Biol Trace Elem Res (2010) 138:346–357 DOI 10.1007/s12011-010-8610-8

Interaction of Zinc, Ascorbic Acid, and Folic Acid in Glycation with Albumin as Protein Model Rashmi Tupe & Vaishali Agte

Received: 24 October 2009 / Accepted: 6 January 2010 / Published online: 9 February 2010 # Springer Science+Business Media, LLC 2010

Abstract Using albumin as model, we conducted series of in vitro glycation experiments to examine role of zinc in glycation using glucose at 4–100 mg/ml, incubations at 37°C or 60°C, duration of 2 or 4 weeks and in presence of zinc or ascorbic acid (AA) or folic acid (FA). Modifications of bovine serum albumin (BSA) were examined by using fluorescence of advanced glycation end products (AGEs) and dityrosine, UV, and Fourier transformed infrared spectroscopy. Adding zinc (0 to 768.5 μmol/l) resulted in significant inhibition of albumin glycation by glucose with a linear fit, y ¼ 0:0895x þ 230:99ðR2 ¼ 0:7676; p ¼ 0:013Þ. The glycation by fructose was greater than that of glucose with stronger inhibitory effect by zinc in fructose–glycation (t=−5.8, p=0.002). Addition of zinc significantly decreased fluorescence as seen in Zn+FA or Zn+AA sets as compared to sets of FA alone (p=0.00056) or AA alone (p=0.037). The fluorescence for dityrosine and AGE had a correlation of 0.897 (p< 0.01). The data from fluorescence, UV, and FTIR spectra collectively suggested inhibitory effect of zinc in BSA glycation alone or in presence of FA and AA, showing new dimension for the protective action of zinc in hyperglycemic conditions. Keywords Zinc . Advanced glycation end products . BSA . Ascorbic acid . Folic acid Abbreviations BSA bovine serum albumin AGEs advanced glycation end products G glucose AA ascorbic acid FA folic acid NA nicotinic acid FTIR Fourier transformed infrared

R. Tupe : V. Agte (*) Agharkar Research Institute, G.G. Agarkar Road, Pune 411004, India e-mail: [email protected]

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Introduction Glycation of structural and enzymatic proteins leading to partial loss of their activity is one of the serious consequences of chronic hyperglycemia. Further, oxidative stress resulting from the metabolic consequence of hyperglycemia leads to increased formation of advanced glycation end products (AGEs) in tissue proteins [1–5]. The formation of AGEs, also known as carbonyl stress, is a key pathophysiological event. Carbonyl stress not only links to diabetic complications but also to a range of important human diseases such as Alzheimer’s disease, decreased skin elasticity, male erectile dysfunction, pulmonary fibrosis, and atherosclerosis [6]. The transition metals (Zn, Cu, Mn, and Fe) play an important role as micronutrients. But, many of the glucose-associated oxidative modifications have been attributed to Fenton chemistry carried out by some transition metals like copper and iron [7]. Although Cu and Fe have two oxidation states, Zn remains in a single Zn+2 state which might differentiate its action from that of Cu and Fe in cellular metabolism during carbonyl stress. Zinc is known to play a critical protective role for structure and function of cell membranes and proteins [8]. Zn also participates in the synthesis, storage and secretion of insulin, as well as conformational integrity of insulin in the hexameric form [9]. However, the role of zinc as a proglycating or antiglycating factor is still not clearly established. Albumin is the major zinc carrier protein of blood plasma and about 90% of circulating zinc is bound to albumin. Experiments conducted in our laboratory as well as those reported by earlier workers indicate high binding potential of albumin for zinc and other metabolites [10–13]. The N terminal zone of albumin is considered as the key binding site of zinc to the protein [14] apart from other five binding sites with the binding constant K ¼ 7:28 M  1 [15, 16]. These binding sites for zinc may have significance since bovine albumin has also been reported to have 83 potential glycation sites including the N terminal [17]. There is however no direct evidence of an association between level of albumin glycation with level of zinc in hyperglycemic conditions. Many of the vitamins in their free form or active dinucleotide form present in blood plasma have functional groups that are capable of binding to zinc. The log stability constants for some of the complexes of vitamins and zinc have been reported earlier [18]. In our previous results, in vitro binding of zinc with albumin was significantly increased by folic acid (FA) in dose–response manner [10]. Therefore, in an effort towards understanding the significance of zinc binding to albumin under hyperglycemic condition and to study potential interplay of zinc with some vitamins in glycation reaction, we initiated in vitro studies on how these factors affect albumin glycation as evident through fluorescence, UV, and Fourier transformed infrared (FTIR) spectroscopy.

Materials and Methods Materials Zinc sulfate, D-glucose, L-AA, and D-fructose were purchased from SRL (India). Bovine serum albumin (Fraction V), KBr (FTIR grade), and FA were procured from Sigma (St. Louis, MO, USA). In Vitro Glycation of BSA Table 1 gives details of the experimental conditions followed for various experiments viz. change in albumin and glucose concentrations, change in temperature, and incubation time. Experimental/reaction mixtures were prepared in 200 mmol/l potassium phosphate buffer (pH 7.4). Sodium azide (1 mmol/l) was used as

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Table 1 Different Experimental Conditions for Glycation Experiments Incubation Temperature Variables Glucose Expt. Albumin (°C) concentration concentration time (mg/ml) (mg/ml)

Remarks

1

20

90

2 weeks

60

Zn, AA, FA. Other micronutrients

Albumin glycation as per Yamaguchi [19]

2

20

90

2 weeks

60

Zn

Albumin glycation as per Yamaguchi [19]

3

40

4

2 weeks

60

Zn, AA, FA, Zn+AA, Albumin glycation Zn+FA (6 levels) at physiological concentrations

4

40

4

2 weeks

37

Zn, AA, FA, Zn+AA, Albumin glycation Zn+FA (6 levels) at physiological concentrations

5

10

100

2 and 4 weeks

37

Zn, AA, FA, Zn+AA, Albumin glycation Zn+FA (6 levels) as per Yasujiro [20]

6

10

100

2 weeks

37

Zn, AA, FA, Zn+AA, Albumin glycation Zn+FA (6 levels) as per Yasujiro [20]

preservative. Experiments were carried out in triplicate. Reaction mixtures were filtered through 0.22 μm Millipore membrane filters into sterile plastic capped vials of an appropriate volume in order to avoid contamination. The solution, containing albumin and glucose or fructose, is referred to as the positive control. The negative nonglycated control contained albumin in buffer alone. All additions and the removal of sample aliquots at various times were done aseptically. After the incubation period, it was ensured that all the solutions were free of microbiological contamination. Measurement of the AGE formation The formation of AGEs was estimated as described by Yamaguchi and Yasujiro [19, 20]. Briefly, after incubation of albumin with glycating agent, the reaction mixture was treated with equal volume of ice cold 10% (w/v) trichloroacetic acid and precipitate was collected by centrifugation. The precipitate was washed twice with 5% (w/v) ice cold trichloroacetic acid and redissolved in 200 mmol/l potassium phosphate buffer (pH 7.4). AGEs fluorescence was measured by using spectroflurometer (Shimadzu, RF-5301PC, Japan) with excitation wavelength of 325-nm and emission wavelengths of 450-nm. The dityrosine formation was estimated as the fluorescence intensity at wavelengths of 335-nm for excitation and 385-nm for emission and compared with the AGEs fluorescence. Fluorescence of glycated albumin was taken as estimate of carbohydrate–protein adducts formation. Background fluorescence of buffer and nonglycated albumin control were subtracted. The fluorescence was expressed as arbitrary units. Measurement of UV Spectra After fluorescence measurement, absorption spectra of protein solutions in the region 250–350 nm were recorded on a spectrophotometer (UV1, Thermo Electron Corp., Merck, Germany). The absorption maxima were recorded at 280 and 325 nm were used for the quantification of total protein and glycation of protein, respectively.

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FTIR Spectra The samples after incubation were lyophilized, mixed in KBr, and spectra were recorded in the range of 500–4,000 cm−1 on FTIR spectrophotometer (Perkin Elmer Spectrum one FTIR, USA). Statistical Analysis All the experiments were done in triplicates for each level of factor and average values were used for assessing their effect. One-way analysis of variance (ANOVA), two-way ANOVA, and the paired or unpaired Student’s t test, as appropriate, were used to evaluate the effect of micronutrient factors on modification of the experimental protein. All the statistical tests were performed using Microsoft Excel under Windows XP professional package. Differences were regarded as significant at p<0.05.

Results Albumin is present in the circulation at relatively high concentrations (35–50 g/l) and is prone to glycation in vivo [21]. Using BSA as a model protein, effect of zinc and other micronutrients on albumin glycation was studied at various parameters such as different incubation periods and temperatures, different glycating agents, and glycation at different albumin:glucose ratios. Effect of Zinc and Micronutrients on AGEs Formation of BSA For assessing effect of micronutrients on albumin glycation, they were added to different set of tubes at following concentrations: Zn (768.5 µM), AA (74 mM), FA (63 µM), nicotinic acid (NA, 41 µM), Mn (55 µM), Cu (200 µM), Se (20 µM). After 2 weeks incubation period at 37°C, fluorescence of glycated BSA with glucose (positive control) was significantly enhanced as compared to only BSA samples (negative control). All the other samples containing micronutrients were compared to positive control (Fig. 1). The results indicated that among eight different micronutrients, zinc and manganese exhibited the significant antiglycation effect (p<0.01). In zinc coincubated samples, the AGEs fluorescence was decreased by 38% against positive control. The glycation was increased by ∼30% in presence of FA (p<0.05), and about 66% 90

**

% Glycation

70

**

**

Cu

Fe

50

* 30 10

+ve control

-10 -30

** -50

** Zn

Mn

NA

Se

Fig. 1 Effect of different micronutrients on BSA glycation

FA

AA

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by coincubation with Cu, AA, and Fe (p<0.01). Selenium and NA did not show any significant influence on extent of glycation. To further investigate effect of Zn and other micronutrients, dose–response curves were generated. For these experiments, zinc, AA, and FA were added in increasing concentration (Zn ¼ 75  375mmol=l, AA ¼ 12:33  74mmol=l, FA ¼ 10:50  63mmol=l) during BSA glycation. The effect of adding zinc on albumin glycation by glucose showed the response curve with a linear fit of y ¼ 0:0895x þ 230:99 (R2 =0.768, Table 2, p=0.013). Both AA and FA increased glycation but without significant trend in dose–response curve (p=0.157 for FA and p=0.065 for AA). Table 2 also gives results of two-way ANOVA which show that addition of zinc significantly decreased the fluorescence of Zn+FA or Zn+AA as compared to FA alone (p=0.00056) or AA alone (p=0.037). Effect of Adding Increasing Levels of Zinc at Three Ratios of Albumin and Glucose The ratio of albumin and glucose can affect the degree of glycation. Therefore, it was of interest to study effect of zinc at different ratios of albumin and glucose. Two reported protocols used in the study had the ratio 0.22 and 0.10 while the ratio at physiological levels was 10 (Table 1). Among different experiments, it was interesting to note that in the control tubes (added zinc=0), glycation was highest, where the ratio of albumin to glucose was also highest (10). Secondly, in spite of the differences in the ratios of albumin:glucose in all the three experiments, the data showed decrease in fluorescence intensity of albumin (y) with increasing levels of zinc (x). The nonlinear relationship were: Expt:2 : y ¼ 23:131e0:0004x ; R2 ¼ 0:7932; Expt:3 : y ¼ 25:391e0:0024x ; R2 ¼ 0:9533; Expt:6 : y ¼ 23:724e0:0037x ; R2 ¼ 0:8991: Effect of Zinc on BSA Glycation at Two Different Incubation Temperatures Nonenzymatic glycation hasten with increase in temperature. To examine this, independent experiments were conducted in presence of zinc, FA, and AA at 60°C and 37°C for 2 weeks (albumin at 40 mg/ml and glucose at 4 mg/ml Experiments 3 and 4; Fig. 2). AGEs formation was higher at 60°C than 37°C (t=−8.31,p=0.0004) but the interactive effects of zinc continued to be seen even at 37°C which represented the physiological temperature. Zn+FA, Zn+AA, and AA alone showed similar but less pronounced responses at two different temperatures. Table 2 Results of One-Way and Two-Way ANOVA for Experiment 4

Experiment 4

F value

p value

One-way ANOVA Zn at six levels

4.64

0.013

AA at six levels

2.82

0.065

FA at six levels

2.09

0.157

Two-way ANOVA FA+Zn and FA at six levels

29.44

0.00056

AA+Zn and AA at six levels

5.03

0.036426

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Fluorescence intensity (AU)

500

37˚C 60˚C

400

300

200

100

0 0

75

150

225 Zn (µM)

300

375

Fig. 2 Effect of zinc on albumin glycation at two incubation temperatures (37°C and 60°C)

Comparison of the Effect of Zinc on Glycation in Presence of Glucose and Fructose Effect of increase in zinc concentration (0–768.5 μmol/l) on BSA glycation (20 mg/ml) was studied using glucose and fructose at higher concentration (90 mg/ml; Fig. 3). In another experiment, the effect of zinc (0–385 μmol/l) during glycation of BSA (40 mg/ml) was studied in presence of glucose and fructose at 4 mg/ml, representing physiological conditions. It was interesting to note that the BSA glycation by fructose was greater than that of glucose. Additionally, with increase of zinc the slope of fructose curve was also higher than glucose curve indicating stronger inhibitory effect by zinc in fructose glycation (t=−5.8, p=0.002) in both experiments. However, since glucose being the sugar of physiological significance, experiments were done mainly using glucose. Effect of Glycation at Two Incubation Periods During experiment 5, glycation was carried out at 37°C for 2 and 4 weeks. In terms of AGEs fluorescence, significant differences were observed between two incubation periods (t=−2.57, p=0.06). However, the inhibitory effect of zinc was seen only at 2 weeks of incubation period; whereas FA and AA showed enhanced AGEs formation at 4 weeks incubation period (Fig. 4).

Fluorescence intensity (AU)

700

Glucose

600

Fructose

500 400 300 200 100 0 0

100

200

300

400 500 Zn (µM)

600

700

800

Fig. 3 Effect of increase in zinc concentration (0–768.5 μmol/l) on autofluorescence of BSA (20 mg/ml) using glucose and fructose at higher concentration (90 mg/ml)

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Fluoresence Intensity (AU)

500

2 Weeks 4 Weeks

400 300 200 100 0 BSA

BSA+G

BSA+Zn

BSA+FA

BSA+AA

Fig. 4 Result of different incubation time (2 and 4 weeks) on glycation in presence of different factors

Effect of Zinc and Vitamins in the Fixed Ratio Finally, the interaction between zinc and two vitamins on albumin glycation was observed at fixed molar ratio (experiment 6). The reaction solutions were incubated at 37°C for 2 weeks. There was no trend in the fluorescence intensity for increasing levels of Zn+FA or Zn+AA. This absence of trend was also noted when FA was increased at constant zinc level. This showed that FA and AA are enhancers of glycation but their action during glycation seems to be like a cofactor. Formation of Dityrosine During Glycation of BSA The dityrosine formation was estimated as the fluorescence intensity at wavelengths of 335 nm for excitation and 385 nm for emission and compared with the AGEs fluorescence. For all the samples except those containing FA, the fluorescence readings for dityrosine and AGEs were changing hand in hand with a correlation of 0.897 (p<0.01). This indicated similar effects of zinc on inhibition of dityrosine formation. When the FA containing samples were included the correlation was reduced (0.71). This effect of FA needs further investigation. UV Spectra of Glycated Proteins The presence of benzene ring in the amino component accelerates glycation. Strong correlation between the intensity of the fluorescence and the absorption at 325 nm has been reported for glycated peptides and amino acids [22]. Figure 5 represents the UV spectra for unglycated BSA, glycated BSA alone, or with three levels of zinc, corrected for protein content (OD at 280 nm). It was observed that with increasing zinc concentration, the extent of glycation (absorption at 325 nm) significantly decreased. The difference between glycated BSA and BSA+G+Zn (375 µM) was highly significant (p<0.01). The results are in concurrence with AGEs fluorescence measurements. FTIR Spectroscopy Infrared spectrometry techniques are extremely valuable to study structural modifications of a protein. The shift of the maximum peak of amide I band from 1,655 cm−1 (alpha-helix), at 1,634 cm-1 has been assigned to beta-antiparallel sheet structure, the shift of peak at 1540 cm-1 to 1527 (1,529) cm−1 in amide II band [23]. To get further evidence of the effect of zinc and two vitamins on structural group modifications during albumin glycation, FTIR spectroscopy data were examined (Figs. 6 and 7). There was no change in the peak at 1,655, indicating no change in amide I band. However, there were shifts in the peak of unglycated BSA at 1,544 cm−1 to 1,540 cm−1 for all the glycated albumins indicating minor changes in amide II bands. In presence of zinc, there was a new peak at 2,036 cm−1 indicating probably the presence of hydrogen bond or ionized compound.

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1.4

BSA

Absorbance

1.2

BSA+G BSA+G+Zn (225 µM)

1

BSA+G+Zn (300 µM) 0.8

BSA+G+Zn (375 µM)

0.6 0.4 0.2 0 250

270

290

310

330

350

Wavelength (nm) Fig. 5 UV spectra for unglycated BSA as well as glycated BSA in presence of increasing amounts of Zn (0–375 μmol/l)

Discussion Role of zinc in glycation of albumin and other body proteins has still to be clearly understood. Using albumin as a model protein, we report that among eight different micronutrients, zinc acted as an antiglycating agent while iron, copper, FA, and AA provoked glycation. It is pertinent to note that the antiglycation effect of zinc was seen in different experiments done at variable temperatures, incubation periods, and different glucose:BSA molar ratios. A consistent inhibitory effect of zinc was observed in BSA glycation alone or in presence of FA and AA, giving a new dimension to zinc in hyperglycemic conditions. In our previous work, we could find that zinc and micronutrients interacted with each other and their interactions played important role in Zn uptake by erythrocytes in presence of vitamins and their dinucleotides [24]. It will be necessary to examine effect of prolonged treatment of zinc, especially in diabetics, on reduction in the glycation of body proteins such as albumin. 120

BSA + G BSA BSA + G + Zn

110 100

%R

90 80 70

New peak

60 50 40 4000

3500

3000

2500

2000

1500

1000

-1

cm

Fig. 6 FTIR spectra for BSA alone, BSA+Glucose, and BSA+Glucose+Zn

500

354

Tupe and Agte BSA + G + FA BSA + G + AA BSA + G + Zn + FA BSA + G + Zn + AA

100 90 80 70

%R

60 50 40 30 20 10 0 4000

3500

3000

2500

2000 -1 cm

1500

1000

500

Fig. 7 FTIR spectra for BSA+Glucose+FA, BSA+Glucose+AA, BSA+Glucose+Zn+FA, BSA+Glucose+ Zn+AA

We find that Cu enhances BSA glycation while Zn inhibits the same. Cu is reported to be the accelerant, stimulating oxidation, lipid peroxidation, and glycation; and copper chelation is instrumental to glycation fighters, including carnosine [25, 26]. Moreover, advanced ageing and particularly advanced age-related chronic degenerative diseases are associated with a significant increase in the copper/zinc ratio and systemic oxidative stress [27]. There are other reports that also explain the differential behavior of zinc in contrast to copper as seen in the present data [28, 29]. Use of UV spectra in monitoring glycation reactions is a simple and complimentary tool used in glycation during the interactions of glucose and fructose with amino acids (Gly, Phe), peptides (Gly-Gly, Gly-Phe, Phe-Gly) and Ac-Lys studied under physiological conditions. Strong correlation between the intensity of the fluorescence and the absorption at 325 nm was found for all reaction systems [30]. This was the basis for taking UV spectra which indicated decrease of absorbance at 325 nm with increase of zinc concentration in concurrence with the results based on fluorescence measurements. FTIR spectroscopy has been used to follow the fate of the model protein BSA. The posttranslational modification of proteins by carbohydrates leads to the formation of new coordination centers for metal ions within a protein chain. But no complexation of Zn (II) with Maillard reaction products Nε fructolysine and Nε carboxymethyl lysine was observed [31]. Similar results of the absence of peak for zinc binding are observed through our FTIR data, except for a new peak at 2,036 cm−1, indicating presence of hydrogen bond or ionized compound in presence of zinc. These results are suggestive of the possibility that zinc may not participate as a substrate but may shift the equilibrium constant in the reverse direction in the glycation reaction. We find that albumin glycation was higher in presence of fructose than glucose. In a similar study, the fructose-modified BSA, almost all of the loss of lysine was attributed to the formation of CML (Carboxy Methyl Lysine), with the yield of CML being up to 17-fold higher than glucose-modified BSA [32]. The next question addressed was whether antiglycation action of zinc differs in presence of other biomolecules like AA and FA, which are also present in the plasma. In fact, with increasing concentrations of AA and FA, the AGEs formation increased. However, when glycation was studied in presence of zinc and both vitamins, zinc continued to show inhibitory effect on albumin glycation. Further, in these experiments, relative concentration of zinc with respect to vitamin was found to be

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important determinant in manifesting inhibitory effect. Micronutrients are generally prescribed as mixtures and not as single compound. Both FA (10 mg/day) and AA (1– 10 g/day) along with iron are prescribed in very high doses during pregnancy, severe iron deficiency anemia, and in other health disorders. In the absence of supplementing sufficient amount of zinc, the glycation promoting effect of FA warrant further investigation since its prescription at therapeutic doses may pose harmful side effects particularly in hyperglycemic conditions. There are contradictory reports of AA on glycation. AA is reported to be much more reactive than glucose at equivalent concentrations in glycation reaction [33]. Its oxidized form can pose increased glyoxidation stress. However, when 18 healthy individuals received 1,000 mg of AA as citrus fruit complex daily for 4 weeks, glycation of serum proteins was decreased by an average of 46.8% [34]. Nevertheless, this source contained AA along with bioflavonoids, which are known for antiglycating action and protecting AA from oxidation. Present experimental conditions used AA alone and support its glyoxidating action under aerobic conditions and indicated risk of long-term consumption of AA in higher amounts. Serum albumin has a half-life of about 18–20 days as against the half-life of 120 days for hemoglobin. Glycosylated hemoglobin is routinely used as a biomarker of status of glucose metabolism. Glycation of serum albumin can also be used as a biomarker of recent status of glucose metabolism [35, 36]. Poor zinc status is a common feature of diabetics which might be increasing the risk of oxidative damage and glycation of albumin which is the crucial protein of body. Our results coupled with these reports indicate albumin to be a link for action of zinc in glucose metabolism. The glycated proteins produce 50-fold more free radicals than nonglycated proteins. Several of such complications of diabetes may be related to increased intracellular oxidants and free radicals associated with decreased intracellular Zn and Zn dependent antioxidant enzymes. There appears therefore a complex interrelationship between Zn and both, Type 1 and Type 2 diabetes. The role of Zn in the clinical management of diabetes, its complications or in its prevention is at best, unclear. Present data supports antiglycation activity to zinc for serum albumin but the exact mechanisms of zinc’s antiglycation action is still not clear. It is likely that the N terminal being the common site of binding of zinc (through coordinate bond) as well as for glycation domain of albumin, zinc hinders the initial steps of AGE formation as shown by presence of new ionic or hydrogen bond. Nevertheless, considering importance of zinc in cellular metabolism, its nontoxic nature from the pharmaceutical viewpoint, it will be useful to further investigate the potential of zinc in developing antiglycation therapies. Acknowledgments We are grateful to Director, A.R.I., Pune for providing necessary facilities to carry out this work. We acknowledge Dr. Bhosle and Dr. Nilegaonkar for their help in spectroflurometric and FTIR analysis.

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