Albumin Abundance and Its Glycation Status Determine Hemoglobin Glycation.

Diabetes diagnosis and management majorly depend upon the measurement of glycated hemoglobin (HbA1c) levels. Various factors influence HbA1c levels such as the use of various analytical methods and the presence of various clinical conditions. Plasma albumin levels were known to be negatively associated with HbA1c. However, the precise mechanism by which they affect HbA1c is not well understood. Therefore, we have studied the influence of albumin levels and its glycation status on hemoglobin glycation using erythrocyte culture experiments. Erythrocytes maintained at low albumin concentration exhibited relatively increased albumin and hemoglobin glycation as compared to that in those maintained at higher albumin concentration. Increase in albumin glycation may decrease its ability to protect hemoglobin glycation. This was demonstrated by treatment of erythrocytes with N(ε)-(carboxymethyl)lysine-modified serum albumin (CMSA), which failed to protect hemoglobin glycation; instead, it increased hemoglobin glycation. The inability of CMSA to reduce hemoglobin glycation was due to the lack of free lysine residues of albumin, which was corroborated by using N(ε)-(acetyl)lysine serum albumin (AcSA) and clinical diabetic plasma. This is the first study which demonstrates that the modification of lysine residues of albumin impairs its ability to inhibit hemoglobin glycation. Furthermore, correlation studies between HbA1c and albumin levels or relative albumin fructosamine from clinical subjects supported our experimental finding that albumin abundance and its glycation status influence hemoglobin glycation. Therefore, we propose albumin level and its glycation status to be quantified in conjunction with HbA1c for better management of diabetes.

Introduction

Poorly controlled diabetes leads to the development of complications such as nephropathy, retinopathy, neuropathy, and cardiovascular diseases.[1] Therefore, good glycemic control is critical to prevent any such complications.[2] Glycemic status monitored by measuring blood glucose provides an instantaneous level measure of its level; however, it varies throughout the day depending upon the diet, work, and antidiabetic medicines.[3] Considering the limitations in the measurement of blood glucose, an International Expert Committee recommended the measurement of glycated hemoglobin (HbA1c), as it provides average glucose level of preceding 120 days, the lifespan of erythrocytes.[4,5] This recommendation of HbA1c measurement was subsequently adopted by the World Health Organization. Furthermore, the usefulness of HbA1c for prediction of diabetic complications was also established by Diabetes Control and Complications Trial in type 1 diabetes[6] and the UK Prospective Diabetes Study in type 2 diabetes.[7] Thus, HbA1c has become a gold standard for the assessment of chronic glycemic status in clinical practice. However, quite a few studies also debate the usefulness of HbA1c because of technical challenges in precise quantification of the N1-deoxyfructosyl valine-β-Hb substance (HbA1c) by routinely used methods, such as ion exchange[8] and phenylboronate affinity,[9] as well as the presence of other clinical conditions including anemia, blood loss, splenomegaly, and iron deficiency, which affect HbA1c levels.[10]

Apart from these factors, human serum albumin (HSA) has been known to influence HbA1c; however, it has not gained required attention perhaps due to its abundance in the plasma, although it has been well established that low albumin levels were negatively correlated with HbA1c in a big cohort study comprising 4158 diabetic subjects.[11] This association was corroborated in 610 Asian Indian diabetic subjects.[12] Furthermore, it has been demonstrated in vitro that albumin can protect glycation of other proteins such as insulin and apomyoglobin by its mere abundance.[13] Additionally, low albumin levels were associated with increased plasma protein glycation including albumin, fibrinogen, apolipoprotein, heptaglobin, etc., as well as HbA1c in a streptozotocin-induced diabetic mice model and in clinical subjects.[14,15] Moreover, patients with low albumin levels are at higher risk of development of cardiovascular complications.[16] Hence, it was proposed that maintenance of albumin levels in diabetes would be helpful to prevent the accumulation of advanced glycation end products and delay the onset of complications.[17] Despite the great significance of albumin in diabetes, it is not routinely quantified in clinical diagnostics. However, glycation of albumin and plasma proteins is quantified as total plasma fructosamine, which is used to predict HbA1c. Discordance has been observed between the predicted HbA1c and observed HbA1c, which is termed as glycation gap, suggesting that HbA1c does not correlate to the total plasma fructosamine levels.[18] Because albumin is the most abundant plasma protein, it is plausible that its glycation status could influence the prediction of HbA1c using the plasma fructosamine content. Moreover, albumin may act as a principal target for glycation as compared with hemoglobin because it is freely circulated in plasma and it has a large number of free lysine and arginine residues accessible for glucose binding. However, hemoglobin is an intracellular protein and for its glycation glucose needs to be transported into erythrocytes through glucose transporter protein 1. Therefore, we hypothesize that albumin glycation precedes hemoglobin glycation and hence albumin concentration and its glycation status influences hemoglobin glycation or HbA1c. To prove this, we have mechanistically demonstrated that albumin levels and its glycation status indeed influence hemoglobin glycation in erythrocyte culture. Furthermore, the association among albumin, plasma fructosamine, and HbA1c was studied in clinical subjects. The overview of complete study design is depicted in Figure .

Figure 1

Overview of the complete study design. The study was conducted to understand the effect of albumin and its glycation status on hemoglobin glycation using both in vitro erythrocyte culture and clinical sample analysis. (A) In vitro lysine modification and mass spectrometric characterization of albumin. (B) Erythrocytes were maintained in Roswell Park Memorial Institute (RPMI) medium containing either low glucose (LG) (5.2 mmol/L) or high glucose (HG) (15.7 mmol/L). For different experiments, erythrocytes were subjected to various treatments such as aminoguanidine (AMG) 10 mM, unmodified serum albumin (SA) or carboxymethylated serum albumin (CMSA) (15, 20, or 25 mg/mL), acetylated serum albumin (AcSA) (25 mg/mL), and control and diabetic plasma (25 mg/mL plasma protein). Hemoglobin glycation was assessed by measuring HbA1c level, AGE-Hb fluorescence, Western blotting, and targeted SWATH-mass spectrometry analysis. (C) Clinical study, Pearson’s correlation analysis between HbA1c and albumin, plasma fructosamine, or relative albumin fructosamine (RAF).

Results

Establishment of Erythrocytes as an In Vitro Model for Studying Glycation

Erythrocytes maintained in normal or relatively low glucose (LG) (5.2 mM) conditions had HbA1c of 6.1%, whereas it increased to about 10.6% in erythrocytes maintained in high glucose (15.7 mM) conditions. Aminoguanidine (AMG), a well-known glycation inhibitor, reduced HbA1c to 6.8% when compared to that in erythrocytes maintained at high glucose (Supporting Information Figure S1A). Protein glycation leads to the formation of both fluorescent and nonfluorescent advanced glycation end products (AGEs).[19] Therefore, fluorescent AGEs were quantified by measuring AGE–hemoglobin fluorescence, and among nonfluorescent AGEs, carboxymethyl lysine (CML) being the predominant AGE, CML modification of Hb was monitored by anti-CML antibody Western blotting.[20] Both AGE–hemoglobin fluorescence and CML modification of Hb showed a similar trend as that of HbA1c (Supporting Information Figure S1A–C).

Unmodified Albumin Protects Hemoglobin Glycation

Previous studies have shown that low albumin levels are associated with increased HbA1c.[11,12,14] To find out the mechanistic insight into this association, erythrocytes were maintained at varying levels of albumin (15, 20, and 25 mg/mL) in both low and high glucose condition and hemoglobin glycation was monitored. Albumin reduced HbA1c in a concentration-dependent manner. A higher level of albumin (25 mg/mL) reduced HbA1c significantly as compared with the lower level of albumin (20 or 15 mg/mL) or absence of albumin in high glucose conditions. However, in low glucose condition, albumin concentration of only 25 mg/mL reduced HbA1c significantly (Figure A). In addition, albumin reduced advanced glycation of hemoglobin in a concentration-dependent manner, as measured by AGE-Hb fluorescence, in both low and high glucose conditions (Figure B). This trend was also observed in Western blotting with anti-CML antibody (Figure C). Furthermore, the effect of albumin on hemoglobin glycation was characterized and quantified using information-dependent acquisition (IDA) and targeted SWATH-MS approach, respectively. By using IDA approaches, a total of 16 glycated peptides of hemoglobin were characterized. SWATH-MS, a label-free quantitative approach allowed targeted extraction and quantification of less abundant glycated peptides post acquisition (Figure A,B; Supporting Information Data 1 and File 1). Among 16 glycated peptides, only 4 were consistently present in all of the mass spectrometric acquisitions and therefore these peptides were selected for targeted quantification (Figure C). In particular, VCMHLTPEEK, a carboxymethylated N-terminal valine-containing peptide of the β subunit of Hb, was found to be the most intense glycated peptide, as reported earlier.[21,22] The area under the curve (AUC) of these glycated peptides was higher in erythrocytes grown in high glucose conditions as compared to that of those maintained in low glucose conditions. Furthermore, in the presence of albumin, the AUC of these glycated peptides was reduced in a concentration-dependent manner (Figure C). All of these data strengthen the fact that the presence of albumin in erythrocyte culture reduces the glycation of hemoglobin.

Figure 2

Unmodified albumin protects hemoglobin glycation: Influence of albumin on hemoglobin glycation was measured in erythrocytes maintained at varying levels of albumin (15, 20, and 25 mg/mL concentrations represented as L-SA, M-SA, and H-SA, respectively) in either low or high glucose conditions. (A) Bar graph depicts HbA1c levels measured using a Nycocard HbA1c analyzer. Albumin reduced HbA1c in a concentration-dependent manner. A higher level of albumin (25 mg/mL) reduced HbA1c significantly as compared with the lower level of albumin (20 of 15 mg/mL) or absence of albumin in high glucose conditions. (B) Bar graph shows AGE-Hb fluorescence, which was monitored using excitation and emission wavelengths of 308 and 345 nm, respectively, and (C) Western blot analysis performed for hemoglobin isolated from erythrocyte culture using anti-carboxymethyl (CML) antibody. CML modification of hemoglobin showed a trend similar to that of HbA1c and AGE-Hb fluorescence.

Figure 3

Glycated hemoglobin peptide analysis was performed using targeted SWATH-MS analysis. (A) Carboxymethyl valine-modified (VCMHLTPEEK) peptide MS/MS spectra of carboxymethy (precursor mass, 1010.5116 Da) of β-hemoglobin. (B) Representative extracted ion chromatogram for the glycated peptide from targeted SWATH-MS analysis using PeakVeiw software. (C) Heat map of the area under the curve (AUC) of glycated peptides of hemoglobin. The AUC of glycated peptides was higher in erythrocytes maintained in high glucose conditions as compared to that of those maintained in low glucose conditions. In addition, upon albumin treatment, the AUC’s of these glycated peptides reduced in a concentration-dependent manner. For example, the VCMHLTPEEK (CMV1-K8 of β-Hb) peptide was found to be the most predominant glycated peptide among all of the other glycated peptides of hemoglobin and albumin treatment reduced the area under the curve (AUC) of glycated peptides in a concentration-dependent manner. More or less all remaining glycated peptides showed a trend similar to that of the VCMHLTPEEK (CMV1-K8 of β-Hb) peptide.

Low Albumin Concentration in Erythrocyte Culture was Associated with Increase in Its Glycation

Unmodified albumin reduced the hemoglobin glycation in a concentration-dependent manner in erythrocyte culture, suggesting that it may competitively inhibit the glycation, by itself getting glycated, because it has more number of freely accessible lysine and arginine residues. To test this possibility, we analyzed glycation of albumin using mass spectrometry. A total of 25 glycated peptides were identified by the IDA approach, and 7 glycation-sensitive peptides were consistently present in all of the acquisitions (Supporting Information Data 2 and File 1). Therefore, these peptides were selected for targeted quantification.[23,24] The area under the curve (AUC) of glycated peptides was higher in high glucose than in low glucose conditions. Furthermore, the area under the curve (AUC) of glycated peptides was more at lower albumin concentration and vice versa (Supporting Information Figure S2). This suggests that (i) at a higher concentration of albumin, relatively more numbers of lysine and/or arginine are accessible for modification and possibly not all of them get modified, thus leading to lower extent of glycation, (ii) whereas at a lower concentration of albumin, relatively lesser numbers of lysine and/or arginine residues are accessible for modification and possibly majority of them get modified, thus leading to higher extent of glycation. Hence, at a lower albumin concentration due to increase in its own glycation, it may lose its ability to protect glycation of other proteins. Conversely, a higher concentration of albumin, due to its lesser glycation, may result in a better capability to protect glycation of other proteins such as hemoglobin.

N(ε)-(Carboxymethyl)lysine-Modified Serum Albumin (CMSA) Increases Hemoglobin Glycation

To corroborate the role of albumin levels and its glycation status, albumin was modified with glyoxylic acid to obtain CMSA and its ability to protect glycation of hemoglobin was evaluated. CMSA was characterized mass spectrometrically. A total of 25 CML-modified peptides of albumin were identified and characterized (Table S1 and Supporting Information Data 3). Erythrocytes maintained in the presence of various concentrations of CMSA at low and high glucose concentrations were evaluated for hemoglobin glycation by various methods. Unlike unmodified albumin, CMSA failed to protect hemoglobin glycation as measured by HbA1c levels, AGE-Hb fluorescence, Western blotting with anti-CML antibody, and mass spectrometry-based quantification of glycated peptides of hemoglobin (Figure A–D). Instead, a strikingly higher HbA1c level was observed with CMSA treatment as compared to that with only low or high glucose treatment (Figure A).

Figure 4

N(ε)-(Carboxymethyl)lysine-modified serum albumin (CMSA) increases hemoglobin glycation: Influence of CMSA on glycation of hemoglobin from erythrocytes maintained at either low or high glucose conditions with varying levels of CMSA (15, 20, and 25 mg/mL concentrations represented as L-CMSA, M-CMSA, and H-CMSA, respectively) was monitored. (A) Bar graph represents HbA1c levels measured using a Nycocard HbA1c analyzer. CMSA increased HbA1c levels in a concentration-dependent manner. A higher level of CMSA (25 mg/mL) increased HbA1c significantly as compared with lower levels of CMSA (20 of 15 mg/mL) or absence of CMSA in high glucose conditions. (B) Bar graph represents AGE-Hb fluorescence, which was monitored using excitation and emission wavelengths of 308 and 345 nm, respectively, and (C) Western blotting analysis of hemoglobin extracted from erythrocyte culture was performed using anti-carboxymethyl antibody. The band intensity of the glycated hemoglobin was higher in high glucose conditions with 25 mg/mL CMSA treatment compared to that in low glucose conditions with 25 mg/mL CMSA treatment. (D) Heat map of area under the curve (AUC) of glycated peptides of hemoglobin. Glycated hemoglobin peptide analysis was performed using targeted SWATH-MS analysis to validate HbA1c analysis, AGE-Hb fluorescence, and Western blot analysis. The AUC’s of glycated hemoglobin peptide was normalized with average total ion count of all individual runs. The AUC of glycated peptides were higher in erythrocytes maintained in high glucose conditions as compared to that of those maintained in low glucose conditions. In addition, upon CMSA treatment, the AUC’s of these glycated peptides increased in a concentration-dependent manner.

Modification of Lysine Residues of Albumin Impairs Its Ability To Protect Hemoglobin Glycation

The above results suggest that modification of lysine residues of albumin may impair its ability to protect hemoglobin glycation, as treatment of CMSA led to an increase in HbA1c levels. To examine the effect of lysine modification of SA on its ability to protect hemoglobin glycation, lysine residues were acetylated with acetyl salicylic acid (ASA) (aspirin). Acetylation of lysine residues was confirmed mass spectrometrically. A total of 24 acetylated peptides with 24 lysine residues modified (Figure A; Table S2 and Supporting Information Data 4). Like CMSA, acetylated serum albumin (AcSA) failed to protect hemoglobin glycation of erythrocytes maintained at high glucose concentration, as measured by HbA1c levels, AGE-Hb fluorescence, and Western blotting with anti-CML antibody, in comparison with unmodified albumin (Figure B–D). However, AcSA was relatively less efficient compared with CMSA in increasing hemoglobin glycation. These results unequivocally demonstrate that albumin requires lysine residues to be maintained in an unmodified state to protect the glycation of other proteins such as hemoglobin.

Figure 5

Modification of lysine residues of albumin impairs its ability to protect hemoglobin glycation. Erythrocytes were maintained in high glucose (HG) media either with or without AcSA (25 mg/mL represented as H-AcSA) or CMSA (25 mg/mL represented as H-CMSA). (A) MS/MS spectra of acetyl lysine-modified (LKAcetylHLVDEPQNLIK) peptide (precursor mass, 1588.906 Da) of albumin. (B) Bar graph represents the HbA1c level. Unlike unmodified albumin, treatment of AcSA failed to reduce HbA1c in the erythrocytes maintained in high glucose media. (C & D) AGE–hemoglobin fluorescence and Western blot analysis performed for hemoglobin isolated from erythrocyte culture using anti-carboxymethyl (CML) antibody showed a trend similar to that of HbA1c data.

Diabetic Plasma has Reduced Ability To Protect Hemoglobin Glycation

Furthermore, we have examined the effect of clinical plasma from healthy and diabetic subjects on hemoglobin glycation in erythrocyte culture. Human serum albumin (HSA) is the most abundant plasma protein and preferentially gets glycated in diabetes owing to its abundance, as well as due to large number lysine and arginine residues accessible for glycation.[17] Therefore, the effect of clinical plasma on hemoglobin glycation can be attributed to mainly albumin and its glycation, although the effect of glucose, other metabolites, and other proteins cannot be ruled out. Individual plasma obtained from healthy control and diabetic subjects was analyzed for fasting and postprandial blood glucose, HbA1c, plasma fructosamine, and albumin levels (Table S3). Three individual plasma samples from healthy control and diabetic subjects were pooled based on fasting and postprandial blood glucose and HbA1c. The mean albumin levels were relatively more in healthy control plasma (48.5 ± 3.6 g/L) than in the diabetic plasma (30.5 ± 4.7 g/L). However, the mean plasma fructosamine in healthy control was 217.56 ± 14.7 μmol/L, whereas in diabetes, it was 410.4 ± 52.6 μmol/L. Further glycation status of albumin was characterized and quantified by IDA and SWATH-MS. Previously reported six glycation-sensitive peptides were used for quantification (Figure A). The details of identification, characterization, and quantification of glycated peptides are shown in Supporting Information Data 5 and File 1.[23,24] The AUCs for these six glycated peptides were significantly higher in diabetic plasma than in healthy control plasma. Further treatment of healthy control plasma to erythrocytes maintained in high glucose conditions reduced HbA1c levels. However, diabetic plasma is relatively inefficient to reducing HbA1c. Although both control and diabetic plasma reduced hemoglobin glycation of erythrocytes maintained in high glucose conditions, as measured by HbA1c levels, AGE-Hb fluorescence, and Western blotting (Figure B–D), the extent of decrease in hemoglobin glycation was more with the treatment of control plasma than the diabetic plasma. This data supports the fact that the albumin levels and its glycation status influence hemoglobin glycation because albumin levels were relatively low in pooled diabetic plasma, whereas its glycation was relatively higher (Table S3).

Figure 6

Diabetic plasma has reduced ability to protect hemoglobin glycation erythrocytes were maintained in high glucose media either with healthy control plasma or diabetic plasma. (A) Heat map represents the relative quantification of AGE-modified peptides in serum albumin from clinical plasma samples. Most of these glycated peptides were higher in diabetic plasma than in control plasma, and, in addition, these were reported as glycation-sensitive residue containing peptides in previous studies. (B) Bar graph depicts HbA1c values. Erythrocytes maintained in the presence of high glucose with healthy control plasma treatment showed a significant decrease in HbA1c level as compared with erythrocytes maintained only in high glucose media. However, treatment of erythrocytes with diabetic plasma resulted in increase in the HbA1c level compared to that from erythrocytes treated with healthy control plasma. (C & D) AGE-Hb fluorescence and Western blot with anti-carboxymethyl antibody showed a trend similar to that of HbA1c data.

HSA and HSA–Fructosamine are Negatively and Positively Associated with HbA1c, Respectively

Next, we investigated whether the results of our in vitro experiments with erythrocyte culture can be extended to clinical settings. A total of 75 blood plasma samples collected from 25 subjects each of healthy control, prediabetes, and diabetes were analyzed for various biochemical parameters such as fasting and postprandial blood glucose, HbA1c, albumin, fructosamine levels (Table S4 and Supporting Information File 1). The average HbA1c levels in healthy control was 5.1 ± 0.2%, whereas in prediabetes and diabetes, it was 5.9 ± 0.2% and 7.4 ± 0.9%, respectively. However, the albumin levels were more in healthy control (46.49 ± 2.7 g/L) than in prediabetes (42.1 ± 2.4 g/L) and diabetes (39.1 ± 3.0 g/L). Quantification of plasma fructosamine revealed that it was maximum (570.3 ± 85.6 μmol/L) in diabetes followed by prediabetes (457.2 ± 34.6 μmol/L) and healthy control (304.1 ± 37.6 μmol/L). As plasma protein fructosamine reflects the total plasma protein glycation, the relative plasma albumin fructosamine (RAF), which is the contribution of albumin to plasma protein fructosamine, was deduced from the ratio of albumin to total protein concentration (Supporting Information File 1). The RAF levels showed a trend similar to that of plasma protein fructosamine. Furthermore, Pearson’s correlation was performed between HbA1c and albumin, plasma fructosamine, or RAF levels, respectively (Figure A–C). HbA1c and albumin levels showed a significant negative correlation (r = −0.6584, n = 75), as observed in previous studies.[11,12,14] However, plasma protein fructosamine (r = 0.7357, n = 75) and RAF (r = 0.6718, n = 75) showed positive correlation with HbA1c. The coefficients of correlation between plasma protein fructosamine and RAF were more or less same, suggesting that RAF contributes predominantly to plasma protein fructosamine. Although it has been well established in many previous studies that plasma protein fructosamine is positively associated with HbA1c, we would like to hypothesize that RAF could determine HbA1c outcome because it contributes predominantly to plasma protein fructosamine.

Figure 7

Pearson’s correlation analysis of HbA1c and levels of albumin, plasma fructosamine, or relative albumin fructosamine (RAF). (A) Correlation analysis showed significant negative correlation between HbA1c and albumin levels (r = −0.6584 and p value (one-tail) <0.0001). (B) Correlation analysis showed significant positive correlation between HbA1c and plasma fructosamine level (r = 0.7357 and p value (one-tail) <0.0001), and (C) Correlation analysis showed significant positive correlation between HbA1c and plasma albumin fructosamine level (r = 0.6718 and p value (one-tail) <0.0001).

Discussion

Glycated hemoglobin (HbA1c) is considered as a gold standard for the assessment of glycemic status in diabetes. It is important to consider the confounding factors that influence HbA1c during treatment and management of the disease. Many previous studies have shown that various factors affect the HbA1c value, for example, the age and lifespan of erythrocytes; intracellular glucose in erythrocytes; conditions like anemia, splenomegaly, and pregnancy; ethnicity and gender; estimation methods; chemical modifications such as glutathiolation and advanced glycation such as carboxymethylation; antiglycation drugs such as aspirin, etc.; iron-containing diet; and supplements (Table S5).[10] Apart from these factors, the plasma albumin level has been shown to be negatively associated with HbA1c in a large cohort of diabetic subjects.[11,12,14] The plausible explanation that albumin could competitively protect hemoglobin glycation was derived from previous studies where low albumin levels were associated with increased glycation of plasma proteins including insulin, fibrinogen, etc.[13−15] In this study, we have unequivocally demonstrated in erythrocyte culture that albumin levels influence hemoglobin glycation, i.e., higher levels of albumin reduce hemoglobin glycation and vice versa. At lower albumin levels, increased albumin glycation was observed, which perhaps decreased its ability to reduce hemoglobin glycation. This was substantiated by treatment of glycated albumin, i.e., CMSA, which failed to reduce hemoglobin glycation; instead, it increased hemoglobin glycation. The inability of CMSA to reduce hemoglobin glycation was due to the lack of availability of free lysine residues of CMSA, which otherwise competitively inhibited the glycation of hemoglobin. This observation was corroborated by modifying lysine residues with acetylation. For the first time, we demonstrate that modification of lysine residues of albumin impairs its ability to inhibit hemoglobin glycation. Furthermore, correlation studies between HbA1c and albumin or RAF supported our in vitro experimental finding that albumin abundance and its glycation status determine hemoglobin glycation in erythrocyte culture. Therefore, it is quite plausible that albumin glycation precedes hemoglobin glycation in vivo because albumin is the most abundant protein in circulation with a large number of free lysine or arginine residues accessible for glycation. Even though hemoglobin is 3–4 times more abundant than albumin, it is an intracellular protein and has a relatively lesser number of lysine/arginine residues accessible for glycation. Also, previous studies have shown that the percent glycated albumin is relatively higher (control range (10–15%) and diabetic range (18–30%)) than the percent glycated hemoglobin, as measured by HbA1c (control 4–6% and diabetic 6.5–15%),[25] which supports the hypothesis that albumin glycation precedes hemoglobin glycation, although it is technically challenging to prove this in clinical setting or in animal experiments. A cartoon showing the relationship among albumin, glycated albumin, and HbA1c levels is depicted in the TOC or graphical abstract figure. In vivo glycated albumin can also increase HbA1c level by interacting with the receptor for AGE, which causes oxidative stress, inflammation, and insulin resistance, forming a vicious cycle.[26]

Conclusions and Outlook

This study demonstrates that not only it is important to maintain normal levels of albumin in diabetes but also this should be with minimal glycation. This is the first study that highlights the role of albumin and its glycation status in regulation of hemoglobin glycation (HbA1c). Thus, we propose that quantification of albumin and glycated albumin in conjunction with HbA1c has a great clinical significance in management of diabetes.

Experimental Section

This study was carried out to investigate the effect of albumin abundance and its glycation status on hemoglobin glycation using in vitro erythrocyte culture and clinical analysis. The overview of the current study is depicted in Figure .

Chemicals

Bovine serum albumin (BSA), glyoxylic acid, sodium cyanoborohydride, dialysis membrane, and Penstrep were procured from Sigma-Aldrich, MO. MS-grade solvents such as acetonitrile and water were obtained from J.T. Baker, PA. Rapigest-SF was procured from Waters Corporation, MA. Membrane cutoff filters of 3 kDa were procured from Millipore, MA. RPMI-1640 medium was procured from GIBCO Invitrogen. All other chemicals and reagents of analytical grade were procured from Sigma-Aldrich unless otherwise stated.

In Vitro Synthesis of CML-Modified Serum Albumin (CMSA) and Acetylated Serum Albumin (AcSA)

Human serum albumin (HSA) and BSA share a considerable amount of homology in their sequence and structure;[17] therefore, in all of the experiments, BSA was used in place of HSA. CMSA was synthesized as described earlier.[27] Briefly, glyoxalic acid (45 mM), BSA (50 mg/mL), and sodium cyanoborohydride (150 mM) were dissolved in 100 mM phosphate buffer (pH 7.4) and incubated for 24 h in the dark at 37 °C. However, AcSA was synthesized as described previously.[28] Briefly, BSA (50 mg/mL) in 100 mM phosphate buffer (pH 7.4) and 10 mM acetyl salicylic acid (ASA) was incubated for 24 h in the dark at 37 °C. Moreover, for unmodified albumin, BSA was dissolved in 100 mM phosphate buffer (pH 7.4) and incubated for 24 h in dark at 37 °C. Consequently, both modified and unmodified albumin were extensively dialyzed, followed by ultrafiltration with 3 kDa cutoff membrane filters to remove the free glyoxalic acid/aspirin/salts against sodium phosphate buffer (0.1 mol/L, pH 7.4, 350 μL). The synthesis of CMSA and AcSA were confirmed by mass spectrometry analysis, and the detailed mass spectrometry analysis procedure is described below.

Erythrocytes Culture

Freshly drawn human blood was obtained from the Blood Bank. Blood was centrifuged (1500 rpm, 5 min at 4 °C), plasma and buffy coat were aspirated, and the pellet containing erythrocytes was washed thrice with 3 volumes of RPMI medium. The washed erythrocytes (approximately 4 × 109 cells/mL) were maintained in RPMI medium containing either low glucose (LG) (5.2 mmol/L) or high glucose (HG) (15.7 mmol/L) concentration in a T25 culture flask with antibiotic 1-X Penstrep.[29−31] For different experiments, erythrocytes were subjected to various treatments such as aminoguanidine (AMG) (10 mM), unmodified serum albumin (SA) or carboxymethylated serum albumin (CMSA) (15, 20, or 25 mg/mL), acetylated serum albumin (AcSA) (25 mg/mL), and control and diabetic plasma (25 mg/mL plasma protein) and were maintained at 37 °C in a 5% CO2 incubator for 7 days.

Measurement of Hemoglobin Glycation

Hemoglobin glycation was monitored by measuring HbA1c levels, AGE-Hb fluorescence, and AGE-Hb by Western blotting with anti-CML antibody and mass spectrometry analysis. The detailed procedure is given below.

HbA1c Analysis

For the measurement of HbA1c, the erythrocyte pellet was obtained by centrifugation at 1500 rpm for 15 min at room temperature to separate the medium. This pellet was used to measure the HbA1c level by a Nycocard HbA1c analyzer as per the manufacturer’s instruction.

AGE–Hemoglobin Fluorescence

Hemoglobin was isolated from the erythrocyte pellet by vortexing with a buffer (7 M urea, 2 M thiourea, and 100 mM DTT in 100 mM ammonium bicarbonate buffer, pH 8.3), followed by incubation on ice for 30 min. Thereafter, lysed erythrocytes were centrifuged at 12 000g for 30 min at 4 °C. The supernatant containing hemoglobin was estimated for protein concentration by Bradford’s assay. Hemoglobin (1.5 mg) was used for the measurement of AGE–hemoglobin fluorescence with excitation/emission wavelength of 308/345 nm, respectively, using a Varioskan flash multimode plate reader (Thermo Scientific) as described earlier.[32]

Western Blot Analysis with Anti-Carboxymethyl Antibody

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blotting was performed with 5 μg of hemoglobin. Hemoglobin was reduced and denatured by dissolving in Laemmli buffer (5% β-mercaptoethanol and 10% SDS) and separated on 12% SDS-PAGE. Then, the protein from the gel was transferred onto the polyvinylidene difluoride (PVDF) membrane. For the confirmation of protein transfer, Ponceau staining was performed and the stain was removed by washing thrice with 1× PBS. The membrane was blocked using 5% skimmed milk (HiMedia, India) in PBS overnight at 4 °C. Then, the membrane was probed with anti-carboxymethyl (1:2000) antibody (Abcam, Cambridge, U.K.) for 2 h at room temperature. Following washing with PBS-T and PBS, the membrane was incubated with secondary antibody conjugated to horseradish peroxidase (1:2500) antibody (Bangalore Genei, India) for 1 h at room temperature. Bands were detected by chemiluminescence using the WesternBright Quantum Western blotting detection kit (Bio-rad) as per the manufacturer’s instructions.

Trypsin Enzymatic Digestion of Proteins

Protein (100 μg); either hemoglobin isolated from erythrocytes or albumin treated to erythrocytes/clinical plasma, or CMSA, AcSA; was diluted to 100 μL with 50 mM ammonium bicarbonate buffer containing 0.1% RapiGest. Proteins were denatured at 80 °C for 15 min, subsequently reduced with dithiothritol (100 mM) at 60 °C for 15 min, and alkylated with iodoacetamide (200 mM) at room temperature in the dark for 30 min. Proteins were digested with 2 μg of proteomic-grade trypsin (1 μg/μL in 50 mmol/L ammonium bicarbonate, Sigma-Aldrich) at 37 °C for 14–16 h. Tryptic digestion was stopped by addition of 2 μL of concentrated HCl. Digested peptides were desalted using C18 Ziptips (Millipore, Billerica, MA) and concentrated in a vacuum concentrator. Peptides were reconstituted in 3% (v/v) aqueous acetonitrile containing 0.1% (v/v) formic acid for mass spectrometric analysis.[27]

Mass Spectrometric Characterization of In Vitro Synthesized CMSA and AcSA

Peptides (1.5 μg) of CMSA or AcSA were loaded onto a reverse-phase C18 column (150 × 2.1 mm2, 1.9 μm) and separated on an UPLC (Accela 1250, Thermo Fisher Scientific) online coupled to a Q-Exactive Orbitrap mass spectrometer (Thermo Fisher Scientific) at a flow rate of 350 μL/min for a duration of 45 min, using five gradient segments (2–40% A for 35 min, 40–98% A for 2 min, held at 98% A for 2 min, 98–2% A for 2 min, and held at 2% A for 4 min), where A and B are solvent A (ACN (100%, v/v) with 0.1% formic acid (0.1%, v/v)) and solvent B (aqueous water (100%, v/v) with 0.1% formic acid (0.1%, v/v)), respectively. The mass spectra of eluted peptides were acquired in a data-dependent manner using a hybrid quadruple Q-Exactive Orbitrap mass spectrometer in a positive acquisition mode, with the precursor mass range of 350–2000 Da m/z (resolution at 70 000 with AGC target 1e6) and scan time of 120 ms. MS acquisition was performed at precursor’s ion selection width of 2 Da m/z, under fill ratio of 0.3% and with dynamic exclusion time of 15 s. The peptide fragmentation was achieved by high-energy collision-induced dissociation (HCD) with 28 eV and fragment ion resolution at 17 500 (MS/MS at m/z 400 Da) with AGC target 1e5 (MS/MS).[27,33]

Acquired mass spectral data was analyzed by Proteome Discover software (PD 1.4.0.288, Thermo Fisher Scientific) with Sequest-HT as a search engine. The data were searched against bovine serum albumin (UniProt IDs: P02769). Search parameters included trypsin as proteolytic enzyme with two missed cleavages and 1% false discovery rate. Peptide and fragment mass tolerance were set at 10 ppm and 0.6 Da, respectively. Search criteria included fixed and variable modifications as carbamidomethylation (C) and oxidation (M), respectively. Additional variable lysine-specific modifications included carboxymethylation ((CML) mass increment of +58.0055 Da) and acetylation (mass increment of +42.0105 Da at lysine residues).[27,28,33,34] Carboxymethylated and acetylated albumin peptides were manually validated for accurate mass shift in the precursor ion due to modification and presence of fragment ions retaining modification, and peptides with more than 1 XCorr value were considered as true modified peptides.

Quantification of Glycated Peptides of Hemoglobin and Albumin by SWATH-MS Analysis

Mass spectral acquisition of glycated peptides of hemoglobin and albumin was performed in using micro LC 200 (Eksigent; Dublin, CA) online coupled Triple-ToF 5600 (SCIEX) in a high-sensitivity mode. For the development of spectral library, mass spectral acquisition was done in an information-dependent manner (IDA) and subsequently SWATH-MS was performed for relative quantification of glycated peptides. Peptides (3 μg) were directly injected onto an Agilent C18-RP HPLC column (100 × 0.3 mm2, 3 μm, 120 Å) and then separated using a 95 min gradient of 3–40% mobile phase (mobile phase A: 100% water (v/v) with 0.1% (v/v) formic acid; mobile phase B: 100% acetonitrile (v/v) with 0.1% (v/v) formic acid) at a flow rate of 8 μL/min. Mass spectrometry parameters were set as follows: mass range from 400 to 1250 Da with an accumulation time of 0.25 ms at the MS level and from 50 to 1999 Da with an accumulation time of 0.01 ms at the MS/MS level. Fragmentation of peptides was done using rolling collision energy of 28 kV.

For relative quantification, hemoglobin and albumin peptides were individually analyzed in SWATH-MS (sequential window acquisition of all theoretical mass spectra) mode in three biological and two technical replicates. Briefly, the peptide (1 μg) was directly injected onto an Agilent C18-RP HPLC column (100 × 0.3 mm2, 3 μm, 120 Å) and then separated as mentioned in IDA data. In SWATH-MS mode, the instrument was specifically optimized, the quadrupole settings for the selection of precursor ion selection windows m/z of 25 Da wide with a 0.5 Da window overlap, a set of 34 overlapping windows was constructed covering the precursor mass range of 400–1250 Da. SWATH-MS/MS spectra were collected from 50 to 1999 Da. The rolling collision energy was optimized for each window with a spread of 25 kV. Dwell time of 70 ms was used for all fragment ion scans in high-sensitivity mode, and for each SWATH-MS cycle acquired in high-resolution mode for 100 ms, resulting in a duty cycle of 3.4.[27]

For the identification of glycated peptides of hemoglobin or albumin peptides listed from IDA acquisitions, the spectral data was analyzed using ProteinPilot software (version 5.0; SCIEX) with respective database by selecting the post-translational modification option. Identified glycated peptides were manually verified for accurate precursor mass and presence of modified fragment ions. Post SWATH-MS, the glycated peptides were quantified extracting peak area using Peakview software (version 2.2; SCIEX) by extracting precursor mass with an error of 0.001 Da and by retention time shift of ±1 min and the presence of modified fragment ions was manually inspected. Considering all of these parameters, the area under the curve (AUC) for glycated peptides was obtained and normalized with average total ion counts for quantification.

Clinical Study

The blood samples were collected from individual volunteers at Chellaram Diabetes Institute, Pune, with an approval of Institutional Ethics Committee and patient written consent, according to standard guidelines of American Diabetes Association (ADA). Subjects with known history of cancer, hypothyroidism, hematuria, and inflammatory diseases or any infection were excluded from the study. This study involved 75 clinical subjects. After blood withdrawal, biochemical parameters such as fasting plasma glucose, postprandial glucose, hemoglobin, HbA1c, and lipid profile for each subject were measured (detailed biochemical measurements are listed in Supporting Information, Table S3). Subjects were divided into three groups on the basis of glucose and HbA1c measurements: (1) 25 controls (mean age 53.37 ± 11.27 years); (2) 25 prediabetes subjects (mean age 56.8 ± 8.78 years); and (3) 25 controlled diabetes subjects (mean age 58.74 ± 8.83 years). Remaining blood collected was used for preparation of plasma using blood collection tubes with K2EDTA (BD Vacutainer Plastic, Thermo Fisher Scientific).

Total Plasma Protein Quantification

Plasma samples were diluted 10-fold with Milli-Q water, and protein concentration was determined by using a Bradford protein assay kit (Bio-Rad Protein Assay: Bradford).[35]

Plasma Albumin Measurement

Plasma albumin was estimated using a bromocresol green-based colorimetric method (Sigma-Aldrich: BCG (bromocresol green) albumin assay kit). Bromocresol green interacts with albumin to form a chromophore, which was measured at 520 nm spectrophotometrically.[11]

Measurement of Plasma Protein Fructosamine and Relative Plasma Albumin Fructosamine (RAF) Level

Plasma protein fructosamine was measured using a colorimetric fructosamine assay (Merck, NJ). This colorimetric assay is based on the ability of ketoamines to reduce nitrotetrazolium blue to formazan in an alkaline solution. The rate of formazan formation is directly proportional to the concentration of plasma protein fructosamine.[11] The relative plasma albumin fructosamine (RAF) was calculated by deducing from the ratio of albumin to total protein concentration.

Statistical Analysis

All in vitro erythrocytes experiments were performed in three biological replicates and mass specta were acquired in two technical replicates for library creation and in technical triplicates for the SWATH-MS-based quantification, and to corroborate the findings of in vitro erythrocyte culture experiments, albumin, plasma fructosamine, relative albumin fructosamine, and HbA1c levels were analyzed in clinical subjects (n = 75, technical triplicates). All of the statistical analyses were performed using GraphPad Prism software (GraphPad Software, Inc.). The values of HbA1c, AGE-Hb fluorescence, and AUCs represent mean with standard deviation. The statistical significance was calculated using two-way analysis of variance (ANOVA) analysis. p values were calculated and the level of significance was set at p < 0.05 (ns—no significance (p > 0.05), *p < 0.05, **p < 0.01, and ***p < 0.001). The correlation between different variables was estimated using Pearson’s correlation coefficient and was examined with an ANOVA model.