Low Plasma Albumin Levels Are Associated with Increased Plasma

Dec 19, 2011 - (RAGE), triggering a cascade of events leading to oxidative stress and proinflammatory pathways.3 Accumulation of AGEs has been found t...
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Low Plasma Albumin Levels Are Associated with Increased Plasma Protein Glycation and HbA1c in Diabetes Hemangi S. Bhonsle,† Arvind M. Korwar,† Sachin S. Kote,† Sandeep B. Golegaonkar,† Ashok D. Chougale,† Mahemud L. Shaik,‡ Nitin L. Dhande,§ Ashok P. Giri,† Kishore M. Shelgikar,§ Ramanamurthy Boppana,‡ and Mahesh J. Kulkarni*,† †

Proteomics Facility, Division of Biochemical Sciences, CSIR-National Chemical Laboratory, Pune, 411008, India National Center for Cell Science, Pune, India § Maharashtra Medical Research Society (MMRS), Pune, India ‡

S Supporting Information *

ABSTRACT: Albumin is one of the most abundant plasma proteins and is heavily glycated in diabetes. In this study, we have addressed whether variation in the albumin levels influence glycation of plasma proteins and HbA1c. The study was performed in three systems: (1) streptozotocin (STZ)-induced diabetic mice plasma, (2) diabetic clinical plasma, and (3) in vitro glycated plasma. Diabetic mice and clinical plasma samples were categorized as diabetic high albumin plasma (DHAP) and diabetic low albumin plasma (DLAP) on the basis of their albumin levels. For the in vitro experiment, two albumin levels, high albumin plasma (HAP) and low albumin plasma (LAP), were created by differential depletion of plasma albumin. Protein glycation was studied by using a combination of two-dimensional electrophoresis (2DE), Western blotting, and LC−MSE. In both mice and clinical experiments, an increased plasma protein glycation was observed in DLAP than in DHAP. Additionally, plasma albumin levels were negatively correlated with HbA1c. The in vitro experiment with differential depletion of albumin mechanistically showed that the low albumin levels are associated with increased plasma protein glycation and that albumin competes for glycation with other plasma proteins. KEYWORDS: albumin, post translational modification, advanced glycation end products (AGEs), glucose

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INTRODUCTION Diabetes is characterized by elevated levels of plasma glucose, which in turn modify the proteins by a nonenzymatic reaction called glycation.1 Protein glycation leads to the formation of heterogeneous fluorescent molecules, “advanced glycation end products” (AGEs), which modify conformation and function of proteins.2 Furthermore, AGEs interact with receptors for AGEs (RAGE), triggering a cascade of events leading to oxidative stress and proinflammatory pathways.3 Accumulation of AGEs has been found to be accelerated in diabetes and to contribute to the pathogenesis of diabetic complications. Plasma proteins are the primary target of glycation as they are directly exposed to higher glucose concentrations in plasma.4 Various plasma proteins including human serum albumin (HSA) and immunoglobulin G were the first few proteins to be characterized as glycated proteins.5,6 Among plasma proteins, albumin is one of the heavily glycated proteins because of its abundance, relatively longer half-life time (21 days), and greater number of lysine and arginine residues, the hotspots of glycation.7 Albumin comprises about 50% of plasma proteins, and any variation in levels of albumin may change the stoichiometry of plasma protein glycation. Albumin levels in © 2011 American Chemical Society

plasma are determined by its expression, secretion, and catabolism. Various factors including diet,8 lifestyle, inflammation,9 disease,10 drugs, etc. can affect these processes, leading to altered levels of albumin in plasma. For example, albumin synthesis and secretion has been reported to be decreased in diabetic rats, which was attributed to insulin deficiency, as replenishment of insulin in diabetic rats restored the albumin levels to that of normal rats.11,12 Therefore, it is expected that albumin levels may vary in diabetes. Patients with decreased albumin levels ascribed to malnutrition were more prone to develop vascular complications in diabetes.13 In our previous study with in vitro experiments, it was observed that higher levels of albumin were associated with decreased glycation of insulin and apomyoglobin, which were used as a model for low abundance proteins and vice versa.14 It is likely that albumin may have a similar role in inhibiting glycation of other plasma proteins in vivo. The fact that hypoalbuminemia ascribed to malnutrition increases the risk of diabetic complication, coupled with our previous study of competitive glycation inhibition by Received: October 15, 2011 Published: December 19, 2011 1391

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using anti-AGE antibody (Millipore, MA). Immunodetection was performed by incubating membranes with streptavidin conjugated horseradish peroxidase using DAB system. Stained gel images were acquired by GS 800 calibrated densitometer (Bio-Rad, Hercules, CA). Image analysis was performed using PDQUEST advanced software (Bio-Rad, Hercules, CA).

albumin, strengthened our hypothesis that albumin regulates the plasma protein glycation in vivo as well. In this study, we particularly show how a variation in plasma albumin levels in diabetes influences the glycation of other plasma proteins and HbA1c, using two-dimensional gel electrophoresis (2DE), Western blot analysis and LC−MSE approach.

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Trypsin Digestion

MATERIALS AND METHODS All chemicals were procured from Sigma-Aldrich unless otherwise mentioned.

Spots were excised and destained. Proteins were reduced and alkylated by 10 mM DTT and 55 mM iodoacetamide respectively. Tryptically digested peptides at 37 °C overnight were extracted with 5% formic acid in 50% ACN, and were reconstituted in 5 μL of 0.1% formic acid in 3% ACN. For insolution digestion, 10 μg of protein was solubilized with 0.1% RapiGest SF (Waters Corporation, MA) in 50 mM NH4HCO3 to enhance proteolytic cleavage. Proteins were reduced and alkylated with 100 mM DTT at 56 °C for 15 min and 200 mM iodoacetamide in the dark for 15 min, respectively. The proteins were digested overnight with trypsin in 10:1 ratio at 37 °C.

Mice Plasma Sample

Male BALB/c strain of mice were injected with 45 mg/kg body weight of streptozotocin (STZ) in 50 mM citrate buffer for five consecutive days to induce hyperglycemia. The control set of mice were injected with 50 mM citrate buffer. The induction of diabetes was confirmed after 15 days by measuring the blood glucose levels with glucometer (Bayer, Germany). Blood samples were collected after two months of STZ injection and were immediately analyzed for blood glucose and HbA1c by using “in2itTM” analyzer (Bio-Rad, Hercules, CA). Animals having a blood glucose level of above 6.12 ± 1.7 mmol/L were selected for further studies. Plasma was obtained by EDTA treatment, which was then centrifuged at 1500g for 15 min, and the supernatant was stored at −80 °C until further use.

In Vitro Glycation of Plasma Proteins

Nondiabetic plasma was differentially depleted for albumin by Cibacron-Blue so as to achieve two concentrations (45 g/L and 35 g/L) of albumin. In vitro glycation of depleted plasma was performed by incubating it with 0.5 M glucose in 0.2 M phosphate buffer (pH 7.4) at 37 °C for 7 days. Glucose was removed by dialyzing the sample against 50 mM Tris-HCl. Samples obtained were further processed for 2DE followed by Western blot analysis and LC−MSE as described above.

Clinical Plasma Sample

Blood samples were collected from diabetic patients through an informed consent from Maharashtra Medical Research Society (MMRS), Joshi Hospital, approved by the Institution Ethics Committee. Blood plasma was collected from 60 diabetic patients and 7 nondiabetic controls, and glucose and HbA1c levels were determined.

LC−MSE, Protein Identification, Database Search, and PTM Analysis

Two microliters of digested peptides with a final concentration of 100 ng/μL were analyzed by using nanoACQUITY UPLC online coupled to a SYNAPT HDMS system (MSE) (Waters Corporation, Milford, MA) as described by Cheng et al.15 After MSE analysis, data were analyzed with Protein Lynx Global Server software (PLGS version 2.4 Waters Corporation, Milford, MA). A preliminary search of processed samples was performed for protein identification against the UniProt human database containing 44 987 protein entries. Glycation modification sites were identified by subjecting PLGS search against individual protein databases that were identified in the preliminary search. Search criteria included fixed and variable modifications as carbamidomethylation and oxidation (M), respectively. Additionally, variable glycation modifications involving lysine specific residues were fructosyl-lysine-2H20 (FL-2H2O) (+126.0317 Da); carboxymethyllysine (CML) (+58.0055 Da); carboxyethyllysine (CEL) (+72.0211 Da); pyrraline (+108.0211 Da). Those involving arginine specific residues were imidazolone-B (+142.03); argpyrimidine (80.0262 Da); Nε-(5-hydro-5-methyl-4-imidazolon-2-yl) ornithine (MG-H1) (+54.0106 Da); Nε-(5-hydro-4- imidazolon-2yl) ornithine (G-H1) (+39.9949 Da). Those involving both lysine and arginine residues were 1-alkyl-2-formyl-3,4-glycosylpyrrole (AFGP) (+270.074 Da); Amadori product (+162.02); imidazolone-A (144.03 Da); methylglyoxal lysine dimer (MOLD) (49.0078 Da); methylglyoxal-derived imidazolium cross-link (MODIC) (36.0179 Da); and crossline (252.11 Da). All of these modifications were included in the search. The PLGS-identified glycated peptides were manually validated with the following criteria: (a) correct parent ion mass shift values should be present, (b) all glycated peptides identified by PLGS

Pooling of Diabetic Plasma Samples

Both mice and clinical plasma samples were analyzed for fructosamine and plasma albumin levels by using the nitroblue tetrazolium colorimetric method (Fructosamine, Merck, NJ) and the BCG method (Innoline Albumin, Merck, NJ), respectively, as per the manufacturer’s instructions. On the basis of plasma albumin levels, diabetic plasma was grouped into two groups: diabetic-high albumin plasma (DHAP) and diabetic-low albumin plasma (DLAP). The nondiabetic plasma had relatively higher levels of albumin than diabetic plasma. To create sufficient material for further proteomic analysis, seven representative samples from each group were pooled. Plasma Protein Sample Preparation

Abundant plasma proteins were depleted either by PROTBA (Sigma Aldrich) for two-dimensional electrophoresis or by Proteominer (Bio-Rad, Hercules, CA) for LC−MSE analysis, as per the manufacturer’s instructions. Protein concentration was estimated by using Bio-Rad protein assay kit (Bio-Rad, Hercules, CA). Two-Dimensional Gel Electrophoresis, Western Blotting, and Image Analysis

Protein (150 μg) was solubilized in 125 μL of rehydration buffer containing 8 M urea, 2 M thiourea, 4% CHAPS, 70 mM DTT, 0.5% C7BzO, and 1 μL of ampholytes (pH 3−10), and 7 cm nonlinear IPG strips (pH 4−7) were rehydrated overnight. Isoelectricfocusing was performed using the PROTEAN IEF Cell (BioRad, Hercules, CA) followed by second dimension separation of proteins on 12.5% SDS-PAGE. Resolved proteins were visualized by CBB-R250 staining or by Western blotting 1392

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Table 1. Characteristics of Plasma Samples Used for 2DE, Western Blot, and LC−MSE Analysisa mice

fasting plasma glucose (mmol/L)

HbA1c (%)

albumin (g/L)

fructosamine (μmol/L)

number of glycated proteins identified by LC−MSE

4.23 ± 1.67 6.12 ± 1.76b 6.31 ± 2.54b,c

6.4 ± 0.08 8.8 ± 0.86b 10.2 ± 0.67b,c

45.54 ± 2.8 34.73 ± 1.5b 26.12 ± 1.7b,c

168.31 ± 57.72 341.51 ± 65.97b 417.22 ± 111.37b,c

6 15 20

4.31 ± 0.49 6.85 ± 1.75b 6.606 ± 0.91b,c

5.2 ± 0.17 8.4 ± 0.9b 10.5 ± 1.6b,c

60.61 ± 3.24 51.33 ± 1.43b 44.11 ± 4.31b,c

186.38 ± 6.51 369.67 ± 16.93b 348.12 ± 49.77b

24 32 36

ND (n = 9) DHAP (n = 9) DLAP (n = 9) clinical ND (n = 10) DHAP (n = 7) DLAP (n = 7) a

Data expressed are in mean ± SD. bP < 0.005 when compared with ND. cP < 0.05 when compared with DHAP.

should have a corresponding unglycated peptide in MSE analysis, (c) glycated peptides should have a minimum of five fragment ions matching with the sequence, and (d) glycated peptides should have a neutral loss of water. Statistical Analysis

All experiments were performed in triplicates. Statistical analysis was performed by Student’s t-test. Data are expressed as means ± SD. A p-value < 0.05 was considered as statistically significant.

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RESULTS AND DISCUSSION In both animal and clinical experiments, the glucose, HbA1c, and fructosamine levels were significantly higher in diabetes than their corresponding control. Further, albumin levels were higher in the nondiabetic plasma than in diabetic plasma. Depending on the albumin levels, diabetic plasma samples were divided in two groups, DHAP and DLAP, in both mice and clinical samples (Table 1). The influence of variation in albumin levels on fructosamine was studied in both mice and clinical plasma samples (Table 1). Fructosamine levels were higher in DLAP than DHAP in mice. However, in clinical plasma, though the levels of fructosamine were higher in diabetes, there was no significant difference between DLAP and DHAP. Fructosamine, the first product of glycation reactions, is eventually converted to heterogeneous AGEs. The formation of AGEs is accelerated by various factors including auto-oxidation of glucose, age, oxidative stress, etc.1 Alternatively, they are enzymatically deglycated by fructosamine-3-kinase and chemically transglycated.16 Moreover, the fructosamine level in the clinical samples may be affected by the medication and glycemic control. Sample preparation for identification of glycated proteins by LC−MSE and characterization of glycation modifications were performed as described in Figure 1. The number of identified glycated proteins was more in diabetes than control. However, in the diabetic group, the number of glycated proteins was more in DLAP than DHAP in both mice and clinical plasma (Table 1) and are listed in Supporting Information Table S1. Many of the identified proteins have been previously reported to be glycated in the diabetic plasma.4,17−19 A similar trend was observed for glycation modified sites. The number of glycation modified sites was higher in DLAP than in DHAP and nondiabetic control in both mice and clinical plasma (Figure 2A and 2B, respectively). Further, 2DE-Western blot analysis with anti-AGE antibodies indicated an increase in the extent of glycation in diabetes than control. Glycated proteins (haptoglobin, vitamin D binding protein, apolipoprotein A IV, and fibrinogen gamma) that were common to mice, clinical, and in vitro glycated plasma are represented in Figure 3. The extent of glycation was higher in plasma proteins of DLAP than

Figure 1. Scheme depicting glycated plasma protein identification from nondiabetic and diabetic samples. Pooled samples from mice, clinical patients, and in vitro glycated plasma were processed and analyzed as outlined in the chart.

in DHAP as revealed by densitometry. These proteins also had a higher number of glycation sites in LC−MSE experiments as shown in Figure 2. The logical explanation for this differential glycation of plasma proteins could be the differential exposure of these proteins to glucose caused by variations in albumin levels. Furthermore, albumin levels showed a negative correlation with glycated hemoglobin HbA1c in both mice and clinical experiments (Figure 4A and 4B). This was also quite evident in a previous study with 4158 participants, where it was shown that more than 50% of the patients with lower albumin had higher HbA1c (>8%), whereas HbA1c was lower among patients with high albumin levels.20 Our study suggests that variation in albumin levels can also influence the differential glycation of HbA1c. It is possible that a similar blood glucose level may lead to different HbA1c depending upon the albumin content in the plasma. Further, HbA1c levels are influenced by various factors, including glucose status over approximately 8 weeks, erythrocyte life span, anemia, hemodialysis, etc.21 Therefore, sometimes HbA1c may not indicate the precise glycemic control. Alternatively, glycated albumin has been suggested to be a better marker for short-term glycemic control.7 Additionally, low serum albumin in diabetes was also associated with increased cardiovascular disease.22 In addition, hypoalbuminemic patients associated with malnutrition were more prone to develop diabetic complications.13,22 To establish the fact that variation in albumin levels influences the glycation of plasma proteins, control human plasma was differentially depleted of albumin and divided into two groups: high albumin plasma (HAP) and low albumin 1393

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Figure 2. Heat map analysis of glycation modification sites on glycated proteins of (A) mice plasma, (B) clinical plasma, and (C) in vitro glycated plasma. The analysis shows an increased number of glycation modifications in DLAP than in DHAP and ND plasma. The expanded form of abbreviated proteins are listed in Supporting Information Table S1.

Figure 3. 2DE and Western blot analysis of glycated proteins in (A) mice plasma, (B) clinical plasma, and (C) in vitro glycated plasma. Abundant plasma proteins were depleted and separated by 2DE. Glycated proteins were visualized by Western blotting using anti-AGE antibody.

trend was also reflected in 2DE-Western blot analysis. These results conclusively prove that variation in albumin levels influence the glycation of plasma proteins. Furthermore, albumin competes for glycation with other plasma proteins as it gets more glycated than the other proteins as revealed by the number of glycation modified sites (Figure 2C). Our previous

plasma (LAP), with albumin levels of 45 g/L and 35 g/L, respectively, which were in vitro glycated. LC−MSE analysis of these plasma samples resulted in the identification of a higher number of glycated proteins as shown in Supporting Information Table S1 and showed that their modification sites were higher in LAP than HAP (Figure 2C). The same 1394

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Figure 4. Correlation between albumin and HbA1c levels in (A) mice plasma [n = 83, diabetic (63) and 20 nondiabetic (20)] and (B) clinical plasma [n = 67, diabetic (60) and nondiabetic (7)]. Albumin and HbA1c levels were estimated from nondiabetic and diabetic plasma. A negative correlation was observed where HbA1c levels were found to be decreased with increased albumin concentration. Vertical lines indicate SE.

clinical trials. Thus, lower levels of albumin in diabetes could be a risk factor for glycation-induced complications.

study shows that albumin competitively inhibits the glycation of insulin, which was used as model for low abundant proteins.14 The probable mechanism by which albumin can inhibit or regulate the glycation of insulin has been depicted in Figure 5.

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ASSOCIATED CONTENT

S Supporting Information *

Table S1 contains information on glycated proteins identified from mice, clinical, and in vitro glycated plasma samples. Table S2A,B,C contains sequences of glycated peptides from mice, clinical, and in vitro glycated plasma samples, respectively. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +91-20-25902541. Fax: +91-20-25902648. E-mail: mj. [email protected]. Figure 5. Mechanism of glycation regulation by albumin. Insulin (model protein) was glycated in vitro by incubating with glucose in presence of either low or high levels of albumin. (A) At low albumin levels, the relative intensity of glycated insulin was higher, and (B) at high albumin levels, the relative intensity of glycated insulin was decreased as albumin competes for glycation because of its abundance. I, insulin; Alb, albumin; G, glucose; GI, glycated insulin; G-Alb, glycated albumin.

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ACKNOWLEDGMENTS

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ABBREVIATIONS

Authors are thankful to Dr. Vidya Gupta, Chair, Biochemical Sciences, for helping us in establishing a collaboration with Joshi Hospital, Dr. Sourav Pal, Director, and Dr. S. Sivaram, Bhatnagar Scientist, CSIR-NCL. We are also thankful to Dr. A. M. Deshpande, Chairman, MMRS, for his support to carry out this study. This work was carried out under CSIR network Project NWP0004. H.S.B. thanks Lady Tata Memorial Trust for a Senior Research Fellowship.

At low albumin levels, the relative intensity of glycated insulin was about 50%, whereas the intensity decreased with higher albumin levels, as albumin gets glycated competitively because of its abundance. In conclusion, all of the evidence presented here reinforces the fact that low levels of albumin are associated with increased plasma protein glycation and HbA1c, and vice versa. This is the first study that shows the importance of albumin levels in the regulation of glycation of plasma proteins and HbA1c. Perhaps maintaining near-normal levels of albumin in diabetes may be helpful by protecting plasma proteins from the adverse effects of glycation. However, this needs to be substantiated with

ND, nondiabetic; DLAP, diabetic low albumin plasma; DHAP, diabetic high albumin plasma; HAP, high albumin plasma; LAP, low albumin plasma; BCG, bromocresolgreen; CHAPS, 3-[(3cholamidopropyl) dimethylammonio]-1-propanesulfonate; DTT, 1,4-dithio-D-threitol; IAA, iodoacetamide; IPG, immobilized pH gradient; CBB, Coommassie brilliant blue; PVDF, polyvinylidene fluoride; TBS, Tris-buffered saline; DAB, 3,3′diaminobenzidine 1395

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(20) Rodriguez-Segade, S.; Rodriguez, R.; Mayan, D.; Camina, F. Plasma albumin concentration is a predictor of HbA1c among type 2 diabetic patients, independently of fasting plasma glucose and fructosamine. Diabetes Care 2005, 28 (2), 437−439. (21) Jeffcoate, S. L. Diabetes control and complications: The role of glycated haemoglobin, 25 years on. Diabetic Med. 2004, 21, 657−665. (22) Folsom, A. R.; Ma, J.; Eckfeldt, J. H.; Nieto, F. J.; Metcalf, P. A.; Barnes, R. W. Low serum albumin. Association with diabetes mellitus and other cardiovascular risk factors but not with prevalent cardiovascular disease or carotid artery intima-media thickness. The Atherosclerosis Risk in Communities (ARIC) Study Investigators. Ann. Epidemiol. 1995, 3, 186−191.

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