Background It has been documented that nitric oxide (NO) donor sodium nitroprusside (SNP) and authentic peroxynitrite are capable of promoting apoptosis in a number of different cell types. Various endonucleases have been proposed as candidates responsible for the internucleosomal cleavage of the genomic DNA observed during apoptosis, but the main effect is attributed to the alkaline-DNases (Mg 2+ - and caspase-dependent) and acid-DNase. The aim of this study was to examine an  in vivo  and  in vitro  possibility for alkaline- and acid-DNases to be activated by SNP and peroxynitrite.
Background
It has been documented that nitric oxide (NO) donor sodium nitroprusside (SNP) and authentic peroxynitrite are capable of promoting apoptosis in a number of different cell types. Various endonucleases have been proposed as candidates responsible for the internucleosomal cleavage of the genomic DNA observed during apoptosis, but the main effect is attributed to the alkaline-DNases (Mg 2+ - and caspase-dependent) and acid-DNase. The aim of this study was to examine an  in vivo  and  in vitro  possibility for alkaline- and acid-DNases to be activated by SNP and peroxynitrite.in vivoin vitroResults The effect on liver tissue alkaline and acid DNase activity together with the markers of tissue and plasma oxidative and nitrosative stress (lipid peroxidation, SH group content, carbonyl groups and nitrotyrosine formation) was investigated in plasma and liver tissue. The activity of liver alkaline DNase increased and that of acid DNase decreased after  in vivo  treatment with either SNP or peroxynitrite. A difference observed between the  in vivo  and  in vitro  effect of oxide donor (i.e., SNP) or peroxynitrite upon alkaline DNase activity existed, and it may be due to the existence of the "inducible" endonuclease. After a spectrophotometric scan analysis of purified DNA, it was documented that both SNP and peroxynitrite induce various DNA modifications (nitroguanine formation being the most important one) whereas DNA fragmentation was not significantly increased.
Results
The effect on liver tissue alkaline and acid DNase activity together with the markers of tissue and plasma oxidative and nitrosative stress (lipid peroxidation, SH group content, carbonyl groups and nitrotyrosine formation) was investigated in plasma and liver tissue. The activity of liver alkaline DNase increased and that of acid DNase decreased after  in vivo  treatment with either SNP or peroxynitrite. A difference observed between the  in vivo  and  in vitro  effect of oxide donor (i.e., SNP) or peroxynitrite upon alkaline DNase activity existed, and it may be due to the existence of the "inducible" endonuclease. After a spectrophotometric scan analysis of purified DNA, it was documented that both SNP and peroxynitrite induce various DNA modifications (nitroguanine formation being the most important one) whereas DNA fragmentation was not significantly increased.in vivoin vivoin vitroConclusion Alkaline DNase activation seems to be associated with the programmed destruction of the genome, leading to the fragmentation of damaged DNA sites. Thus, the elimination of damaged cells appears to be a likely factor in prevention against mutation and carcinogenesis.
Conclusion
Alkaline DNase activation seems to be associated with the programmed destruction of the genome, leading to the fragmentation of damaged DNA sites. Thus, the elimination of damaged cells appears to be a likely factor in prevention against mutation and carcinogenesis.

Background In its response to tissue damage and inflammation induced by a variety of xenobiotics, endotoxins and disease states (such as viral hepatitis), post-ischemic and regenerative injury, the liver produces a large quantity of nitric oxide (NO). Nearly all cell types in liver tissue, including hepatocytes, Kupffer cells, stellate cells and endothelial cells, have the capacity for generating NO. It has been documented that NO is capable of promoting apoptosis in a number of different cell types, generally classified as cGMP-dependent or cGMP-independent [ 1 - 4 ]. The potential of chemical NO donor sodium nitroprusside (SNP) to induce apoptosis directly from NO liberation has been established  in vitro  [ 5 ]. The fact that NO is capable of triggering apoptosis is consistent with its ability to induce DNA damage, the inhibition of DNA synthesis and cell cycle arrest [ 6 , 7 ]. The reaction product formed between NO •  and superoxide [i.e., peroxynitrite (ONOO - )] plays a critical role in the induction of inflammatory reaction and apoptosis, but is also associated with tumor promotion and/or progression. Potentially toxic levels of peroxynitrite can be achieved whenever NO •  and O 2 .-  production is stimulated, due to the fact that a 100-fold increase in the rate of peroxynitrite formation occurs for every 10-fold increase in NO •  and O 2 .-  concentration [ 8 ]. Apoptosis, frequently termed "programmed cell death", is the form of cell death that occurs in normal liver in the course of its development and organogenesis, and in adult liver during the renewal of hepatocytes. In addition, apoptosis can be triggered by several hepatotropic viruses and toxic drugs, as well as in various liver diseases and experimental liver conditions such as hepatic allograft rejection. Degradation of the nuclear DNA, a common phenomenon observed in many organisms throughout the evolutionary scale, is one of the best-characterized biochemical features of apoptotic cell death. It has been established that the cell undergoes epigenetic reprogramming in the D 1  phase of programmed cell death, the result of which is the activation of double-stranded DNA fragmentation in the F phase during which the nuclear morphology dramatically changes [ 9 ]. The cleavage of DNA may have a protective function in that it reduces the likelihood for genes in a potentially active site to be transferred from dying cells to the nuclei of viable neighboring cells. It is possible that various endonucleases exert a DNA degrading activity, as well as that many proteins can receive DNA degrading properties upon change of pH conditions [ 11 ]. The most important aspect of apoptosis is the universal property of some proteins to exert a dual function: the protection against proteolysis and the maintenance of the structure and function of normal cells. Being free from the inhibitory complex, however, these proteins may also contribute to protein or chromatin cleavage during apoptosis [ 12 , 13 ]. Changes in DNA degradation may lead to the pathogenesis in various disorders, such as liver cancer [ 14 , 15 ]. On the basis of the pharmacological data supporting the critical role of NO and peroxynitrite in apoptosis, current research studies have evaluated the activity of alkaline and acid DNase during the administration of SNP or of peroxynitrite, as well as the changes in numerous susceptible parameters of nitrosative stress, including SH group oxidation, carbonyl group formation, lipid peroxidation and DNA modification. An assay of enzyme activity was performed using liver tissue after  in vivo  administration and  in vitro  treatment of isolated rat hepatocytes or purified commercial enzymes either with SNP or authentic peroxynitrite.
Background
In its response to tissue damage and inflammation induced by a variety of xenobiotics, endotoxins and disease states (such as viral hepatitis), post-ischemic and regenerative injury, the liver produces a large quantity of nitric oxide (NO). Nearly all cell types in liver tissue, including hepatocytes, Kupffer cells, stellate cells and endothelial cells, have the capacity for generating NO. It has been documented that NO is capable of promoting apoptosis in a number of different cell types, generally classified as cGMP-dependent or cGMP-independent [ 1 - 4 ]. The potential of chemical NO donor sodium nitroprusside (SNP) to induce apoptosis directly from NO liberation has been established  in vitro  [ 5 ]. The fact that NO is capable of triggering apoptosis is consistent with its ability to induce DNA damage, the inhibition of DNA synthesis and cell cycle arrest [ 6 , 7 ]. The reaction product formed between NO •  and superoxide [i.e., peroxynitrite (ONOO - )] plays a critical role in the induction of inflammatory reaction and apoptosis, but is also associated with tumor promotion and/or progression. Potentially toxic levels of peroxynitrite can be achieved whenever NO •  and O 2 .-  production is stimulated, due to the fact that a 100-fold increase in the rate of peroxynitrite formation occurs for every 10-fold increase in NO •  and O 2 .-  concentration [ 8 ].in vitroApoptosis, frequently termed "programmed cell death", is the form of cell death that occurs in normal liver in the course of its development and organogenesis, and in adult liver during the renewal of hepatocytes. In addition, apoptosis can be triggered by several hepatotropic viruses and toxic drugs, as well as in various liver diseases and experimental liver conditions such as hepatic allograft rejection. Degradation of the nuclear DNA, a common phenomenon observed in many organisms throughout the evolutionary scale, is one of the best-characterized biochemical features of apoptotic cell death. It has been established that the cell undergoes epigenetic reprogramming in the D 1  phase of programmed cell death, the result of which is the activation of double-stranded DNA fragmentation in the F phase during which the nuclear morphology dramatically changes [ 9 ]. The cleavage of DNA may have a protective function in that it reduces the likelihood for genes in a potentially active site to be transferred from dying cells to the nuclei of viable neighboring cells. It is possible that various endonucleases exert a DNA degrading activity, as well as that many proteins can receive DNA degrading properties upon change of pH conditions [ 11 ]. The most important aspect of apoptosis is the universal property of some proteins to exert a dual function: the protection against proteolysis and the maintenance of the structure and function of normal cells. Being free from the inhibitory complex, however, these proteins may also contribute to protein or chromatin cleavage during apoptosis [ 12 , 13 ]. Changes in DNA degradation may lead to the pathogenesis in various disorders, such as liver cancer [ 14 , 15 ].On the basis of the pharmacological data supporting the critical role of NO and peroxynitrite in apoptosis, current research studies have evaluated the activity of alkaline and acid DNase during the administration of SNP or of peroxynitrite, as well as the changes in numerous susceptible parameters of nitrosative stress, including SH group oxidation, carbonyl group formation, lipid peroxidation and DNA modification. An assay of enzyme activity was performed using liver tissue after  in vivo  administration and  in vitro  treatment of isolated rat hepatocytes or purified commercial enzymes either with SNP or authentic peroxynitrite.in vivoin vitroResults There are few data concerning the  in vivo  susceptibility of liver tissue to NO donor SNP and authentic peroxynitrite. A considerable attention has been paid to the establishment of  in vivo  tolerability and to the markers of apoptotic effects. Both NO and peroxynitrite can directly react with aromatic and sulfhydryl nucleophiles and nitrate aromatic residues. Sulfhydryl groups oxidation was documented in the plasma and, almost equally, in liver tissue. Peroxynitrite administration led to a more pronounced decrease in the concentration of other plasma free radical scavengers such as uric acid, and was followed by an increase in plasma nitrate concentration (Tables  1  and  2 ). According to the data suggesting that peroxynitrite decomposes rapidly to OH •  and NO 2  • -like species at physiological pH, it was assumed that the carbonyl groups and the lipid peroxidation product [i.e., malondyaldehyde (MDA)] may play a significant role in liver cell toxicity. Neither plasma nor liver protein carbonyls showed any significant increase. This may be a likely consequence of the significant increase in aromatic amino acids nitration, presumably tyrosine the spectral contribution of which was substracted from the samples treated with 2,4-dinitrophenylhidrazine. Plasma and liver MDA concentrations were not significantly changed, either. The obtained results do not support the data suggesting that oxygen radicals, probably generated during cellular SNP metabolism, may mediate cell toxicity and apoptosis, but do confirm previous  in vitro  observations [ 16 ]. Plasma alanine aminotranferase (ALT) activity, used as the standard liver functional test, decreased in the peroxynitrite-treated group (Table  1 ). The activity of liver alkaline DNase increased and that of acid DNase decreased after  in vivo  treatment with either SNP or peroxynitrite (Table  2 ). Table 1 Plasma levels of investigated parameters of Sprague-Dawley rats after  in vivo  treatment with nitric oxide donor (SNP) and peroxynitrite. Data expressed as Mean (SD); n = 8 per group. Parameters Control SNP Peroxynitrite Lipid peroxidation (MDA) (μmol/l) 7.10 (0.86) 6.78 (2.11) 6.64 (2.85) SH groups (μmol/l) 180.47 (36.08) 150.84 (4.78) 110.40 (11.96)* NO 2 +NO 3  (μmol/l) 45.47 (18.80) 66.82 (4.12) 81.94 (10.18) Uric acid (μmol/l) 64.82 (4.18) 22.34 (8.32) 17.50 (3.21)* Carbonyl groups (μmol/g protein) 179.59 (28.50) 144.90 (67.40) 169.06 (14.88) Nitrotyrosine (nmol/g protein) 96.02 (26.53) 136.17 (27.47) 135.11 (4.64) ALT (U/l) 31.9 (4.15) 34.44 (4.96) 28.5 (5.15) Male Sprague-Dawley rats three months old were allocated into three different groups. Either peroxynitrite (0.5 ml/kg BW of 30 mmol solution) or SNP (10 mg/kg BW) in a volume of 100 μl were administrated in bolus in systemic circulation. The control group received physiological saline solution at the same volume. All procedures were carried out as described in Methods. Asterisk: significantly different from the control (p < 0.05). Table 2 Liver levels of investigated parameters of Sprague-Dawley rats after  in vivo  treatment with nitric oxide donor (SNP) and peroxynitrite. Data expressed as Mean (SD); n = 8 per group. Parameters Control SNP Peroxynitrite Lipid peroxidation (MDA) (μmol/g protein) 54.42 (8.68) 52.5 (4.34) 58.2 (6.11) SH groups (μmol/g protein) 636.04 (74.58) 538.67 (82.87) 505.52 (41.43) NO 2 +NO 3  (μmol/g protein) 6.15 (2.91) 6.04 (2.61) 5.74 (1.03) Carbonyl groups (μmol/g protein) 4.14 (0.13) 4.25 (0.12) 4.20 (0.26) Nitrotyrosine (nmol/g protein) 10.26 (2.62) 11.40 (1.24) 16.62 (3.58) DNA (mg/g tissue) 10.71 (1.75) 12.65 (1.01) 12.15 (1.25) DNA fragmentation (%) 11.99 (0.33) 13.21 (1.43) 13.0 (0.89) Alkaline-DNase (U/g protein) 11.95 (0.57) 15.30 (0.72) 16.45 (1.04) Acid-DNase (U/g protein) 15.28 (3.42) 12.34 (0.35) 9.34 (0.76) Male Sprague-Dawley rats three months old were allocated into three different groups. Either peroxynitrite (0.5 ml/kg BW of 30 mmol solution) or SNP (10 mg/kg BW) in a volume of 100 μl were administrated  in bolus  in systemic circulation. The control group received physiological saline solution at the same volume. All procedures were carried out as described in Methods. The ultraviolet spectra of DNA were obtained by spectrophotometric scanning between 230–500 nm on a scan detecting system. According to the data by Yermilov et al. [ 17 ], the appearance of a peak between 375 and 405 nm (depending on pH) corresponds to 8-nitroguanine. In the scan analysis (Figure  1 ), the peak was between 390 and 410 nm with a maximum absorbance at 405 nm in alkaline conditions (DNA extract was adjusted to pH 10). This peak may correspond to the formation of nitro-derivatives, most probably of 8-nitroguanine. The nitroguanine peak at 405 nm was particularly apparent in peroxynitrite-treated samples. Figure 1 The peak appearance of isolated liver DNA . The extraction oftissue DNA was performed according to the method of Wannemacher et al. [50], modified by Setaro & Morley [51], with the protein and nucleic acid precipitation by using ice-cold trichloroacetic acid after lipid extraction. DNA was separated from proteins by hydrolisis of resulting pellet at 96 ± 1°C for 45 min. Samples were analyzed for DNA concentration by ultraviolet absorption difference at 260 and 290 nm. Purified DNA was employed for spectral changes, monitored by using Beckman spectrophotometer. On the basis of the data obtained by Yermilov et al. [13], the appearance of a peak between 375 and 405 nm (depending on pH) corresponds to 8-nitroguanine. The peak appearance was between 390 and 410 nm with the maximum absorbance at 405 nm, obtained in alkaline conditions (DNA extract was adjusted to pH 10). In vivo  administration of SNP or peroxynitrite tended to increase the rate of DNA fragmentation, but it was not statistically significant. The rate was estimated according to the percentage of DNA resisting centrifugation at 27 000 g (Table  2 ). After  in vitro  exposure of isolated hepatocytes to SNP or peroxynitrite, the activity of both alkaline and acid DNase decreased in a dose-dependent fashion (Figure  2 ). During  in vitro  incubation of purified enzymes DNase I and DNase II with SNP or peroxynitrite, a dose-dependent decrease of enzyme activity was also documented (Figure  3 ). Figure 2 The activity of alkaline and acid DNase after  in vitro  treatment of isolated hepatocytes with NO donor (SNP) or peroxynitrite . The isolation of hepatocytes was done according to the method already published [42], by using a 1% collagenase dissolved in RPMI 1640 medium. Hepatocytes, isolated from 8 Male Sprague-Dawley rats, were dissolved in a physiological saline solution in a concentration of approximately 10 8 cells/ml. They were divided into seven groups (each comprising 8 samples), exposed to either SNP (0.1, 1 and 10 mmol) or peroxynitrite (0.03, 0.3 and 3 mmol) for a period of 1 hour at 37°C. Given  in vitro  concentrations were calculated according to the literature data [38]. The activity of alkaline and acid-DNase was measured by the methods of Bartholeyns et al. [43] and acid soluble nucleotides were determined spectrophotometrically at 260 nm. The enzyme activity was expressed as U/g protein. Data (n = 8) in graph is putted as: Mean + SD. Figure 3 The activity of commercial DNase I and DNase II after in vitro treatment with NO donor (SNP) or peroxynitrite.  Purified enzymes DNase I (E.C. 3.1.21.1) and DNase II (E.C. 3.1.22.1) were dissolved in physiological saline solution. Hepatocytes, isolated from 8 Male Sprague-Dawley rats, were dissolved in a physiological saline solution in a concentration of approximately 10 8  cells/ml. They were divided into seven groups (each comprising 8 samples) exposed to either SNP (0.1, 1 and 10 mmol) or peroxynitrite (0.03, 0.3 and 3 mmol). The reducing agent (cysteine 1 mmol) was added to SNP to induce  in vitro  NO release [39]. The activity of alkaline and acid-DNase was measured by the methods of Bartholeyns et al. [43] and acid soluble nucleotides were determined spectrophotometrically at 260 nm. The defined units for purified DNase I and DNase II (increase in absorbance of 0.001/min in a sample containing 0.132 mg DNA, pH 7.4 or pH 5 and 3 ml of reaction mixture) were obtained from the Sigma catalogue label. Data (n = 8) in graph is putted as: Mean + SD.
Results
There are few data concerning the  in vivo  susceptibility of liver tissue to NO donor SNP and authentic peroxynitrite. A considerable attention has been paid to the establishment of  in vivo  tolerability and to the markers of apoptotic effects. Both NO and peroxynitrite can directly react with aromatic and sulfhydryl nucleophiles and nitrate aromatic residues. Sulfhydryl groups oxidation was documented in the plasma and, almost equally, in liver tissue. Peroxynitrite administration led to a more pronounced decrease in the concentration of other plasma free radical scavengers such as uric acid, and was followed by an increase in plasma nitrate concentration (Tables  1  and  2 ). According to the data suggesting that peroxynitrite decomposes rapidly to OH •  and NO 2  • -like species at physiological pH, it was assumed that the carbonyl groups and the lipid peroxidation product [i.e., malondyaldehyde (MDA)] may play a significant role in liver cell toxicity. Neither plasma nor liver protein carbonyls showed any significant increase. This may be a likely consequence of the significant increase in aromatic amino acids nitration, presumably tyrosine the spectral contribution of which was substracted from the samples treated with 2,4-dinitrophenylhidrazine. Plasma and liver MDA concentrations were not significantly changed, either. The obtained results do not support the data suggesting that oxygen radicals, probably generated during cellular SNP metabolism, may mediate cell toxicity and apoptosis, but do confirm previous  in vitro  observations [ 16 ]. Plasma alanine aminotranferase (ALT) activity, used as the standard liver functional test, decreased in the peroxynitrite-treated group (Table  1 ). The activity of liver alkaline DNase increased and that of acid DNase decreased after  in vivo  treatment with either SNP or peroxynitrite (Table  2 ).in vivoin vivoin vitroin vivoPlasma levels of investigated parameters of Sprague-Dawley rats after  in vivo  treatment with nitric oxide donor (SNP) and peroxynitrite. Data expressed as Mean (SD); n = 8 per group.in vivoParametersControlSNPPeroxynitriteMale Sprague-Dawley rats three months old were allocated into three different groups. Either peroxynitrite (0.5 ml/kg BW of 30 mmol solution) or SNP (10 mg/kg BW) in a volume of 100 μl were administrated in bolus in systemic circulation. The control group received physiological saline solution at the same volume. All procedures were carried out as described in Methods. Asterisk: significantly different from the control (p < 0.05).Liver levels of investigated parameters of Sprague-Dawley rats after  in vivo  treatment with nitric oxide donor (SNP) and peroxynitrite. Data expressed as Mean (SD); n = 8 per group.in vivoParametersControlSNPPeroxynitriteMale Sprague-Dawley rats three months old were allocated into three different groups. Either peroxynitrite (0.5 ml/kg BW of 30 mmol solution) or SNP (10 mg/kg BW) in a volume of 100 μl were administrated  in bolus  in systemic circulation. The control group received physiological saline solution at the same volume. All procedures were carried out as described in Methods.in bolusThe ultraviolet spectra of DNA were obtained by spectrophotometric scanning between 230–500 nm on a scan detecting system. According to the data by Yermilov et al. [ 17 ], the appearance of a peak between 375 and 405 nm (depending on pH) corresponds to 8-nitroguanine. In the scan analysis (Figure  1 ), the peak was between 390 and 410 nm with a maximum absorbance at 405 nm in alkaline conditions (DNA extract was adjusted to pH 10). This peak may correspond to the formation of nitro-derivatives, most probably of 8-nitroguanine. The nitroguanine peak at 405 nm was particularly apparent in peroxynitrite-treated samples.In vivo  administration of SNP or peroxynitrite tended to increase the rate of DNA fragmentation, but it was not statistically significant. The rate was estimated according to the percentage of DNA resisting centrifugation at 27 000 g (Table  2 ).In vivoAfter  in vitro  exposure of isolated hepatocytes to SNP or peroxynitrite, the activity of both alkaline and acid DNase decreased in a dose-dependent fashion (Figure  2 ). During  in vitro  incubation of purified enzymes DNase I and DNase II with SNP or peroxynitrite, a dose-dependent decrease of enzyme activity was also documented (Figure  3 ).in vitroin vitroDiscussion NO • , a free radical gaseous molecule is one of the simplest compounds found to be continuously produced in humans and animals. It can be derived from L-arginine through the enzyme nitric oxide synthase (NOS) and by different NO donors, including SNP. NO has been shown to play an unprecedented range of roles in biological systems, acting as a universal intracellular and transcellular signaling molecule and the regulator of vascular tone, cell proliferation and apoptosis [ 18 - 20 ]. Peroxynitrite is a strong, relatively long-lived oxidant with a half-life of approximately 0.5–1 s under physiological conditions. Our study confirmed that in both plasma and liver tissue peroxynitrite causes a rapid oxidation of sulfhydryl groups and thioethers, as well as the nitration and hydroxylation of aromatic compounds (Tables  1  and  2 ). A chronic exposure of hepatocytes to reactive nitrogen species exhibits a cytotoxic and cytostatic activity leading to functional and morphological alterations [ 8 , 21 ]. Cell death after exposure to different NO-donors such as SNP has been to date established through the expression of tumor suppressor gene p53 and pro-apoptotic genes such as bax, cyclin-dependent kinase inhibitor p21, the inhibited expression of anti-apoptotic protein bcl-2, the inhibited NF-κB binding activity, ERK and p-38-dependent cytochrome c release, and caspase-3 activation [ 22 - 24 ]. In contrast, the anti-apoptotic effects of NO may be mediated through the mechanisms such as blockade of the recruitment of pro-caspase-9 to the Apaf-1 apoptosome, stimulation of c-GMP-dependent protein kinase, control of mitochondrial permeability transition, induction of the heat shock protein HSP 70, and interaction with the ceramide pathway [ 25 , 26 ]. The prolonged damage of p53 gene by peroxynitrite has been associated with tumor formation. Recent results by Vincent and Maiese [ 3 ] indicate that NO donor SNP (at 300 μmol concentration) is capable of inducing strong apoptotic effects via DNA fragmentation and induction of Mg 2+ -dependent endonuclease activity in the culture of neuronal cells. In our  in vivo  study (Table  2 ), the activity of alkaline DNase increased within 24 h after exposure to SNP (achieving approximately a similar blood concentration of about 250 μmol) or to authentic peroxynitrite. Several molecules involved in nuclear DNA fragmentation have been detected and characterized based on their ionic sensitivity. Besides the presence of constitutive Ca 2+ /Mg 2+ -dependent endonucleases, a great deal of endonuclease activity within a 7.2–8.0 pH range most probably represents the inducible form of DNase. The molecular weights of the constitutive (NO-independent) and inducible (NO-dependent) endonuclease are similar, as well as their optimum pH range (7.5–8.0). A likely conclusion is that Mg 2+ -dependent endonuclease seems to be a result of  de novo  synthesized or the pre-existing Ca 2+ /Mg 2+ -dependent endonuclease activation. Up to now, several Mg 2+ - or Ca 2+ /Mg 2+ -dependent alkaline DNases (DNase I) with an optimum activity within the range of 7.5–9.5 have been purified. Some of them, including specific caspase3-activated DNase (CAD), are active upon release of the specific inhibitor ICAD [ 27 , 28 ]. DNase gamma has been documented as a critical component of apoptotic machinery, in that it cleaves the chromosomal DNA into nucleosomal units, thus leading to DNA ladder formation [ 29 ]. The alkaline DNase, active only during apoptosis, has been documented to be inherent to cyclophilins (A, B and C) as well, irrespective of their protein folding (peptidylprolyl cis-trans-isomerase) activity. All of them have the ability to degrade the supercoiled, single stranded and double stranded DNA [ 30 , 31 ]. Besides alkaline DNases, the cation-independent endonuclease with an optimum activity at pH 5, known as acid or DNase II, was identified. One leucocyte elastase inhibitor (LEI) can also exert an acid DNase activity after post-translational modification through the proteolytic cleavage [ 32 ]. The specific involvement of DNase II in physiological nuclear degradation during apoptosis could not be excluded upon decrease of intracellular pH values below 7 with a proton ionophore. Three potential  N -nitrosylation sites are important for DNase II regulation [ 32 , 33 ]. Since our experimental data indicated a decrease in acid DNase activity 24 h after exposure to SNP or peroxynitrite (Table  2 ), the inhibition of DNase II may be explained by the nitrosylation of its susceptible sites. Indeed, when isolated hepatocytes were exposed to SNP or peroxynitrite for 1 h, a dose-dependent inhibition of DNase II was also documented (Fig.  2 ). The same result was obtained after exposure of purified enzyme to SNP (in the presence of the reducing agent cysteine 1 mmol) or peroxynitrite (Fig.  3 ). The formation of 8-nitroguanine, 8-oxo-deoxyguanine and oxazolone and the oxidative modification of 2'-deoxyribose into TBA-responsive compounds are the most prominent nucleotide modifications after reactive nitrogen species attack [ 34 , 35 ]. A highly potential mutagenic product 8-nitroguanine can be depurinated yielding apurinic sites capable of inducing GC→TA transversions, GC→CG transversions and deletions [ 17 , 36 ]. The appearance of the nitroguanine peak during the scan analysis of purified DNA at 405 nm was documented in our study (Fig.  1 ). The rate of DNA fragmentation tended to be increased, but the difference was not significant (Table  2 ).
Discussion
NO • , a free radical gaseous molecule is one of the simplest compounds found to be continuously produced in humans and animals. It can be derived from L-arginine through the enzyme nitric oxide synthase (NOS) and by different NO donors, including SNP. NO has been shown to play an unprecedented range of roles in biological systems, acting as a universal intracellular and transcellular signaling molecule and the regulator of vascular tone, cell proliferation and apoptosis [ 18 - 20 ]. Peroxynitrite is a strong, relatively long-lived oxidant with a half-life of approximately 0.5–1 s under physiological conditions. Our study confirmed that in both plasma and liver tissue peroxynitrite causes a rapid oxidation of sulfhydryl groups and thioethers, as well as the nitration and hydroxylation of aromatic compounds (Tables  1  and  2 ). A chronic exposure of hepatocytes to reactive nitrogen species exhibits a cytotoxic and cytostatic activity leading to functional and morphological alterations [ 8 , 21 ]. Cell death after exposure to different NO-donors such as SNP has been to date established through the expression of tumor suppressor gene p53 and pro-apoptotic genes such as bax, cyclin-dependent kinase inhibitor p21, the inhibited expression of anti-apoptotic protein bcl-2, the inhibited NF-κB binding activity, ERK and p-38-dependent cytochrome c release, and caspase-3 activation [ 22 - 24 ]. In contrast, the anti-apoptotic effects of NO may be mediated through the mechanisms such as blockade of the recruitment of pro-caspase-9 to the Apaf-1 apoptosome, stimulation of c-GMP-dependent protein kinase, control of mitochondrial permeability transition, induction of the heat shock protein HSP 70, and interaction with the ceramide pathway [ 25 , 26 ]. The prolonged damage of p53 gene by peroxynitrite has been associated with tumor formation. Recent results by Vincent and Maiese [ 3 ] indicate that NO donor SNP (at 300 μmol concentration) is capable of inducing strong apoptotic effects via DNA fragmentation and induction of Mg 2+ -dependent endonuclease activity in the culture of neuronal cells. In our  in vivo  study (Table  2 ), the activity of alkaline DNase increased within 24 h after exposure to SNP (achieving approximately a similar blood concentration of about 250 μmol) or to authentic peroxynitrite. Several molecules involved in nuclear DNA fragmentation have been detected and characterized based on their ionic sensitivity. Besides the presence of constitutive Ca 2+ /Mg 2+ -dependent endonucleases, a great deal of endonuclease activity within a 7.2–8.0 pH range most probably represents the inducible form of DNase. The molecular weights of the constitutive (NO-independent) and inducible (NO-dependent) endonuclease are similar, as well as their optimum pH range (7.5–8.0). A likely conclusion is that Mg 2+ -dependent endonuclease seems to be a result of  de novo  synthesized or the pre-existing Ca 2+ /Mg 2+ -dependent endonuclease activation. Up to now, several Mg 2+ - or Ca 2+ /Mg 2+ -dependent alkaline DNases (DNase I) with an optimum activity within the range of 7.5–9.5 have been purified. Some of them, including specific caspase3-activated DNase (CAD), are active upon release of the specific inhibitor ICAD [ 27 , 28 ]. DNase gamma has been documented as a critical component of apoptotic machinery, in that it cleaves the chromosomal DNA into nucleosomal units, thus leading to DNA ladder formation [ 29 ]. The alkaline DNase, active only during apoptosis, has been documented to be inherent to cyclophilins (A, B and C) as well, irrespective of their protein folding (peptidylprolyl cis-trans-isomerase) activity. All of them have the ability to degrade the supercoiled, single stranded and double stranded DNA [ 30 , 31 ]. Besides alkaline DNases, the cation-independent endonuclease with an optimum activity at pH 5, known as acid or DNase II, was identified. One leucocyte elastase inhibitor (LEI) can also exert an acid DNase activity after post-translational modification through the proteolytic cleavage [ 32 ]. The specific involvement of DNase II in physiological nuclear degradation during apoptosis could not be excluded upon decrease of intracellular pH values below 7 with a proton ionophore. Three potential  N -nitrosylation sites are important for DNase II regulation [ 32 , 33 ]. Since our experimental data indicated a decrease in acid DNase activity 24 h after exposure to SNP or peroxynitrite (Table  2 ), the inhibition of DNase II may be explained by the nitrosylation of its susceptible sites. Indeed, when isolated hepatocytes were exposed to SNP or peroxynitrite for 1 h, a dose-dependent inhibition of DNase II was also documented (Fig.  2 ). The same result was obtained after exposure of purified enzyme to SNP (in the presence of the reducing agent cysteine 1 mmol) or peroxynitrite (Fig.  3 ).in vivode novoNThe formation of 8-nitroguanine, 8-oxo-deoxyguanine and oxazolone and the oxidative modification of 2'-deoxyribose into TBA-responsive compounds are the most prominent nucleotide modifications after reactive nitrogen species attack [ 34 , 35 ]. A highly potential mutagenic product 8-nitroguanine can be depurinated yielding apurinic sites capable of inducing GC→TA transversions, GC→CG transversions and deletions [ 17 , 36 ]. The appearance of the nitroguanine peak during the scan analysis of purified DNA at 405 nm was documented in our study (Fig.  1 ). The rate of DNA fragmentation tended to be increased, but the difference was not significant (Table  2 ).Conclusions In vivo  administrated SNP and peroxynitrite increase the activity of alkaline DNase. They also induced DNA modifications, such as nitroguanine formation. The obtained DNase activation seems to be associated with the programmed destruction of the genome and cell death. Given the above results and observations, the elimination of damaged hepatic cells appears to be a likely factor in prevention against mutation and carcinogenesis.
Conclusions
In vivo  administrated SNP and peroxynitrite increase the activity of alkaline DNase. They also induced DNA modifications, such as nitroguanine formation. The obtained DNase activation seems to be associated with the programmed destruction of the genome and cell death. Given the above results and observations, the elimination of damaged hepatic cells appears to be a likely factor in prevention against mutation and carcinogenesis.In vivoMethods Chemicals SNP, DNA, DNase I (E.C. 3.1.21.1.) and DNase II (E.C. 3.1.22.1) were obtained from Sigma-Aldrich Company. RPMI-1640, fetal calf serum (FCS) and collagenase were purchased from ICN (Costa Mesa, CA). Authentic peroxynitrite was freshly synthesized by the quench-flow technique [ 37 ] and its concentration was monitored in alkaline solution before use in each experiment by measuring the extinction coefficient at 302 nm [ 38 ]. All other chemicals were of the highest purity range. In vivo  study Twenty-four male Sprague-Dawley rats, three months old, were divided into three different groups, each comprising 8 animals. Either SNP (10 mg/kg BW) or peroxynitrite (0.5 ml/kg BW of 30 mmol solution) in a volume of 100 μl were administrated  in bolus  in systemic circulation by intraventricular injection under penthobarbital sodium anesthesia. The concentrations were calculated according to the literature data concerning their  in vivo  tolerability and in vitro ability to induce apoptotic effects [ 39 , 40 ]. The calculation of peroxynitrite intra-arterial concentration (6 nmol) was done according to its biological half-life of about 0.6 s, cardiac output of 40 ml/min/100 g and circulating volume of 20 ml and 250 g of rat BW [ 41 ]. The corresponding control group received physiological saline solution in the same volume. The rats were killed 24 h afterwards, under the same anesthesia. Blood was collected from the abdominal aorta and livers were quickly removed, frozen and homogenised on ice. Isolation of hepatocytes The isolation of hepatocytes was done according to a method already published [ 42 ], by using a 1% collagenase dissolved in RPMI 1640 medium. Collagenase was inhibited by using 10% FCS and cells were washed twice in physiological saline solution. Hepatocytes were isolated from 8 Male Sprague-Dawley rats. They were dissolved in a physiological saline solution in a concentration approximately 10 8  cells/ml. They were divided into seven groups (each comprising 8 samples), exposed to either SNP (0.1, 1 and 10 mmol) or peroxynitrite (0.03, 0.3 and 3 mmol) for a period of 1 hour at 37°C. Given  in vitro  concentrations were calculated according to the literature data [ 38 ]. Purified enzymes DNase I and DNase II were dissolved in physiological saline solution, exposed to the same concentrations of SNP and peroxynitrite, except that the reducing agent (cysteine 1 mmol) was added to SNP to induce  in vitro  NO release [ 39 ]. Methods for alkaline and acid-DNase The activity of alkaline and acid-DNase was measured by the methods of Bartholeyns et al. [ 43 ] and acid soluble nucleotides were determined spectrophotometrically at 260 nm. The enzyme activity was expressed as U/g protein, for tissue and cell samples. The defined units for purified DNase I and DNase II (increase in absorbance of 0.001 / min in a sample containing 0.132 mg DNA, pH 7.4 or pH 5 and 3 ml of reaction mixture) were obtained from the Sigma catalogue label. Extraction of DNA and proteins The extraction of tissue DNA and proteins was performed according to the method of Wannemacher et al. [ 44 ] modified by Setaro & Morley [ 45 ] by protein and nucleic acid precipitation using ice-cold trichloroacetic acid (TCA), 0.6 N, after lipid extraction. RNA and DNA were isolated by using cold 60% perchloric acid (PCA). DNA was separated from proteins by hydrolysis of resulting pellet at 96 ± 1°C for 45 min after adding 0.5 N PCA. Tissue protein content was measured according to the Lowry et al. procedure [ 46 ]. Samples were analysed for DNA concentration by an ultraviolet absorption difference at 260 and 290 nm. Purified DNA was employed for spectral changes monitored by using Beckman DU 530 spectrophotometer. Protein carbonyls and protein nitrotyrosine were measured in plasma proteins and the remaining protein pellet according to the method of Oliver et al. [ 47 ] modified by Tien et al. [ 48 ]. DNA fragmentation assay was performed according to the method of Jones et al. [ 49 ] based on the percentage of DNA resisting centrifugation at 27 000 g for 20 min. The proportion is expressed as percentage of the total DNA in the uncentrifugated sample. Protein carbonyls were quantified by spectrophotometric measurement of their 2,4 dinitrophenylhydrazone derivatives (ε 370 nm = 22000 M-1 cm -1 ). The difference between the spectrum of the DNPH-treated sample and that of the HCl control was determined and expressed as μmol DNPH/g protein. As nitrotyrosine also absorbs at 370 nm, it was measured according to its spectral contribution at 370 nm. Plasma and tissue SH groups were measured by using DTMB according to the Elman method [ 50 ]. Plasma and tissue lipid peroxidation product MDA was measured according to the method of Ohkava et al. [ 51 ]. Nitrates were measured according to the method of Navarro-Gonzales et al. [ 52 ]. Plasma uric acid and ALT were measured using the Synchron analyzer. Statistics Statistical analysis was made with the software SPSS. The effect of treatments was firstly evaluated by one-way ANOVA. If there was a significant effect, experimental data sets were compared against the control group by the Dunnett post hoc test. Significance level was set at α = 0.05. Data were normally distributed with equal variances among groups.
Methods
Chemicals SNP, DNA, DNase I (E.C. 3.1.21.1.) and DNase II (E.C. 3.1.22.1) were obtained from Sigma-Aldrich Company. RPMI-1640, fetal calf serum (FCS) and collagenase were purchased from ICN (Costa Mesa, CA). Authentic peroxynitrite was freshly synthesized by the quench-flow technique [ 37 ] and its concentration was monitored in alkaline solution before use in each experiment by measuring the extinction coefficient at 302 nm [ 38 ]. All other chemicals were of the highest purity range.
Chemicals
SNP, DNA, DNase I (E.C. 3.1.21.1.) and DNase II (E.C. 3.1.22.1) were obtained from Sigma-Aldrich Company. RPMI-1640, fetal calf serum (FCS) and collagenase were purchased from ICN (Costa Mesa, CA). Authentic peroxynitrite was freshly synthesized by the quench-flow technique [ 37 ] and its concentration was monitored in alkaline solution before use in each experiment by measuring the extinction coefficient at 302 nm [ 38 ]. All other chemicals were of the highest purity range.In vivo  study Twenty-four male Sprague-Dawley rats, three months old, were divided into three different groups, each comprising 8 animals. Either SNP (10 mg/kg BW) or peroxynitrite (0.5 ml/kg BW of 30 mmol solution) in a volume of 100 μl were administrated  in bolus  in systemic circulation by intraventricular injection under penthobarbital sodium anesthesia. The concentrations were calculated according to the literature data concerning their  in vivo  tolerability and in vitro ability to induce apoptotic effects [ 39 , 40 ]. The calculation of peroxynitrite intra-arterial concentration (6 nmol) was done according to its biological half-life of about 0.6 s, cardiac output of 40 ml/min/100 g and circulating volume of 20 ml and 250 g of rat BW [ 41 ]. The corresponding control group received physiological saline solution in the same volume. The rats were killed 24 h afterwards, under the same anesthesia. Blood was collected from the abdominal aorta and livers were quickly removed, frozen and homogenised on ice.
In vivo  study
In vivoTwenty-four male Sprague-Dawley rats, three months old, were divided into three different groups, each comprising 8 animals. Either SNP (10 mg/kg BW) or peroxynitrite (0.5 ml/kg BW of 30 mmol solution) in a volume of 100 μl were administrated  in bolus  in systemic circulation by intraventricular injection under penthobarbital sodium anesthesia. The concentrations were calculated according to the literature data concerning their  in vivo  tolerability and in vitro ability to induce apoptotic effects [ 39 , 40 ]. The calculation of peroxynitrite intra-arterial concentration (6 nmol) was done according to its biological half-life of about 0.6 s, cardiac output of 40 ml/min/100 g and circulating volume of 20 ml and 250 g of rat BW [ 41 ]. The corresponding control group received physiological saline solution in the same volume. The rats were killed 24 h afterwards, under the same anesthesia. Blood was collected from the abdominal aorta and livers were quickly removed, frozen and homogenised on ice.in bolusin vivoIsolation of hepatocytes The isolation of hepatocytes was done according to a method already published [ 42 ], by using a 1% collagenase dissolved in RPMI 1640 medium. Collagenase was inhibited by using 10% FCS and cells were washed twice in physiological saline solution. Hepatocytes were isolated from 8 Male Sprague-Dawley rats. They were dissolved in a physiological saline solution in a concentration approximately 10 8  cells/ml. They were divided into seven groups (each comprising 8 samples), exposed to either SNP (0.1, 1 and 10 mmol) or peroxynitrite (0.03, 0.3 and 3 mmol) for a period of 1 hour at 37°C. Given  in vitro  concentrations were calculated according to the literature data [ 38 ]. Purified enzymes DNase I and DNase II were dissolved in physiological saline solution, exposed to the same concentrations of SNP and peroxynitrite, except that the reducing agent (cysteine 1 mmol) was added to SNP to induce  in vitro  NO release [ 39 ].
Isolation of hepatocytes
The isolation of hepatocytes was done according to a method already published [ 42 ], by using a 1% collagenase dissolved in RPMI 1640 medium. Collagenase was inhibited by using 10% FCS and cells were washed twice in physiological saline solution. Hepatocytes were isolated from 8 Male Sprague-Dawley rats. They were dissolved in a physiological saline solution in a concentration approximately 10 8  cells/ml. They were divided into seven groups (each comprising 8 samples), exposed to either SNP (0.1, 1 and 10 mmol) or peroxynitrite (0.03, 0.3 and 3 mmol) for a period of 1 hour at 37°C. Given  in vitro  concentrations were calculated according to the literature data [ 38 ]. Purified enzymes DNase I and DNase II were dissolved in physiological saline solution, exposed to the same concentrations of SNP and peroxynitrite, except that the reducing agent (cysteine 1 mmol) was added to SNP to induce  in vitro  NO release [ 39 ].in vitroin vitroMethods for alkaline and acid-DNase The activity of alkaline and acid-DNase was measured by the methods of Bartholeyns et al. [ 43 ] and acid soluble nucleotides were determined spectrophotometrically at 260 nm. The enzyme activity was expressed as U/g protein, for tissue and cell samples. The defined units for purified DNase I and DNase II (increase in absorbance of 0.001 / min in a sample containing 0.132 mg DNA, pH 7.4 or pH 5 and 3 ml of reaction mixture) were obtained from the Sigma catalogue label.
Methods for alkaline and acid-DNase
The activity of alkaline and acid-DNase was measured by the methods of Bartholeyns et al. [ 43 ] and acid soluble nucleotides were determined spectrophotometrically at 260 nm. The enzyme activity was expressed as U/g protein, for tissue and cell samples. The defined units for purified DNase I and DNase II (increase in absorbance of 0.001 / min in a sample containing 0.132 mg DNA, pH 7.4 or pH 5 and 3 ml of reaction mixture) were obtained from the Sigma catalogue label.Extraction of DNA and proteins The extraction of tissue DNA and proteins was performed according to the method of Wannemacher et al. [ 44 ] modified by Setaro & Morley [ 45 ] by protein and nucleic acid precipitation using ice-cold trichloroacetic acid (TCA), 0.6 N, after lipid extraction. RNA and DNA were isolated by using cold 60% perchloric acid (PCA). DNA was separated from proteins by hydrolysis of resulting pellet at 96 ± 1°C for 45 min after adding 0.5 N PCA. Tissue protein content was measured according to the Lowry et al. procedure [ 46 ]. Samples were analysed for DNA concentration by an ultraviolet absorption difference at 260 and 290 nm. Purified DNA was employed for spectral changes monitored by using Beckman DU 530 spectrophotometer. Protein carbonyls and protein nitrotyrosine were measured in plasma proteins and the remaining protein pellet according to the method of Oliver et al. [ 47 ] modified by Tien et al. [ 48 ]. DNA fragmentation assay was performed according to the method of Jones et al. [ 49 ] based on the percentage of DNA resisting centrifugation at 27 000 g for 20 min. The proportion is expressed as percentage of the total DNA in the uncentrifugated sample. Protein carbonyls were quantified by spectrophotometric measurement of their 2,4 dinitrophenylhydrazone derivatives (ε 370 nm = 22000 M-1 cm -1 ). The difference between the spectrum of the DNPH-treated sample and that of the HCl control was determined and expressed as μmol DNPH/g protein. As nitrotyrosine also absorbs at 370 nm, it was measured according to its spectral contribution at 370 nm. Plasma and tissue SH groups were measured by using DTMB according to the Elman method [ 50 ]. Plasma and tissue lipid peroxidation product MDA was measured according to the method of Ohkava et al. [ 51 ]. Nitrates were measured according to the method of Navarro-Gonzales et al. [ 52 ]. Plasma uric acid and ALT were measured using the Synchron analyzer.
Extraction of DNA and proteins
The extraction of tissue DNA and proteins was performed according to the method of Wannemacher et al. [ 44 ] modified by Setaro & Morley [ 45 ] by protein and nucleic acid precipitation using ice-cold trichloroacetic acid (TCA), 0.6 N, after lipid extraction. RNA and DNA were isolated by using cold 60% perchloric acid (PCA). DNA was separated from proteins by hydrolysis of resulting pellet at 96 ± 1°C for 45 min after adding 0.5 N PCA. Tissue protein content was measured according to the Lowry et al. procedure [ 46 ]. Samples were analysed for DNA concentration by an ultraviolet absorption difference at 260 and 290 nm. Purified DNA was employed for spectral changes monitored by using Beckman DU 530 spectrophotometer. Protein carbonyls and protein nitrotyrosine were measured in plasma proteins and the remaining protein pellet according to the method of Oliver et al. [ 47 ] modified by Tien et al. [ 48 ]. DNA fragmentation assay was performed according to the method of Jones et al. [ 49 ] based on the percentage of DNA resisting centrifugation at 27 000 g for 20 min. The proportion is expressed as percentage of the total DNA in the uncentrifugated sample. Protein carbonyls were quantified by spectrophotometric measurement of their 2,4 dinitrophenylhydrazone derivatives (ε 370 nm = 22000 M-1 cm -1 ). The difference between the spectrum of the DNPH-treated sample and that of the HCl control was determined and expressed as μmol DNPH/g protein. As nitrotyrosine also absorbs at 370 nm, it was measured according to its spectral contribution at 370 nm. Plasma and tissue SH groups were measured by using DTMB according to the Elman method [ 50 ]. Plasma and tissue lipid peroxidation product MDA was measured according to the method of Ohkava et al. [ 51 ]. Nitrates were measured according to the method of Navarro-Gonzales et al. [ 52 ]. Plasma uric acid and ALT were measured using the Synchron analyzer.Statistics Statistical analysis was made with the software SPSS. The effect of treatments was firstly evaluated by one-way ANOVA. If there was a significant effect, experimental data sets were compared against the control group by the Dunnett post hoc test. Significance level was set at α = 0.05. Data were normally distributed with equal variances among groups.
Statistics
Statistical analysis was made with the software SPSS. The effect of treatments was firstly evaluated by one-way ANOVA. If there was a significant effect, experimental data sets were compared against the control group by the Dunnett post hoc test. Significance level was set at α = 0.05. Data were normally distributed with equal variances among groups.Authors' contributions GK carried out the  in vivo  and  in vitro  experiments, culture experiments and wrote the paper. RP and GN carried out DNA spectral analysis. TC performed measurement of SH groups and lipid peroxides. IS performed the measurement of nitrates and nitrites. TJ assisted during  in vivo  and  in vitro  experiments and did the graphical presentation. DS performed statistical analysis and assisted during  in vivo  experiments. DP and RK assisted during  in vitro  experiments and participated in the design of the study. All the authors read and approved the final manuscript.
Authors' contributions
GK carried out the  in vivo  and  in vitro  experiments, culture experiments and wrote the paper. RP and GN carried out DNA spectral analysis. TC performed measurement of SH groups and lipid peroxides. IS performed the measurement of nitrates and nitrites. TJ assisted during  in vivo  and  in vitro  experiments and did the graphical presentation. DS performed statistical analysis and assisted during  in vivo  experiments. DP and RK assisted during  in vitro  experiments and participated in the design of the study. All the authors read and approved the final manuscript.in vivoin vitroin vivoin vitroin vivoin vitro


Background The Steno hypothesis states that changes in basement membrane anionic charge leads to diabetic microvascular complications. In diabetic nephropathy, loss of basement membrane glycosaminoglycans and the association between glomerular basement membrane heparan sulphate and proteinuria has been documented. A correlation between erythrocyte surface and the glomerular capillary wall charges has also been observed. The aim of this study is to evaluate the relationship between retinopathy and erythrocyte anionic charge and urinary glycosaminoglycan excretion in type 2 diabetic patients.
Background
The Steno hypothesis states that changes in basement membrane anionic charge leads to diabetic microvascular complications. In diabetic nephropathy, loss of basement membrane glycosaminoglycans and the association between glomerular basement membrane heparan sulphate and proteinuria has been documented. A correlation between erythrocyte surface and the glomerular capillary wall charges has also been observed.The aim of this study is to evaluate the relationship between retinopathy and erythrocyte anionic charge and urinary glycosaminoglycan excretion in type 2 diabetic patients.Methods 49 subjects (58 ± 7 yrs, M/F 27/22) with type 2 diabetes with proliferative retinopathy (n = 13), nonproliferative retinopathy (n = 13) and without retinopathy (n = 23) were included in the study. 38 healthy subjects were selected as control group (57 ± 5 yrs, M/F 19/19). Erythrocyte anionic charge (EAC) was determined by the binding of the cationic dye, alcian blue. Urinary glycosaminoglycan and microalbumin excretion were measured.
Methods
49 subjects (58 ± 7 yrs, M/F 27/22) with type 2 diabetes with proliferative retinopathy (n = 13), nonproliferative retinopathy (n = 13) and without retinopathy (n = 23) were included in the study. 38 healthy subjects were selected as control group (57 ± 5 yrs, M/F 19/19). Erythrocyte anionic charge (EAC) was determined by the binding of the cationic dye, alcian blue. Urinary glycosaminoglycan and microalbumin excretion were measured.Results EAC was significantly decreased in diabetic patients with retinopathy (255 ± 30 ng alcian blue/10 6  RBC, 312 ± 30 ng alcian blue/10 6  RBC for diabetic and control groups respectively, p < 0.001). We did not observe an association between urinary GAG and microalbumin excretion and diabetic retinopathy. EAC is found to be negatively corralated with microalbuminuria in all groups.
Results
EAC was significantly decreased in diabetic patients with retinopathy (255 ± 30 ng alcian blue/10 6  RBC, 312 ± 30 ng alcian blue/10 6  RBC for diabetic and control groups respectively, p < 0.001). We did not observe an association between urinary GAG and microalbumin excretion and diabetic retinopathy. EAC is found to be negatively corralated with microalbuminuria in all groups.Conclusions We conclude that type 2 diabetic patients with low erythrocyte anionic charge are associated with diabetic retinopathy. Reduction of negative charge of basement membranes may indicate general changes in microvasculature rather than retinopathy. More prospective and large studies needs to clarify the role of glycosaminoglycans on progression of retinopathy in type 2 diabetic patients.
Conclusions
We conclude that type 2 diabetic patients with low erythrocyte anionic charge are associated with diabetic retinopathy. Reduction of negative charge of basement membranes may indicate general changes in microvasculature rather than retinopathy. More prospective and large studies needs to clarify the role of glycosaminoglycans on progression of retinopathy in type 2 diabetic patients.

Background Diabetic retinopathy is the leading cause of blindness in diabetic adults [ 1 , 2 ]. During the first two decades of the disease, nearly all patients with type 1 diabetes and > 60 % of type 2 diabetes have retinopathy. The duration of the diabetes is probably the strongest predictor for development and progression of retinopathy [ 3 ]. The incidence of retinopathy positively correlates with HbA 1c  [ 4 ]. Diabetic patients with evidence of nephropathy are characterized by a 5 to 10 times higher incidence of proliferative retinopathy [ 5 ]. Albuminuria not only is associated with kidney disease but, is also a strong predictor of cardiovascular disease and proliferative retinopathy, suggesting it reflects a generalized vascular disease [ 6 ]. Thus, the coincidence of generalized vascular dysfunction, albuminuria, mesangial expansion, proliferative retinopathy and accelerated development of atherosclerosis suggests a common cause of abnormalities in susceptible diabetic patients. The Steno hypothesis proposed that increased loss of anionic charge from basement membranes leads to diabetic microangiopathy [ 7 ]. This hypothesis has been proven for diabetic nephropathy but it's not clear for diabetic retinopathy. Anionic content of basement membranes predominantly produced by glycosaminoglycan molecules (GAG). They constitute the charge selective barrier of the glomerul basement membrane [ 8 ]. Several clinical studies indicate that loss of the GAG is associated with diabetic albuminuria in diabetic and experimental models [ 9 , 10 ]. Although abnormal GAG metabolism is a well known phenomen in diabetic nephropathy, it is still debated whether it has a pathogenic role in diabetic retinopathy. The aim of this study was to evaluate the relationship between diabetic retinopathy and erythrocyte anionic charge as well as the urinary glycosaminoglycan excretion in type 2 diabetic patients.
Background
Diabetic retinopathy is the leading cause of blindness in diabetic adults [ 1 , 2 ]. During the first two decades of the disease, nearly all patients with type 1 diabetes and > 60 % of type 2 diabetes have retinopathy. The duration of the diabetes is probably the strongest predictor for development and progression of retinopathy [ 3 ]. The incidence of retinopathy positively correlates with HbA 1c  [ 4 ].Diabetic patients with evidence of nephropathy are characterized by a 5 to 10 times higher incidence of proliferative retinopathy [ 5 ]. Albuminuria not only is associated with kidney disease but, is also a strong predictor of cardiovascular disease and proliferative retinopathy, suggesting it reflects a generalized vascular disease [ 6 ]. Thus, the coincidence of generalized vascular dysfunction, albuminuria, mesangial expansion, proliferative retinopathy and accelerated development of atherosclerosis suggests a common cause of abnormalities in susceptible diabetic patients.The Steno hypothesis proposed that increased loss of anionic charge from basement membranes leads to diabetic microangiopathy [ 7 ]. This hypothesis has been proven for diabetic nephropathy but it's not clear for diabetic retinopathy.Anionic content of basement membranes predominantly produced by glycosaminoglycan molecules (GAG). They constitute the charge selective barrier of the glomerul basement membrane [ 8 ]. Several clinical studies indicate that loss of the GAG is associated with diabetic albuminuria in diabetic and experimental models [ 9 , 10 ].Although abnormal GAG metabolism is a well known phenomen in diabetic nephropathy, it is still debated whether it has a pathogenic role in diabetic retinopathy. The aim of this study was to evaluate the relationship between diabetic retinopathy and erythrocyte anionic charge as well as the urinary glycosaminoglycan excretion in type 2 diabetic patients.Methods 49 outpatients (27 male, 22 female) with type 2 diabetes mellitus diagnosed after the age of 30, were included and divided into 3 subgroups according to severety of retinopathy. Diabetes was diagnosed according to American Diabetes Association criteria. The onset of diabetes was after the age of 30 in these patients. The study was approved by the institutional ethics committee and patients gave written informed consent. Exclusion criteria included hypertension, myocardial infarction, cerebrovascular disease, heart failure, treatment with antiaggregants, steroids or other drugs that effect blood pressure and glucose, serum creatinine > 200 μmol/l, connective tissue disorders and other systemic disease. Renal and bladder infection diseases of patients and controls were excluded biochemically and microbiologically. The ocular fundus was examined by an ophthalmologist after dilation of the pupils and classified. The patients were divided into three groups according to the severity of retinopathy. Group 1 consisted of 23 patients without retinopathy (R 0 ). Group 2 consisted of 13 patients with nonproliferative retinopathy (R 1 -hemorrhages, microaneurysms, cotton-wool exudates). The 13 patients in group 3 (R 2 ) had proliferative retinopathy (R 2 -neovascularisations, vitreous hemorrhages, ablatio of the retina). Age and sex matched 38 healthy volunteers were included as healthy controls. Demographic characteristics of the groups are shown in Table  1 . Venous samples were collected after an overnight fast and 24 hour urine samples collected. The charge on erythrocytes (RBC) was measured by means of cationic dye alcian blue 8GX (Sigma catalogue no: A 5268) according to the method of Levin with minor modifications as follows: [ 11 ]. From citrated venous blood samples, platelets and leukocytes were removed using the method of Beutler et al. [ 12 ]. After removal of platelets and leucocytes erythrocytes were washed 3 times in phosphate buffered saline (PBS), and subsequently a fraction of RBC was resuspended in PBS containing alcian blue at a final concentration of 250 mg/l. After a 30 min incubation at 37°C, the RBC suspension was centrifuged, and the remaining alcian blue concentration was measured in the supernatant with a Shimadzu UV 1201 V spectrophotometer (Shimadzu, Japan) at a wavelength of 650 nm. Each determination represents the mean of 2 assays. The quantity of alcian blue bound to RBCs was expressed as nanograms of alcian blue per 10 6  RBCs. In our experimental conditions the intraassay and interassay coefficients of variation were 5.8 and 7.6 % for 100 nanograms of alcian blue per 10 6  RBCs. Urinary glycosaminoglycan (U GAG ) excretion was determined in 24 hr urine samples spectrophotometrically at 520 nm by the addition of dimethylmethylene blue (Aldrich Chem Co., USA) and bovine kidney heparan sulfate as standard (Sigma catalogue no H 7640) [ 13 ]. In our experimental conditions the intraassay and interassay coefficients of variation were 1.5 and 2.4 % for 10 mg/l glycosaminoglycan, respectively. Serum sialic acid was measured in serum with a colorimetric method described by Svennerholm with a Shimadzu UV-1201 spectrophotometer (Shimadzu, Japan) with a wavelength of 525 nm. Using N-acetyl neuraminic acid (Sigma catalogue no: A 3007) as standard [ 14 ]. Intra assay and interassay coefficients of variations were 6.6 and 9.2 % for 1 mmol/l sialic acid respectively. Urinary albumin excretion was measured by an immunoturbidemetric assay (Roche diagnostics, USA) on automated clinical chemistry analyzer (Hitachi 902). In all patients, the annual level was determined as the mean of urinary albumin excretions in three 24 h urine collections taken at home during normal physical activity. The intraassay and interassay coefficients of variation were 1.3 and 4.3 % for a mean value of 25 mg/l albumin concentration. Serum glucose, cholesterol, triglyceride and HDL (Dade Behring, USA) were evaluated with enzymatic methods (Dade Behring, USA) on an automated clinical chemistry analyzer (Dimension RxL). Creatinin was assayed by the Jaffe reaction on an automated clinical chemistry analyzer (Dimension RxL). HbA 1C  (reference range 4.4–6.0 %) was measured by a turbidimetric inhibition immunoassay technique (Roche Diagnostics, USA) on automated clinical chemistry analyzer (Hitachi 902). The intraassay and interassay coefficients of variation were 1.8 and 3.0 % for a mean value of 10.5 % HbA 1C  concentration. The analysis of the data was performed with a PC compatible Instat-II programme. Paired t test and ANOVA were used where appropriate. Correlation analysis was performed with Spearman rank test. The differences were considered significant when the probability was p < 0.05. The results were given as mean ± SD.
Methods
49 outpatients (27 male, 22 female) with type 2 diabetes mellitus diagnosed after the age of 30, were included and divided into 3 subgroups according to severety of retinopathy. Diabetes was diagnosed according to American Diabetes Association criteria. The onset of diabetes was after the age of 30 in these patients.The study was approved by the institutional ethics committee and patients gave written informed consent. Exclusion criteria included hypertension, myocardial infarction, cerebrovascular disease, heart failure, treatment with antiaggregants, steroids or other drugs that effect blood pressure and glucose, serum creatinine > 200 μmol/l, connective tissue disorders and other systemic disease.Renal and bladder infection diseases of patients and controls were excluded biochemically and microbiologically.The ocular fundus was examined by an ophthalmologist after dilation of the pupils and classified. The patients were divided into three groups according to the severity of retinopathy. Group 1 consisted of 23 patients without retinopathy (R 0 ). Group 2 consisted of 13 patients with nonproliferative retinopathy (R 1 -hemorrhages, microaneurysms, cotton-wool exudates). The 13 patients in group 3 (R 2 ) had proliferative retinopathy (R 2 -neovascularisations, vitreous hemorrhages, ablatio of the retina).Age and sex matched 38 healthy volunteers were included as healthy controls. Demographic characteristics of the groups are shown in Table  1 .Venous samples were collected after an overnight fast and 24 hour urine samples collected.The charge on erythrocytes (RBC) was measured by means of cationic dye alcian blue 8GX (Sigma catalogue no: A 5268) according to the method of Levin with minor modifications as follows: [ 11 ]. From citrated venous blood samples, platelets and leukocytes were removed using the method of Beutler et al. [ 12 ]. After removal of platelets and leucocytes erythrocytes were washed 3 times in phosphate buffered saline (PBS), and subsequently a fraction of RBC was resuspended in PBS containing alcian blue at a final concentration of 250 mg/l. After a 30 min incubation at 37°C, the RBC suspension was centrifuged, and the remaining alcian blue concentration was measured in the supernatant with a Shimadzu UV 1201 V spectrophotometer (Shimadzu, Japan) at a wavelength of 650 nm. Each determination represents the mean of 2 assays. The quantity of alcian blue bound to RBCs was expressed as nanograms of alcian blue per 10 6  RBCs. In our experimental conditions the intraassay and interassay coefficients of variation were 5.8 and 7.6 % for 100 nanograms of alcian blue per 10 6  RBCs.Urinary glycosaminoglycan (U GAG ) excretion was determined in 24 hr urine samples spectrophotometrically at 520 nm by the addition of dimethylmethylene blue (Aldrich Chem Co., USA) and bovine kidney heparan sulfate as standard (Sigma catalogue no H 7640) [ 13 ]. In our experimental conditions the intraassay and interassay coefficients of variation were 1.5 and 2.4 % for 10 mg/l glycosaminoglycan, respectively.Serum sialic acid was measured in serum with a colorimetric method described by Svennerholm with a Shimadzu UV-1201 spectrophotometer (Shimadzu, Japan) with a wavelength of 525 nm. Using N-acetyl neuraminic acid (Sigma catalogue no: A 3007) as standard [ 14 ]. Intra assay and interassay coefficients of variations were 6.6 and 9.2 % for 1 mmol/l sialic acid respectively.Urinary albumin excretion was measured by an immunoturbidemetric assay (Roche diagnostics, USA) on automated clinical chemistry analyzer (Hitachi 902). In all patients, the annual level was determined as the mean of urinary albumin excretions in three 24 h urine collections taken at home during normal physical activity. The intraassay and interassay coefficients of variation were 1.3 and 4.3 % for a mean value of 25 mg/l albumin concentration.Serum glucose, cholesterol, triglyceride and HDL (Dade Behring, USA) were evaluated with enzymatic methods (Dade Behring, USA) on an automated clinical chemistry analyzer (Dimension RxL). Creatinin was assayed by the Jaffe reaction on an automated clinical chemistry analyzer (Dimension RxL).HbA 1C  (reference range 4.4–6.0 %) was measured by a turbidimetric inhibition immunoassay technique (Roche Diagnostics, USA) on automated clinical chemistry analyzer (Hitachi 902). The intraassay and interassay coefficients of variation were 1.8 and 3.0 % for a mean value of 10.5 % HbA 1C  concentration.The analysis of the data was performed with a PC compatible Instat-II programme. Paired t test and ANOVA were used where appropriate. Correlation analysis was performed with Spearman rank test. The differences were considered significant when the probability was p < 0.05. The results were given as mean ± SD.Results The clinical characteristics of the study groups are shown in Table  1 . There were no difference in age and BMI between the groups. Duration of diabetes mellitus was slightly longer, although not significantly different than that of the patients with proliferative retinopathy. Results of laboratory parameters are shown in Table  2 . Patients with proliferative retinopathy had higher concentrations of triglyceride and lower concentrations of HDL cholesterol as compared to diabetics without evidence of retinopathy and control group. Serum cholesterol level was the same in all study groups. Blood glucose and HbA 1C  levels were not different in subgroups of our diabetic population. Distribution of erythrocyte anionic charges of all study groups are demonstrated in figure  1 . EAC was 312 ± 30 ng alcian blue/10 6  RBC in healthy controls. Diabetic patients both with nonproliferative and proliferative retinopathy had a significantly lower alcian blue binding to RBCs compared with diabetic patients without retinopathy (p < 0.001). A marked (p < 0.05) difference was found between type 2 diabetic patients (8.3 ± 4.1, 8.7 ± 4.8, 8.4 ± 3.3 mg/24 h, Table  2 ) and controls (5.0 ± 2.4 mg/24 h) regarding urinary GAG excretion. We did not observe a correlation between urinary GAG excretion and diabetic retinopathy. A significant difference (p < 0.05) was observed in serum sialic acid values in diabetic patients compared with healthy controls. Serum sialic acid level was higher in the nonproliferative retinopathy group, but the difference was statistically insignificant (p > 0.05). Microalbumin level was significantly p < 0.05 higher in diabetic patients (73 ± 40 [35–250], 75 ± 21 [50–202], 85 ± 45 [56–224] mg/24 h, Table  2 ) compared to healthy controls (10 ± 6 mg/24 h). But there wasn't a correlation between the severity of retinopathy and microalbuminuria (p > 0.05). Results of the Spearman rank correlation test are shown on table  3 . EAC is found to be negatively corralated with microalbuminuria in all groups.
Results
The clinical characteristics of the study groups are shown in Table  1 . There were no difference in age and BMI between the groups. Duration of diabetes mellitus was slightly longer, although not significantly different than that of the patients with proliferative retinopathy.Results of laboratory parameters are shown in Table  2 . Patients with proliferative retinopathy had higher concentrations of triglyceride and lower concentrations of HDL cholesterol as compared to diabetics without evidence of retinopathy and control group. Serum cholesterol level was the same in all study groups. Blood glucose and HbA 1C  levels were not different in subgroups of our diabetic population.Distribution of erythrocyte anionic charges of all study groups are demonstrated in figure  1 . EAC was 312 ± 30 ng alcian blue/10 6  RBC in healthy controls. Diabetic patients both with nonproliferative and proliferative retinopathy had a significantly lower alcian blue binding to RBCs compared with diabetic patients without retinopathy (p < 0.001).A marked (p < 0.05) difference was found between type 2 diabetic patients (8.3 ± 4.1, 8.7 ± 4.8, 8.4 ± 3.3 mg/24 h, Table  2 ) and controls (5.0 ± 2.4 mg/24 h) regarding urinary GAG excretion. We did not observe a correlation between urinary GAG excretion and diabetic retinopathy.A significant difference (p < 0.05) was observed in serum sialic acid values in diabetic patients compared with healthy controls. Serum sialic acid level was higher in the nonproliferative retinopathy group, but the difference was statistically insignificant (p > 0.05).Microalbumin level was significantly p < 0.05 higher in diabetic patients (73 ± 40 [35–250], 75 ± 21 [50–202], 85 ± 45 [56–224] mg/24 h, Table  2 ) compared to healthy controls (10 ± 6 mg/24 h). But there wasn't a correlation between the severity of retinopathy and microalbuminuria (p > 0.05).Results of the Spearman rank correlation test are shown on table  3 . EAC is found to be negatively corralated with microalbuminuria in all groups.Discussion Our study demonstrates a statistically significant quantitative reduction of alcian blue binding to RBCs in diabetic patients than healthy subjects, which is in accordance with literature [ 15 , 16 ]. Previously we have shown a statistically significant quantitative reduction of alcian blue binding to RBCs in streptozosin diabetic rats [ 17 ]. Alcian blue is a complex amphoteric molecule that binds to acidic glycoproteins which represent a large class of cell surface anionic molecules [ 18 ]. As alcian blue binding is an expression of the anionic charge on the cell surface, this result indicates that RBC anionic charge is reduced in diabetic patients. Erythrocyte anionic charge by itself is unlikely to be important in the pathogenesis of diabetic retinopathy but reduced RBC alcian blue binding was found to be associated with the loss of glomerular basement membrane anionic charges in diabetic rats and patients [ 19 ]. Changes in the composition of basement membranes are likely to be responsible for functional disturbances and hence for the development of capillary disease. Chakrabarti et al. reported a decreased density of anionic sites in the retinal basement membrane of BB rats [ 20 ]. A similar decrease in anionic density was also demonstrated in the Bruch's membrane of BB and streptozosin diabetic rats [ 21 ]. Several authors have questioned the validity of the alcian blue binding assay. The major objections concern the incomplete alcian blue dissolution in PBS buffer and its tendency to precipitate with time [ 22 , 23 ]. We prepared a fresh alcian blue solution for each experiment. Each experiment was considered valid when no change in the optical density of the blank alcian blue solution occurred over the duration of the experiment. In addition, the experiments were done in batches with cells from a healthy person. Therefore any flaw in the methodology would not affect only one group and thus distort the results. Diabetic retinopathy is characterized by gradually progressive alterations in the retinal microvasculature leading to vascular hyperpermeability, capillary occlusion and ultimately neovascularization. Thickening of the retinal microvascular basement membrane and loss of anionic content are well documented morphological features of diabetic retinopathy [ 20 , 21 , 24 ]. These changes causes breakdown of the blood-retinal barrier resulting in capillary hyperpermeability and leakage of proteins into the deep and superficial layers of the retina [ 1 , 2 ]. Glycosaminoglycan primarily heparan sulfate has been implicated in permeability properties of basement membrane of glomerular basement membrane as well as the retinal basement membrane [ 25 ]. Kahaly et al. reported that urinary GAG excretion is increased in diabetic patients with microangiopathy [ 26 ]. Williamson et al and Ginn et al. have shown that albumin permeation is increased into the diabetic compared to normal retina [ 27 , 28 ]. Loss of heparan sulfate from the retinal basement membrane in addition to its thickening may cause increased vascular permeation of albumin and other substances into the retina and possibly into the optic nerve. Systemic treatment of IDDM patients with proteinuria with danaparoid sodium, a glycosaminoglycan, not only reduced proteinuria, but also decreased the number of hard exudates in the retina [ 29 ]. Studies in diabetic rats have shown reduced activity of the key enzyme in the biosynthesis of heparan sulfate, N-deacetylase, which results in impaired heparan sulfate biosynthesis in experimental diabetes [ 30 , 31 ]. In this study we found that urinary GAG excretion is increased in diabetic patients than healthy controls, but there was no difference in GAG excretion between nonproliferative and proliferative retinopathy patients. We couldn't find a correlation between GAG excretion and EAC but we found a correlation between albumin excretion and EAC. Although our working hypothesis is that the reduction in anionic charge in basement membranes may be due to an abnormal GAG metabolism, we couldn't confirm it with this study. Our study did not directly address the pathophysiology of diabetic retinopathy but a reduction in erythrocyte charge perhaps connected with the properties of retinal basement membrane. Several reports suggest that elevated serum sialic acid levels in type 1 and type 2 diabetic patients indicate microvascular damage [ 32 , 33 ]. In our study, diabetic patients were found to have elevated serum sialic acid levels in comparison with healthy controls. Therefore, the increase in serum sialic acid level may indicate a coincidence between the structural alterations in the retina and general vascular damage.
Discussion
Our study demonstrates a statistically significant quantitative reduction of alcian blue binding to RBCs in diabetic patients than healthy subjects, which is in accordance with literature [ 15 , 16 ]. Previously we have shown a statistically significant quantitative reduction of alcian blue binding to RBCs in streptozosin diabetic rats [ 17 ]. Alcian blue is a complex amphoteric molecule that binds to acidic glycoproteins which represent a large class of cell surface anionic molecules [ 18 ]. As alcian blue binding is an expression of the anionic charge on the cell surface, this result indicates that RBC anionic charge is reduced in diabetic patients.Erythrocyte anionic charge by itself is unlikely to be important in the pathogenesis of diabetic retinopathy but reduced RBC alcian blue binding was found to be associated with the loss of glomerular basement membrane anionic charges in diabetic rats and patients [ 19 ]. Changes in the composition of basement membranes are likely to be responsible for functional disturbances and hence for the development of capillary disease. Chakrabarti et al. reported a decreased density of anionic sites in the retinal basement membrane of BB rats [ 20 ]. A similar decrease in anionic density was also demonstrated in the Bruch's membrane of BB and streptozosin diabetic rats [ 21 ].Several authors have questioned the validity of the alcian blue binding assay. The major objections concern the incomplete alcian blue dissolution in PBS buffer and its tendency to precipitate with time [ 22 , 23 ]. We prepared a fresh alcian blue solution for each experiment. Each experiment was considered valid when no change in the optical density of the blank alcian blue solution occurred over the duration of the experiment. In addition, the experiments were done in batches with cells from a healthy person. Therefore any flaw in the methodology would not affect only one group and thus distort the results.Diabetic retinopathy is characterized by gradually progressive alterations in the retinal microvasculature leading to vascular hyperpermeability, capillary occlusion and ultimately neovascularization. Thickening of the retinal microvascular basement membrane and loss of anionic content are well documented morphological features of diabetic retinopathy [ 20 , 21 , 24 ]. These changes causes breakdown of the blood-retinal barrier resulting in capillary hyperpermeability and leakage of proteins into the deep and superficial layers of the retina [ 1 , 2 ].Glycosaminoglycan primarily heparan sulfate has been implicated in permeability properties of basement membrane of glomerular basement membrane as well as the retinal basement membrane [ 25 ]. Kahaly et al. reported that urinary GAG excretion is increased in diabetic patients with microangiopathy [ 26 ]. Williamson et al and Ginn et al. have shown that albumin permeation is increased into the diabetic compared to normal retina [ 27 , 28 ]. Loss of heparan sulfate from the retinal basement membrane in addition to its thickening may cause increased vascular permeation of albumin and other substances into the retina and possibly into the optic nerve.Systemic treatment of IDDM patients with proteinuria with danaparoid sodium, a glycosaminoglycan, not only reduced proteinuria, but also decreased the number of hard exudates in the retina [ 29 ]. Studies in diabetic rats have shown reduced activity of the key enzyme in the biosynthesis of heparan sulfate, N-deacetylase, which results in impaired heparan sulfate biosynthesis in experimental diabetes [ 30 , 31 ].In this study we found that urinary GAG excretion is increased in diabetic patients than healthy controls, but there was no difference in GAG excretion between nonproliferative and proliferative retinopathy patients. We couldn't find a correlation between GAG excretion and EAC but we found a correlation between albumin excretion and EAC. Although our working hypothesis is that the reduction in anionic charge in basement membranes may be due to an abnormal GAG metabolism, we couldn't confirm it with this study.Our study did not directly address the pathophysiology of diabetic retinopathy but a reduction in erythrocyte charge perhaps connected with the properties of retinal basement membrane.Several reports suggest that elevated serum sialic acid levels in type 1 and type 2 diabetic patients indicate microvascular damage [ 32 , 33 ]. In our study, diabetic patients were found to have elevated serum sialic acid levels in comparison with healthy controls. Therefore, the increase in serum sialic acid level may indicate a coincidence between the structural alterations in the retina and general vascular damage.Conclusions We conclude that type 2 diabetic patients with low erythrocyte anionic charge are associated with diabetic retinopathy. Reduction of negative charge of basement membranes may indicate general changes in microvasculature rather than retinopathy. More prospective and large studies needs to clarify the role of glycosaminoglycans on progression of retinopathy in type 2 diabetic patients.
Conclusions
We conclude that type 2 diabetic patients with low erythrocyte anionic charge are associated with diabetic retinopathy. Reduction of negative charge of basement membranes may indicate general changes in microvasculature rather than retinopathy. More prospective and large studies needs to clarify the role of glycosaminoglycans on progression of retinopathy in type 2 diabetic patients.List of abbrevations BMI: Body mass index EAC: Erythrocyte anionic charge GAG: Glycosaminoglycan PBS: Phosphate buffered saline RBC: Erythrocytes U GAG  : Urinary glycosaminoglycan
List of abbrevations
BMI: Body mass indexEAC: Erythrocyte anionic chargeGAG: GlycosaminoglycanPBS: Phosphate buffered salineRBC: ErythrocytesU GAG  : Urinary glycosaminoglycanCompeting interests The authors declare that they have no competing interests.
Competing interests
The authors declare that they have no competing interests.Authors' contributions YB :  Evaluated laboratory parameters. Designed the study. Wrote the paper. HD:  Diagnosed outpatients according to American Diabetes Association criteria. MA:  Examined ocular fundus after dilation of the pupils and classified according to the severity of retinopathy. DY:  Performed the analysis of the data with a PC compatible Instat-II programme. Improved the manuscript.
Authors' contributions
YB :  Evaluated laboratory parameters. Designed the study. Wrote the paper.YB :HD:  Diagnosed outpatients according to American Diabetes Association criteria.HD:MA:  Examined ocular fundus after dilation of the pupils and classified according to the severity of retinopathy.MA:DY:  Performed the analysis of the data with a PC compatible Instat-II programme. Improved the manuscript.DY:Pre-publication history The pre-publication history for this paper can be accessed here:
Pre-publication history
The pre-publication history for this paper can be accessed here:


A new study settles a long-standing dispute about the genesis of an endangered species. With scant fossil evidence supporting a prehistoric presence, scientists could not say for sure where Borneo's elephants came from. Did they descend from ancient prototypes of the Pleistocene era or from modern relatives introduced just 300–500 years ago? That question, as Fernando et al. report in this issue, is no longer subject to debate.Applying DNA analysis and dating techniques to investigate the elephants' evolutionary path, researchers from the United States, India, and Malaysia, led by Don Melnick of the Center for Environmental Research and Conservation at Columbia, demonstrate that Borneo's elephants are not recent arrivals. They are genetically distinct from other Asian elephants and may have parted ways with their closest Asian cousins when Borneo separated from the mainland, effectively isolating the Borneo elephants some 300,000 years ago.In the 1950s, Borneo elephants had been classified as a subspecies of Asian elephants (either Indian or Sumatran) based on anatomical differences, such as smaller skull size and tusk variations. This classification was later changed, partly because of the popular view that these animals had descended from imported domesticated elephants. Until now, there was no solid evidence to refute this belief and no reason to prioritize the conservation of Borneo elephants.Their new status, as revealed by this study, has profound implications for the fate of Borneo's largest mammals. Wild Asian elephant populations are disappearing as expanding human development disrupts their migration routes, depletes their food sources, and destroys their habitat. Recognizing these elephants as native to Borneo makes their conservation a high priority and gives biologists important clues about how to manage them.


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