Sodium Sulfite and N-Acetylcysteine: New Additives to Dialysate for Inhibiting Formation of Glucose Degradation Products and Advanced Glycation End-Products

Asahi Sakai,1 Masaaki Nakayama,2 Miwako Numata,2 Shingo Takesawa,3 Masahiko Nakamoto4
From: 1Japan Science and Technology Corporation, 2Tokyo Jikeikai University School of Medicine, 3Suzuka University of Medical Science, and 4Saiseikai Yahata Hospital, Tokyo, Japan.

The present study evaluated the inhibiting effect of various chemicals on the advanced glycation end-product (AGEs) cross-linking caused in protein by glucose degradation products (GDPs). We evaluated a few dozen organic and inorganic chemicals—in addition to previously reported AGE inhibitors, such as thiazolium derivatives and aminoguanidine—for their inhibiting effect.
Collagen IV (from human placenta) or human serum albumin (HSA) was incubated with an AGE accelerator and one of the selected chemicals in phosphate buffer solution at 37°C for as long as 14 days. Fluorescence intensity (440 nm) was determined after a given incubation time.
Among 36 chemicals tested, 8 new chemicals and 5 previously known AGE inhibitors significantly suppressed the increase in fluorescence intensity seen after incubation of HSA with methylglyoxal. We believe that 6 chemicals may effectively quench GDPs and inhibit AGE cross-link formation, in a manner different from that of aminoguanidine and thiazolium.

Key words

Introduction

Glucose degradation products (GDPs) such as 3-deoxyglucosone, glyoxal, and methylglyoxal are formed during heat sterilization (1,2) of glucose-containing peritoneal dialysis fluids and are much more deleterious than glucose itself for peritoneum (1–8).
Efforts have been made to reduce GDPs in peritoneal dialysate by dividing the solution into two compartments, with glucose in acid solution, and bicarbonate and other electrolytes held separately during autoclave steaming (9–11). Still, the improved dialysate is reported to contain some GDPs, as compared with filter-sterilized dialysate, which has an extremely low GDP content (11).
Formation of AGEs in diabetes mellitus has been reported to be prevented with aminoguanidine (12,13). Thiazolium derivatives have also been reported to inhibit, and even to break, AGE cross-linking in protein (14).
We have been aiming at recovering albumin from PD effluent and reusing it as the osmotic agent in dialysate (15–17). By fluorescence intensity under sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), we determined that the recovered albumin was insignificantly cross-linked. However after repeated reuse in dialysate, the albumin is likely to become oxidized and polymerized—no longer a physiological osmotic agent.
In healthy plasma, glutathione (that is, tri-peptide comprising glutamine–cysteine–glycine) is known to change oxidized albumin into reduced albumin (18). We earlier reported that N-acetylcysteine is effective in reducing the formation of (carboxymethyl)lysine (19).
When we started looking for AGE inhibitors to add to peritoneal dialysate, our first target was the glutathione and cysteine neighborhood. A commonly active residue from these chemicals is mercapto; for others, the residue is thiol or sulfhydryl (–SH). We therefore selected several other mercapto compounds with greater aqueous solubility: 2-mercaptoethanol and dithiothreitol, among others. For comparison, vitamin C and vitamin E were tested.
We also tested some antioxidants from groups such as medicines, food ingredients, and food and drink additives, and a few oncotic agents that have been tried as substitutes for glucose in dialysate, for example, N-acetylglucosamine and chondroitin sulfate (20,21). Further, we synthesized N-phenacylthiazolium bromide and submitted it to the evaluation. Because this chemical is not soluble in water, we dissolved it in a 50:50 methanol and water mixture. A few thiazolium derivatives that have higher solubility in water were also evaluated.
A U.S. patent suggests that aldehydes may prevent AGE formation. Some aldehydes were therefore evaluated for their effectiveness.

Materials and Methods

Human serum albumin (HAS) or collagen IV (from human placenta) was incubated in phosphate buffer solution with an AGE accelerator (glyoxal, methylglyoxal, or 3-deoxyglucosone), alone or with one of the test chemicals at 37°C for as long as 14 days.
After a given incubation time, fluorescence intensity [(FI) excitation at 370 nm, measurement at 440 nm) was determined.
Suppression rate (SR%) was estimated by this formula:

SR% = 100 × ( B – C ) / ( B – A )

where A = FI at day 0; B = FI of AGE accelerator alone at day 7; C = FI of AGE accelerator plus tested chemical at day 7 (Figure 1).
In the case of C > B, the value becomes negative.

figure 1 Estimation of fluorescence suppression rate. G = glyoxyl; A = fluorescence intensity (FI) at day 0; B = FI of advanced glycation end-product (AGE) accelerator alone at day 7; C = FI of AGE accelerator plus test chemical at day 7.

Results

Figure 2 shows some typical results. Table I lists the suppression rate for every chemical tested.

figure 2 Typical suppression effect on fluorescence intensity.

table i Suppression rate for each tested chemical.

Discussion

We determined that N-phenacylthiazolium bromide is as effective as previously reported (5). However, this chemical is not easily soluble in water (PBS) and may not be a suitable additive for peritoneal dialysate.
Residue of thiazolium seems to affect its suppression rate to a great extent.
Aminoguanidine HCl was effective, probably because its two or more amino residues block carbonyl residue in glucose degradation products (GDPs). Aminoguanidine H2CO3 was not soluble in PBS.
Both glutathione and N-acetylcysteine were effective.
Some mercapto compounds were effective, probably because their –SH residue shows reducing activity. Not every mercapto compound was effective, however. To elucidate the mechanism of suppression, further study is required.
Catechin and epicatechin indicated good suppression of fluorescence intensity increase. However their dense color might affect FI emission at 440 nm. Further verification, by SDS-PAGE for example, is required to confirm the suppression effect.
Vitamin C showed no suppression at all. Vitamin E was not water-soluble. Acetylsalicylic acid was quite effective.
Sodium sulfite and bisulfite were effective. Sulfur dioxide has been added to wine in concentrations as high as 50 – 100 mg/L (or more) to protect the flavor against oxidation.
Aldehydes showed acceleration, not suppression, as predicted.
Among soluble thiazolium derivatives, N-(2-thiazolyl)sulfanilamide was effective.

Conclusion

In addition to previously reported AGE inhibitors [glutathione, N-acetylcysteine, N-phenacylthiazolium bromide, N-(2-thiazolyl)sulfanilamide], six new chemicals (sodium sulfite, sodium bisulfite, 2-mercaptoethanol, dithiothreitol, S-acetylmercapto succinic anhydride, and acetylsalicylic acid) significantly suppressed fluorescence intensity. The mercapto compounds and sodium sulfite may work in a manner different from that of thiazolium derivatives and aminoguanidine.

Acknowledgment

Japan Science and Technology Corporation, a government organization, supported this research.

References

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Corresponding author:

Asahi Sakai, md, Peritoneal Dialysis Research Project, Japan Science and Technology Corporation, c/o Tokyo Metropolitan Industrial Technology Research Institute, 11-1 Fukazawa 2-chome, Setagaya-ku, Tokyo 158-0081 Japan.