Association of serum and vitreous homocysteine and uric acid concentrations with post-vitrectomy prognosis in patients with proliferative diabetic retinopathy

Abstract

BACKGROUND

Proliferative diabetic retinopathy (PDR) is a major cause of vision loss, often requiring pars plana vitrectomy (PPV). Systemic and intraocular metabolic alterations, including dysregulation of homocysteine (Hcy) and uric acid (UA), may influence surgical outcomes. While prior studies suggest associations between these biomarkers and retinal pathology, the role of these biomarkers in postoperative prognosis remains unclear. This study hypothesized that elevated serum and vitreous Hcy and UA levels are associated with visual, structural, and microvascular changes following PPV in patients with PDR.

AIM

To evaluate the associations between serum and vitreous Hcy/UA concentrations with postoperative outcomes in patients with PDR following PPV.

METHODS

In this prospective observational study at a tertiary care center, 44 patients with PDR and 46 non-diabetic controls undergoing PPV between June 2021 and December 2022 were enrolled. Serum and vitreous Hcy and UA levels were measured. Best-corrected visual acuity, multimodal retinal imaging, and capillary density metrics were evaluated preoperatively and postoperatively. Correlation analyses assessed the relationships between biomarkers and clinical outcomes.

RESULTS

Patients with PDR showed significantly higher serum and vitreous Hcy and UA concentrations compared to those of controls. Serum Hcy and UA levels correlated with vitreous levels. In patients with PDR, elevated vitreous Hcy correlated with worse best-corrected visual acuity at 1 day and reduced peripapillary retinal nerve fiber layer thickness at 7 days and 90 days. It also correlated with foveal avascular zone enlargement at 90 days and inferior superficial capillary plexus (SCP) width density at 7 days. Vitreous UA had negative correlations at 30 days with nasal SCP length density and temporal/inner ring SCP width density.

CONCLUSION

Vitreous, but not serum, Hcy predicts post-PPV impairment, underscoring the prognostic value of the local ocular environment over systemic factors in PDR.

Key Words: Proliferative diabetic retinopathy; Homocysteine; Uric acid; Pars plana vitrectomy; Prognosis; Optical coherence tomography; Optical coherence tomography angiography; Diabetic retinopathy

Core Tip: This study demonstrates that vitreous homocysteine and uric acid are significantly elevated in patients with proliferative diabetic retinopathy, reflecting localized ocular pathology. Unlike their serum counterparts, elevated vitreous homocysteine specifically correlates with worse early visual acuity, progressive retinal nerve fiber layer thinning, and delayed microvascular impairment after pars plana vitrectomy, positioning it as a superior, compartment-specific predictor of multidimensional postoperative outcomes.

INTRODUCTION

Diabetic retinopathy (DR), a severe microvascular complication of diabetes mellitus, represents a primary cause of blindness in the working-age population worldwide[1-5]. Epidemiological studies estimate 103.1 million global cases of DR in 2020, with projections rising to 191 million by 2030[6-8], imposing substantial socioeconomic burdens. The disease progresses from non-proliferative DR (PDR) to sight-threatening PDR, characterized by neovascularization, vitreous hemorrhage, and tractional retinal detachment[2-4,9]. Current interventions, including pars plana vitrectomy (PPV), exhibit variable outcomes and complications[9,10], highlighting the urgent need for deeper pathophysiological insights to improve prognosis.

Emerging evidence implicates serum homocysteine (Hcy) - a sulfur-containing amino acid formed during methionine demethylation - as a critical mediator in DR progression. Elevated Hcy levels disrupt both inner[11,12] and outer[13] blood-retinal barrier (BRB) integrity through mechanisms involving retinal ischemia, pathological angiogenesis, and vascular leakage, as evidenced in experimental models[11-13]. Clinical studies confirm that elevated serum Hcy in patients with diabetes correlates with BRB breakdown and visual impairment[14-17], mechanistically linked to retinal vascular endothelial growth factor (VEGF) upregulation, driving neovascularization[18]. Crucially, Hcy dysregulation extends to ocular compartments: Elevated levels are documented in retinal tissues of experimental DR models[19,20] and vitreous humor of patients with PDR[21,22]. Preclinical models recapitulating type 2 diabetes mellitus, including streptozotocin-induced mice, Akita mice, and streptozotocin rats, consistently demonstrate increased Hcy levels in serum, vitreous, and retinal tissues[14]. Intravitreal Hcy administration exacerbates retinal pathology in diabetic mice, revealing synergistic toxicity between hyperglycemia and hyperhomocysteinemia[14]. Clinically, case-control studies confirm significantly higher geometric mean Hcy concentrations in the plasma, vitreous, and aqueous humor of patients with PDR vs non-diabetic controls (P < 0.05)[21,22]. Therefore, both experimental and clinical evidence consistently demonstrate that systemically and ocularly elevated Hcy levels are critical in the pathological mechanisms of DR.

Uric acid (UA), the terminal byproduct of purine metabolism in the human body, is primarily excreted by the kidneys. It exhibits multifaceted roles, including antioxidant, pro-oxidative, pro-inflammatory, nitric oxide (NO)-modulating, and immune effects, which are significant in both physiological and pathological contexts[23-26]. Although the relationship between serum UA and DR remains controversial[27-29], accumulating evidence indicates a correlation. Experimental and clinical research further reveals that UA contributes to DR progression through oxidative stress and inflammatory responses, which induce microvascular damage[30]. Mechanistically, UA reduces NO bioavailability, activates the renal renin-angiotensin system, and promotes endothelial dysfunction and aberrant endothelial cell proliferation, ultimately leading to retinal microvascular injury[31-34]. A strong association exists between UA and DR progression[27,35-38], particularly PDR. This may be attributed to UA exacerbating vascular endothelial injury via disturbances in glucose and lipid metabolism, thereby inducing retinal neovascularization and PDR pathogenesis[38-40]. Notably, studies have detected elevated vitreous UA levels in patients with DR or animal models compared to those of controls[39,41], suggesting its involvement in localized ocular pathophysiology.

Notably, intraocular Hcy and UA predominantly originate from systemic metabolic dysregulation and reflect compromised BRB integrity[16,26,42]. The vitreous humor serves as a reservoir for pro-angiogenic, inflammatory, and oxidative mediators driving PDR pathogenesis, including VEGF, Hcy, and UA[18,41]. While epidemiological studies have explored serological risk factors for surgical outcomes[43], the relationship between vitreous biomarkers and post-PPV prognosis remains uninvestigated. Therefore, this prospective study examines the correlations between serum/vitreous Hcy/UA concentrations and multidimensional postoperative outcomes, including visual function, retinal/choroidal structural parameters, and microvascular density in patients with PDR undergoing PPV, using non-diabetic vitrectomy patients as controls to elucidate the prognostic utility of these biomarkers in PDR management.

MATERIALS AND METHODS

Study population

The present single-center prospective cohort study adhered to the principles of the Helsinki Declaration. The study was approved by the Ethics Committee of West China Hospital of Sichuan University, No. 2020 (834), and was registered in the Chinese Clinical Trial Registry (Registration number: No. ChiCTR2000039698, registration date: November 6, 2020). All study participants, or their legal guardian, provided informed written consent prior to study enrollment.

Patients admitted to the Ophthalmology Department of West China Hospital, Sichuan University, between June 2021 and December 2022, were recruited. A description of the PDR population can be found in the literature[44-46]. Non-diabetic age-matched patients requiring PPV for epiretinal membrane or macular hole served as controls (control group). All patients who met the inclusion and exclusion criteria were enrolled and underwent PPV surgery. The inclusion criteria for the PDR group were: (1) Vitreous hemorrhage > 1 month, that responded poorly to conservative treatment, with no prior PPV in the affected eye; (2) No absolute contraindications to surgery; (3) Fasting glucose < 8 mmol/L and postprandial glucose < 11 mmol/L, with the glucose levels maintained for at least 1 week; and (4) Provision of informed consent. The inclusion criteria for the control group were: (1) Non-diabetic with epiretinal membranes or macular holes and clear surgical indications, with no prior vitrectomy in the affected eye; (2) No absolute contraindications to surgery; and (3) Provision of informed consent.

The exclusion criteria for the PDR group were: (1) Communicable diseases, such as acquired immunodeficiency syndrome, syphilis, and leukemia; (2) Type 1 diabetes mellitus; (3) Iris neovascularization or neovascular glaucoma, uveitis, retinal artery occlusion, retinal vein occlusion, paracentral acute middle maculopathy, age-related macular degeneration, ocular trauma, endophthalmitis; (4) History of intravitreal injections, retinal laser photocoagulation, and intraocular surgery in the 6 months prior to PPV; (5) Postoperative ocular conditions (postoperative complications), including postoperative ocular hypertension, postoperative vitreous hemorrhage, epiretinal membrane, macular hole, retinal/macular detachment, and neovascular glaucoma; and (6) Administration of medications (anti-VEGF agents, anti-inflammatory agents) via intravitreal injection for postoperative macular edema.

Sample collection and processing

The demographic and clinical variables we recorded included body mass index, glycated hemoglobin (HbA1c) (as a marker of glycemic control/insulin resistance), and sex. Fasting venous blood samples were collected from patients on admission, from which serum was separated. Renal function markers, such as blood urea nitrogen (BUN), cystatin C (CysC), creatinine (Crea), and estimated glomerular filtration rate (eGFR), were also tested. Additionally, urine was collected for the detection of albumin and ultimately used to calculate the urinary albumin-to-Crea ratio (UACR).

Standard three-port 25-gauge PPV was performed by the same skilled retinal surgeon (Zhang MX) in all patients, under retrobulbar/general anesthesia. Undiluted vitreous samples (0.2-0.5 mL) from the central vitreous cavity were obtained from each subject through a 25-gauge needle before opening the infusion port at the start of PPV, as described in previously published literature[21]. The samples were collected into sterile plastic tubes and immediately transferred to ice in the laboratory, where they were centrifuged. Before biochemical analysis, the vitreous sample was centrifuged at 4000 r/minute for 10 minutes at 5 °C, and the supernatant was collected for testing and stored in a -80 °C freezer until further use. Serum and vitreous Hcy and UA concentrations were measured using an automated biochemical analyzer (Roche701, Switzerland). Hcy was measured using a capture-based enzymatic assay, and UA was measured using an enzymatic colorimetric assay.

Follow-up and ophthalmic examinations

Non-mydriatic refractometry was performed before surgery and at each follow-up visit, with best-corrected visual acuity (BCVA) recorded in the standard logarithmic minimum angle of resolution (logMAR) units. Since the BCVA may change immediately after surgery, we followed up the BCVA of the patients at 1, 7, 30, and 90 days after the PPV. Imaging assessments were performed at 7, 30, and 90 days postoperatively and included intraocular pressure, slit-lamp examination, enhanced depth imaging optical coherence tomography (EDI-OCT), and optical coherence tomography angiography (OCTA).

Assessment of outcomes

After mydriasis, compound tropicamide eye drops (Mydrin-P; Santen, Osaka, Japan) were used for EDI-OCT. Standardized EDI-OCT images were obtained in the afternoon by technicians using spectral-domain OCT (Spectralis OCT; Heidelberg Engineering, Heidelberg, Germany) to avoid diurnal variations. The quantitative measurements, such as central macular thickness (CMT) and subfoveal choroid thickness (SFCT), were acquired using EDI-OCT with the horizontal/vertical scans intersecting the fovea center (ART 100 frames, high speed). CMT was measured automatically. Using the digital calipers that the Heidelberg Eye Explorer software supplied, the SFCT was measured manually. CMT was defined as the vertical distance from the inner limiting membrane to the RPE at the fovea. The SFCT was defined as the vertical distance from the outer surface of the retinal pigment epithelium to the chorioscleral interface in the macula. The quantitative measurement of peripapillary retinal nerve fiber layer (pRNFL) thickness was automatically measured with the glaucoma mode using spectral-domain OCT. During the follow-up period, all EDI-OCTs were performed in follow-up mode.

Macular microvasculature was assessed using OCTA performed on a Zeiss Cirrus HD-OCT 5000 system (Carl Zeiss Meditec, Inc., Germany), acquiring 3 mm × 3 mm scans centered on the fovea. Signal strength was evaluated for each scan to ensure data quality. Blood flow density within the superficial capillary plexus (SCP) was quantified using the instrument’s automated analysis software (with manual adjustments applied when necessary) based on a standardized Early Treatment Diabetic Retinopathy Study grid centered on the fovea. Specifically, two key SCP metrics were automatically calculated: Vessel width density (also termed perfusion density, reflecting the area covered by perfused vessels and indicative of blood flow volume) and vessel length density (also termed vascular density, representing the total length of vessels per unit area and sensitive to changes in vessel density and branching). Additionally, the area of the foveal avascular zone (FAZ) was automatically measured in square millimeters (mm²) from the en face OCT images. The blood flow density of the SCP was divided into three Early Treatment Diabetic Retinopathy Study sectors: Fovea, inner ring (defined as the 0.5-1.5 mm annulus centered at the fovea), and full area.

Statistical analysis

Data analysis was performed using SPSS 22.0 statistical software. Normally distributed continuous variables were expressed as the mean ± SD, and group comparisons were performed using independent t-tests. Non-normally distributed variables were expressed as interquartile ranges and compared using the Mann-Whitney U test. Pearson/Spearman correlation coefficients were used for correlation analysis, which was performed specifically within the PDR group to assess the relationship between biomarkers and postoperative outcomes. Analysis was performed on available complete data for each variable and time point; no imputation for missing data was performed. P value < 0.05 was considered statistically significant.

Sample size calculation

A formal sample size calculation was not performed a priori; the sample size was determined based on feasibility and the number of eligible patients presenting during the study period, aiming to be comparable to previous similar biomarker studies in the vitreous humor.

RESULTS

Demographic and biochemical characteristics

A total of 90 patients were included, and the characteristics of the participants are summarized in Table 1. The PDR group consisted of 44 patients, of whom 27 (61.4%) were male. The control group consisted of 46 patients; 8 (17.4%) were male. The difference in sex composition was significant (P < 0.001), but that of age was not (P = 0.099). The PDR group exhibited significantly higher serum Hcy levels compared to those of controls (15.10 ± 6.92 μmol/L vs 11.21 ± 2.76 μmol/L; P = 0.005), though serum UA differences did not achieve statistical significance (397.79 ± 97.55 μmol/L vs 287.67 ± 85.77 μmol/L; P = 0.381). In vitreous analyses, both Hcy (2.66 ± 1.46 μmol/L vs 0.82 ± 0.49 μmol/L; P < 0.001) and UA concentrations (198.84 ± 76.86 μmol/L vs 45.17 ± 37.18 μmol/L; P < 0.001) were markedly elevated in the PDR group relative to controls. Serum BUN (P = 0.010), Cys-C (P < 0.001), and HbA1c (P = 0.003) levels differed significantly between the PDR and control groups. No significant differences were observed in the remaining parameters (P > 0.05)

Table 1 Baseline characteristics, n (%)/mean ± SD.

Variables	Control group (n = 46)	PDR group (n = 44)	T	P value
Male	8 (17.4)	27 (61.4)	20.624	< 0.001a
Age (years)	59.5 ± 4.95	54.59 ± 9.26	49.590	0.099
Serum
FBG (mmol/L)	5.59 ± 0.72	5.78 ± 1.35	72.682	0.905
HbA1c	6.04 ± 0.70	7.30 ± 1.22	47.781	0.003a
Hcy (μmol/L)	11.21 ± 2.76	15.10 ± 6.92	72.136	0.005a
UA (μmol/L)	287.67 ± 85.77	397.79 ± 97.55	71.468	0.381
BUN (mmol/L)	5.11 ± 1.52	10.85 ± 6.54	65.065	0.01a
Crea (μmol/L)	68.3 ± 12.73	165.09 ± 195.37	54.824	0.263
eGFR (mL/minute/m2)	85.4 ± 13.94	65.63 ± 36.46	78.432	0.396
UACR (mg/g)	15.7 ± 17.74	842.50 ± 1347.98	47.424	0.075
CysC (mg/L)	0.97 ± 0.12	1.97 ± 1.50	67.702	< 0.001a
Vitreous
Hcy (μmol/L)	0.82 ± 0.49	2.66 ± 1.46	30.935	< 0.001a
UA (μmol/L)	45.17 ± 37.18	198.84 ± 76.86	0.811	< 0.001a

^aP < 0.05.

FBG: Fasting blood glucose; HbA1c: Hemoglobin A1c; Hcy: Homocysteine; UA: Uric acid; BUN: Blood urea nitrogen; Crea: Creatinine; eGFR: Estimated glomerular filtration rate; UACR: Urinary albumin-to-creatinine ratio; CysC: Cystatin C.

Correlation between vitreous Hcy and UA, and serum indicators in PDR

In the subsequent correlation analysis, vitreous Hcy levels correlated significantly with serum Hcy, BUN, Crea, eGFR, and CysC, and vitreous UA levels correlated significantly with serum Hcy, UA, BUN, Crea, eGFR, CysC, and UACR in PDR (Table 2).

Table 2 Correlation analysis of vitreous homocysteine and uric acid, and serum indicators within the proliferative diabetic retinopathy group (n = 44).

Serum variables	Vitreous Hcy		Vitreous UA
	Pearson	P value	Pearson	P value
FBG (mmol/L)	-0.108	0.492	-0.135	0.338
HbA1c (%)	0.049	0.759	0.097	0.542
Hcy (μmol/L)	0.554	< 0.001a	0.472	0.001a
UA (μmol/L)	0.157	0.309	0.480	< 0.001a
BUN (mmol/L)	0.496	< 0.001a	0.591	< 0.001a
Crea (μmol/L)	0.570	< 0.001a	0.552	< 0.001a
eGFR (mL/minute/m2)	-0.521	< 0.001a	-0.588	< 0.001a
CysC (mg/L)	0.584	< 0.001a	0.530	< 0.001a
UACR (mg/g)	0.318	0.071	0.407	0.019a

^aP < 0.05.

Hcy: Homocysteine; UA: Uric acid; FBG: Fasting blood glucose; HbA1c: Hemoglobin A1c; BUN: Blood urea nitrogen; Crea: Creatinine; eGFR: Estimated glomerular filtration rate; UACR: Urinary albumin-to-creatinine ratio; CysC: Cystatin C.

Correlation between serum Hcy, UA, and postoperative outcomes in PDR

No significant correlation was observed between serum Hcy/UA levels and postoperative BCVA (logMAR) or SCP microvascular parameters (vessel length/width density) in patients with PDR at any follow-up visit (Table 3).

Table 3 Correlation of serum homocysteine, uric acid, and best-corrected visual acuity, superficial capillary plexus within the proliferative diabetic retinopathy group (n = 44).

Variables	Time	Serum Hcy (μmol/L)		Serum UA (μmol/L)
		Pearson	P value	Pearson	P value
BCVA (logMAR)	7 days	-0.029	0.866	-0.079	0.643
	30 days	-0.150	0.474	0.068	0.749
	90 days	-0.046	0.877	0.040	0.893
SCP vessel length density (mm)	7 days	-0.214	0.063	-0.056	0.632
	30 days	-0.116	0.332	-0.154	0.196
	90 days	-0.183	0.115	-0.172	0.141
SCP vessel width density (mm2)	7 days	-0.223	0.053	-0.009	0.936
	30 days	-0.042	0.724	-0.053	0.656
	90 days	-0.126	0.282	-0.170	0.144

Hcy: Homocysteine; UA: Uric acid; BCVA: Best-corrected visual acuity; SCP: Superficial capillary plexus; logMAR: Logarithmic minimum angle of resolution.

Correlation between vitreous Hcy, UA, and postoperative outcomes in PDR

Analysis of vitreous Hcy in patients with PDR revealed moderate positive correlations with BCVA (logMAR) at 1 day (r = 0.594, P = 0.006) post-PPV. EDI-OCT imaging showed vitreous Hcy negatively correlated with superior temporal pRNFL thickness at 90 days (r = -0.399, P = 0.036), inferior nasal pRNFL thickness at 7 days (r = -0.354, P = 0.043) and at 90 days (r = -0.716, P < 0.001), but no associations with CMT or SFCT. No significant correlation was observed between vitreous UA levels and BCVA, pRNFL thickness, CMT, or SFCT in patients with PDR at any postoperative follow-up visit (Table 4). Vitreous Hcy was correlated negatively with inferior SCP width density at 7 days postoperatively (r = -0.303, P = 0.014) and positively with FAZ area at 90 days (r = 0.343, P = 0.032), but showed no association with SCP length density. Vitreous UA had negative correlations at 30 days with nasal SCP length density and temporal/inner ring SCP width density (all P < 0.05; Table 5).

Table 4 Correlation analysis of vitreous homocysteine, uric acid, and best-corrected visual acuity, optical coherence tomography parameters within the proliferative diabetic retinopathy group (n = 44).

Variables	Time	Vitreous Hcy (μmol/L)		Vitreous UA (μmol/L)
		Pearson	P value	Pearson	P value
BCVA (logMAR)	1 day	0.594	0.006a	-0.064	0.787
	7 days	0.261	0.113	0.027	0.871
	30 days	0.156	0.448	-0.086	0.676
	90 days	0.264	0.361	0.093	0.753
pRNFL
Optic daysisc	7 days	-0.164	0.354	-0.107	0.548
	30 days	-0.149	0.440	-0.102	0.597
	90 days	-0.382	0.054	-0.297	0.141
Temporal	7 days	-0.105	0.553	-0.046	0.794
	30 days	0.032	0.869	0.057	0.771
	90 days	-0.085	0.631	-0.376	0.058
Superior temporal	7 days	-0.029	0.866	-0.216	0.199
	30 days	0.046	0.804	-0.243	0.180
	90 days	-0.399	0.036a	-0.285	0.142
Inferior temporal	7 days	-0.155	0.361	-0.055	0.745
	30 days	-0.039	0.818	0.053	0.754
	90 days	-0.057	0.747	-0.042	0.812
Nasal	7 days	0.112	0.530	-0.033	0.852
	30 days	-0.113	0.559	-0.132	0.494
	90 days	-0.107	0.603	-0.131	0.523
Superior nasal	7 days	0.118	0.506	-0.002	0.993
	30 days	0.179	0.353	0.032	0.871
	90 days	-0.054	0.761	-0.058	0.743
Inferior nasal	7 days	-0.354	0.043a	0.177	0.323
	30 days	-0.268	0.160	0.065	0.737
	90 days	-0.716	< 0.001a	-0.343	0.086
CMT and SFCT
CMT	7 days	-0.105	0.531	-0.078	0.642
	30 days	-0.114	0.526	-0.043	0.812
	90 days	-0.166	0.400	-0.085	0.669
SFCT	7 days	-0.023	0.894	-0.059	0.727
	30 days	-0.059	0.726	-0.210	0.248
	90 days	-0.012	0.953	-0.080	0.693

^aP < 0.05.

Hcy: Homocysteine; UA: Uric acid; BCVA: Best-corrected visual acuity; LogMAR: Logarithmic minimum angle of resolution; OCT: Optical coherence tomography angiography; pRNFL: Peripapillary retinal nerve fiber layer; CMT: Central macular thickness; SFCT: Subfoveal choroid thickness.

Table 5 Correlation analysis of vitreous homocysteine and uric acid with optical coherence tomography angiography parameters within the proliferative diabetic retinopathy group (n = 44).

Variables	Time	Vitreous Hcy (μmol/L)		Vitreous UA (μmol/L)
		Pearson	P value	Pearson	P value
SCP length density (mm-1)
Superior	7 days	0.015	0.927	0.18	0.261
	30 days	-0.025	0.882	-0.179	0.288
	90 days	0.021	0.898	0.021	0.9
Inferior	7 days	-0.15	0.348	-0.089	0.581
	30 days	-0.013	0.941	0.246	0.143
	90 days	-0.059	0.72	0.018	0.915
Nasal	7 days	-0.063	0.697	0.133	0.407
	30 days	0.255	0.128	-0.413	0.011a
	90 days	-0.058	0.726	0.141	0.392
Temporal	7 days	-0.199	0.212	-0.114	0.476
	30 days	0.06	0.725	0.178	0.292
	90 days	-0.101	0.542	-0.163	0.323
Fovea	7 days	-0.084	0.604	0.066	0.681
	30 days	0.132	0.443	-0.232	0.174
	90 days	-0.22	0.183	-0.009	0.959
Inner ring	7 days	-0.199	0.213	-0.106	0.51
	30 days	0.024	0.883	0.252	0.108
	90 days	-0.196	0.22	-0.013	0.935
Full area	7 days	-0.158	0.307	0.221	0.15
	30 days	-0.01	0.949	0.195	0.215
	90 days	-0.12	0.941	0.1	0.543
SCP width density (mm-2)
Superior	7 days	-0.255	0.108	0.032	0.84
	30 days	-0.006	0.974	-0.083	0.624
	90 days	0.006	0.973	-0.005	0.978
Inferior	7 days	-0.303	0.014a	0.00	1.00
	30 days	0.032	0.85	0.057	0.737
	90 days	-0.07	0.67	-0.009	0.957
Nasal	7 days	-0.157	0.326	0.134	0.405
	30 days	0.18	0.287	0.313	0.06
	90 days	0.027	0.869	-0.007	0.967
Temporal	7 days	-0.237	0.136	0.134	0.403
	30 days	0.278	0.096	-0.391a	0.017a
	90 days	0.143	0.385	-0.205	0.21
Fovea	7 days	-0.238	0.134	0.026	0.874
	30 days	-0.074	0.662	-0.109	0.522
	90 days	-0.096	0.56	-0.015	0.93
Inner ring	7 days	-0.291	0.065	-0.067	0.667
	30 days	-0.151	0.372	-0.341	0.039a
	90 days	0.136	0.408	0.197	0.23
Full area	7 days	-0.3	0.057	-0.073	0.651
	30 days	0.139	0.412	0.315	0.057
	90 days	-0.001	0.997	0.115	0.485
FAZ
FAZ (mm2)	7 days	-0.157	0.326	-0.008	0.959
	30 days	0.177	0.294	0.134	0.428
	90 days	0.343a	0.032a	0.041	0.803
The perimeter of FAZ (mm)	7 days	-0.158	0.325	-0.108	0.503
	30 days	0.084	0.621	-0.013	0.941
	90 days	0.208	0.204	0.03	0.858

^aP < 0.05.

Hcy: Homocysteine; UA: Uric acid; OCTA: Optical coherence tomography angiography; SCP: Superficial capillary plexus; FAZ: Foveal avascular zone.

DISCUSSION

This prospective cohort study provides compelling evidence for the differential prognostic capacity of systemic vs intraocular biomarkers in patients with PDR undergoing PPV. These findings confirm significant elevations in serum Hcy and vitreous Hcy/UA concentrations in patients with PDR compared to those of non-diabetic controls, extending prior clinical observations by Lim et al[21] and Aydemir et al[22]. Critically, we demonstrate that vitreous Hcy - unlike its serum counterpart - exhibits multidimensional correlations with postoperative structural and functional outcomes, positioning it as a pivotal mediator of surgical prognosis.

The strong correlations between vitreous Hcy/UA levels and renal function markers (serum BUN, CysC, eGFR, UACR) suggest a pathophysiological triad linking diabetic kidney disease, BRB disruption, and intraocular biochemical dysregulation. This aligns with epidemiological evidence demonstrating that microvascular complications in diabetes manifest concurrently across renal and retinal tissues through shared mechanisms of endothelial dysfunction and inflammation[47]. Vitreous Hcy elevation likely reflects both systemic metabolic derangement and localized BRB compromise, as evidenced by a preclinical study where intravitreal Hcy administration exacerbated retinal vascular leakage and neuronal damage in diabetic models[14]. Notably, while serum Hcy demonstrated no association with postoperative BCVA (logMAR), vitreous Hcy exhibited a significant positive correlation at 1 day post-PPV (r = 0.594, P = 0.006). This phenomenon may involve local surgical trauma effects, where physical disruption and inflammatory stimulation of intraocular tissues (e.g., retina, vasculature, BRB) induce edema and stimulate additional Hcy release.

Progressive neuroretinal degeneration was evidenced by negative correlations between vitreous Hcy and pRNFL thickness, particularly inferior nasal pRNFL thickness at 7 days (r = -0.354, P = 0.043) and 90 days (r = -0.716, P < 0.001), and superior temporal pRNFL thickness at 90 days (r = -0.399, P = 0.036). These correlations reflect Hcy’s direct neurotoxic impact on retinal ganglion cells (RGCs), where elevated Hcy - whether endogenous (e.g., cystathionine β-synthase knockout mice) or exogenous (intravitreal injection) - promotes RGC loss via N-methyl-D-aspartate receptor (NMDAR) activation[48-52]. Structurally analogous to L-glutamate, Hcy binds NMDARs on RGCs, triggering cytoplasmic calcium overload, oxidative stress, and mitochondrial dysfunction[53-56], culminating in caspase-dependent apoptosis that correlates with reduced pRNFL thickness in patients with diabetes[57].

The temporal progression of pRNFL correlations (e.g., r = -0.354 at 7 days and r = -0.716 at 90 days) aligns with Hcy-mediated pathological processes. Short-term hyperhomocysteinemia exposure activates RGCs and upregulates VEGF, initiating inflammatory damage[58], whereas chronic Hcy elevation disrupts tight junctions (e.g., claudin-5) in the BRB and promotes mitochondrial damage via NMDAR overactivation[59]. In cystathionine β-synthase-deficient mice, altered NMDAR subunit expression (e.g., NR2A/B upregulation) exacerbates retinal functional deficits, further linking Hcy to progressive RNFL thinning[60]. The intensified correlation at 90 d suggests cumulative Hcy-induced neurodegeneration, as persistent calcium influx and oxidative stress progressively degrade RGC axons comprising the RNFL.

Delayed microvascular compromise was reflected in reduced SCP vessel width density at 7 days (r = -0.303, P = 0.014) and enlarged FAZ area at 90 days (r = 0.343, P = 0.032). This progression implicates Hcy-driven endothelial dysfunction and BRB disruption. While direct evidence linking Hcy to SCP/FAZ alterations in DR remains limited, clinical studies substantiate its pathogenic relevance: Hcy elevation correlates with DR severity[61] and reduced deep capillary plexus vessel density[62], paralleling the observed SCP rarefaction. Mechanistically, early SCP rarefaction may stem from Hcy-induced retinal capillary cell apoptosis and acellular capillary formation[63], potentially amplified by endotoxic effects on pericytes[64,65]. Subsequent FAZ expansion at 90 days suggests chronic vascular remodeling, possibly through Hcy-mediated VEGF upregulation[18], promoting pathological angiogenesis and vascular leakage, compounded by extracellular matrix destabilization via lysyl oxidase inhibition[66-69]. Critically, Hcy orchestrates microvascular collapse through interconnected cascades: NMDAR-mediated calcium dysregulation and oxidative stress disrupt endothelial-pericyte signaling[11,12,70,71], whereas diminished NO bioavailability[72-74] impairs perfusion regulation. These processes, synergizing with Hcy’s pro-thrombotic[75,76] and pro-inflammatory[77] actions, may drive capillary dropout and FAZ enlargement - mirroring BRB breakdown patterns in hyperhomocysteinemia models[11-17].

Consistent with prior reports[39,41], our study confirmed elevated vitreous UA levels in patients with diabetes vs non-diabetic controls, whereas serum UA showed no significant intergroup difference. This divergence suggests vitreous UA accumulation occurs independently of systemic concentrations, highlighting the ocular microenvironment as a critical reservoir for oxidative mediators[39,78,79]. Vitreous UA exhibited selective and transient involvement, distinct from Hcy’s broad microvascular impact, demonstrating negative correlations exclusively with nasal SCP vessel length density (r = -0.413, P = 0.011) and temporal region/inner ring SCP width density (r = -0.391/-0.341, P ≤ 0.039) at 30 days postoperatively. This compartmentalized and temporally specific effect aligns with clinical observations linking systemic hyperuricemia to impaired retinal microvasculature[31,80] and mirrors regional susceptibility patterns consistent with UA’s dual role in oxidative stress[23-26]. Mechanistically, hyperuricemia promotes DR both by activating the nod-like receptor protein 3/NACHT, LRR, and PYD domains-containing protein 3 inflammasome to increase inflammatory factors (tumor necrosis factor alpha, interleukin-6, C-reactive protein)[25], inducing pathological changes such as vessel dilation, retinal edema, and platelet aggregation[81], and by enhancing Notch signaling pathway activity specifically under hyperglycemic conditions, further driving retinal inflammation and DR progression[26]. The transient postoperative peak at 30 days reflects preclinical evidence that short-term hyperuricemia induces reversible retinal microvascular impairment and glial activation, reversible by urate-lowering agents[82]. This temporal specificity implies mitigation via compensatory antioxidant responses or acute inflammatory waves, distinct from chronic remodeling. Regional selectivity may also relate to sex-dependent vascular vulnerability[31,38,80]. Collectively, vitreous UA’s localized effects underscore its role in inflammation-driven vascular dropout[26] and its influence on surgical outcomes.

Considering the accessibility of blood samples and their freedom from ethical or surgical constraints, we investigated the relationship between circulating Hcy/UA levels and intraocular outcomes in patients with PDR. While previous studies have examined associations between serum Hcy/UA and disease progression in non-vitrectomy DR cohorts, research simultaneously linking serum Hcy/UA, vitreous Hcy/UA, and postoperative prognosis in vitrectomized patients with PDR remains scarce. This study uniquely addresses this tripartite relationship. Key findings indicate that neither serum Hcy/UA correlated with postoperative outcomes in patients with PDR undergoing vitrectomy. Vitreous UA demonstrates transient microvascular involvement without structural or functional sequelae. In contrast, vitreous Hcy demonstrated significant associations with clinical prognosis. While these preliminary results provide compelling evidence for vitreous Hcy as a potential prognostic biomarker, larger prospective studies adjusting for key confounders are needed to establish its independent predictive value.

This study has several limitations that warrant consideration. First, although we analyzed key clinical confounders like renal function and HbA1c, the lack of assessment for potential confounders such as vitamin B12/folate deficiencies - known to artificially elevate Hcy levels - and detailed dietary or lifestyle data may introduce bias into the observed associations. Second, while the cohort size (n = 90) was sufficient for preliminary analyses, it limited subgroup stratification and generalizability across diverse diabetic populations. The statistical analysis relied on correlation tests at individual time points. While Linear Mixed-Effects Models would have been more robust for analyzing longitudinal data and adjusting for surgical confounders, our sample size was insufficient to support such a complex model without risk of overfitting. Future studies with larger cohorts should employ these advanced statistical techniques to confirm the predictive value of vitreous Hcy. Third, the follow-up period was restricted to 90 days, precluding evaluation of long-term outcomes of elevated Hcy. Fourth, heterogeneity in the control group (non-diabetic patients with epiretinal membranes/macular holes) may subtly alter vitreous biochemistry, complicating comparisons. Finally, the clinical applicability of vitreous Hcy/UA measurements is constrained by the invasiveness of vitreous humor sampling. Future studies should validate findings in multiethnic cohorts with a longitudinal design (> 12 months), integrate vitreous proteomics to map Hcy-associated pathways, and explore Hcy-lowering interventions in patients with PDR.

CONCLUSION

This study demonstrates that patients with PDR exhibit significant elevations in serum Hcy and vitreous Hcy/UA concentrations, reflecting systemic metabolic dysregulation compounded by BRB failure. Crucially, prognostic utility is compartment-specific: (1) Serum biomarkers (Hcy/UA) show minimal association with postoperative outcomes, limiting clinical applicability; (2) Vitreous Hcy independently predicts early visual impairment, progressive neuroretinal degeneration, and delayed microvascular compromise, positioning it as a surrogate for ischemia-driven pan-retinal damage; and (3) Vitreous UA demonstrates transient microvascular involvement without structural or functional sequelae, reflecting its niche role in inflammatory cascades. These findings implicate ocular Hcy dysregulation as a critical driver of postoperative deterioration in vitrectomized patients with PDR.

ACKNOWLEDGEMENTS

We are grateful to members of our laboratories for their input and discussions. The authors also want to thank all patients and their families who consented to participate in the study.