONS: A limited duration of FO appears to be safe and effective in reversing IFALD.

Fish-oil fat emulsion supplementation reduces the risk of retinopathy in very low birth weight infants: a prospective, randomized study.

BACKGROUND: Preliminary studies suggest that fish-oil lipid emulsion given parenterally to very preterm infants reduces the severity of retinopathy (ROP) and cholestasis. METHODS: Infants weighing <1250 g at birth were randomly allocated to 2 groups: an experimental group of 60 infants that received an intravenous (IV) soybean, olive oil, and fish oil emulsion, and a control group of 70 infants that was given a parenteral soybean and olive oil emulsion. Plasma and erythrocyte concentrations of docosahexaenoic acid (DHA) were determined using a high-performance liquid chromatography-mass spectrometry analysis. RESULTS: Nine infants in the fish oil group required laser therapy for ROP compared with 22 infants in the standard intralipid group (risk ratio [RR], 0.48; 95% confidence interval [CI], 0.24-0.96). Three infants in the fish oil group developed cholestasis compared with 20 infants in the standard intralipid group (RR, 0.18; 95% CI, 0.055-0.56). The mean plasma DHA concentrations in treated infants were 2.9-fold higher in the fish oil group than in control infants on the 7th and 14th days of life. The mean DHA content in erythrocytes of treated infants was 4.5-fold and 2.7-fold higher compared with controls at 7 and 14 days of age. CONCLUSIONS: Premature infants receiving an IV fat emulsion containing fish oil had less ROP requiring laser treatment and less cholestasis than those receiving a standard lipid emulsion. These infants also had higher plasma and erythrocyte DHA levels at 7 and 14 days, suggesting potential long-term neurodevelopmental benefits.

Effect of decreased parenteral soybean lipid emulsion on hepatic function in infants at risk for parenteral nutrition-associated liver disease: a pilot study.

PURPOSE: We performed a pilot trial to compare reduced dose versus standard soybean lipid emulsion in neonates at risk for parenteral nutrition-associated liver disease. METHODS: A prospective randomized controlled trial was performed (2009-2011) enrolling surgical patients ≥ 26 weeks' gestation anticipated to require >50% of daily caloric intake from parenteral nutrition (PN) for at least 4 weeks. Randomization occurred into either reduced (1.0 g/kg/day) or standard (3g/kg/day) groups. Co-primary outcomes for interpretation of the results were conjugated bilirubin and total bile acids. Additional outcomes included ALT, AST, GGT, alkaline phosphatase, growth, and essential fatty acid levels. Outcomes were compared between treatment groups using Wilcoxon rank sums tests. RESULTS: Twenty-eight patients (47% enrollment rate) were included in the study with an average treatment duration of 5.4 weeks. Groups had similar PN calories and protein intake throughout the study. Total increase from baseline was smaller in the reduced vs. standard group for conjugated bilirubin (p=0.04) and total bile acids (p=0.02). Weight z-score increased more in the standard group, and no patient experienced essential fatty acid deficiency. CONCLUSION: Markers of cholestasis rose at a slower rate using reduced lipid doses. This pilot study demonstrates feasibility and need for a larger study evaluating the effects of reduced lipids in patients at risk for developing parenteral nutrition-associated liver disease.