Comparison of preoperative urinary Titin levels and serum Titin levels in patients with gastrointestinal malignancy

Introduction

Titin is a giant elastic protein specific to striated muscle, consisting of approximately 34,350 amino acids with a molecular weight of about 3 MDa (1). Fragmented titin has been detected in biological fluids under conditions associated with skeletal or cardiac muscle injury (2-4). Although urinary titin (U-Titin) has been investigated in several clinical settings, evidence regarding serum titin (S-Titin) remains limited. In particular, data on S-Titin levels in patients with gastrointestinal malignancies (GIMs) are scarce. Given recent technical advances that enable reliable quantification of S-Titin, we aimed to measure preoperative S-Titin concentrations in patients undergoing surgery for GIM, compare them with U-Titin levels, and explore their clinical relevance as biomarkers of muscle and nutritional status. We present this article in accordance with the REMARK reporting checklist (available at https://jgo.amegroups.com/article/view/10.21037/jgo-2026-1-0176/rc).

Methods

Patients

Patients were consecutively enrolled from those who underwent surgical treatment during the study period.

A total of 104 patients who underwent surgical treatment for GIMs in our department between May 2024 and November 2025 were enrolled in this study.

The study protocol adhered to the ethical principles outlined in the Declaration of Helsinki and its subsequent amendments and received approval from the Medical Ethics Committee of Tokyo Medical University (No. IB1761). Written informed consent was obtained from all adult participants prior to study participation.

Measurement of bioelectrical impedance analysis (BIA) using InBody 770^® (InBody Co., Ltd., Seoul, Korea)

Body composition was assessed using a multifrequency bioelectrical impedance analyzer (InBody 770^®) (5). Standardized measurement conditions were applied, including restrictions on exercise, food intake, caffeine, and smoking before testing. Skeletal muscle mass index (SMI), percent body fat (PBF), fat mass index (FMI), extracellular water to total body water ratio (ECW/TBW), and whole-body phase angle (Ph A) were derived according to the manufacturer’s algorithms and established formulas (6).

SMI was determined by summing the skeletal muscle mass of the bilateral upper and lower extremities and normalizing this value to height squared (kg/m²).

ECW/TBW, PBF and FMI were calculated as below: first, a current was passed at frequencies between 1 and 1,000 kHz to measure TBW. Subsequently, the weight of non-fat tissue was calculated from the water content, and body fat mass was determined as body weight minus fat-free mass. PBF (%) = body fat mass (kg)/body weight (kg) × 100, FMI (kg/m²) = body fat mass (kg)/height (m²).

PhA was derived at 50 kHz using raw resistance (R) and reactance (Xc) values and was calculated as the arctangent of the ratio of Xc to R, multiplied by 180° divided by π (7).

Handling of U-Titin and S-Titin samples

Serum and urine samples were collected simultaneously. Because a delay between sample collection and processing was anticipated, stability tests were performed to assess appropriate storage conditions. For urine samples, marked decreases in measured values were observed within 2 weeks when samples were stored at −20 ℃, indicating instability under these conditions. In contrast, urine samples stored at −80 ℃ remained stable for long-term storage (up to 6 months). Therefore, all urine samples were stored at −80 ℃ until analysis.

For serum samples, stable results were obtained even after 3 months of storage at −20 ℃; accordingly, serum samples were stored at −20 ℃. All specimens were retrieved and processed within 3 months of collection.

Measurement of U-Titin N-terminal fragment concentration

For the spot urine (2.0 mL) measurement of U-Titin, collected urine samples were stored at −80 ℃ until measurement. U-Titin was measured by Titin N-Fragment Assay IBL, ELISA kit (IBL Co., Ltd., Gunma, Japan) (8). Measured values were corrected with urinary creatinine in consideration of concentrated or diluted urine as follows: Titin-N fragment value (pmol/mg/creatinine) = measured Titin-N fragment value (nmol/L) × 100 ÷ urinary creatinine value (mg/dL) (8). In spot urine samples, both U-Titin concentrations and urinary creatinine levels were found to be influenced by variations in urine volume; therefore, normalization using urinary creatinine was performed. In contrast, no such sources of variability were present for serum samples, and thus no normalization was applied.

Serum samples and measurement of S-Titin

Serum samples were collected from patients who had given their informed consent and stored at −20 ℃ until analysis. S-Titin levels were measured using the Human Titin-N Fragment (serum) enzyme-linked immunosorbent assay (ELISA) kit (IBL Co., Ltd., Gunma, Japan) according to the manufacturer’s instructions (8). All samples were assayed in duplicate under blinded conditions, and the mean values were used for analysis.

Furthermore, for S-Titin measurements, serum concentrations are not influenced by variations in water volume; therefore, no normalization was performed.

Diurnal variations in serum and U-Titin

To our knowledge, no studies have reported clinically relevant diurnal variations in S-Titin levels. In addition, creatinine-corrected U-Titin concentrations are considered to be stable and exhibit minimal diurnal fluctuation, provided that patients are not engaged in vigorous physical activity. Furthermore, U-Titin levels (creatinine-corrected values) are considered to be stable and exhibit minimal diurnal variation when subjects are not engaging in vigorous exercise or similar activities.

Sample size calculation

The sample size for this study was determined based on the primary objective of evaluating the correlation between S-Titin and U-Titin levels in patients with GIM.

Pearson’s correlation coefficient was selected as the primary statistical measure.

Previous reports investigating titin-related biomarkers in clinical populations have shown moderate correlation coefficients (r≈0.40). Therefore, assuming an expected correlation coefficient of r=0.40, a two-tailed significance level of α=0.05, and a statistical power of 80%, the minimum required sample size was calculated using a standard formula for correlation studies:

n=(Zα/2+Zβ)2[ln(1+r1−r)/2]2+3[1]

Substituting expected values yielded a minimum required sample size of approximately 47 participants.

To account for heterogeneity among GIM subtypes and potential missing data, the target enrollment was set at 100 or more patients.

Ultimately, 104 patients were included, meeting and exceeding the statistical requirement for sufficient power.

Statistical analysis

All statistical analyses were carried out using SPSS software, version 29 (SPSS Inc., Chicago, IL, USA).

Medians were used for each parameter. Correlations were presented as scatterplots and were analyzed using the Pearson test. The absolute value of “r” was evaluated as follows: <0.20, slight, almost negligible relationship; 0.20–0.40, low correlation, definite but small relationship; 0.40–0.70, moderate correlation, substantial relationship; 0.70–0.90, high correlation, marked relationship; >0.90, very high correlation, very dependable relationship (9). P<0.05 was considered a statistically significant difference between each parameter.

Results

Patient characteristics

The study cohort comprised 104 individuals, including 76 men and 28 women, with a median age of 68 (range, 54–89) years. Among these patients, 36 were diagnosed with colorectal malignancies (CRMs), 42 with hepatobiliary-pancreatic malignancies (HPBMs), and 26 with malignancies of the upper gastrointestinal tract (UGIM).

Analysis of the correlation between S-Titin and U-Titin (Figure 1)

Figure 1 Analysis of all 104 cases revealed a strong positive correlation between S-Titin and U-Titin values. Cr, creatinine; S, serum; U, urine.

S-Titin and U-Titin levels showed a strong positive correlation (r=0.78, y=0.19x+1.40, P<0.001).

Analysis of the correlation between S-Titin and U-Titin in patients with HPBM, CRM, and UGIM (Figure 2)

Figure 2 CRM and UGIM showed a strong positive correlation with S-Titin and U-Titin values, while HPBM demonstrated a relatively strong correlation. Cr, creatinine; CRM, colorectal malignancy; HPBM, hepatobiliary-pancreatic malignancy; S, serum; U, urine; UGIM, upper gastrointestinal tract malignancy.

CRM and UGIM showed a strong positive correlation with S-and U-Titin levels, while HPBM demonstrated a relatively strong correlation.

Analysis of the correlation between S-Titin and U-Titin and various parameters (SMI, creatinine, PBF, FMI, Ph A, ECW/TBW, and age) (Table 1)

Age, PBF, FMI, and Ph A showed weak negative correlations with serum and U-Titin, respectively, while ECW/TBW showed a weak positive correlation; however, no correlation was observed for SMI. Furthermore, no differences were observed in the correlations between S-Titin and U-Titin among the respective parameters. Creatinine showed a positive correlation with S-Titin, but no correlation was observed with U-Titin.

Discussion

While the utility of U-Titin has been reported primarily to date (1-8), Nambu et al.’s first report was to utilize S-Titin (10). However, their report employed the S-Titin/creatinine ratio; otherwise, the previous report was the first to investigate pure S-Titin (10).

This study measured preoperative S-Titin and U-Titin in 104 patients with GIM, examining their correlation and association with body composition indices. The results demonstrated a strong positive correlation between S-Titin and U-Titin overall, suggesting that Titin can be measured in both serum and urine in patients with GIM and that the two forms are interchangeable. Furthermore, it has been demonstrated that Titin’s circulating form can be detected in serum, enabling the assessment of muscle injury status using Titin even in dialysis patients and those with impaired renal function, for whom measurement in urine was previously impossible.

In analysis by GIM tumor location, a strong correlation was observed between CRM and UGIM, whereas HPBM showed a tendency towards a slightly lower correlation compared to CRM and UGIM. In upper and lower GIM, a more pronounced deterioration in nutritional status than in HPBM is conceivable, and it is also possible that the dynamics of Titin fragments released into the blood and renal clearance may fluctuate, potentially widening the concentration gradient between body fluids. These points were considered to require further consideration in the future.

BIA was used for the purpose of comparing muscle mass and nutritional indicators, measuring age, ECW/TBW, PBF, FMI, Ph A, and SMI, and these were examined in relation to S-Titin and U-Titin. In all cases, S-Titin showed a weak negative correlation with U-Titin. Advanced age, high ECW ratio, high fat mass, and low Ph A are indicators suggesting a tendency towards sarcopenia; furthermore, it is thought to reflect a decline in muscle mass and muscle quality. On the other hand, the absence of correlation with SMI suggests that Titin may be a marker reflecting muscle quality, such as necrosis and breakdown, or muscle damage more strongly than muscle mass (a quantitative indicator). Going forward, we plan to accurately identify GIM patients with sarcopenia or malnutrition, measure S-Titin levels, and investigate the significance of Titin.

A comparative analysis with serum creatinine was also carried out in this study. Serum creatinine showed a positive correlation with S-Titin levels, whereas no association was observed with U-Titin. Creatinine is a metabolic byproduct that primarily depends on SMI and tends to be higher in individuals with preserved muscle mass. In contrast, Titin is a biomarker reflecting the degradation or damage of myofibrillar structural proteins. The observed positive correlation between these S-Titin and creatinine suggests that, in patients with GIM, there may be a subset of individuals in whom muscle injury or accelerated muscle metabolic turnover is already present, even at a stage when muscle mass is relatively preserved. This phenomenon may be attributable to factors such as aging, chronic undernutrition, or fluid imbalance. In contrast, U-Titin represents titin fragments that have appeared in the circulation and are subsequently excreted via the kidneys. Therefore, it might serve as a more sensitive marker reflecting short-term and dynamic changes, such as the onset or exacerbation of muscle injury. In preoperative patients with GIM, who often experience substantial fluctuations in fluid status, U-Titin may not correlate with creatinine, a marker of muscle mass, for this reason.

This study had several limitations as follows: this is a single-center study with a limited number of cases. Postoperative measurement of Titin levels has not been evaluated. Multivariate analysis with inflammatory markers, renal function, and nutritional indicators has not been performed. We believe that further analyses, including investigations into the association with postoperative complication rates, the association with long-term prognosis, and analyses adjusted for the effects of inflammation and renal function, will enable us to clarify the clinical utility of S-Titin more definitively.

Conclusions

S-Titin is a promising biomarker reflecting muscle quality and nutritional vulnerability in GIM patients. Its feasibility and physiologic relevance support its potential integration into perioperative risk assessment frameworks.

Acknowledgments

We thank all clinical staff for their contributions to data collection and patient care.

Footnote

Reporting Checklist: The authors have completed the REMARK reporting checklist. Available at https://jgo.amegroups.com/article/view/10.21037/jgo-2026-1-0176/rc

Data Sharing Statement: Available at https://jgo.amegroups.com/article/view/10.21037/jgo-2026-1-0176/dss

Peer Review File: Available at https://jgo.amegroups.com/article/view/10.21037/jgo-2026-1-0176/prf

Funding: None.

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://jgo.amegroups.com/article/view/10.21037/jgo-2026-1-0176/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study protocol adhered to the ethical principles outlined in the Declaration of Helsinki and its subsequent amendments and received approval from the Medical Ethics Committee of Tokyo Medical University (No. IB1761). Written informed consent was obtained from all adult participants prior to study participation.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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