Background

3-Deoxyglucosone (3-DG) accumulating in uremic serum plays an important role in the formation of advanced glycation end products (AGEs). To determine if 3-DG is involved in the formation of intracellular AGEs, we measured the erythrocyte levels of 3-DG and AGEs such as imidazolone and N-carboxymethyllysine (CML) in hemodialysis (HD) patients with diabetes. Further, to determine if the polyol pathway is involved in the formation of erythrocyte 3-DG and AGEs, an aldose reductase inhibitor (ARI) was administered to these patients.

Methods

The erythrocyte levels of sorbitol, 3-DG, imidazolone, and CML were measured in ten diabetic HD patients before and after treatment with ARI (epalrestat) for eight weeks, and were compared with those in eleven healthy subjects. 3-DG was incubated in vitro with hemoglobin for two weeks to determine if imidazolone and CML are formed by reacting 3-DG with hemoglobin.

Results

The erythrocyte levels of sorbitol, 3-DG, imidazolone, and CML were significantly elevated in diabetic HD patients as compared with healthy subjects. The erythrocyte levels of 3-DG significantly decreased after HD, but sorbitol, imidazolone or CML did not. The administration of ARI significantly decreased the erythrocyte levels of sorbitol, 3-DG and imidazolone, and tended to decrease the CML level. Imidazolone was rapidly produced in vitro by incubating 3-DG with hemoglobin, and CML was also produced, but less markedly as compared with imidazolone.

Conclusion

The erythrocyte levels of 3-DG and AGEs are elevated in diabetic HD patients. The administration of ARI reduces the erythrocyte levels of 3-DG and AGEs, especially imidazolone, as well as sorbitol. Thus, 3-DG and AGEs, especially imidazolone, in the erythrocytes are produced mainly via the polyol pathway. ARI may prevent diabetic and uremic complications associated with AGEs.

METHODS

Patients

Blood samples were obtained from ten HD patients with diabetes mellitus (7 males, 3 females; 58.2 15.4 years old, mean SD) before and after HD, and eleven healthy subjects (6 males, 5 females). The patients had been treated by HD for 3.5 1.7 years. HD was performed for four hours three times a week. ARI (epalrestat; Ono Pharmaceutical Co., Osaka, Japan) was administered to the patients at 150 mg/day for 8 weeks, and the blood samples were collected from the patients before and after ARI treatment. Epalrestat is approved by the Japanese government only for the treatment of diabetic complication, and is widely used in Japan to treat diabetic patients. Thus, the ARI could not be administered to non-diabetic HD patients. The heparinized blood was centrifuged to separate erythrocytes from plasma. After removing plasma and buffy coat, erythrocyte fraction was kept at -20°C until sample preparation.

Sample preparation for gas chromatography/ mass spectrometry

3-DG levels in erythrocytes were measured by using gas chromatography/mass spectrometry (GC/MS), according to the method of Niwa et al^6,9,10. ¹³C₆-3-DG (1.19 nmol), as an internal standard, was added to hemolyzed erythrocytes (100 l), and was diluted with distilled water (900 l). The mixture was deproteinized by addition of ethanol (2 ml), and subsequently centrifuged at 1,000 g for 10 minutes. The supernatant was applied to a Bond Elut SCX cartridge (cation exchange, 100 mg in 1 ml; Analytichem International, Harbor City, CA, USA), and eluted with distilled water (3 ml). The collected eluate was then applied to a Bond Elut SAX cartridge (anion exchange, 100 mg in 1 ml; Analytichem International), and eluted with distilled water (3 ml). The eluate was collected, and lyophilized. The carbonyl groups of the residue were transformed to their methoxime derivatives at 70°C for 30 minutes with 1% methoxylamine hydrochloride (Sigma Chemical, St. Louis, MO, USA) in pyridine (200 l).

GC/MS of erythrocyte 3-DG

After evaporation over nitrogen stream, hydroxyl groups were converted to their trimethylsilyl derivatives at 60°C for 20 minutes with N,O-bis(trimethylsilyl)trifluoroacetoamide (20 l) containing 1% trimethylchlorosilane (Pierce, Rockford, IL, USA). After cooling to room temperature, the sample (2 l) was subjected to GC/MS (GCMS-QP5000; Shimadzu, Kyoto, Japan) equipped with a capillary column (30 m 0.25 mm i.d., 0.23 m thick membrane; J&W Scientific, Folsom, CA, USA). The column temperature was programmed at 10°C/min from 100°C to 250°C. For chemical ionization, isobutane was used as a reactant gas.

Sample preparation for enzyme-linked immunosorbent assay (ELISA)

Erythrocyte contents of imidazolone and CML were measured by ELISA using monoclonal anti-imidazolone (AG-1) and anti-CML (AG-10) antibodies, according to the method of Niwa et al¹⁹. Hemolyzed erythrocytes (100 l) were mixed with distilled water (300 l) and toluene (300 l). After centrifugation at 1,500 g for 30 minutes, the supernatant lipid layer was removed. The water layer (100 l) was reduced with 50 mM NaBH₄ (2.8 ml) at 37°C for one hour. After cooling with ice, ice-cold 20% (wt/vol) trichloroacetic acid (1.2 ml) was added to the reduced solution, and the mixture was kept cool with ice for 30 minutes. After centrifugation at 1,500 g for 45 minutes, the supernatant was removed and the precipitated protein was dissolved at 4°C for two days by adding 0.1 M NaOH (450 l). After neutralizing the solution with 0.5 M boric acid, the neutral solution was diluted two or four times with PBS, and then the diluted solutions were assayed for imidazolone and CML using competitive ELISA. The protein concentration of the solution was measured by the Lowry method. Then, the imidazolone and CML contents were expressed as arbitrary units (AU) per mg protein.

Competitive sandwich ELISA of erythrocyte imidazolone and CML

Sample solution (25 l) or standard AGE-HSA (0 to 200 mg/liter) was incubated at room temperature for one hour in AGE-HSA (10 mg/liter)-coated microplate with PBS containing 0.25% BSA (50 l), and peroxidase-conjugated anti-imidazolone antibody or peroxidase-conjugated anti-CML antibody. After washing four times with PBS, o-phenylenediamine (100 l) was added to the microplate. The reaction was stopped by adding 1.6 M H₂SO₄ (100 l), and absorption at 492 nm was measured using 620 nm as the control.

Fluorescence spectrophotometry of erythrocyte sorbitol

Erythrocyte fraction was diluted two times with PBS. The hemoglobin contents were determined by Hemoglobin Test Wako kit (Wako Pure Chemical Industries, Osaka, Japan) in hemolyzed erythrocytes (100 l) diluted with saline (100 l). The diluted erythrocyte sample (200 l) was stirred with 10% perchloric acid (200 l). The mixture was kept at 4°C for 30 minutes, and then centrifuged at 1,000 g for 10 minutes. The pH of the supernatant (300 l) was adjusted between 8.2 to 9.0 with 2 M K₂CO₃. After standing overnight, the sample was centrifuged at 1,000 g at 4°C for 10 minutes. The supernatant (75 l) was diluted with distilled water (50 l), and the sample solution was incubated with 0.25 M glycine buffer, pH 9.5 (100 l; Wako Pure Chemical Industries), 12 mM NAD (25 l; Sigma Chemical Co.), in the presence or absence of 22 U/ml sorbitol dehydrogenase (5 l; Sigma Chemical Co.) at 37°C for 40 minutes. The fluorescence of NADH produced in the solution was detected by a fluorescence spectrophotometer 850 (Hitachi, Tokyo, Japan) at excitation 350 nm and emission 440 nm.

In vitro incubation of hemoglobin with 3-DG

Human hemoglobin (15 mg/ml) (Sigma Chemical) was incubated with 3-DG (1 mM) in distilled water at 37°C for 14 days. The levels of imidazolone and CML in the mixture were measured by ELISA.

Statistics

A non-paired t-test was applied to the comparison between the groups of healthy subjects and diabetic HD patients. The paired t-test was used in the comparisons between before and after HD, and between pre- and post-ARI in the group of diabetic HD patients. Significance was found in cases where the P value was less than 0.05.

RESULTS

Figure 3 demonstrates the production of imidazolone and CML during incubation of human hemoglobin with 3-DG. Although the formation of imidazolone was obviously marked as compared to CML, the amounts of these compounds increased time-dependently, demonstrating that 3-DG rapidly formed AGEs, especially imidazolone.

Figure 3.

Time course of the formation of AGE-hemoglobin by incubating 3-DG at 37°C with human hemoglobin. The experiments were done in duplicate, and the data are expressed as means. The amounts of AGEs (imidazolone and CML) increased time-dependently, although the formation of imidazolone is obviously marked compared with CML.

Full figure and legend (13K)

Table 1 shows the erythrocyte levels of 3-DG and the other parameters in diabetic HD patients and in healthy subjects. The diabetic HD patients showed significantly elevated erythrocyte levels of sorbitol, 3-DG, imidazolone, and CML as compared with the normal subjects. In the diabetic HD patients, erythrocyte 3-DG levels did not show any significant correlation with HbA1c, sorbitol, imidazolone, CML, or the other parameters. Among the other combinations, the significant correlations were found only between sorbitol and HbA1c , between CML and HbAlc , and between CML and imidazolone .

Table 1 - Erythrocyte levels of sorbitol, 3-deoxyglucosone (3-DG), imidazolone, N-carboxymethyllysine (CML), and the other biochemical parameters in diabetic hemodialysis (HD) patients and healthy subjects, and effects of aldose reductase inhibitor (ARI) on their levels.

Full table

The erythrocyte levels of 3-DG decreased after HD with a mean reduction rate of 60%, because 3-DG is a small molecule with the molecular weight of 162. Even after HD, the erythrocyte levels of 3-DG in HD patients were significantly higher than those in normal subjects. However, erythrocyte sorbitol, CML, or imidazolone could not be removed by HD.

Furthermore, we treated the patients with ARI for eight weeks to suppress polyol pathway in erythrocytes. As shown in Table 1, the erythrocyte levels of sorbitol and 3-DG were significantly decreased after ARI therapy. The erythrocyte levels of imidazolone were significantly decreased by the ARI treatment, and the erythrocyte levels of CML tended to be decreased after the ARI treatment. However, the mean erythrocyte levels of both imidazolone and CML, which were averaged before and after HD respectively, were also significantly decreased after ARI treatment.

DISCUSSION

The erythrocyte levels of sorbitol, 3-DG, and AGEs in diabetic HD patients were markedly elevated in diabetic HD patients as compared with healthy subjects. The oral administration of ARI to the diabetic HD patients reduced the erythrocyte levels of 3-DG and AGEs, especially imidazolone, as well as sorbitol. This evidence clearly demonstrates that erythrocyte 3-DG and AGEs, especially imidazolone, are mainly derived through fructose from sorbitol produced via the polyol pathway.

The mean level of erythrocyte 3-DG in healthy subjects was 6.2 mol/liter, which is three times higher than that in normal plasma (1.9 mol/liter)⁹. In the other reports, 3-DG levels in the plasma of healthy subjects were much lower compared to our results⁹: 62 nmol/liter in a study by Knecht et al²², and 79 nmol/liter in Hamada et al's²³ study. To resolve this disagreement, Lal et al deproteinized the plasma samples by ultrafiltration or by the addition of ethanol²⁴, as described by Niwa et al⁹, and measured the normal plasma level of 3-DG by GC/MS. The normal plasma level of 3-DG deproteinized by ultrafiltration was 58 nmol/liter, whereas the levels deproteinized by ethanol was 1.7 mol/liter, similar to our data. Thus, Lal et al demonstrated that this disagreement was due to the difference in the methods for deproteinization of the samples, and they considered that the greatly increased 3-DG observed upon ethanol extraction is likely to be due to an ethanol-mediated release of 3-DG bound to plasma macromolecules, probably proteins. In the present study, we extracted 3-DG using ethanol to obtain total amounts of 3-DG, including free and protein-bound forms in the erythrocytes.

Although erythrocyte 3-DG could be removed by HD with the reduction rate of 60%, erythrocyte sorbitol and AGEs could not be removed at all. The reduction rate of erythrocyte 3-DG by HD was similar to that of plasma 3-DG as reported by Niwa et al^6,10. Fujii et al demonstrated that 3-DG easily passes through erythrocyte membranes from the incubation medium²⁵. The production of 3-DG in erythrocytes may be very rapid, because there is a marked difference between erythrocyte and plasma 3-DG levels despite the easy transition of 3-DG across the erythrocyte membranes. However, the presence of a specific mechanism to transport 3-DG across the membranes is not yet known. In contrast, sorbitol hardly passes through the erythrocyte membranes, and consequently can hardly be removed by HD. Because erythrocyte imidazolone and CML are covalently bound mainly to hemoglobin, they cannot be removed at all by HD.

3-DG was considered to be produced mainly via a degradation of Amadori products in the Maillard reaction until another possibility was pointed out by the discovery of fructose-3-phosphate in rat lens¹² and in human erythrocytes²⁶. Fructose-3-phosphate, a potent crosslinking intermediate, yields 3-DG non-enzymatically¹⁴. Fructose is not utilized in vivo except for its metabolism into fructose 6-phosphate in the glycolytic pathway, and that hexokinase scarcely phosphorylates fructose in a glucose-rich condition because of its substrate affinity. Recently, Petersen et al reported that erythrocyte fructose is phosphorylated to fructose 3-phosphate in an incubation solution including glucose at 5 mM²⁷. We consider that fructose and its metabolite, fructose-3-phosphate, derived from the polyol pathway play an important role in the formation of erythrocyte 3-DG, especially in diabetic patients.

3-DG is detoxified mainly to 3-deoxyfructose by reducing enzymes or to 2-keto-3-deoxygluconic acid by oxoaldehyde dehydrogenase²⁵. Some reductases were identified as aldehyde reductases^28,29 and aldose reductases^29,30, which are included in the aldo-keto reductase superfamily³¹ and dihydrodiol dehydrogenases³². 3-Deoxyfructose has been detected in the human urine and plasma³³. The ability to detoxify 3-DG by these enzymes may provide a genetic basis for the differences in the severity of age-related pathologies and diabetic complications. The elevated levels of serum 3-DG in diabetic patients may be due to the increased production of 3-DG via the Maillard reaction resulting from hyperglycemia, while the increased levels of erythrocyte 3-DG is due to the enhanced polyol pathway.

3-DG stimulates the formation of AGEs, which then show a number of biological activities and are involved in the pathogenesis of diseases such as diabetes and aging³⁴. Recently, however, 3-DG itself also has been demonstrated to have some biological activities. 3-DG induced heparin-binding epidermal growth factor-like growth factor in rat aortic smooth muscle cells³⁵, and may be involved in the development of atherosclerosis. 3-DG induces apoptotic cell death in macrophage-derived cell lines³⁶, and suppresses cell-cycle progression during the S phase of rat fibroblasts³⁷. Thus, 3-DG is thought to be a glycotoxin or a uremic toxin, and may be involved in the pathogenesis of diabetic and/or uremic complications.

The polyol pathway is enhanced not only in erythrocytes, but also in other tissues such as peripheral nerves, lens, retina, aortas, and kidneys in diabetic patients, because these tissues contain aldose reductase³⁸. The enhanced polyol pathway leads to the intracellular accumulation of sorbitol and fructose in these tissues. As we observed in erythrocytes, and contents of intracellular 3-DG and AGEs, especially imidazolone, in these tissues may also be increased in diabetic HD patients, because they are produced via the polyol pathway. The administration of ARI may also decrease the intracellular 3-DG and AGEs in these tissues by reducing the sorbitol concentration.

In conclusion, the high levels of 3-DG and AGEs, especially imidazolone, in the erythrocytes of diabetic HD patients are due mainly to the enhanced polyol pathway. ARI may prevent the development of diabetic and/or uremic complications associated with AGEs.

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