Intravascular hemolysis accompanies diverse diseases, yet blood-based markers (eg, plasma-free hemoglobin [PFH], haptoglobin, lactate dehydrogenase [LDH], and bilirubin) are variably sensitive, nonspecific, or impractical for point-of-care use. We evaluated urinary carbonic anhydrase 1 (CA1) as a mechanistically grounded, urine-based marker of hemolysis in a multicenter study spanning the United Kingdom, Bangladesh, and Peru. We enrolled 234 participants: healthy adults and adults with inherited anemias (Oxford), newborns in intensive care (London), children and adults with complicated or uncomplicated malaria (Bangladesh), and adults attending rural clinics for various medical reasons (Peru). Urine CA1 and hemoglobin (Hb) were quantified by enzyme-linked immunosorbent assay and immunoblot, with CA1:Hb stoichiometry used to distinguish intravascular hemolysis from urogenital blood contamination. Relationships with PFH, LDH, bilirubin, Hb, C-reactive protein (CRP), and clinical variables were tested using regression, principal component analysis, and decision tree. Reference urine samples were predominantly CA1-negative/Hb-negative. In inherited anemias, urinary CA1 was highest in sickle cell disease and correlated with serum LDH and inversely with blood Hb. In neonates, longitudinal CA1 trajectories stratified infants into physiological, transient, and sustained hemolysis groups; higher CA1 levels were associated with prematurity, elevated CRP and lower white blood cell count. Urinary CA1 was detected in Bangladeshi patients with elevated PFH and in cases of intravascular hemolysis not identified by PFH. Among Peruvian participants, urinary CA1 correlated with raised CRP level, consistent with inflammation being a prohemolytic trigger. To enable rapid and cost-effective testing, a lateral flow device was developed and verified for excellent sensitivity and specificity. Urinary CA1 provides a sensitive and practical readout of intravascular hemolysis suitable for point-of-care testing globally.

Dubin-Johnson Syndrome (DJS; OMIM 237500) is a rare autosomal recessive disorder caused by pathogenic variants in the ABCC2 gene, encoding for the multidrug resistance protein 2 (MRP2), resulting in impeded biliary excretion of bilirubin metabolites. It is typically characterized by chronic or intermittent jaundice and conjugated hyperbilirubinemia. We report the case of a 54-year-old male with hyperbilirubinemia (mostly conjugated) and hypertransaminasemia. Hypertransaminasemia was due to presence of Metabolic dysfunction-associated fatty liver disease (MASLD), while whole-genome sequencing revealed a homozygous missense variant affecting ABCC2 (c.3893G > A, p.Gly1298Asp), a previously undescribed variant likely linked to hyperbilirubinemia. It is an extremely rare genetic variant (allele frequency = 6.2 × 10-7). In silico analyses predicted the variant to be highly pathogenic (CADD score 29; AlphaMissense score 0.978; PhyloP 8.87; DynaMut ΔΔG = -0.765 kcal·mol-1 and ΔΔSVib = -0.234 kcal·mol-1·K-1). Structural modeling suggested no gross conformational changes but potential effects local conformation and overall function of the protein. We describe a novel ABCC2 mutation associated with Dubin-Johnson Syndrome. This finding expands the spectrum of ABCC2 variants to evaluate in case of hyperbilirubinemia and highlights the importance of genetic testing in unexplained cases of conjugated hyperbilirubinemia.

Fermented products derived from Lactobacillus have demonstrated therapeutic potential in the management of metabolic disorders, particularly liver diseases. This study aimed to evaluate the hepatoprotective effects of Lactobacillus-fermented products in a carbon tetrachloride (CCl4)-induced murine model of liver damage, with a focus on analyzing the influence of bioactive compounds on key liver function biomarkers. Two Lactobacillus strains (Lactobacillus acidophilus LB-CARS1 and L. acidophilus ST-CARS2) were isolated from commercial yogurts, and milk was fermented using each strain. A total of 40 male Swiss Albino mice were allocated into 5 experimental groups: control, CCl4-only, CCl4 + silymarin, and 2 treatment groups receiving CCl4 plus 1 of the 2 lactic acid bacteria (LAB)-fermented products. After 6 wk, serum concentrations of aspartate aminotransferase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP), bilirubin, and creatinine were measured. A protein-protein network was constructed using the proteins responsible for hepatoprotection. The cellular pathways of these proteins were also visualized by the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway. Molecular docking was performed to assess interactions between identified bioactive compounds and target proteins (superoxide dismutase, catalase, transforming growth factor-β 1, peroxisome proliferator-activated receptor γ, IL-6, and tumor necrosis factor-α). Treatment with LAB-fermented products significantly reduced (P < 0.05) serum AST, ALT, ALP, bilirubin, and creatinine concentrations in the CCl4-treated mice. The 2 products reduced the liver enzymes, though their intensity was different. The interactive pathways of the protein network revealed the most common genes responsible for hepatoprotective activity. Molecular docking revealed strong binding affinities between bioactive compounds and proteins related to inflammation and oxidation. Furthermore, absorption, distribution, metabolism, excretion, and toxicity analysis suggested the potential drug candidates among the docked compounds. The results indicate that LAB-fermented products contain bioactive compounds capable of attenuating liver injury, likely through anti-inflammatory and antioxidant mechanisms, highlighting their potential as functional food interventions in hepatoprotection.

Background Wilson disease (WD) is a rare autosomal recessive disorder of copper metabolism and an important treatable cause of hepatic dysfunction in young adults. Data from South Asian populations remains limited. Hence, the current study aimed to assess the demographic characteristics, clinical features, and diagnostic profile of patients with WD presenting at a tertiary care hospital in Peshawar, Pakistan. Methods This retrospective descriptive study was conducted in the Department of Medicine, Hayatabad Medical Complex, Peshawar, between January 2023 and December 2024. Medical records were systematically reviewed to identify all patients diagnosed with WD during this period. 21 patients with a confirmed WD diagnosis based on the modified Leipzig scoring system (score ≥ 4) and complete diagnostic workup were included. Complete diagnostic data was defined as documented serum ceruloplasmin, 24-hour urinary copper excretion, liver function tests, slit-lamp examination for Kayser-Fleischer (KF) rings, and negative screening for viral and autoimmune hepatitis. Demographic, clinical, and biochemical data were extracted from hospital records and analyzed using SPSS version 25. Post-hoc ethical approval was obtained from the institutional review board. Results The mean age at presentation was 23.2 ± 4.8 years (range 18-35), with a male-to-female ratio of 2:1. 15 patients (71.4%) were between 18 and 25 years of age. Jaundice was the predominant symptom, observed in 21 (100%) patients, while hepatomegaly and KF rings were observed in 18 (85.7%) and 12 (57.1%) of patients, respectively. The mean 24-hour urinary copper excretion was 1,450.3 ± 420.7 µg/day, and the mean serum ceruloplasmin level was 8.4 ± 3.2 mg/dL. Four patients (19%) were from two consanguineous families. A positive correlation between urinary copper and total bilirubin levels was observed (r = 0.74, p < 0.001). Conclusion WD in this internal medicine cohort primarily affected young adults presenting with hepatic manifestations, most commonly jaundice. Elevated urinary copper excretion and reduced serum ceruloplasmin levels were consistent diagnostic findings. Familial clustering, particularly in consanguineous families, was also observed, indicating a pattern consistent with autosomal recessive inheritance within this population.

Bilirubin is a breakdown product of erythrocytes and plays a crucial role in elimination of heme-containing proteins. After its synthesis in the reticuloendothelial system, unconjugated bilirubin is released into plasma and taken up into the liver. In hepatocytes, bilirubin is conjugated and excreted into the gastrointestinal tract via bile, where it is further converted to urobilinoids. There are various genetic factors causing abnormal bilirubin levels in plasma, such as Gilbert syndrome, Crigler-Najjar syndrome, Dubin-Johnson syndrome, and Rotor syndrome. To better understand bilirubin metabolism and its disorders, this study develops a physiologically based computational model incorporating published literature as well as real-world clinical data from the Explorys database. The model simulates bilirubin levels in both healthy individuals and patients with disorders of bilirubin metabolism. Population simulations show that Gilbert syndrome requires a substantial reduction in UDP-glucuronosyltransferase 1A1 activity, while Crigler-Najjar syndrome requires near-complete loss of its function. In contrast, Dubin-Johnson syndrome is characterized by a significant impairment of multidrug resistance-associated protein 2 activity. To also illustrate model behavior under targeted perturbations, we simulated administration of atazanavir in healthy individuals and patients with Gilbert syndrome to investigate its effect on bilirubin levels. Relative to baseline, unconjugated bilirubin maximum concentration (Cmax) increased by 34% in healthy individuals but by 67% in Gilbert syndrome. Overall, this study provides a conceptual and mechanistically informed framework for studying bilirubin homeostasis and the functional consequences of drug administration in health and disease.