**2. Methodology**

A number of 17 subjects with high-risk proliferative diabetic retinopathy (HRPDR), 14 subjects with mild non-proliferative DR (MNPDR), 11 age and gender-matched diabetic subjects without clinically evident retinopathy (DNR), and 5 healthy controls (HCs), whose clinical condition independently indicated for vitrectomy were enrolled in the present cross-sectional study. Subjects with hypertension (systolic BP > 140 mm Hg and or diastolic BP > 90 mm Hg), cardiovascular diseases, neuropathy (assessed by Michigan Neuropathy Screening Instrument), nephropathy (serum creatinine level > 1.5 mg/dl and or urinary albumin creatinine ratio 300 μg/mg), deficiency of B vitamins, any other ocular diseases (glaucoma, optic neuropathy, cataract, branch retinal vein occlusion, and Eales disease) were excluded from the study. The subjects were chosen consecutively from the 'Outdoor Patient Department' of 'Regional Institute of Ophthalmology, Calcutta Medical College, West Bengal, and Kolkata, India. The institutional ethical committee was approved the study and informed consent was collected from all the study subjects according to the Helsinki guideline.

Subjects with type 2 DM were diagnosed according to the guideline of the American Diabetes Association (2010). The fasting plasma glucose (FPG), postprandial plasma glucose (PPG), and glycated hemoglobin (HbA1c %) levels were used for the assessment of the glycaemic status of each subject. None of the study subjects were taking insulin or lipid-lowering drugs during the study period.

#### **2.1 Comprehensive ophthalmological examinations**

The subjects enrolled in the study had undergone different ophthalmological examinations which included slit-lamp biomicroscopy (by ±90 diopters and Goldman 3 mirror lens), seven fields of digital fundus photography with fluorescein angiography, and spectral-domain optical coherence tomography (SD-OCT). Visual functions were evaluated by measuring VA. The subjects with different grades of DR were diagnosed according to the modified guideline of 'Early Treatment of Diabetic Retinopathy Study' [13].

#### **2.2 Collection of vitreous sample**

Vitreous samples from study subjects were drawn by 3-port parsplana vitrectomy during surgery of vitreous hemorrhage, of idiopathic macular hole or removal of a dropped nucleus which occurred accidentally after blunt trauma. Vitreous was also collected during management of preoperative complication of phacoemulsification. Undiluted vitreous gel (500 μL) was excised from midvitreous by vitreous cutter and carefully aspirated into the hand-held sterile syringe attached to the suction port of the vitrectomy probe. Immediately after collection, the vitreous samples were taken in micro centrifuged tube and centrifuged at 3000 rpm for 5 minutes. The clear solution without any precipitate was then collected in another tube and preserved in - 80° C for farther use.

### **2.3 Measurement of NADPH oxidase activity**

NADPH oxidase activity was measured in vitreous using Nitrobluetetrazolium (NBT) as the substrate. Briefly, 100 μl plasma/ vitreous was mixed with NBT (4 mg/ml in water) and incubated for 20 minutes at 37°C. 1 M HCl was used to terminate the reaction. Then the samples were centrifuged at 3500 rpm for 5 minutes. 400 μl Dimethylsulfoxide (DMSO) was added to form a stable triphenylmethyl ester whose absorbance was measured at 550 nm using a microplate reader (MerilyzerEiaquant, Meril Diagnostics Pvt. Ltd., Vapi, Gujarat). OD 550 is directly proportional to NADPH oxidase activity [14].

#### **2.4 Measurement of vitamin C level**

Vitamin C level was measured by the protocol of Kyaw et al. [15]. Briefly, the colored reagent was prepared using Sodium Tungstate, DisodiumHydrogen Phosphate, H2SO4 and distilled water. Plasma/vitreous sample (1 ml) was thoroughlymixed with 2 ml colored reagent. After 30 minutes incubation at room temperature (RT) the tubes were centrifuged at 3000 pm for 10 minutes. The absorbance was measured at 700 nm from the supernatant, without disturbing the precipitate. Standard curve was prepared using oxalic acid and distilled water is used as substrate blank in the experiment. The vitamin C concentration in samples was expressed in mg/dl.

#### **2.5 Measurement of protein carbonylation (PCC)**

Protein carbonylation was measured from vitreous by spectrophotometric method by protein derivatization with 2, 4-dinitrophenyl-hydrazine (DNPH). Briefly, protein lysates from vitreous (50 μl) were incubated in dark for 30 minutes with DNPH (10 mM in 2 N HCl, 100 μl). After that TCA (20%, 100 μl) was used to precipitate proteins and free DNPH was removed by washing with ethanol-ethyl acetate (1:1). The resultant pellet was dissolved in 350 μl of sodium dodecyl sulfate (2% SDS) and protein-hydrazone complex's absorbance was measured at 370 nm using spectrophotometer. The carbonyl concentration was calculated using the extinction coefficient of the protein-hydrazone complex (22,000 M−1 cm−1) from the specific absorption (relative to the reagent blank). The final concentration was expressed as nanomoles of carbonyl groups per milligram protein [16–18].

#### **2.6 Estimation of malondialdehyde (MDA)**

The MDA level in vitreous was measured by thiobarbituric acid (TBA) assay method. In the assay procedure, the plasma samples were first reacted with trichloroacetic acid (TCA) to remove proteins. Then chromogenic adducts of MDA was precipitated using TBA. Finally the precipitated MDA was extracted using n-butyl alcohol, by vigorous shaking. Then the chromogenic adduct was measured spectrophotometrically at 532 nm; the results were expressed as mM/L [19].

*Role of Lipid, Protein-Derived Toxic Molecules, and Deficiency of Antioxidants… DOI: http://dx.doi.org/10.5772/intechopen.99904*

#### **2.7 Measurement of HNE**

Human vitreous HNE was estimated by competitive inhibition enzyme immunoassay technique (ELISA) using research kit from CUSABIO (cat no: CSB-E16214h). In the assay, an antibody specific for human HNE was coated on the well plate. A series of standards ranging from 39 pg/ml to 2500 pg/ml and samples (vitreous samples were run in 5 fold diluted form respectively) were added into the wells with Horseradish Peroxide (HRP) conjugated HNE. The competitive inhibition reaction was launched between HRP conjugated HNE and HNE in the samples. Then a substrate solution was added to the wells and the color developed is inversely proportional to the amount of HNE in the sample. The color development was stopped using stop solution and the intensity of the color was measured colorimetrically by using 450 nm filter in an ELISA plate reader MerilyzerEiaquant (Meril Diagnostics Pvt. Ltd., Vapi, Gujarat).

#### **2.8 Measurement of HLY**

Human vitreous HLY was estimated also by competitive enzyme immunoassay technique using commercial kits (MyBiosource, Catalog no: MBS753480) and utilizing a polyclonal anti-HLY antibody and an HLY-HRP conjugate. At first the assay sample and buffer were incubated together with HLY-HRP conjugate in precoated plate for one hour. After the incubation period, the wells were decanted and washed five times. The wells were then incubated with a substrate for HRP enzyme. The product of the enzyme-substrate reaction forms a blue colored complex. Then, a stop solution was added to stop the reaction, which will then turn the solution yellow. The intensity of color was measured spectrophotometrically at 450 nm in a microplate reader. The intensity of the color was inversely proportional to the HLY concentration present in the sample. A standard curve was also plotted relating the intensity of the color (or O.D.) to the concentration of standards. The HLY concentration in each sample was interpolated from this standard curve.

#### **2.9 Measurement of VEGF**

Human vitreous VEGF was estimated by sandwich enzyme-linked immune sorbent assay (ELISA) method using Ray Biotech kit (cat no: ELH-VEGF-001, Norcross USA). In the assay, an antibody specific for human VEGF was coated on the well plate. A series of standards ranging from 8.23 pg/ml to 6000 pg/ml and samples (vitreous samples were run in a half diluted form respectively) were added into the wells. VEGF protein present in the sample was bound to the wells by the immobilized antibody. The wells were washed and a biotinylated anti-human VEGF antibody was added. After buffer washing, HRP –conjugated streptavidin was pipette to wash and TMB substrate solution was added into the wells and was placed in incubation at room temperature for 30 minutes. The intensity of the final color product was proportional to the concentration of VEGF protein present in the samples and absorbance of the color product was measured colorimetrically by using 450 nm filter in an ELISA plate reader MerilyzerEiaquant (Meril Diagnostics Pvt. Ltd., Vapi, Gujarat). The concentration of VEGF was determined by a standard curve and the assay detects less than 10 pg/ml of VEGF from the sample.

## **3. Results**

As shown in the **Table 1**, different study groups enrolled in the present study showed no statistically significant differences for age, gender distributions, body

#### *Diabetic Eye Disease - From Therapeutic Pipeline to the Real World*


*HC, healthy control; DNR, diabetic subjects without clinically evident retinopathy, MNPDR, early non-proliferative diabetic retinopathy; HRPDR, high-risk proliferative diabetic retinopathy; BMI, body mass index; FPG, fasting plasma glucose, PPG, postprandial plasma glucose; HbA1C, glycated haemoglobin. The Kruskal Wallis nonparametric ANOVA followed by Dunn's multiple comparisons test was administrated to find out significant differences between the groups. A value of p < 0.05 was considered as statistically significant.*

*\* HC vs DNR, p < 0.05.*

*†††HC vs MNPDR, p < 001.*

*††HC vs MNPDR, p < 0.01. !!!!HC vs HRPDR, p < 0001.*

*!!HC vs HRPDR, p < 0.01.*

*! , HC vs HRPDR, p < 0.05.*

#### **Table 1.**

*Demographic and clinical characteristics of study subjects.*

mass index (BMI), and duration of diabetes, systolic and diastolic blood pressure. Glycaemic parameters like FPG level were found to be increased significantly in the DNR (p < 0.05), MNPDR (p < 0.01), and HRPDR (p < 0.01) subjects compared to the HCs. Similarly, the PPG level were found to be increased significantly in the DNR (p < 0.05), MNPDR (p < 0.01), and HRPDR (p < 0.0001) subjects compared to the HCs. Regarding, HbA1C DNR (p < 0.05), MNPDR (p < 0.001), and HRPDR (p < 0.05) subjects showed significantly higher values of HbA1c compared to HCs. However, statistical analysis showed no significant differences in FPG, PPG, and HbA1C levels between DNR, MNPDR, and HRPDR subjects.

Vitreous NADPH oxidase activity was found to be increased significantly among DNR (0.217 ± 0.031 OD550/100 μL, vitreous, p < 0.0001), MNPDR (0.286 ± 0.033 OD550/100 μL, vitreous, p < 0.0001), and HRPDR (0.365 ± 0.032 OD550/100 μL, vitreous, p < 0.0001) subjects compared to HC (0.121 ± 0.018 OD550/100 μL, vitreous) subjects. Again, both the MNPDR (p < 0.01) and HRPDR subjects (p < 0.0001) showed significantly higher level of NADPH oxidase than DNRs. Further, the HRPDR subjects showed a higher NADPH oxidase level (p < 0.0001) than the former (**Figure 1A**).

Regarding vitamin-C concentration in vitreous, the DNR (0.807 ± 0.043 mg/dl, p < 0.01), MNPDR (0.874 ± 0.061 mg/dl, p < 0.0001), and HRPDR (0.970 ± 0.110 mg/dl, p < 0.0001) subjects showed lower vitamin-C level compared to HC (0.682 ± 0.038 mg/dl) subjects. Again, both the MNPDR (p < 0.05) and HRPDR subjects (p < 0.0001) showed significantly lower level of vitamin-C than DNRs. HRPDR subjects showed a lower vitamin-C level (p < 0.01) than the MNPDR ones (**Figure 1B**).

*Role of Lipid, Protein-Derived Toxic Molecules, and Deficiency of Antioxidants… DOI: http://dx.doi.org/10.5772/intechopen.99904*

#### **Figure 1.**

*Comparison of vitreous level NADPH oxidase, vitamin-C and protein carbonyl compound among study groups. [A] Comparison of vitreous level NADPH oxidase, [B] Comparison of vitreous level vitamin-C, [C] Comparison of vitreous level protein carbonyl compound. The one way ANOVA followed by Tuky's comparisons test was administrated to find out significant differences between the groups. A value of p < 0.05 was considered as statistically significant.*

Vitreous PCC concentration was found to be increased significantly among DNR (41.57 ± 19.96 nmol/mg protein, p < 0.05), MNPDR (65.43 ± 17.31 nmol/mg protein, p < 0.001), and HRPDR (88.65 ± 24.93 nmol/mg protein, p < 0.0001) subjects compared to HC (18.46 ± 7.18 nmol/mg protein) subjects. Again, both the MNPDR (p < 0.05) and HRPDR subjects (p < 0.0001) showed significantly higher level of PCC than DNRs. Further, the HRPDR subjects showed a higher PCC level (p < 0.01) than the former (**Figure 1C**).

Vitreous MDA level was found to be increased significantly among DNR (2.814 ± 0.482 μM/L, p < 0.01), MNPDR (4.58 ± 0.655 μM/L, p < 0.0001), and HRPDR (8.51 ± 1.23 μM/L, p < 0.0001) subjects compared to HC (1.129 ± 0.579 μM/L) subjects. Again, both the MNPDR (p < 0.0001) and HRPDR subjects (p < 0.0001) showed significantly higher level of MDA than DNRs. Further, the HRPDR subjects showed a higher MDA level (p < 0.0001) than MNPDR subjects (**Figure 2A**).

Regarding HNE concentration in vitreous, the DNR (3936 ± 457.2 pg/ml, p < 0.0001), MNPDR (8643 ± 771.8 pg/ml, p < 0.0001), and HRPDR (7407 ± 622.3 pg/ml, p < 0.0001) subjects showed higher HNE level compared to HC (2092 ± 1201 pg/ml) subjects. Again, both the MNPDR (p < 0.0001) and HRPDR subjects (p < 0.0001) showed significantly higher level of HNE than DNRs. HRPDR subjects showed a higher HNE level (p < 0.0001) than the MNPDR ones (**Figure 2B**).

Vitreous HLY level was found to be increased significantly among DNR (70.93 ± 16.29 nmol/L, p < 0.0001), MNPDR (113.0 ± 10.56 nmol/L, p < 0.0001), and HRPDR (132.1 ± 16.22 nmol/L, p < 0.0001) subjects compared to HC (30.68 ± 7.29 nmol/L) subjects. Again, both the MNPDR (p < 0.01) and HRPDR subjects (p < 0.0001) showed significantly higher level of HLY than DNRs. Further, the HRPDR subjects showed a higher HLY level (p < 0.01) than MNPDR subjects (**Figure 2C**).

Vitreous VEGF level was found to be increased significantly among DNR (90.53 ± 5.611 pg/ml, p < 0.05), MNPDR (117.0 ± 17.09 pg/ml, p < 0.0001), and HRPDR (131.3 ± 12.21 pg/ml, p < 0.0001) subjects compared to HC (72.06 ± 5.109 pg/ml) subjects. Again, both the MNPDR (p < 0.0001) and HRPDR subjects (p < 0.0001) showed significantly higher level of VEGF than DNRs. Further, the HRPDR subjects showed a higher VEGF level (p < 0.05) than MNPDR subjects (**Figure 2D**).

In the DNR and DR group, vitreous NADPH activity showed significant positive correlations with PCC, MDA, HNE, and HLY respectively. However, the study showed no significant correlation with the same in the HC group (**Table 2**).

#### **Figure 2.**

*Comparison of vitreous level MDA, HNE, HLY and VEGF among study groups. [A] Comparison of vitreous level MDA, [B] Comparison of vitreous level HNE, [C] Comparison of vitreous level HLY, [D] Comparison of vitreous level VEGF. The one way ANOVA followed by Tuky's comparisons test was administrated to find out significant differences between the groups. A value of p < 0.05 was considered as statistically significant.*


#### **Table 2.**

*Correlation of vitreous NADPH oxidase activity with PCC, MDA, HNE and HLY in HC, DNR and DR (MNPDR+HRPDR) group.*

The VEGF level of vitreous showed a significant negative correlation with vitamin-C and positive correlations with PCC, MDA, HNE, and HLY levels respectively in both DNR and DR groups. However, the study showed no significant correlation with the same in the HC group (**Table 3**).


#### **Table 3.**

*Correlation of vitamin-C, PCC, MDA, HNE and ALE with VEGF in different study groups.*

*Role of Lipid, Protein-Derived Toxic Molecules, and Deficiency of Antioxidants… DOI: http://dx.doi.org/10.5772/intechopen.99904*
