Letter to the Editor: Indirect bilirubin: A potential predictive biomarker for diabetic retinopathy and its clinical translational potential

Abstract

Diabetic retinopathy (DR), a major microvascular complication of type 2 diabetes mellitus (T2DM), is a leading cause of blindness and visual impairment. Elucidating risk factors for DR progression is critical in reducing the incidence of preventable blindness and enhancing patient outcomes. Recent research has highlighted an independent inverse relationship between indirect bilirubin (IBIL) levels and DR risk in a cohort of 6993 T2DM individuals. This finding underscores the potential of IBIL as an inexpensive, non-invasive biomarker for DR risk prediction with considerable translational prospects. This article synthesizes clinical and experimental evidence to evaluate the role of IBIL in DR risk prediction, discusses its potential and limitations as a predictive biomarker, and explores its integration with established risk factors as well as practical considerations for clinical implementation.

Key Words: Biomarker; Diabetic retinopathy; Indirect bilirubin; Type 2 diabetes mellitus

Core Tip: Diabetic retinopathy (DR) remains a leading cause of vision loss and blindness among individuals with type 2 diabetes. A critical need exists for reliable biomarkers that can facilitate early identification and diagnosis of DR, as the absence of such markers significantly impedes timely intervention and effective management. The study by Lin et al reveals that "IBIL levels are independently and inversely associated with DR risk, suggesting its potential as a low-cost, non-invasive, and readily applicable predictive biomarker". To facilitate a comprehensive understanding of this novel predictive indicator, this review first evaluates the potential of indirect bilirubin in DR risk prediction from both basic science and clinical perspectives. It subsequently addresses the advantages and challenges associated with its application as a predictive biomarker and concludes with projections on its future role in DR screening strategies.

TO THE EDITOR

We fully support the published in the current issue of the World Journal of Diabetes by Lin et al conclusion that indirect bilirubin (IBIL) levels are inversely correlated with the risk of diabetic retinopathy (DR) and that IBIL holds promise as a potential predictive biomarker for this condition. The analysis of data from 6993 patients with type 2 diabetes confirmed that this significant association remains robust even after comprehensive adjustment for multiple confounding factors. This finding highlights the substantial potential of IBIL as a low-cost and non-invasive tool for the early detection of DR, which could significantly enhance the efficacy of clinical screening and risk assessment protocols. We commend the authors for their groundbreaking work in elucidating the role of IBIL in DR prediction, which opens new avenues for clinical intervention and preventive care in the diabetic population.

Overview of the role of IBIL in DR risk

Bilirubin, a metabolic byproduct of hemoglobin derived from senescent red blood cells, circulates in two principal forms: IBIL and direct bilirubin (DBIL). IBIL, the unconjugated form, represents the major fraction bilirubin[1,2]. Being water-insoluble, it requires hepatic conjugation with glucuronic acid to be converted into DBIL, which is subsequently excreted via the bile[3]. Historically regarded merely as a metabolic waste product, the physiological role of bilirubin has been reevaluated in recent years, with growing evidence underscoring its protective functions against a variety of diseases, including diabetes, metabolic syndrome, and atherosclerosis[4,5]. Accumulating studies have demonstrated that bilirubin possesses significant antioxidant and anti-inflammatory properties. In a high-fat diet-induced non-alcoholic fatty liver disease animal model, bilirubin administration significantly reduced fasting blood glucose, body weight, liver weight, liver-specific enzyme activities, and lipid profiles in rats. Furthermore, bilirubin notably attenuated hepatic steatosis, fibrosis, inflammation, and necrosis. Additionally, bilirubin induced substantial changes in the expression levels of autophagy-related genes and the Beclin-1 protein[6]. Another animal study revealed that bilirubin improved renal function in type 2 diabetic rats induced by a high-fat diet, achieved through alleviating endoplasmic reticulum stress and suppressing renal inflammation[7]. In lipopolysaccharide-treated Raw264.7 macrophages and chondrocytes, bilirubin was shown to slow the progression of osteoarthritis induced by oxidative stress, mediated through the activation of the Nrf2/HO-1 pathway and inhibition of the NF-κB signaling cascade[8].

Bilirubin has also demonstrated promise in the early diagnosis and prognostic assessment of diabetes-related complications. An 11-year observational study enrolled 1167 Korean participants aged ≥ 20 years with type 2 diabetes mellitus (T2DM) and an estimated glomerular filtration rate (eGFR) > 60 mL/min/1.73 m². Cox regression analysis identified a significant association between serum bilirubin levels and a ≥ 40% decline in eGFR. This finding suggests the potential of bilirubin as a prognostic marker for the onset and progression of diabetic kidney disease[9]. Furthermore, a clinical investigation involving 1652 pediatric patients hospitalized for type 1 diabetic ketoacidosis further corroborated the value of serum bilirubin as an early diagnostic indicator[10]. In addition, a prospective study assessing serum bilirubin in patients with ischemic stroke comorbid with T2DM found a correlation between stroke severity at admission (measured using the National Institutes of Health Stroke Scale) and functional status at discharge (assessed using the modified Rankin Scale). The results indicated a correlation between initial total bilirubin levels and stroke severity, underscoring the critical importance of early management of total bilirubin levels for the treatment and prognosis of ischemic stroke in T2DM patients[11,12].

IBIL as a promising predictive biomarker for DR

Substantial clinical evidence supports the association between serum bilirubin levels and DR risk[13-15]. IBIL, the predominant circulating bilirubin form, may have protective effects on retinal health in diabetic individuals. The significant inverse correlation between higher IBIL levels and lower DR risk further supports its potential use as a biomarker for predicting this complication[16]. Although the exact mechanisms behind this association are not fully understood, several biologically plausible pathways have been suggested. Bilirubin is a powerful endogenous antioxidant and plays an essential role in plasma antioxidant capacity[17,18]. Experimental studies have shown that bilirubin reduces oxidative stress by scavenging reactive oxygen species, inhibiting NADPH oxidase activity, and enhancing additional antioxidant systems[19-21]. Thus, mildly elevated IBIL levels may decrease oxidative damage to retinal microvessels. Oxidative stress significantly contributes to endothelial dysfunction and pericyte loss in DR. Additionally, bilirubin modulates immune responses by inhibiting pro-inflammatory cytokine production and reducing T-cell activation[22]. This immunomodulatory activity is particularly relevant given the role of chronic low-grade inflammation in retinal microvascular injury[23]. Beyond antioxidant and immunomodulatory properties, recent research suggests bilirubin may serve as an endogenous ligand for peroxisome proliferator-activated receptor alpha (PPARα). At physiological levels, bilirubin activates genes involved in fatty acid β-oxidation and metabolic homeostasis[24]. Supporting this, fenofibrate, a pharmacological agonist of PPARα, has reduced the need for laser treatment by 31% in patients with vision-threatening DR[25]. Importantly, DBIL does not interact with PPARα. This distinction may explain why IBIL exhibits a stronger association with DR risk compared to DBIL. Critically, the antioxidant, anti-inflammatory, and PPARα-associated properties of IBIL likely function via interconnected rather than independent pathways. These pathways may form a synergistic network that maintains retinal vascular homeostasis. By reducing oxidative stress, IBIL indirectly suppresses inflammatory signaling. Concurrent activation of PPARα additionally regulates metabolic and inflammatory processes. This combined mechanism provides a plausible explanation for the inverse relationship between IBIL levels and DR risk, although experimental validation remains necessary. Future studies should employ targeted experimental models to establish causality. For example, gene-modified animal models or in vitro retinal cell co-culture systems could validate IBIL's roles in oxidative stress, inflammation, and PPARα signaling.

The relationship between bilirubin and DR risk may not be linear. Studies suggest a U-shaped curve, where moderately elevated bilirubin levels offer protection against DR, but higher concentrations might increase risk[26-29]. Additionally, discrepancies among studies may partially arise from variations in baseline bilirubin concentrations and liver metabolic functions. Bilirubin is closely associated with hepatic activity; thus, differences in liver enzyme function, subclinical liver disorders, or bilirubin metabolism could influence the observed IBIL-DR association. Medication use, particularly lipid-lowering agents, antidiabetic medications, or drugs that affect hepatic metabolism, might also alter circulating bilirubin levels, complicating comparisons across studies. Furthermore, population heterogeneity, including differences related to sex, ethnicity, bilirubin metabolism, and genetic factors, may further contribute to inconsistent findings. Despite substantial evidence supporting IBIL as a biomarker for predicting DR, significant limitations exist[13,15,30-33]. The predominance of cross-sectional studies restricts causal inference. Variations in study populations, sample sizes, and analytical approaches may also lead to inconsistent outcomes. Additionally, inadequate control of confounding factors, such as liver metabolic status, concurrent medication use, and ethnic differences, could obscure the true nature of the IBIL-DR relationship. These factors potentially account for divergent associations reported across studies, highlighting the importance of comprehensive phenotyping and standardized statistical adjustments in future research. Collectively, these considerations emphasize the necessity of conducting well-designed prospective longitudinal studies and multi-center clinical trials to clarify IBIL’s predictive and mechanistic role in DR across different populations.

Clinical integration of IBIL into multivariable DR risk prediction

IBIL shows promise as an independent biomarker; however, its clinical value may be maximized when combined with established DR risk factors. Traditional predictors, such as glycemic control, duration of diabetes, and blood pressure, remain central to current DR risk assessment frameworks. In clinical practice, incorporating IBIL into multivariable prediction models alongside these factors is a reasonable progression, consistent with prior efforts to improve DR risk prediction using composite approaches[34,35]. In this setting, the added value of IBIL can be evaluated using standard performance metrics, including discrimination and reclassification indices. These metrics assess whether IBIL offers meaningful improvement beyond existing variables. Such analyses would help determine whether IBIL functions primarily as an adjunct rather than a standalone predictor. Beyond model performance, practical considerations related to IBIL measurement must also be addressed. Although IBIL testing is routinely available in clinical laboratories, measurement reliability may be affected by pre-analytical factors, including fasting status, sample handling, blood draw timing, and coexisting liver dysfunction[36,37]. Therefore, establishing standardized measurement protocols is essential to ensure consistency across studies and clinical settings. Furthermore, differences in laboratory infrastructure, analytical techniques, and clinical workflows across healthcare systems may limit broad implementation. Addressing these issues is critical for translating IBIL from a promising biomarker into a practical and widely applicable component of DR risk prediction strategies.

CONCLUSION

T2DM has emerged as a critical global public health challenge. DR, a prevalent and debilitating microvascular complication of T2DM, remains a leading cause of visual impairment and blindness worldwide. Identifying reliable biomarkers for predicting DR risk is therefore essential to reduce preventable vision loss and improve the quality of life of affected individuals. In this context, the study entitled “Indirect bilirubin is inversely associated with diabetic retinopathy risk and is a potential predictive biomarker” is of substantial clinical relevance. We concur with the authors’ central conclusion that IBIL levels are independently and inversely associated with DR risk in a large cohort of patients with T2DM. We also commend the authors for their transparent discussion of the study’s limitations. Several constraints should be acknowledged. The cross-sectional design limits causal inference, while the single-center, hospital-based population restricts generalizability. In addition, reliance on single time-point measurements and the presence of potential liver comorbidities may confound the interpretation of the observed associations. Building upon these findings and limitations, several priorities should be emphasized for future research. First, multi-center, prospective longitudinal studies involving multi-ethnic and outpatient populations are needed to enhance external validity and clarify the temporal relationship between IBIL levels and DR development or progression. Second, standardized protocols for IBIL measurement should be established, with repeated assessments to capture intra-individual variability and dynamic changes over time. Third, the incremental predictive value of IBIL should be systematically evaluated by integrating it with traditional risk factors, quantitative retinal imaging markers, and emerging machine-learning models, while rigorously controlling for liver-related confounders. Finally, further mechanistic investigations are warranted to elucidate the role of IBIL in oxidative stress, inflammatory pathways, and PPARα-related signaling, thereby clarifying its biological contribution to diabetic microvascular complications. With the accumulation of robust clinical and experimental evidence, IBIL holds considerable promise as a scalable, cost-effective biomarker that may be seamlessly incorporated into clinical pathways for DR risk prediction and screening.

ACKNOWLEDGEMENTS

We are grateful to all the members of the Ophthalmology Society of China Association of Chinese Medicine for their invaluable guidance and assistance.