Early Detection of Liver Injuries by the Serum Enhanced Binding Test Sensitive to Albumin Post-Transcriptional Modifications

Bibliographic info

• Authors: Souleiman El Balkhi*, Mohamad Ali Rahali, Roy Lakis, François Ludovic Sauvage, Marving Martin, Angelika Janaszkiewicz, Roland Lawson, Ruben Goncalves, Paul Carrier, Véronique Loustaud-Ratti, Anne Guyot, Pierre Marquet, Florent Di Meo, Franck Saint-Marcoux
• Journal: Scientific Reports, 2024, vol. 14, art. 1434
• DOI: 10.1038/s41598-024-51412-0
• Institution: P&T UMR1248 INSERM / CHU Limoges
• File: raw/my_work/Sci Rep El Balkhi et al.pdf

Key question

Can a simple binding-based functional assay (SEB test) detect HSA PTMs from hepatocyte injury earlier than standard liver enzyme tests (ALT, AST), and can this be validated in an animal model of hepatotoxicity?

The SEB test

The Serum Enhanced Binding (SEB) test is a novel functional assay for detecting HSA PTMs:

• Principle: serum is spiked ex vivo with specific ligands whose binding sites on HSA are affected by the most clinically relevant PTMs; the unbound (free) fraction of each ligand is measured
• Logic: if the binding site is modified (e.g., cysteinylated, glycated, oxidized), the ligand cannot bind → higher free fraction → positive signal
• Ligands tested: Gold (Au), Copper (Cu), Cadmium (Cd) — metals with specific binding sites related to different PTMs
• Compared to: IMA/ACB test (Ischemia Modified Albumin — FDA-approved for cardiac ischemia, based on cobalt binding to N-terminus), SEB is broader and liver-specific

Methods

• Model: Male albino Wistar rats
• Hepatotoxicity induction:
  • Group 1: high daily doses of ethanol (EtOH)
  • Group 2: CCl₄ (carbon tetrachloride — direct hepatotoxin)
  • Controls: vehicle
• Sample type: Rat plasma at serial timepoints (D0, D3, D5, D7)
• Assays:
  • HSA isoform quantification by LC-HR-MS (top-down)
  • Classical liver tests: ALT, AST, ALP, GGT, bilirubin
  • SEB test (ex vivo ligand binding + free fraction measurement)
  • Liver histology
  • Structural analysis + molecular dynamics simulation (palmitate binding sites, RMSD)

Human cohort (in the same paper)

The paper is not animal-only: alongside the rat model it reports a human application cohort.

• Primary human cohort: 90 subjects — 45 patients with cirrhosis (various stages, incl. Child–Pugh C) vs 45 controls without hepatic dysfunction (used to set >90%-binding thresholds per ligand and report discrimination)
• Independent confirmation set: a separate 12 cirrhotic vs 12 controls, distinct from the analytical-development subjects
• Ligands in humans: Au (Cys34), Cu (N-terminal site), Cd (multi-metal site), L-thyroxine (Sudlow I), dansylsarcosine
• Result: SEB ligands discriminated cirrhotic from control patients with individually high sensitivity/specificity (Fig. 2). On a combined threshold, 17/45 (37.8%) cirrhotic flagged ≥1 ligand above threshold vs only 4/45 controls

Main findings

1. Early detection (rat): albumin PTMs (HSA isoforms by LC-HR-MS) and SEB test positivity detected at D3 in both EtOH and CCl₄ groups
2. Classical tests lag (rat): ALT, AST, ALP significantly elevated only at D7 — 4 days later
3. Histological correlation (rat): liver lesions appeared at D3, confirming albumin isoforms and SEB test are detecting real early injury
4. Human discrimination: SEB distinguishes cirrhotic patients from controls (45 vs 45), establishing first human proof-of-concept for the functional assay
5. Ligand behavior supported by structural/MD analysis: specific ligands (Au, Cu, Cd) bind to sites that are disrupted by specific PTMs; MD simulation of palmitate binding sites (FABS) confirmed structural basis
6. SEB test feasibility: simpler to implement clinically than full LC-HR-MS isoform profiling — no mass spectrometer required

PTMs detected

PTM type	Marker	Detection method	Timepoint
Cysteinylation / oxidation (Cys34)	HNA1, HNA2	LC-HR-MS + SEB (Cu)	D3
Glycation	HSA+GLYC	LC-HR-MS	D3
N-term truncation	HSA-DA	LC-HR-MS	D3
General PTM (binding site disruption)	SEB positive	SEB test (Au, Cu, Cd)	D3

Clinical context

• Disease: DILI and early liver injury (animal model as proof-of-concept)
• Clinical need: current standard (ALT/AST) lacks early sensitivity — injury must be severe before enzymes leak; HSA PTMs and SEB test detect at the onset of hepatocyte dysfunction, not necrosis
• Translation pathway: SEB test is simpler than LC-HR-MS profiling → feasible in routine labs → potential companion or screening assay

Limitations

• Human cohort is cirrhosis vs controls (cross-sectional, single-centre, 45 v 45 + 12 v 12) — the early-detection/DILI claim rests on the rat model, not a human DILI cohort
• Large multicentric human validation still pending → addressed by MALAHBAR (560 patients / 8 French CHUs, readout ~2027)
• Ligand panel (Au, Cu, Cd) needs further optimization for human application
• Mechanism of individual ligand sensitivity to specific PTMs not fully characterized
• Comparison to IMA test not directly performed

Connections

• DILI — primary clinical context (early detection)
• HSA — sole protein studied
• SEB test — method page for this assay
• Cysteinylation / Oxidation — PTMs driving SEB positivity at Cys34
• Glycation — PTM detected early in EtOH model
• Top-down proteomics — isoform characterization method
• el-balkhi-2025 — companion paper applying isoform profiling to CLD staging (same group, next step)
• lakis-2024 — foundational LC-MS quantification method used here

My notes

• This paper establishes the key timeline argument: HSA PTMs are leading indicators of hepatocyte dysfunction while enzyme leakage is a lagging indicator of cell death. This is the mechanistic rationale for using HSA isoforms as early biomarkers.
• The SEB test is positioned as the clinically deployable version — human proof-of-concept exists (45 cirrhotic vs 45 controls); what remains is large multicentric validation (MALAHBAR) and regulatory development.
• The molecular simulation angle (how specific ligand binding sites are disrupted by specific PTMs) is an important structural foundation for designing future targeted assays.
• Connection to the IMA/ACB test history is important: shows SEB test fits an established regulatory paradigm (FDA-approved functional albumin assay).