Scandinavian Journal of Clinical & Laboratory Investigation, 2010; 70: 122–127

ISSN 0036-5513 print/ISSN 1502-7686 online © 2010 Informa UK Ltd. (Informa Healthcare, Taylor & Francis AS)DOI: 10.3109/00365511003624137

Correspondence: Mohammad Ali Ghaffari, Department of Clinical Biochemistry, Physiology Research Center & Cellular and Molecular Research Center, Ahwaz Jundishapour University of Medical Sciences, Ahwaz, Iran. Fax: �98 611 333 2036. E-mail: ghaffari@ajums.ac.ir

(Received 22 July 2009; accepted 30 December 2009)

ORIGINAL ARTICLE

In vitro inhibition of low density lipoprotein carbamylation by vitamins, as an ameliorating atherosclerotic risk in uremic patients

MOHAMMAD ALI GHAFFARI & MEHRNOOSH SHANAKI

Cellular and Molecular Research Center & Physiology Research Center, Ahwaz Jundishapour University of Medical Sciences, Ahwaz, Iran

AbstractIntroduction: Previous studies have shown that the increase of carbamylated LDL (cLDL), a product of nonenzymatic modifi cation of LDL in human serum by urea-derived cyanate, may cause cardiovascular complications in patients with chronic renal insuffi ciency. This study examined the inhibitory effect of ascorbic acid, α-tocopherol and lycopene on LDL carbamylation in an in vitro model system. Methods: After isolation of LDL from plasma using an ultracentrifuge technique, cyanate was added to it and then LDL carbamylation was measured in both the absence and presence of ascorbic acid, α-tocopherol and/or lycopene by the colorimetric method at 530 nm Results: The fi ndings indicated that these vitamins inhibit LDL carbamylation and the most effective vitamin of the three is lycopene. Moreover, the effect of lycopene on this process increased in the presence of ascorbic acid and α-tocopherol. Conclusion: This study indicated that ascorbic acid, α-tocopherol and lycopene with antioxidant activity can probably inhibit LDL carbamylation and therefore may have a role in ameliorating atherosclerotic risk of patients with kidney failure. However in vitro and in vivo investigations are required to confi rm the exact effects of these vitamins on patients suffering from uremic disorders.

Key Words: Carbamylation , ascorbic acid , a-tocopherol, lycopene, low density lipoprotein (LDL)

Introduction

Chronic kidney diseases are worldwide medical and public health problems which are beginning to assume epidemic proportions. Chronic kidney diseases can result from a wide range of disorders (including dia-betes, hypertension and glomerulonephritis) and account for about 10% of the mortality in the world [1]. A large body of evidence supports the fact that kidney problems are independent risk factors for the development of cardiovascular disorders [1,2]. Cardiovascular disorders are considered as one of the leading causes of mortality in patients with chronic renal insuffi ciency. Mortality resulting from cardio-vascular disorders which is secondary to an advanced renal problem is much higher than in patients who only suffer from a renal disorder [2]. The increased prevalence of coronary artery disorders in patients with kidney problems cannot be explained entirely by well-known cardiovascular risk factors.

Carbamylation results from the non-enzymatic post-translational modifi cation of proteins by urea-

derived cyanate which is normally present in human blood plasma and is elevated in patients with chronic kidney disorders [3]. Several studies revealed that the protein component of the low density lipoprotein (LDL) particle, apolipoprotein B 100 (apo-B 100), may be carbamylated at lysine or the terminal protein amino acids [1,4]. It is also revealed that carbamyla-tion of LDL is a nontraditional risk factor for car-diovascular diseases [5]. It is commonly believed that endothelial cell injury is an initial event in atheroscle-rosis. Injured endothelial cells attract monocytes, which are burrowed beneath the endothelial cell layer and ingest modifi ed LDL to form ‘foam cells’ [6]. This process leads to the formation of atherosclerotic plaque (consisting of ‘foam cells’ and macrophages covered by a fi brous cap) which protrudes into the vessel lumen over the proliferating vascular smooth muscular cells [6].

Fruits and vegetables are important dietary sources of vitamins, especially ascorbic acid, α-tocopherol and lycopene [7]. These vitamins are of

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LDL carbamylation and vitamins 123

current interest in research due to their important biological and pharmacological properties attributed to their antioxidant properties. Nevertheless, data concerning the effect of these vitamins in preventing carbamylate modifi cation of proteins seem to be insuffi cient. Therefore, in this study, we examined the inhibitory effect of three vitamins (ascorbic acid, α-tocopherol and lycopene) on LDL carbamylation using an in vitro model system .

Materials and methods

Materials

Ascorbic acid, α-tocopherol and lycopene were pur-chased from Sigma (St, Louis, MO, USA) and used without further purifi cation. Ethylene diamidine tet-raacetic acid (EDTA), dimethyl sulfoxide (DMSO), bovin serum albumin (BSA), agarose, proteinase K, homocitrullin and potassium cyanate (KOCN) were obtained from Merck Chemical Co. (Darmstadt, Germany). Solutions were freshly prepared with double deionized water.

Methods

LDL isoforms. Native human LDL (nLDL) was obtained from the plasma of healthy volunteers after overnight fasting ( n � 20, age 25 � 5 years, all males, non-smokers, non-uremic, and not taking any drugs at least 2 weeks prior to the beginning of the study) by ultracentrifugation using a single step discontinu-ous gradient as described previously [8,9]. nLDL was kept under nitrogen at 4�C, away from light and was used within three weeks after preparation. All proce-dures were approved by international guidelines and the Ethics and Institute Research Committee of Ahwaz Jundishapour University of Medical Sciences.

Carbamylated LDL (cLDL) was prepared by in vitro modifi cation on nLDL as described by Weisgraber et al. [10]. In this study, the best potas-sium cyanate concentration for carbamylation of LDL was investigated by incubation of a range of cyanate concentration (0–30 μmol/L) with nLDL (0.6 mg protein/ml) in PBS (10 mmol/L, pH 7.4) at 35�C for 4 h. The best incubation time for LDL carbamylation was also investigated by incubating 0.6 mg protein/ml of LDL with 20 μmol/L cyanate for 1–7 hours at 35�C. In each step potassium cyanate was removed by extensive dialysis (100-fold volume of dialysis buffer, repeated three times) against 0.15 mol/L NaCl, 0.01% EDTA, pH 7.0, for 36 h at 4�Cunder sterile conditions.

Assessment of carbamylation. A colorimetric method using diacetyl monoxime was used to measure the degree of carbamylation in LDL preparations [11]. The process is briefl y outlined below: the LDL

suspension (0.6 mg protein/ml) was digested in 160 μl phosphate buffered saline (PBS; 10 mmol/L sodium phosphate buffer, pH 7.4, containing 140 mmol/L NaCl), then 30 μl sodium dodecyl sulfate (10%) and 6 μg proteinase K were added to it and incubated at 37�C for 2 h. Afterwards 800 μl of urea nitrogen reagent (0.83 mol/L sulfuric acid, 1.13 mol/L ortho-phosphoric acid, 0.55 mmol/L thiosemicarbazide and 2.6 mmol/L cadmium sulfate) and 160 μl diacetyl monoxime (3%) were added to the reaction mixture and the incubation continued at 97�C for 30 min The precipitate was removed by centrifuge at 3500 g for 10 min at room temperature. The supernatant absorption was measured at 530 nm. A standard curve was generated using homocitrullin (ε-amino-carbamyllysine, 0–30 nmol). The results were expressed in nmol of homocitrulline per mg of LDL protein.

Effect of vitamins on LDL carbamylation. The effect of the three vitamins; ascorbic acid, α-tocopherol and lycopene; on the carbamylation of LDL was exam-ined by incubation of nLDL (0.6 mg protein/ml) with potassium cyanate (20 μmol/L) and varying concentrations of the vitamins (0–40 μmol/L) in PBS, pH 7.4 at 35�C for 4 h. The ascorbic acid was dissolved in distilled water and α-tocopherol and lycopene were dissolved in 10% DMSO in PBS, pH 7.4. Degree of LDL carbamylation was determined at 530 nm as described above [11].

LDL electrophoresis. Isolation of native LDL (nLDL) from plasma was checked by electrophoresis on 0.8% agarose gel in 50 mmol/L sodium barbital buffer, pH 8.6, as described by Noble [12]. After applying the sample (7 μg LDL protein per well), electro-phoresis was run at 12 V/cm for 1 h. The gel was fi xed in an ethanol–acetic acid–water mixture (60: 10: 30, vol: vol: vol) and stained in 0.2% Sudan Black B dissolved in 60% ethanol.

The electrophoretic mobility of nLDL and mod-ifi ed LDLs (carbamylated LDL) in the absence and/or presence of 40 μmol/L vitamins were also compared by electrophoresis on 5% polyacrylamide gel. The gels were stained with Coomassie blue stain [13].

Protein measurement. The LDL protein content was measured by the Bradford method, using bovine serum albumin (BSA) as the standard [14].

Statistical analysis

Statistical analysis was performed with the analysis of variance (ANOVA). Results were expressed as mean � standard deviation of the mean. A value of p � 0.05 was considered signifi cant.

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124 M. A. Ghaffar & M. Shanaki

Results

The isolation of native LDL (nLDL) from plasma was confi rmed by electrophoresis on agarose gel. As shown in Figure 1, the nLDL fraction (lane 2) was compared with plasma LDL (lane 1). In this study incubation of cyanate (0–30 μmol/L) with nLDL in different times (1–7 h) showed that the best cyanate concentration was 20 μmol/L and the best incubation time for LDL carbamylation was 4 h (Figure 2). The degree of carbamylation of nLDL in the absence of the vitamins was assessed by mea-surement of homo citrulline, which indicated that after 4 h of carbamylation, cLDL contained 146 �12 homocitrulline nmol/mg LDL protein (Figure 3A). The extent of LDL carbamylation in the pres-ence of 10–40 μmol/L concentrations of ascorbic acid, α-tocopherol and lycopene, however, showed that these vitamins as having a dose-dependent decrease on LDL carbamylation (Figure 3A). In this study, compared to the control (without vita-mins) the vitamins showed a signifi cant inhibition of LDL carbamylation by the ANOVA test, p �0.001. According to this study 40 μmol/L concen-tration of ascorbic acid, α-tocopherol and lycopene can reduce LDL carbamylation to 38%, 54% and 76%, respectively (Figure 3B). Moreover, co-incu-bation of nLDL with cyanate and 40 μmol/L con-centrations of the three vitamins indicated that cLDL contained 20 � 5 homocitrulline nmol/mg LDL protein, compared to 146.25 � 12 nmol/mg protein in the absence of vitamins (results not shown). Therefore, the co-presence of these vitamins decreased LDL carbamylation to 86%.

Finally, we investigated electrophoretic mobility of 20 μmol/L cyanate treated LDL in the absence and/or presence of 40 μmol/L ascorbic acid, α-tocopherol and/or lycopene on polyacrylamide gel (Figure 4). During carbamylation, urea under-goes a spontaneous nonenzymatic transformation to cyanate, the active form of which, isocyanic acid, reacts with free nonprotonated amino groups of proteins. Carbamylation results in neutralization of the positive charge of the modifi ed lysine and enhances the mobility of the LDL in polyacrylam-ide. In these experiments, the electrophoretic mobility of cLDL was accelerated after carbamyla-tion of nLDL with cyanate, however, in the pres-ence of the vitamins (40 μmol/L) this migration was decreased (Figure 4). These observations sug-gest that these vitamins may decrease LDL car-bamylation in the presence of cyanate.

Discussion

Based on studies of oxidized LDL and LDL with other modifi cations, it is commonly accepted that endothe-lial cell injury by modifi ed LDLs is an initial event in atherosclerosis [6,15]. Although carbamylated LDL (cLDL) was shown to be elevated in uremic patients [3,16], no study was found about the effects of vita-mins on the process of LDL carbamylation. There exists a need for the identifi cation of factors support-ing or enhancing the process of progressive decrease in the LDL carbamylation rate. This investigation pro-vides evidence for in vitro carbamylation of LDL and this process decreases in the presence of the three vita-mins ascorbic acid, α-tocopherol and lycopene.

Figure 1. Electrophoresis of plasma (Lane 1) and nLDL fraction (Lane 2) on 0.8% agarose gel. LDL, Low density lipoprotein; VLDL, Very low density lipoprotein; HDL, High density lipoprotein.

Figure 2. Cyanate concentration (▲) and incubation time (■) effect on LDL carbamylation. Values are represented as the means �

SD of triplicate determinations.

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LDL carbamylation and vitamins 125

Urea-derived cyanate is a reactive molecule that can react irreversibly with the free epsilon amino groups lysine and N-terminal amino acids within proteins to form epsilon-carbamyllysine (homoc-itrulline) and thus alters the charge and structure of amino acids and proteins, as well as their func-tion [17,18]. Evidence of the adverse effects of in vivo carbamylation was found when cyanate was administered as a therapeutic agent in sickle cell anemia caused by peripheral neuropathy [19] and cataracts [20]. cLDL was shown to interact with cell surface receptors in human fi broblasts and prevent the binding of nLDL in human fi broblasts [10]. Horkko et al. [21] reported that clearance of uremic patients’ LDL as well as LDLs that are chemically modifi ed to cLDL decrease in rabbit plasma. We indicated that when cyanate, in a time- (0–4 hours) and dose- (0–20 μmol/L) dependent manner, incu-bated with nLDL (0.6 mg protein/ml), the carbam-ylation process of this lipoprotein increased signifi cantly. However, this phenomenon decreased with the increase of cyanate concentrations (� 20 μmol/L) and/or increase of incubation times (� 4 h) this phenomenon was decreased. Therefore our results showed that cyanate with 20 μmol/L concen-tration at 4 hhad the greatest effect on carbamyla-tion of 0.6 mg protein per ml of LDL concentration, in vitro. Given the above discussion, attempts to prevent LDL carbamylation and/or reduce the effects of carbamylation may provide novel treat-ments for the prevention of atherosclerosis in ure-mic patients. Therefore in the next step the authors investigated the effect of these vitamins on LDL carbamylation by cyanate. This effect was measured with assay of homocitrulline levels. Four different

concentrations of each vitamin were used. Results of this experiment revealed that these vitamins with concentrations of 10 μmol/L, 20 μmol/L, 30 μmol/L and 40 μmol/L in a dose-dependent manner ( n � 3, ANOVA test, p � 0.001) the susceptibility of LDL to carbamylation reduced signifi cantly. A marked reduction of LDL carbamylation was observed in 40 μmol/L lycopene treatment. According to our previous studies and studies conducted by other researchers the inhibitory effect of ascorbic acid, α-tocopherol and lycopene was reported on some LDL modifi cations, as oxidation and glycation [9,22–24], however, few quantitative experimental studies can be found that investigated the inhibition application of these vitamins within LDL carbamy-lation. Several reports suggest that uremia is a state of oxidative stress. These reports showed an increase in lipid peroxidase of red blood cell membranes and a decrease in serum antioxidant activity of uremic patients [25,26]. Thus, the authors hypothesized that antioxidant vitamins might possess anti-car-bamylation characteristics. These investigations showed that these vitamins suppressed LDL car-bamylation in the following manner: Lycopene (76.1%) � α-tocopherol (54%) � ascorbic acid (38%). Lycopene is a red color pigment, unlike most other carotenoids that are widely distributed among a great variety of fruits and vegetables – tomatoes and tomato by-products are the main sources of lycopene [27]. Lycopene has unique structural and chemical features that contribute to its specifi c biological properties. Because of the presence of high number of conjugated double bonds in lycopene, it exhibits higher singlet oxygen quenching ability as compared to other carotenoids

Figure 3. (A) The effect of 10–40 μmol/l concentrations of ascorbic acid (♦), α-tocopherol (■) and lycope`ne (▲) on carbamylation of LDL (0.6 mg protein/ml) by cyanate (20 μmol/l); (B) The comparison of inhibition percent of LDL carbamylation in the presence of 10 μmol/l, 20 μmol/l, 30 μmol/l and 40 μmol/l concentrations of ascorbic acid, α- tocopherol and lycopene. Values are represented as the mean � SD of triplicate determinations. ∗p � 0.001 compared with control (in the absence of vitamins).

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126 M. A. Ghaffar & M. Shanaki

[28]. α-tocopherol and ascorbic acid lack the con-jugated double bonds. Therefore, it can be con-cluded that conjugated double bonds are necessary for carbamylation inhibition and due to this fact lycopene probably has the most effectiveness on the inhibition of LDL carbamylation. Fuhrman et al. found that the combination of lycopene and vita-mine E, glabridin, rosarnic acid, carnosic acid, or garlic, produced a synergistic antioxidant effect on LDL oxidation [29]. In this study we also examined the effect of lycopene, ascorbic acid and α-tocopherol mixture on LDL carbamylation. The results indi-cated that the mixture of these vitamins (40 μmol/L concentration for each vitamin) has a greater effect on LDL carbamylation inhibition as compared with each of the vitamins alone. The inhibition rate of LDL carbamylation in the presence of lycopene alone and in lycopene combined with ascorbic acid and α-tocopherol was 76% and 86%, respectively. Fuhrman et al. [29] reported that if the inhibition ratio of the two mixtures (LDL�vitamins and LDL�lycopene) is more than 1, it would indicate a synergic effect. Because we also obtained a ratio of greater than 1, our fi ndings suggest that the mix-ture of these vitamins as synergist probably is more effective at reducing LDL carbamylation. Sies et al.

[30] and Frei [31] also showed a synergistic effect for antioxidant function of vitamins in protecting LDL against oxidative damages. They reported that lycopene and α-tocopherol are incorporated into the hydrophobic core of the LDL, where they play an important role in terminating the free radical-mediated reactions [30]. Ascorbic acid is also an important aqueous-phase antioxidant which can regenerate lycopene [31]. Thus a molecular mecha-nism has been proposed to partially explain the syn-ergistic effect observed with the mixture of α-tocopherol, ascorbic acid and β-carotene [30,32]. This mechanism describes the transfer of electrons from the carotenoid to the α-tocopheroxyl radical to regenerate α-tocopherol and from ascorbic acid to the resulting carotenoid radical cation to regener-ate the carotenoids. According to these reports we concluded that the synergistic effect of these vita-mins may appear to be the reason for their ability in converting the antioxidant free radicals to their native form.

The results of this study also indicated that the migration of carbamylated LDL increases on poly-acrylamide gel, which probably results from the increase of negative charge by the modifi cation of lysine amino groups. This increase of electrophoretic migration is in accordance with previous fi ndings [17,18]. In the presence of these vitamins, migration of carbamylated LDL (cLDL) decreased on gel and the smallest migration was observed in the presence of lycopene. Therefore, this fi nding supports the sug-gestion that the three vitamins play important roles in the inhibition of LDL carbamylation by cyanate. According to the results of this study and other reports we suggest that anti-carbamylation activity of ascorbic acid, α-tocopherol and lycopene is correlated with their antioxidant abilities.

In conclusion, the results obtained in the present study show that these vitamins, especially lycopene, have inhibitory effects on LDL carbamylation. In addition, mixture of lycopene with ascorbic acid and α-tocopherol showed a synergistic effect that had more effectiveness than lycopene alone in inhibiting this process. Therefore, these vitamins may have a pro-tective effect against hyperurecemia-mediated LDL damage by reducing the amount of LDL carbamyla-tion and/or reducing the effects of carbamylation. In other words, the results of this study and other similar studies may lead to new therapeutic options in pre-venting cardiovascular complications and mortality in patients with kidney disorders. Further in vitro and in vivo investigations , however, are required to elucidate the exact effects of these vitamins on patients with renal disorders.

Acknowledgements

This research was supported by a grant from the Physiology Research Center of Ahwaz Jundishapour

Figure 4. Electrophoresis migration of carbamylated LDL (cLDL) in the absence (Lane 2) and presence of a 40 μmol/L concentration of lycopene (Lane 3), ascorbic acid (Lane 4) and α-tocopherol(Lane 5) compared to native LDL (nLDL) (Lane 1) on 5% polyacrylamide gel.

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LDL carbamylation and vitamins 127

University of Medical Sciences (Ahwaz, Iran), Project No. PRC20.

Declaration of interest: The authors report no confl icts of interest. The authors alone are respon-sible for the content and writing of the paper.

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