Evaluation of Amino Acids Profile as Non-Invasive Biomarkers of Hepatocellular Carcinoma in Egyptians.

BACKGROUND: Hepatocellular carcinoma (HCC) is the most dangerous complication of chronic liver disease. It is a multifactorial complicated disease. Hepatitis C and hepatitis B viruses (HCV and HBV, respectively) represent the main causes of HCC in Egypt. Early diagnosis is very important to aid in early intervention. OBJECTIVES: The goal of this research is to evaluate the metabolic role of different amino acids as non-invasive biomarkers over the course of HCC. METHODS: This study included 302 participants with 97 diagnosed, untreated HCC patients, 81 chronic HCV patients, 56 chronic HBV patients, 18 co-infected patients, and a control group of 50 normal age and gender-matched individuals. All participants provided complete medical histories and underwent complete clinical examinations, abdominal ultrasonography and/or computed tomography, routine laboratory investigations, estimation of serum α-fetoprotein, and determination of amino acid levels using ultra-performance liquid chromatography (UPLC MS/MS). RESULTS: This work revealed a decline in branched chain amino acids (BCAA) and increase in aromatic amino acids (AAA) among infected groups (HCC, HBV, HCV, and co-infected patients) compared to control subjects and a marked change in Fisher's and the BCAAs/tyrosine molar concentration ratios (BTR) between controls and infected groups. CONCLUSION: Different amino acids could be used as non-invasive markers to discriminate and follow chronic hepatitis patients to predict the course of HCC.

Value of Bile Acids in Diagnosing Hepatitis C Virus-Induced Liver Cirrhosis and Hepatocellular Carcinoma.

Background: Metabonomic studies have related bile acids to hepatic impairment, but their role in predicting hepatocellular carcinoma still unclear. The study aimed to examine the feasibility of bile acids in distinguishing hepatocellular carcinoma from post hepatitis C virus-induced liver cirrhosis. Methods: An ultra-performance liquid chromatography coupled with mass spectrometry measured 14 bile acids in patients with noncirrhotic post hepatitis C virus disease (n = 50), cirrhotic post hepatitis C virus disease (n = 50), hepatocellular carcinoma (n = 50), and control group (n = 50). Results: The spectrum of liver disease was associated with a significant increase in many conjugated bile acids. The fold changes in many bile acid concentrations showed a linear trend with hepatocellular carcinoma > cirrhotic disease > noncirrhotic disease > healthy controls (p < 0.05). Receiver operating characteristic curve analysis revealed five conjugated acids TCA, GCA, GUDCA, TCDCA, GCDCA, that discriminated hepatocellular carcinoma from noncirrhotic liver patients (AUC = 0.85-0.96) with a weaker potential to distinguish it from chronic liver cirrhosis (AUC = 0.41-0.64). Conclusion: Serum bile acids are associated primarily with liver cirrhosis with little value in predicting the progress of cirrhotic disease to hepatocellular carcinoma.

Vilazodone-phospholipid mixed micelles for enhancing oral bioavailability and reducing pharmacokinetic variability between fed and fasted states.

Despite the effectiveness and high tolerability of vilazodone (VLZ) as an antidepressant, its use is still limited due to its poor solubility and food dependent absorption. This study aims to load VLZ-phospholipid complex into self-assembled micelles forming VLZ-PL mixed micelles (VLZ-PL-MM), that can enhance VLZ solubility, improve its bioavailability and reduce the pharmacokinetic variability between the fed and fasting conditions. The effect of surfactant type and concentration was assessed using four different non-ionic surfactants (Brij 58, Tween 80, Labrasol and Pluronic F127) in four different weight ratios between the drug-complex and surfactant (1:0.5, 1:1, 1:2 and 1:3 w/w). Two VLZ-PL-MM formulae prepared using Brij 58 and Labrasol in 1:3 w/w ratio were selected as optimised ones since they have the highest encapsulation efficiency (100.83 and 93.87%, respectively), a particle size below 250 nm (206.73 and 221.33 nm, respectively) and negative zeta potential values (-29.63, -17.20 mV, respectively). Lyophilisation of these formulations using 3% sucrose was successful with no statistical changes in particle size and zeta potential upon rehydration. Both formulations elicited faster and higher in-vitro drug release profiles compared to the pure drug and the marketed tablet. In addition, both selected formulae improved ex-vivo permeation across rabbit intestinal membrane compared to the pure drug and the marketed tablet, with marked superiority of the one prepared using Brij 58. The results of the in-vivo study in male albino rabbits revealed similar AUC0-24 values after the oral administration of the best achieved VLZ-PL-MM system under fed and fasted conditions (769.89 and 741.55 ng.h mL-1, respectively). On the other hand, the marketed product showed significantly lower values of the AUC0-24 relative to the VLZ-PL-MM system and there was a marked enhancement of absorption of drug from the marketed product in presence of food (244.24 and 174.96 ng.h mL-1 under fed and fasted conditions, respectively). In addition, VLZ concentrations in the brain after 24 h obtained from the selected VLZ-PL-MM were significantly higher than those obtained from marketed tablet under fed and fasted conditions. Thus, the phospholipid mixed micelles formulation enhances the oral bioavailability of the poorly soluble drug and reduces the pharmacokinetic variability between fasting and fed conditions.

Predictors of azathioprine toxicity in children with autoimmune hepatitis.

AIM OF THE STUDY: Azathioprine (AZA) is an important steroid-sparing drug in the management of autoimmune hepatitis (AIH). Avoidance of its adverse events that could be severe and carry a risk of mortality in a few cases is important, preferably with cheap and easy assessments that could be feasible in developing countries with the unavailability of molecular assays. Assessment of thiopurine methyltransferase (TPMT), the key enzyme for the inactivation of AZA, as a predictor of AZA toxicity had been a matter of conflict. This work aimed to study the role of TPMT serum level assessment and other host-, disease-, and treatment-related factors in predicting AZA toxicity. MATERIAL AND METHODS: Sixty-six children with AIH, divided into two groups, were recruited. Group 1 included twelve children with AZA toxicity and group 2 included fifty-four children without AZA toxicities. Both groups were compared for demographic, clinical, laboratory, histopathological, and treatment-related factors, and serum TPMT level, measured by ELISA. RESULTS: TPMT serum level was comparable in both groups (p = 0.363). Duration of treatment until enzyme normalization and duration of AZA therapy were significantly associated with AZA toxicity (p = 0.007 and p = 0.01, respectively). At the first follow-up treatment with AZA, total leucocyte count (TLC) and neutrophil counts were significantly lower in group 1 (p = 0.005 and p = 0.002, respectively). Moreover, the percentage reduction of TLC and neutrophil counts were significantly higher in group 1 (p < 0.001, for both). CONCLUSIONS: Monitoring for AZA adverse events in those with the defined predictors of AZA-related adverse events is more important than TPMT assessment.

Sugar Alcohols Have a Key Role in Pathogenesis of Chronic Liver Disease and Hepatocellular Carcinoma in Whole Blood and Liver Tissues.

The major risk factors for hepatocellular carcinoma (HCC) are hepatitis C and B viral infections that proceed to Chronic Liver Disease (CLD). Yet, the early diagnosis and treatment of HCC are challenging because the pathogenesis of HCC is not fully defined. To better understand the onset and development of HCC, untargeted GC-TOF MS metabolomics data were acquired from resected human HCC tissues and their paired non-tumor hepatic tissues (n = 46). Blood samples of the same HCC subjects (n = 23) were compared to CLD (n = 15) and healthy control (n = 15) blood samples. The participants were recruited from the National Liver Institute in Egypt. The GC-TOF MS data yielded 194 structurally annotated compounds. The most strikingly significant alteration was found for the class of sugar alcohols that were up-regulated in blood of HCC patients compared to CLD subjects (p < 2.4 × 10-12) and CLD compared to healthy controls (p = 4.1 × 10-7). In HCC tissues, sugar alcohols were the most significant (p < 1 × 10-6) class differentiating resected HCC tissues from non-malignant hepatic tissues for all HCC patients. Alteration of sugar alcohol levels in liver tissues also defined early-stage HCC from their paired non-malignant hepatic tissues (p = 2.7 × 10-6). In blood, sugar alcohols differentiated HCC from CLD subjects with an ROC-curve of 0.875 compared to 0.685 for the classic HCC biomarker alpha-fetoprotein. Blood sugar alcohol levels steadily increased from healthy controls to CLD to early stages of HCC and finally, to late-stage HCC patients. The increase in sugar alcohol levels indicates a role of aldo-keto reductases in the pathogenesis of HCC, possibly opening novel diagnostic and therapeutic options after in-depth validation.

Remodeling Lipids in the Transition from Chronic Liver Disease to Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is a worldwide health problem. HCC patients show a 50% mortality within two years of diagnosis. To better understand the molecular pathogenesis at the level of lipid metabolism, untargeted UPLC MS-QTOF lipidomics data were acquired from resected human HCC tissues and their paired nontumor hepatic tissues (n = 46). Blood samples of the same HCC subjects (n = 23) were compared to chronic liver disease (CLD) (n = 15) and healthy control (n = 15) blood samples. The participants were recruited from the National Liver Institute in Egypt. The lipidomics data yielded 604 identified lipids that were divided into six super classes. Five-hundred and twenty-four blood lipids were found as significantly differentiated (p < 0.05 and qFDR p < 0.1) between the three study groups. In the blood of CLD patients compared to healthy control subjects, almost all lipid classes were significantly upregulated. In CLD patients, triacylglycerides were found as the most significantly upregulated lipid class at qFDR p = 1.3 × 10-56, followed by phosphatidylcholines at qFDR p = 3.3 × 10-51 and plasmalogens at qFDR p = 1.8 × 10-46. In contrast, almost all blood lipids were significantly downregulated in HCC patients compared to CLD patients, and in HCC tissues compared to nontumor hepatic tissues. Ceramides were found as the most significant lipid class (qFDR p = 1 × 10-14) followed by phosphatidylglycerols (qFDR p = 3 × 10-9), phosphatidylcholines and plasmalogens. Despite these major differences, there were also common trends in the transitions between healthy controls, CLD and HCC patients. In blood, several mostly saturated triacylglycerides showed a continued increase in the trajectory towards HCC, accompanied by reduced levels of saturated free fatty acids and saturated lysophospatidylcholines. In contrast, the largest overlaps of lipid alterations that were found in both HCC tissue and blood comparisons were decreased levels of phosphatidylglycerols and sphingolipids. This study highlights the specific impact of HCC tumors on the circulating lipids. Such data may be used to target lipid metabolism for prevention, early detection and treatment of HCC in the background of viral-related CLD etiology.

First report of glycogen storage disease type 111a in a Nigerian child.

Glycogen storage disease (GSD) is a rare inborn error of metabolism with an incidence of 1/20,000-40,000 live births. Some of the presenting clinical features can mimic diseases commonly seen in the tropics and subtropics. We report a 14-month-old Nigerian child who presented at our institution with GSD Type 111a to alert physicians on the need to consider and recognise this rare disorder. The child presented with progressive abdominal swelling due to marked hepatomegaly. From the clinical history, the only clue to hypoglycaemia was that she eats very frequently. Her random blood sugar was normal; however, fasting blood sugar was low. The diagnosis was further entertained with laboratory results showing hypercholesterolaemia and uricaemia and confirmed by histology of biopsied liver tissue. GSD should be suspected in a child with unexplained hepatomegaly and investigated accordingly.

Chromatographic determination of some biomarkers of liver cirrhosis and hepatocellular carcinoma in Egyptian patients.

Metabolomics has been shown to be an effective tool for disease diagnosis, biomarker screening and characterization of biological pathways. A total of 140 subjects were included in this study; urine metabolomes of patients with liver cirrhosis (LC, n = 40), patients with hepatocellular carcinoma (HCC; n = 55) and healthy male subjects (n = 45) as a control group were studied. Gas chromatography/mass spectrometry-based urine metabolomics profiles were investigated for all participants. Diagnostic models were constructed with a combination of marker metabolites, using principal components analysis and receiver operator characteristic curves. A total of 57 peaks could be auto-identified of which 13 marker metabolites (glycine, serine, threonine, proline, urea, phosphate, pyrimidine, arabinose, xylitol, hippuric acid, citric acid, xylonic acid and glycerol) were responsible for the separation of HCC group from healthy subjects. Also, eight markers metabolites (glycine, serine, threonine, proline, citric acid, urea, xylitol and arabinose) showed significant differences between the LC group and healthy subjects. No significant difference was detected between HCC and LC groups regarding all these metabolites. Metabolomic profile using GC-MS established an optimized diagnostic model to discriminate between HCC patients and healthy subjects; also it could be useful for diagnosis of LC patients. However, it failed to differentiate between HCC and LC patients.

Effect of different therapeutic modalities on systemic, renal, and hepatic hemodynamics and short-term outcomes in cirrhotic patients with spontaneous bacterial peritonitis.

BACKGROUND: Spontaneous bacterial peritonitis (SBP) is a major risk factor for hepatorenal syndrome. Albumin infusion has been shown to prevent renal impairment and reduce mortality in SBP. The study aimed to compare the effect of different therapeutic modalities on hemodynamics and short clinical outcomes in high-risk patients with SBP. METHODS: Two hundred cirrhotic patients with SBP and bilirubin greater than 4 mg[Fraction Slash]dl or creatinine more than 1 mg[Fraction Slash]dl were enrolled. Patients were randomized to receive albumin, terlipressin, low-dose albumin plus terlipressin, or midodrine. Systemic, renal, and hepatic hemodynamics were estimated at baseline, 3, and 10 days of treatment. Renal impairment was diagnosed when the blood urea nitrogen or serum creatinine levels increased by more than 50% of the pretreatment value. RESULTS: SBP resolved in most of patients in all groups (P>0.05). Cardiac output and portal flow decreased, whereas systemic vascular resistance increased significantly in terlipressin and albumin plus terlipressin groups compared with the albumin group after 3 and 10 days. After 10 days, plasma renin activity, renal, and hepatic arteries resistive index were significantly higher in the midodrine group compared with the albumin group. The midodrine group did not show any significant changes in the heart rate, mean arterial pressure, cardiac output, and portal blood flow compared with the albumin group after 3 or 10 days. There was no significant difference in renal impairment or mortality between any of the groups. CONCLUSION: Terlipressin and low-dose albumin plus terlipressin could be used as a therapeutic alternative to standard-dose albumin in high-risk SBP patients.

Urinary urobilinogen in biliary atresia: A missed, simple and cheap diagnostic test.

AIM: Early diagnosis of biliary atresia (BA) is of utmost importance for good outcome; however, it is sometimes difficult due to the overlapping diagnostic test results with other causes of neonatal cholestasis. Moreover, many diagnostic tests are costly, invasive and not available in all centers, especially in developing countries. So, we aimed to investigate the diagnostic performance of urinary urobilinogen; an easy, cheap test that was not tested before in BA. METHODS: Seventy-five infants divided into three age- and sex-matched groups (BA, non-BA cholestasis and healthy control group) were recruited for the study. Each group comprised 25 infants. Urinary urobilinogen was measured for all infants using the modified Ehrlich's method. RESULTS: Urinary urobilinogen was significantly lower in the BA group (0.31 ± 0.25 mg/dL) than both of the non-BA cholestasis (2.08 ± 3.48 mg/dL) and healthy control (0.53 ± 0.64 mg/dL) groups at P < 0.0001 and P < 0.001, respectively. Urinary urobilinogen at a cut-off value of 0.32 mg/dL or less can differentiate BA from other non-BA cholestasis with a sensitivity of 88% and a specificity of 72%. When this cut-off value was combined with γ-glutamyltransferase (γ-GT) at a cut-off value of 363 U/L or more, BA could be differentiated from other cholestatic disorders with a sensitivity of 80% and specificity of 100%. On the other hand, dipstick test could not differentiate between BA and non-BA cholestasis (P = 0.396). CONCLUSION: Urinary urobilinogen is a simple, non-invasive, cheap, sensitive and specific marker, especially if combined with γ-GT, which can be used in diagnosis of BA, especially in developing countries.

Impact of nitazoxanide on sustained virologic response in Egyptian patients with chronic hepatitis C genotype 4: a double-blind placebo-controlled trial.

BACKGROUND: Nitazoxanide, approved for the treatment of Cryptosporidium parvum and Giardia lamblia, was found to inhibit hepatitis C virus replication. AIM: The aim of this study was to assess the impact of nitazoxanide as an add-on therapy to pegylated interferon α-2a and ribavirin on sustained virologic response (SVR) in patients with chronic hepatitis C. PATIENTS AND METHODS: A total of 200 patients with chronic hepatitis C were enrolled in the study, assigned randomly in a 1 : 1 ratio to two groups: group A (placebo group) and group B (nitazoxanide group). Five patients withdrew from the study after they signed the consent form.A total of 195 patients were evaluated: 97 patients in group A versus 98 patients in group B at a dose of 500 mg twice daily. Placebo and nitazoxanide were administered as an add-on therapy to pegylated interferon α-2a plus ribavirin following a 12-week lead-in phase. SVR was evaluated. Statistical analysis was carried out using the SPSS software. RESULTS: The mean age of the patients in group A was 46.5 versus 45.7 years in group B. In group A, 85 out of 97 (87.6%) patients were men and in group B, 84 out of 98 (85.7%) patients were men.In group A, 59 out of 97 (60.82%) patients achieved an SVR versus 57 out of 98 (58.16%) patients in group B (P=0.70); this difference was not significant. CONCLUSION: Our data did not show any significant impact of nitazoxanide on SVR.

Neutrophil Gelatinase-Associated Lipocalin: A New Marker of Renal Function in C-Related End Stage Liver Disease.

Background/Aims. Renal impairment is a common complication of cirrhosis. Serum creatinine is less sensitive in these patients. Measurement of the glomerular filtration rate (GFR) is the gold standard but time consuming. The aim is to validate plasma NGAL (pNGAL) and urinary NGAL (uNGAL) as markers of renal function in patients with HCV related cirrhosis. Patient and Methods. One hundred HCV related end stage liver cirrhosis patients were randomized into two groups: Group I (n = 35), patients with GFR < 60 mL/m measured by isotope scanning of the kidney (Renogram), and Group II (n = 65), patients with GFR ≥ 60 mL/m. The pNGAL and uNGAL were measured within 2 days of the Renogram. Results. Both groups were matched with age, sex, and Child Pugh score. There was statistically significant difference between both groups regarding serum creatinine (1.98 ± 1.04 versus 1.38 ± 0.88 mg/dL; p = 0.003) and pNGAL level (5.79 ± 2.06 versus 7.25 ± 3.30 ng/dL; p = 0.019). Both groups were comparable (p > 0.05) for the uNGAL (6.00 ± 0.78 versus 6.03 ± 0.96 ng/mL). Unlike uNGAL, the pNGAL positively correlated with total GFR by Renogram (r = 0.3; p = 0.001). With a cutoff ≥4 ng/mL, pNGAL had 94.3% sensitivity and 1.5% specificity and PPV = 34, NPV = 33.3, LR+ = -175.1, and LR- = -60.6. Conclusion. The pNGAL is a promising marker of the renal function in patients with cirrhosis.

A comparative study of variants of pegylated interferon alpha in treatment of chronic HCV patients.

HCV infection presents a vast burden in the regions of high prevalence such as Egypt, where most HCV isolates are genotype 4b. Combined treatment of three variants of pegylated interferon and ribavirin is still the standard of care in Egypt. However, no conclusive data confirming their efficacy are available. Here, 60 chronic HCV patients were randomized for ribavirin plus Peg Intron (PEG-IFNα-2b), Pegasys (PEG-IFNα-2a) or Reiveron Retard (PEG-IFNα-2a). Serum interferon and antibody (Ab) levels were measured, and responses and costs were compared. Serum interferon levels were higher in Pegasys group (1625.1 ng/mL) followed by Reiveron Retard (1076.5 ng/mL), and Peg Intron group (857.72 ng/mL). Moreover, Ab levels were the lowest in Reiveron Retard group (318.4 ng/mL), followed by Peg Intron (439.93 ng/mL), and Pegasys cases (610.83 ng/mL). The best 24-week response rates were detected in the Pegasys group (73.3%), followed by Peg Intron (66.67%), and Reiveron Retard (40%). Treatment with both Pegasys and Peg Intron were most cost-effective. Furthermore, Pegasys was superior in both 6-month response and serum interferon, despite having higher Ab levels (more antigenicity). Our data have notable clinical implications and suggest that Pegasys may be a superior choice of interferon therapy for chronic HCV under low socioeconomic conditions.

miR-210 is a prognostic marker in clear cell renal cell carcinoma.

Accurate assessment of prognosis of clear cell renal cell carcinoma (ccRCC) is key in optimizing management plans to fit individual patient needs. miRNAs are short noncoding single-stranded RNAs that control the expression of target genes and may act as cancer biomarkers. We analyzed the expression of miR-210 in 276 cases of primary ccRCC and compared its expression in 40 pairs of adjacent normal and cancerous tissues. We assessed its expression in primary and metastatic tumors, in the common RCC subtypes, and the benign oncocytoma. The results were validated with an independent data set from The Cancer Genome Atlas. miR-210 was significantly overexpressed in ccRCC compared with normal kidney. miR-210(+) patients had a statistically higher chance of disease recurrence [hazard ratio (HR), 1.82; P = 0.018] and shorter overall survival (HR, 2.46; P = 0.014). In multivariate analysis, miR-210 lost its statistically significant association with shorter disease-free survival and overall survival after adjusting for tumor size and tumor, node, metastasis stage. Papillary RCC showed comparable miR-210 overexpression, whereas decreased up-regulation was seen in chromophobe RCC and oncocytoma. A number of predicted targets that might be involved in carcinogenesis and aggressive tumor behavior were identified. miR-210 is a potential therapeutic target and independent marker of poor prognosis of ccRCC.

Kallikreins as microRNA targets: an in silico and experimental-based analysis.

microRNAs (miRNAs) are non-coding RNAs that target specific mRNAs. They have been shown to control many biological processes including cancer pathogenesis. Kallikreins (KLKs) are a family of serine proteases that are attracting interest as cancer biomarkers. The mechanism of regulation of kallikrein expression is largely unknown. We investigated the potential roles of miRNAs in regulating KLK expression. Using a bioinformatics approach, we identified 96 strong KLK/miRNA interactions. KLK10 is the most frequently targeted kallikrein, followed by KLK5 and KLK13. KLK1, KLK3, KLK8 and KLK12 do not have strongly predicted miRNA/KLK interactions. Ten miRNAs are predicted to target more than one KLK. KLK2, KLK4, KLK5 and KLK10 have multiple miRNA-targeting sites on their transcript. Chromosomes 19 and 14 harbor significantly more KLK-targeting miRNAs. Many KLK-targeting miRNAs have been shown to be dysregulated in malignancy. We experimentally verified our bioinformatics data for the let-7f miRNA in a cell line model. let-7f transfection led to a significant decrease in secreted KLK6 and KLK10 protein levels. Co-transfection of let-7f and anti-let-7f inhibitor was able to partially rescue these protein levels. We conclude that miRNAs play a role in the regulation of KLK expression. Further studies are needed to investigate whether this regulation is altered in cancer.

Plasma protein advanced glycation end products, carboxymethyl cysteine, and carboxyethyl cysteine, are elevated and related to nephropathy in patients with diabetes.

In Diabetes Mellitus (DM), glucose and the aldehydes glyoxal and methylglyoxal modify free amino groups of lysine and arginine of proteins forming advanced glycation end products (AGEs). Elevated levels of these AGEs are implicated in diabetic complications including nephropathy. Our objective was to measure carboxymethyl cysteine (CMC) and carboxyethyl cysteine (CEC), AGEs formed by modification of free cysteine sulfhydryl groups of proteins by these aldehydes, in plasma proteins of patients with diabetes, and investigate their association with the albumin creatinine ratio (ACR, urine albumin (mg)/creatinine (mmol)), an indicator of nephropathy. Blood was collected from forty-two patients with type 1 and 2 diabetes (18-36 years) and eighteen individuals without diabetes (17-35 years). A liquid chromatography-mass spectrophotometric method was developed to measure plasma protein CMC and CEC levels. Values for ACR and hemoglobin A1C (HbA1C) were obtained. Mean plasma CMC (microg/l) and CEC (microg/l) were significantly higher in DM (55.73 +/- 29.43, 521.47 +/- 239.13, respectively) compared to controls (24.25 +/- 10.26, 262.85 +/- 132.02, respectively). In patients with diabetes CMC and CEC were positively correlated with ACR, as was HbA1C. Further, CMC or CEC in combination with HbA1C were better predictors of nephropathy than any one of these variables alone. These results suggest that glucose, glyoxal, and methylglyoxal may all be involved in the etiology of diabetic nephropathy.