Detection of Circulating Auto‐Antibodies Against Ribosylated‐LDL in Diabetes Patients

Abstract

Background

This study analyzes effect of glycation on ApoB‐100 residues by D‐ribose as D‐ribosylated‐glycated LDL might be responsible for the cause of diabetes mellitus because of its far higher antigenic ability. The binding characteristics of circulating auto‐antibodies in type 1 and type 2 diabetes patients against native and modified LDL were assessed.

Methods

T1 Diabetes (n = 43), T2 diabetes patients (n = 100) were examined by direct binding ELISA as well as inhibition ELISA, were compared with healthy age‐matched controls (n = 50).

Results

High degree of specific binding was observed by 74.42% of T1 diabetes and 45.0% of T2 diabetes patient's sera toward glycated LDL, in comparison to its native analog. Competitive inhibition ELISA reiterates the direct binding results. Furthermore, ketoamine content, Hydroxymethylfurfural (HMF) content and carbonyl content were also estimated in patient's sera healthy subjects. The increase in total serum protein carbonyl levels in the diabetes patients was largely due to an increase in oxidative stress. The increase in ketoamine as well as HMF content inpatients sera than healthy subjects is an agreement of induced glycation reaction in patients than healthy subjects.

Conclusion

D‐ribosylated‐LDL has resulted in structural perturbation causing generation of neo‐antigenic epitopes that are better antigens for antibodies in T1 and T2 diabetes patients.

Introduction

Non‐enzymatic glycation response adjusts the organic exercises of DNA and protein, prompting their brokenness 1. A few studies have set up that lipoproteins are promptly glycated In Vivo and In Vitro to frame lipoprotein propelled advanced lipoxidation end‐products (ALEs) 2, 3, 4. Advanced glycation end‐products (AGEs) amass on serum proteins and in different tissues, especially amid aging, diabetes and renal failure 5. Elevated AGEs levels contribute in the development of diabetes and uremic complications, such as atherosclerosis, nephropathy, and retinopathy 6, 7. The lipoprotein glycation happens together on the apoprotein B (apoB) and on phospholipid segment of LDL, prompting both functional changes in LDL clearance and expanded susceptibility to oxidative alterations 8. A few studies have reported the pathogenic part of the humoral reaction to changed lipoproteins 9, 10. This is principally because of the way that changed LDL and the relating antibodies form immune complexes (mLDL‐IC), which can trigger phagocytic cells through engagement of Fc (Fragment, crystallizable) receptors 11. Engagement of Fc receptors by mLDL‐IC is especially critical in light of the fact that it conveys more grounded enacting signs to phagocytic cells than engagement of scavenger receptors by changed LDL 12. It has additionally been accounted for that raised amounts of glycated LDL (AGE‐LDL) were found in patients with diabetes and recommended to fit in with the components of dyslipidemia identified with diabetes mellitus and renal deficiency 13, 14. Figure 1 schematically represents the probable mechanism of Apo‐B100 of lysine residue with D‐ribose as well as role of intermediate glycation products such as glyoxal (GO), methyl glyoxal (MGO), and 3‐deoxyglucosone (3‐DG) to form immune complex (AGEs‐IgG), responsible to cause diabetes and their secondary complications.

Figure 1.

Schematic representation of the probable mechanism of Apo B100 of lysine residue with D‐ribose as well as role of intermediate glycation products to form immune complex (AGEs‐IgG), responsible to cause diabetes and their secondary complications.

Sugars other than glucose can contribute essentially to the glycation responses In Vivo, in light of their far higher reactivity contrasted with glucose 15. Numerous investigators have reported that glucose, being the most bounteous sugar in cells of about every single living life form got most consideration in studies on protein and nucleic acid glycation. Among all the reducing sugars, D‐ribose is a standout among the most reactive sugar which assumes the critical part in the glycation of biomolecules. In addition, the bioavailability of D‐ribose makes this carbonyl species entirely reactive and harming, hence having direct ramifications in disease. D‐ribose has been found to have a strangely high concentration in the urine of type 2 diabetics 16, proposing that the diabetic patients are experienced glucose digestion system issue as well as from D‐Ribose digestion system issue. Intraperitoneal infusion of D‐Ribose into mice essentially expands their glycated serum protein and blood AGEs, in spite of that the grouping of glucose turned out to be marginally diminished, demonstrating that D‐Ribose can more readily impel AGEs than glucose In Vivo 17.

The evidence that AGEs have antigenic properties has led to a hypothesis that the glucose glycated biomolecular structure found In Vivo may exert an autoimmune response 18. Few studies have revealed the role of glycated LDL in production of auto‐antibodies in experimental animal but no study stated the immunogenicity of D‐ribose glycated LDL in patient's sera. Therefore, it is necessary to determine the antigenic effects of ribose glycated LDL in diabetes patients. So the present study was completed to investigate the immunogenic part of D‐ribose glycated LDL in type 1 diabetes mellitus (T1DM) and type 2 diabetes mellitus (T2DM) patients. In Vitro glycated D‐ribose‐LDL may demonstrate the vicinity and specificity against immunogen particular antibodies present in T1DM and T2DM patients.

Materials and Methods

Materials

LDL, Agarose, anti‐human IgG alkaline phosphatase (ALP) conjugate and para‐nitrophenyl phosphate (pNPP) were obtained from Calbiochem (San Diego, CA). Polystyrene plates were obtained from Nunc (Roskilde, Denmark). D‐ribose, Tween‐20, Tris HCl, EDTA were obtained from Sigma (St. Louis, MO). Every other compound utilized as a part of this study was of the most astounding scientific evaluation accessible.

Study Population

The study at total of 193 (normal and patients) sera samples in which 43 sera samples were of T1 diabetes, 100 were of T2 diabetes patients and 50 sera samples were of normal healthy subjects, as defined by the World Health Organization (WHO) criteria.

Inclusion/Exclusion Criteria

Inclusion criteria:

• Patients with T1DM and T2DM (based on WHO criteria).

• Age group between 25 and 70 years for all patients.

• Subject or subject's representative has signed the informed consent form.

Exclusion criteria:

• Subject is suffering from any chronic disease.

• Subject is suffering from any acute infection.

• Patients with diabetes on treatment (oral antidiabetes agents or insulins).

• Conditions in which phlebotomy is contra‐indicated.

Anthropometric Measurements

Body mass index

Body mass index (BMI) was considered using the formula: observed weight divided by height squared (kg/m²).

Waist and hip ratio

Waist and hip ratio (WHR) was obtained by dividing the waist circumference (cm) by the hip circumference (cm).

Collection of Blood Sample and Serum Separation

The patients attending the OPD/IPD in Integral Institute of Medical Sciences and Research (IIMS&R), Lucknow, India, previously diagnosed and marked as T1DM or T2DM were selected for the present study. Blood samples of T1 diabetes patients (N = 43), T2 diabetes patients (N = 100) and normal human sera (N = 50), were collected in which T1 Diabetes patients included 26 females with a mean age of 23 ± 14.8years and 17 males of 14 ± 10.2 years, T2 Diabetes patients included 34 females with a mean age of 36 ± 8.3 years and 66 males with a mean age of 42 ± 9.2 years. Samples from healthy individuals (N = 50) included 25 females with a mean age of 32 ± 8.3 years and 25 males with a mean age of 35 ± 11.2 served as negative control. Five milliliter of blood samples were obtained between 09.00 and 11.00 hr after fasting from 22.00 hr the previous day from each healthy, T1DM and T2DM human volunteer after the informed consent and transferred to plain vials. The research examination has been completed as per the Declaration of Helsinki after the informed consent to the volunteer. The blood samples were allowed to clot and sera were isolated by centrifugation at 2,000 g for 15 min at 4°C within 2 hr of collection and were maintained at that temperature until further use. To inactivate the complement proteins, all sera were warmed at 56°C for 30 min and put away at −20°C with 0.01% sodium azide as an additive after legitimate marking 19. The serum samples from normal and healthy individuals served as control. The Institutional Ethics Committee has affirmed the study protocol (Ethical approval no. 999).

Biochemical Investigations

All biochemical parameters data like plasma glucose, glycosylated hemoglobin (HbA1c), total cholesterol (TC), triglycerides (TG), high density lipoprotein cholesterol (HDL‐C), and low‐density lipoprotein cholesterol (LDL‐C) were collected from clinical laboratory IIMS&R. The conducted tests were standardized. All measurements were performed by the same technician with an extensive experience in a neutral environment with a constant temperature to minimize the influence of stress.

Preparation of Glycation Modified LDL

LDL was modified by utilizing different concentrations of D‐ribose sugar (5–80 mM) and by escalating the incubation time period as previously illustrated 1. In brief, LDL (62.5 μg/ml) were altogether mixed with 5 mM, 10 mM, 20 mM, 40 mM, and 80 mM of ribose sugar in 100 mM phosphate support saline (PBS), pH 7.4 and incubated with native LDL (without ribose) at 37°C for 3 weeks, followed by extensive dialysis against PBS to remove unbound constituents. Native LDL was served as control.

Enzyme Linked Immunosorbent Assay (ELISA)

Direct binding ELISA was performed on polystyrene plates with slight adjustment 20, 21, 22. Polystyrene immunoplates were covered with 100 μl of the native or glycated LDL (10 μg/ml) in 0.05 M carbonate–bicarbonate buffer with pH 9.6. The plates were incubated for 2 hr at 37°C and overnight at 4°C. Every sample was covered in duplicate and half of the plate without antigen, served as control. Unbound antigen was washed three times with tris buffer saline and Tween 20 (TBS‐T) (20 mM Tris, 150 mM NaCl, pH 7.4 containing 0.05% Tween‐20), and unoccupied spot were blocked with 1.5% BSA in TBS (10 mM Tris, 150 mM NaCl, pH 7.4) for 4–5 hr at 37°C. After incubation, the plates were again washed three times with TBS‐T. Test serum (1:100 dilution), IgG (40 μg/ml) was added to antigen‐coated wells and re‐incubated for 2 hr at 37°C and overnight at 4°C. Bound antibodies were measured with anti‐human IgG‐soluble phosphatase (IgG‐ ALP) conjugate utilizing p‐nitrophenyl phosphate (pNPP) as substrate. Absorbance of every well was observed at 410 nm on a programmed microplate reader (Electronic Enterprise of India Ltd., Mumbai, India), and the mean of copy readings for every specimen was recorded. Results have been communicated as a mean of A_test ‐ A_control.

Determination of Protein‐Bound Carbonyl Groups

Carbonyl contents were determined in T1, T2 diabetes patients sera as well as in healthy subjects using 2,4‐dinitrophenylhydrazine. An aliquot (0.5 ml) of serum samples was treated with an equal volume of 0.1% (wt/vol) 2,4‐dinitrophenylhydrazine (DNPH) in 2 M HCl and incubated for 1 hr at room temperature. This mixture was treated with 0.5 ml of 20% trichloroacetic acid (wt/vol, final concentration), and after centrifugation, the precipitate was extracted three times with ethanol/ethyl acetate (1:1, vol/vol). The protein sample was then dissolved with 2 ml of 8 M guanidine hydrochloride, 13 mM EDTA, and 133 mM Tris solution (pH 7.4). The absorbance was read at 360 nm and the carbonyl content was determined using extinction coefficient of 22,000 M⁻¹ cm⁻¹ 23.

NBT Reduction Assay

The ketoamine moieties were determined in patients as well as in healthy control by nitroblue tetrazolium (NBT) reduction assay as described previously 24. The serum samples (20 μl) were mixed with 200 μl of 100 mM sodium carbonate–bicarbonate buffer (pH 10.8) containing NBT 0.25 mM and incubated at 37°C for 6 min. The absorbance of protein samples were recorded at 525 nm and the content of ketoamine moieties (μmol mg⁻¹) were determined using an extinction coefficient of 12,640 M⁻¹ cm⁻¹.

Thiobarbituric Acid Assay

Hydroxymethylfurfural (HMF) was determined in patients as well as in healthy control serum samples were estimated by mixing 1 ml serum sample with 1 M of oxalic acid. The mixture was kept in water bath for 1 hr. Forty percent of trichloroacetic acid was mixed in the solution after cooling at room temperature. Thiobarbituric acid (TBA) was mixed after removing precipitate by filtration. Incubate this mixture for half an hour. The color was developed and the amount of HMF (nmol/ml) was calculated using molar extinction coefficient value of 4 × 104 M⁻¹ cm⁻¹ at 443 nm 24.

Purification of Antibodies

Immunoglobulin G (IgG) was affinity purified from normal human sera (NHS) and T1DM, T2DM patient's sera on a protein A‐Sepharose column 20. Serum (0.5 ml) diluted with equivalent volume of PBS (pH 7.4), was applied to the column pre‐equilibrated with above buffer. The wash through was recycled 2–3 times. Unbound IgG was evacuated by washing with PBS (pH 7.4). The bound IgG was eluted with 0.58% acetic acid in 0.85% sodium chloride. Three milliliter parts were gathered in a measuring cylinder already containing 1 ml of 1 M Tris–HCl, pH 8.5, and absorbance was recorded. The IgG fixation was resolved considering 1.4 OD280 = 1.0 mg human IgG/ml. The isolated IgG was then dialyzed against PBS (pH 7.4) and put away at −80°C. The homogeneity of isolated IgG was ascertained by 7.5% SDS–polyacrylamide gel electrophoresis.

Competitive ELISA

The antigenic specificity of serum antibodies was calculated by competition ELISA 21. The ELISA plates were coated with 100 μl of glycated LDL (10 μg/ml) for 2 hr at room temperature and overnight at 4°C. Varying amount of inhibitor (glycated LDL; 0–20 μg/ml), was blended with a constant amount of sera of chosen subjects of various age bunches. The blend was incubated at 37°C for 2 hr and overnight at 4°C. Immune complexes were coated in the wells rather than serum and the remaining steps were same as in direct binding ELISA. Percent inhibition was computed utilizing the equation:

Percent Inhibition = 1− (A_inhibited/A_uninhibited) ×100.

Statistical Analysis

Data are presented as mean ± SD and statistical significance of the data was determined by INSTAT Plus 3.3 software. A value of P < 0.05 was considered statistically significant.

Results and Discussions

Biochemical Data Estimation in Patients and Healthy Subjects

The clinical and metabolic characteristics of the study subjects healthy control (HC), T1DM and T2DM patients are showing in Tables 1 and 2. The blood pressure systolic (SST) and diastolic (DST) of T1DM, T2DM as moderately higher in comparison to HC but was within the normal range (132 ± 11, 127 ± 13 vs. 121 ± 16 mmHg and 75 ± 13, 77.5 ± 11 vs. 67 ± 8 mmHg). While there was significantly differ and higher BMI of T1DM and T2DM in comparison to HC (26.72 ± 2.24 and 28.01 ± 1.04 vs. 21.1 ± 1.7 kgm⁻²). Furthermore, %HbA1c in T1DM and T2DM was significantly higher 7.1 ± 2.12 and 6.7 ± 2.12 than HC 4.3 ± 0.12. TC, LDL and TG were moderately higher in T1DM and T2DM than HC (194 ± 3.4 and 179 ± 3.4 vs. 160 ± 2.2 mgdl⁻¹; 151 ± 6.1 and 148 ± 0.1 vs. 145 ± 4.21 mgdl⁻¹; 181 ± 9.45 and 186 ± 7.2 vs. 178 ± 4.2 mgdl⁻¹) while HDL was lower in both T1DM and T2DM than HC (36.2 ± 4.7 and 38.9 ± 4.7 vs. 48 ± 2.1). The TC/HDL ratio in T1 DM and T2 DM was also moderately greater than HC (4.53 ± 0.63 and 4.35 ± 0.55 vs. 3.33 ± 0.05, respectively).

Table 1.

General Clinical and Metabolic Characteristic for the Selection of T1DM and T2DM patients and Healthy Subjects

Type of patient	Selected patients
HC	Age: 25–70 years
	Glucose level:
	Fasting: 80–126 mg/dl
	Postprandial (P.P.): 110–200 mg/dl
	HbA1c: 4.0–5.6% (20–38 mmol/mol)
	BMI: 18.5–24.9 kg/mt2
	WHR: <1.0
T1DM	Age: 25–70 years
	Glucose level:
	Fasting: >126 mg/dl
	Postprandial (P.P.): >200 mg/dl
	HbA1c: ≥6.5% (48 mmol/mol)
	BMI: >25 kg/mt2
	WHR: >1.0
T2DM	Age: 25–70 years
	Glucose level:
	Fasting: >126 mg/dl
	Postprandial (P.P.): >200 mg/dl
	HbA1c: ≥6.5% (48 mmol/mol)
	BMI: >25 kg/mt2
	WHR: >1.0

BMI, Body mass index; HC, healthy control; WHR; Waist and hip ratio.

Table 2.

General Clinical and Metabolic Characteristic of the Study Objects

Characteristic	HC	T1 DM	T2 DM
N	50	43	205
Age (years)	33.5 ± 9.75	35.5 ± 17.5	39 ± 11.65*
Gender (M/F)	25/25	17/26	68/32
BMI (Kg/mt−2)	21.1 ± 1.7	26.72 ± 2.24**	28.01 ± 1.04*
BP (SST) mmHg	121 ± 16	132 ± 11	127 ± 13
BP (DST) mmHg	67 ± 8	75 ± 13	77.5 ± 11
HbA1c % (mmol/mol)	4.3 ± 0.12 (23 ± 4.12)	7.1 ± 2.12 (54 ± 8.12) *	6.7 ± 2.12 (50 ± 6.22) *
TC (mml−1)	160 ± 2.2	194 ± 3.4**	179 ± 3.4*
LDL (mml−1)	145 ± 4.21	151 ± 6.1*	148 ± 0.1***
HDL (mml−1)	48 ± 2.1	36.2 ± 4.7	38.9 ± 4.7
TG(mml−1)	178 ± 4.2	181 ± 9.45*	186 ± 7.2**
TC/HDL	3.33 ± 0.05	4.53 ± 0.63*	4.35 ± 0.55

Values are expressed as mean ± SD.

HC, Healthy control; M/F, Male/Female; BMI, Body mass index; BP (SST), Blood pressure (systolic); BP (DST), Blood pressure (diastolic); DD, Duration of disease; HbA1c, Glycosylated hemoglobin; TC, Total cholesterol; LDL, Low‐density lipoprotein; HDL, High density lipoprotein; TG, Triglycerides.

Significance/non significance: P > 0.05 (not‐significant), *P < 0.05 (significant), **P < 0.01 (very significant), and ***P < 0.001 (extremely significant), with respect to healthy control subjects.

Determination of Carbonyl Content in Patients and HC Sera

Oxidation of lipoproteins logically results in an increase in protein carbonyl contents, a recognized biomarker of oxidative stress. The average carbonyl content (±SD) of three independent assays of patients sera (T1DM and T2DM) were 30 ± 13.91 and 29.72 ± 18.83 μmol mg⁻¹ protein, respectively (Fig. 2), whereas HC sera had almost negligible level of carbonyl content (9.34 ± 3.43 μmol mg⁻¹ protein). This corresponds to almost three to four fold increases in carbonyl contents in patient's sera and HC sera as compared to HC. This shows that higher product concentration in patient's sera in comparison to HC, which shows a very low or negligible level of carbonyl content concentration.

Figure 2.

Level of carbonyl content in serum sample of T1 diabetes mellitus, T2 diabetes mellitus as well as in normal healthy subjects. Significance/non significance: P > 0.05 (not‐significant), P < 0.05 (significant) and P < 0.01 (very significant) with respect to healthy control subjects.

Nitroblue Tetrazolium Reduction Assay for LDL Amadori Products Determination in Patient's and HC Sera

For the detection of early glycation products, the ketoamine moieties formed in patients sera were measured calorimetrically by NBT assay. HC alone showed almost negligible amount of ketoamine content (11.21 ± 3.43 μmol mg⁻¹ protein), whereas patient's sera had higher concentration of ketoamine content. The average ketoamine content of three independent assays in patients sera of T1DM and T2DM were 69.59 ± 36.34, 67.01 ± 16.61 μmol ml⁻¹ protein (Fig. 3). This shows that intermediate product or LDL Amadori product concentration was higher in patient's serum which is an agreement of the privileged D‐ribose‐LDL glycation in patients (T1DM and T2DM) than HS.

Figure 3.

Level of ketoamine content in serum sample of T1 diabetes mellitus, T2 diabetes mellitus as well as in normal healthy subjects. Significance/non significance: P > 0.05 (not‐significant) and P < 0.05 (significant) with respect to healthy control subjects.

Determination of HMF Content in HC and Patient's Sera

Similarly, HMF that might have formed in the early glycation of LDL was determined as thiobarbituric acid reactive substance after hydrolysis. The HMF content in T1DM, and T2DM patients sera were 39.44 ± 12.78, 23.09 ± 9.86 μmol ml⁻¹, respectively, whereas HC had 8.21 ± 2.16 μmol ml⁻¹ (Fig. 4). This shows that early glycation product concentration was higher in patient's sera while there was almost negligible level of HMF concentration in HC. The higher yield of HMF in patient's serum sample is in agreement with the NBT assay result. Ketoamines converted to protein carbonyl compounds via a protein enediol generating superoxide radical 25. Protein carbonyl content is most commonly used biomarker of protein oxidation and AGEs formation 26.

Figure 4.

Level of HMF content in serum sample of T1 diabetes mellitus, T2 diabetes mellitus as well as in normal healthy subjects. Significance/non significance: P > 0.05 (not‐significant), P < 0.05 (significant) and P < 0.01 (very significant) with respect to healthy control subjects. HMF, Hydroxymethylfurfural.

Binding of Antibodies from Patients to Native and Glycated LDL

The pilot study was performed to screen out the positive sera tests (Sera indicating higher tying with immunogen) from T1DM and T2DM subjects. Our study included aggregate 193 serum tests of T1DM, T2DM and HS. Control serum tests from age and sex coordinated people were gotten from 50 normal healthy subjects. All sera were diluted to 1:100 in TBS‐T and subjected to direct binding ELISA on solid phase independently coated with equivalent amounts of native LDL and D‐ribose glycated LDL. Out of 43 sera from T1DM, 32 samples (74.42%) and 100 sera from T2DM, 45 sample (45.0%), tested, showed enhanced binding with the D‐ribosylated‐LDL as compared to the native form (Fig. 5). These results indicate substantial recognition of the glycated LDL by serum antibodies in diabetes patients.

Figure 5.

Direct binding ELISA of serum antibodies from T1 diabetes mellitus (T1DM) and T2 diabetes mellitus (T2DM) patients to native LDL and glycated LDL. Serum from normal human subjects (NHS) served as control. The microtiter plates were coated with the respective antigens (10 μg/ml). Significance/non significance: P > 0.05 (not‐significant), P < 0.05 (significant) and P < 0.001 (extremely significant), with respect to healthy control subjects.

Specificity of Circulating Antibodies in Patient's Sera Against Native and D‐Ribose Glycated LDL

Competition ELISA was carried out to analyze the binding specificity of circulating antibodies in patients to native and glycated LDL. For this, we selected serum samples from patients group showing high binding in direct binding ELISA. In the T1DM and T2DM group, the observed maximum inhibition with glycated LDL ranged from 53.7% to 81.2% and 51.3% to 76.4%, respectively, while the observed maximum inhibition with native LDL ranged from 12.8% to 34.2% and 17.3% to 34.0%, respectively. Mean inhibition of the T1DM and T2DM samples tested with native LDL were 24.96 ± 5.21% and 25.87 ± 4.05% while for glycated LDL it were 66.1 ± 7.83% and 63.77 ± 5.74%, respectively (Fig. 6 A & B). These results indicate substantial recognition of the glycated LDL by serum antibodies in both T1DM and T2DM patients but in T1DM there was significantly higher recognition were observed in comparison to T2DM. It might be possible that duration of the disease may be having an effect on the quantity/titer of anti‐LDL antibodies. These antibodies may be further contributing to other immune factors that are involved in the destruction of the β‐cells of the islet of Langerhans of the pancreas. It is also desirable to find out the effect of age, sex, duration of diabetes, level of blood glucose, and the presence or absence of complications on the level of antibodies to LDL. It may be possible that the above mentioned factors have a role in the development of antibodies that can gradually and eventually help the other auto immune factors in the destruction of the β‐cells of the islet of Langerhans of the pancreas 27. Immunogenicity of LDL has been increased two to three folds upon glycation with D‐ribose, which may lead to the formation of antibodies, immune complexes and development of other complications in diabetes patients.

Figure 6.

Maximum percent inhibition of serum antibodies from T1 diabetes mellitus (T1DM) (A) and T2 diabetes mellitus (T2DM) patients at 20 μg/ml of each native LDL (■) and D‐ribose glycated LDL (□). The microtiter plates were coated with the respective antigens (10 μg/ml). Significance/non significance: P < 0.001 (extremely significant).

All reducing sugars whether aldoses or ketoses 28 and even molecules related to sugars, such as ascorbic acid 29 can initialize the reaction In Vivo. In Vitro studies suggest that D‐ribose, compared with glucose, is a much more potent initiator of the Maillard reaction 30, 31. Because the Maillard reaction may be involved in the aging process 32, there are several main reasons why we expect that D‐ribose, through non‐enzymatic ribosylation, may have a vital effect on the health of normal and diabetes subjects. Ribose is a normally happening pentose monosaccharide present in every single living cell including the blood and is a key part of numerous vital bio‐molecules, such as, riboflavin, RNA and ATP 33. Our study has demonstrated that D‐ribose causes structural deformations in LDL particle bringing about the generation of neo‐antigenic epitopes. This is perceived as non‐self by the immune system, thus breaking the immune tolerance existing normally to self‐antigens. It has been demonstrated that oxidized type of the LDL is exceptionally immunogenic and indicates potential recognition of auto‐antibodies raised against modified LDL 34.

T1 DM is also one such issue which is linked to the autoimmunity state of the disease is the glycation and oxidation reaction of the bio‐macromolecules leading to the formation of the AGEs. The study of the role of glycation of plasma proteins like LDL in the development of autoimmunity is the need of the hour. AGE, which is non‐enzymatic, adducts of proteins, lipids, and nucleic acids, accumulate in the body with advanced age. Auto‐antibodies against oxidative neo‐epitopes are present in humans and other species and their titer may be an indicator of the extent of diabetes. Present study also showing boosting immune responses in D‐ribose glycated LDL induce T1DM and T2DM.

Conclusion

The present study investigated the immunogenic part of D‐ribose glycated LDL. Our results suggest that the D‐ribose glycated LDL produce AGEs which cause damage to the LDL molecule. In this study, the presence of anti‐D‐ribose‐LDL autoantibodies in T1 and T2 diabetic sera may be as a consequence of auto immune response against persistent LDL–AGEs. It might be possible that duration of the disease may be having an effect on the quantity/titer of anti‐LDL antibodies 35 Immunogenicity of LDL has been increased several folds upon glycation with D‐ribose which may lead to the formation of antibodies, immune complexes and development of other complications in diabetes patients.

Present study indicates that LDL–AGEs and autoantibodies against glycated LDL in diabetic subjects may help as additional biomarkers for evaluation of chronic glycemia. Our outcomes additionally point toward the possible inclusion of this pentose sugar glycated LDL in the impelling of antibody response against in T1 DM and T2 DM patients. Thus, it is judicious to infer that at present, the careful pathophysiological part of glycation of apo B is still dark and needs advance study. We trust our discoveries would likewise help the route for the treatment of diabetes and its optional intricacies like retinopathy, neuropathy, cardiomayopathy, and nephropathy. A large prospective and multicentric study will be fruitful in ascertaining the role of anti‐glycated LDL antibodies in the secondary complications associated with diabetes.

Conflict of Interest

The authors declare no conflict of interests exists.