GB2407571A - Treatment of sugar solutions - Google Patents

Abstract

A process for treating a solution containing sugar and a -oxoaldehydes, comprising the step of adding a catalyst which comprises an optionally substituted histidine amino acid, such that the a -oxoaldehydes are catalytically converted to aldonic acids. This process is especially but not exclusively applicable to the treatment of heat-treated sugar solutions for peritoneal dialysis fluids.

Description

240757 1 - ]

TREATMENT OF SUGAR SOLUTIONS

Field of the Invention

This invention relates to treatment of sugar solutions and is especially but not exclusively applicable to treating peritoneal dialysis fluids.

Review of the Art known to the Applicant Clinical renal failure affects 32,000 people in the UK and about 0.1% of the global population. It is managed in pad by renal replacement therapy. This involves kidney transplantation where appropriate and a donor organ is available, and renal dialysis in all other cases and at all other times. Dialysis procedures are ineffective and mortality of patients with renal disease is high - the median life expectancy is about 8 years from the diagnosis of renal failure. Dialysis procedures used are haemodialysis (HD) and peritoneal dialysis (PD). PD is patient preferred, but accounts for only 15% of global dialysis currently but should increase to 30-40% globally in the future. A survival advantage with PD therapy is expected relative to HD, but this is not currently realised so because of poor biocompatibility of PD fluids. This is due to the formation of toxic aldehydes (a-oxoaldehydes) during heat sterilization of the PD fluids.

Severe impairment of kidney function leads to renal failure, the clinical syndrome called end-stage renal disease (ESRD). The causes of ESRD are many and varied but diabetes (20-30%) and inflammatory disorders (20%) are major etiological factors. There are ca. 34,200 people with ESRD in the U.K. (300,000 in the USA) and a further 5, 500 people develop ESRD per year. This is expected to increase due to the increasing incidence and earlier onset of diabetes and increasing elderly population. The current annual growth rate of subjects with ESRD is 7%. Treatment of ESRD requires renal replacement therapy (RRT), preferably, this involves kidney replacement by transplantation but 63% of ESRD patients are unsuitable for kidney transplantation. There is also a shortage of to suitable donors, significant transplantation failure (25% after 5 years) and associated costs. About 47% of patients with ESRD eventually receive a kidney transplant, there are about 2000 kidney transplantations performed per year in the UK. Alternatively, for 53% of subjects in the UK, removal of waste products (renal toxins) from the body is achieved by dialysis [1].

Two main dialysis techniques are employed: (i) haemodialysis (HD) - where blood from the peripheral circulation is circulated out of the body over a semi-permeable membrane that allows renal toxins to diffuse into a second circulating dialysis fluid, and (ii) continuous ambulatory peritoneal dialysis (PD) - where dialysis fluid (typically 2 litres) is infused into the peritoneal cavity, left to dwell there for 4 h or overnight and then so drained out- renal toxins flow across the peritoneal membrane and are thereby removed.

PD is done 4 - 5 times daily. The major problem of dialysis therapy is that renal toxins are not eliminated effectively and complications mainly cardiovascular disease produce morbidity and mortality. The 4-year survival rate for RRT patients on dialysis in the UK in 200] was 48% [1].

There is no clear benefit for morbidity and mortality in ESRD of HD over PD therapy.

Currently, worldwide the use of HD and PD is split 85% to 15%. PD is patient preferred and it is thought that global PD will increase to 3040% of dialysis therapy. In the UK, 55% of ESRD patients receive HD and 45% PD therapy; recently 50% of all new ESRD so subjects have received PD therapy. In continental Europe and the USA, most ESRD patients (90%) are on HD; in some developing countries (e.g. Mexico) 90% of patients receive PD therapy [2;3]. There is an emerging consensus that HD and PD therapies in ESRD are not competitive but are rather complementary. PD is a particularly suitable technique to start RRT as it preserves residual renal function, has a lower risk of hepatitis infection than HD, allows better control of blood pressure, has less severe effects on cardiac output, achieves a higher clearance of high molecular mass toxins and improves early kidney survival after transplantation. It is also more economical that HD allowing more patients to be treated. After prolonged PD therapy, the ultrafiltration properties of the peritoneal membrane are impaired - related to poor biocompatibility of PD fluids. At this point, a timely switch to HD therapy is required or kidney transplantation (or before PD therapy becomes in effective). This is the current approach of "integrated care" for ESRD patients [4].

to HD involves patients visiting a renal clinic 3 times per week and remaining attached to the mechanical dialysis machine for 4 h. Interaction of leukocytes in the circulating blood with the dialysis membrane causes a pro-inflammatory response and may increase the production of renal toxins. HD is inconvenient and expensive - the average cost is 29,000 per patient year in the UK ($63,000 per patient year in the USA).

Improvement in patient survival on HD is achievable by daily dialysis sessions [5] but there would be an associated 2-3 fold increase in cost of patient care provision.

PD is associated currently with patient outcomes with respect to mortality and morbidity similar to those achieved by HD. PD is less expensive - ca 20,000 per patient year in the UK ($45,000 per patient year in the USA). It is associated with a lower inflammatory response since immune cells do not come into contact with exogenous, synthetic membranes. Pro-inflammatory responses initiate and sustain vascular complications of dialysis therapy [6]. It is surprising that PD does not have significantly better clinical outcomes than HD. The explanation for poor performance of PD therapy appears to be due to the poor biocompatibility of commercial dialysis fluids. High concentrations of glucose (1-4%, 76 - 214 mM) are included in PD fluids to create an osmotic flow across the peritoneal membrane to drive the ultrafiltration of uraemic toxins into the peritoneal cavity (typical compositions of peritoneal dialysis fluids are shown in table] ). Heat sterilization of the glucose-containing PD fluids leads to loss of biocompatibility. Filter sterilization is not a viable alternative because of high cost. The major dialysis companies worldwide have research programmer to improve the biocompatibility of PD fluids.

Table 1. Typical composition of peritoneal dialysis fluids, the composition of the Dianeal_ and Physioneal_ dialysis fluids are given below: Ingredient: ll Dianeal_ PD4 solutioll Glucose (% w/v) | 1.36% 11 2. 27% 1 3.86% (g/L) (mM) | (g/L)(mM)(g/L)(mM) Glucose (Mr 180) 13.6 75.6 122.7 126 38. 6 2]4 NaCI (Mr 58.4) 5.4 92 15.4 92 5.4 92 Sodium lactate (Mr 112) 4. 5 40 14.5 40 4.5 40 CaCI2.2H2O (Mr 147) 0.184 1.25 10.184 1.25 0.184 1.25 MgCI2.6H2O (Mr 203) 0.051 0.25 10.051 0.25 0.051 0.25 Sodium Na+ | 132 132 132 Calcium Ca2+ 1.25 1.25 1.25 Magnesium Mg2+ 0.25 0.25 0.25 Chloride Cl- 95 95 95 Lactate 40 40 40 Osmolarity (mOs/L) 344 395 483 i l Ingred ient: PhysionealTM solution Glucose (% w/v) ll 1.36% 11 2.27% ll 3. 86% (g/L) (mM) (g/L)(mM) (g/L) (mM) Compaffment A Glucose.H2O (Mr 198) 41. 25 208 68.85348 117.4 593 Equivalent of glucose (Mr 180) 37 5 208 62. 6348 106.5 593 CaCI2.2H20 (Mr 147) . 0.507 3.45. 0.5073.45 0.507 3.45 MgCI2.6H2O(Mr203) 0.1400.69 10.1400.69 10.1400.69 Compartment B NaCI (Mr 58.4) 8.43144 8.43144 8.43144 NaHCO3 (Mr 84.0) 3.2939.2 3.2939.2 3.2939.2 Sodium lactate (Mr 112) 2.6323.5 2.6323.5 2.6323.5 Final solution after mixing Glucose 13.675.5 22.7126 38.6214 NaCI 5.3892.1 5.3892.1 5.3892.1 CaCI2.2 H20 0.1841.25 0.1841.25 0.184] .25 MgCI2.6 H20 0.0510.25 0.0510. 25 0.0510.25 NaHCO3 2.125.0 2.125.0 2.125.0 Sodium lactate i1.6815.0;1. 6815.0 i 1.6815.0 Sodium Na+ 132 132 132 Calcium Ca2+ 1.25 1.25 1.25 Magnesium Mg2+ 0.25 0.25 0.25 Chloride Cl- 95 95 95 Bicarbonate HCO3- 25 25 25 Lactate Osmolarity (mOs/L) 344 395 i 483 One litre of the final solution is made by mixing 362.5 ml of solution A with 637.5 ml solution B. The final pH is 7.4.

Biocompatibility as applied to peritoneal dialysis refers to the ability of the PD fluid to remove renal toxins with minimal damage to the peritoneal cavity and the peritoneal membrane ultrafiltration capacity. Damage to the peritoneal membrane and the mesothelial cells lining it decreases the efficiency and effectiveness of the PD process, with resulting reliance on haemodialysis and frank uraemia.

to The composition of currently available commercial dialysis fluids typically has high concentrations of glucose to increase the osmolality of the PD fluid to > 500 mOsm/kg and lactate buffer (40 mM); the pH of the medium is ca. 5.2-5.5, the PD fluid is sterilized by heating to 121 C for I h. The most important cause of poor biocompatibility of PD fluids is, however, the formation of a range of reactive a,5-dicarbonyl compounds (or- oxoaldehydes) by fragmentation of a minor fraction of the glucose osmolyte during heat sterilization - Figure I shows a range of the dicarbonyl compounds produced. Some of these compounds are physiological metabolites - glyoxal, methylglyoxal and 3- deoxyglucosone (3-DG) but the concentration in PD fluids is 30 - 2000 fold higher than the normal concentration in blood plasma (see figure 2). A novel strategy to minimize the formation of a-oxoaldehydes has been to use glucose polymers - for example, the to branched chain glucose polymer Icodextrin. However, sterilsation of Icodextrin solutions also forms x- oxoaldehydes [7].

a-Oxoaldehydes (cc,0-dicarbonyl compounds) are toxic because they bind and irreversibly modify proteins, nucleotides and basic phospholipids. The adducts thereby formed are called advanced glycation endproducts (AGEs) examples of the AGEs formed by methylglyoxal are shown in figure 3. These are formed physiologically in cellular and extracellular proteins, nucleotides and phospholipids. The AGEs are released during protein, nucleotide and phospholipid turnover, and are excreted as waste products.

AGEs are, in fact, a class of uremic toxin. Their accumulation to abnormally high levels o in ESRD, along with oxidative stress, is thought to promote the development of macrovascular disease [8]. It was recently found that oxidative markers accumulate 1-2 fold in ESRD subjects but certain AGEs increased by up to 50-fold in ESRD patients [9] and were decreased partially by HD and PD therapy. At high concentrations, AGEs bind cell surface receptors in endothelial cells, monocytes and macrophages - the receptor for advanced glycation endproducts (RAGE) [10; 11]. Signal transduction processes hlitiated by RAGE stimulates the expression of pro-inflammatory, atherosclerosis and thrombosis mediators (slCAM-I, sVCAM, von Willebrand factor and monocyte chemoattractant protein-1 MCP-I)) [12]. These are increased in ESRD and predispose ESRD subjects to cardiovascular disease [13]. The critical feature of poor biocompatibility of PD fluids ao containing a-oxoaldehydes is, therefore, that patients who are already over-burdened with AGEs as a result of ESRD are then infused with up to 8 - 10 litres per day of relatively high concentrations of exogenous oc-oxoaldehydes that produce more AGEs in the peritoneal cavity and elsewhere, recent research shows that PD subjects develop abnormally high levels of AGEs in the peritoneal cavity [9]. AGEs interact with RAGE receptors on mesothelial cells in the peritoneum and promote deterioration of the peritoneal membrane [14].

The major efforts to improve the biocompatibility of PD fluids have been directed to minimising the formation of a-oxoaldehydes during heat sterilisation - a-oxoaldehydes concentrations can be decreased by sterilisation of PD fluids in two compartment bags that keep the buffer separated from the glucose solution during sterilisation and adjusting to the glucose solution compartment to pH 3 before sterilisation; the two compartments are mixed just prior to use. Figure 2 shows that the level of the a-oxoaldehydes, methylglyoxal and 3-deoxyglucosone, is lower in the PD fluid using a 2-compartment bag of Physioneal_ as compared to a single compartment bag of Dianeal_ (Dianeal_ is a single compartment PD fluid bag prepared with 1.36 - 3.86% glucose. Physioneal_ is a 2-compartment PD fluid bag where the buffer and glucose solutions (1.36 - 3.86%) are separated during heat sterilisation and mixed just prior to use. The normal physiological concentrations of glyoxal, methylglyoxal and 3-DG in blood plasma are 100 - 150 Nm).

The concentration of glyoxal was not decreased in Physioneal_ fluids, however, and the concentrations of a-oxoaldehydes still remain relatively high - 70 - ]61 1M 3 o deoxyguclosone, for example. This two compartment system has been optimised and further improvement on this basis seems unlikely [15].

Addition of scavengers of a-oxoaldehydes, aminoguanidine and phenacylthiazollwn bromide, has been considered but these compounds are unstable - especially during thermal sterilisation and are toxic and/or form toxic adducts, and must be added in stoichiometric amounts [16-18] A new strategy to decrease a-oxoaldehydes during heat sterilisation of PD fluids is therefore required.

Similar problems occur in other situations where thermal treatment of solutions containing glucose is required. For example to prevent the generation of a-oxoaldehydes encountered during thermal processing, it is normal to filter rather than thermally process sugar solutions which are to be used for microbial and cell growth media. Typically microbial and cell growth media are supplied in the form of powders, to which an appropriate amount of water is added prior to sterilisation of the resulting solution by autoclaving. This can result in the generation of aoxoaldehydes from the sugar contained in the powder which can have deleterious effects on the quality of the growth medium.

The addition of a catalyst of the type described herein to the powder used would result in the a-oxoaldehydes produced during the autoclaving step being converted to aldonic acids.

The same problems as regards the generation of a-oxoaldehydes are also encountered to during the sterilization of parenteral fluids. (Parenteral fluids are fluids administered to the body by any route except via the alimentary canal and so such fluids need to be sterilised prior to administration. As they contain solutions of reducing sugars, aoxoaldehydes are produced during thermal processing).

Summary of the invention

According to the present invention, there is provided a process for treating a solution containing sugar and a-oxoaldehydes, comprising the step of adding a catalyst which comprises an optionally substituted histidine amino acid, such that the a-oxoaldehydes are catalytically converted to aldonic acids.

Preferably the process involves a thermal processing step which is suitable for the sterilization of the solution.

Preferably the optionally substituted histidine comprises a histidine amino acid substituted with at least one further amino acid and fewer than four further amino acids Preferably the optionally substituted histidine comprises a peptide comprising 2 to 4 amino acids, one of which is histidine.

Preferably the at least one further amino acid comprises a single amino acid chosen from the following; tyrosine, aspartic acid, histidine, arginine, glutamic acid or Iysine.

Preferably the sugar comprises a reducing sugar.

Preferably the reducing sugar is glucose, ribose or reducing sugar polymer.

Preferably the catalyst comprises a plurality of various optionally substituted histidine amino acids.

Preferably the sterilised solution is a peritoneal dialysis fluid.

to Brief description of the drawings

An embodiment of the present invention will now be described by way of example only, with reference to the following drawings of which: Figure I of the drawings illustrates the reactive dicarbonyl compounds formed from glucose during heat sterilization of dialysis fluids.

Figure 2 of the drawings illustrates the concentrations of a-oxoaldehydes in clinical peritoneal dialysis fluids.

Figure 3 shows Advanced Glycation Endproducts (AGEs) formed by methylglyoxal.

Figure 4 shows the catalytic cycle by which the optionally substituted histidine catalyst is envisaged to catalyse the conversion of aoxoaldehydes to aldonic acids.

Figure 5 (a, b) shows the enhancement of the rate of decomposition of methylglyoxal by histidyl dipeptides.

Figure 6 (a-g) shows the decrease in a-oxoaldehyde concentrations in heat sterilised so peritoneal dialysis fluid by histidine and histidyl peptides.

Figure 7 (a-g) shows the decrease of oc-oxoaldehyde concentrations in heat sterilized peritoneal dialysis fluid as catalysed by His-His i.e. activity of His-His catalyst in model heat sterilization of peritoneal dialysis fluid.

Description of the Preferred Embodiment

The invention of the current application relates to a group of compounds which can catalyse the intramolecular disproportionation of aoxoaldehydes to the corresponding to aldonic acids: RCOCHO + H2O RCH(OH) CO2- + H+. The catalyst used to catalyse this reaction comprises an optionally substituted histidine amino acid, the catalyst may comprise a di-, tri- or tetra-peptide comprising at least one histidine and a further amino acid chosen from the group comprising tyrosine, aspartic acid, histidine, arginine, glutamic acid or Iysine.

The catalyst may also comprise a single histidine amino acid wherein no substitution of the amino acid has been carried out.

Alternatively the catalyst used may comprise a mixture of optionally substituted histidine, such as an unsubstituted histidine used in conjunction with substituted histidine amino acids of the type previously described.

The sugar contained within the solution of the current invention is any reducing sugar (i.e. monosaccharide) although the specific examples provided herein are glucose and ribose, or a reducing sugar polymer (e.g. icodextrin)).

Reference is now made to figure I of the drawings which shows the aoxoaldebydes formed from glucose during heat sterilization of dialysis fluids.

A typical process using the catalyst of the present invention would hlvolve adding the so catalyst to a solution containing a sugar such as sucrose (or ribose) and then thermally processing the mixture. Typically in the production of peritoneal dialysis fluids a dialysis fluid is heated at 121 C for upwards of one hour during which time a-oxoaldehydes are generated from the degradation of the sugar such as glucose as shown in Figure 1. The a oxoaldehydes produced by the degradation of the glucose are catalytically converted by the catalyst to the corresponding aldonic acids.

Reference is now made to Figure 4 of the drawings which shows the catalytic cycle by which the optionally substituted histidine catalyst is envisaged to catalyse the conversion of a-oxoaldehydes (oc,0-dicarbonyl substrate) to aldonic acids. These catalyst are stable at high temperatures and so continue to function during thermal sterilization and show activity with a wide range of or-oxoaldehydes. A catalytic cycle is envisaged mechanistically as illustrated by figure 4. The structural loop between the imidazole and to the basic (-B:) groups is variable that is it may constitute a histidine amino acid alone, or one or more amino acids joined to the optionally substituted histidine as previously described.

These catalysts that can catalyse the conversion of a-oxoaldehydes to aldonic acids during the thermal sterilization of reducing sugar solutions, have the advantage over previous compounds used in that only catalytic amounts are required and the degradation products of the o,oxoaldehydes, (i.e. aldonic acids), have no significant toxicity.

When the catalyst is added to model dialysis fluids, it decreases the concentrations of a oxoaldehydes under the experimental sterilization conditions used. They are active at concentrations as low as 1-10 I1M for all the detectable dicarbonyl compounds produced during the thermal sterilization of glucose containing dialysis fluid i.e. glyoxal ((C110)2), methylglyoxal (MeCOCHO), erythrosone (HOCH2CHOHCOCHO), 3-deoxyerythrosone (HOCH2CH2COCHO), 3-deoxyribosone (HOCH2CHOHCH2COCHO), 3 deoxygl ucosone (3 -DG,(HOCH2(C HOH)2CH2COCHO)).

The basis of the invention claimed herein is the discovery of a catalyst (small molecular weight molecules) that when added to solutions containing reducing sugars (such as glucose, ribose or a reducing sugar polymer (e.g. icodextrin)) and in particular peritoneal so dialysis fluid prior to sterilization, they catalytically decompose the dicarbonyl compounds produced and thereby improve the biocompatibility of the fluid. Given this improved peritoneal dialysis fluid, decreased risk of cardiovascular disease and decreased risk of peritoneal dialysis failure is expected.

Similarly if the catalyst is added to parenteral fluid containing a reducing sugar prior to sterilization, the catalyst would catalytically decompose the dicarbonyl compounds produced and thereby improve the biocompatibility of the sterilized parenteral fluid.

Parenteral fluids are fluids administered to the body by any route except via the alimentary canal.

Reference is now made to figure 5 of the drawings which shows the decrease in the level of methylglyoxal as catalysed by a range of llistidyl dipeptides. Imidazole-containing histidyl peptides were screened for their catalytic activity in converting a-oxoaldehydes to aldonic acids, initially by incubation with 10 mM methylglyoxal in sodium phosphate buffered saline at pH 7.4 and 37 C for 24 h, assaying the initial and final concentrations of methylglyoxal by derivatisation with 1,2-diamino-4,5-dimethoxybenzene and HPLC of the resultant quinoxaline adducts with fluorimetric detection [19-21].

In this example histidyl peptides (50 I1M) were incubated with methylglyoxal (10 M) in phosphate buffered saline, pH 7.4 at 37 C, for 24 h. Methylglyoxal concentrations were determined initially (t = 0) and after the incubations. Data are mean + SD (n = 3). *, ** and ***, P<0.05, <0.01 and <0.001 with respect to the control. The histidyl di-peptides o studied were His-His, Glu-His, Lys-His, Arg-His, His-Glu, His-Lys and HisArg. In all cases a decrease in the level of methylglyoxal present in the solution was shown, as compared to the control, due to the catalytic action of the di-peptide resulting in the conversion of the methylglyoxal to the corresponding aldonic acid. The greatest decrease in the level of methylglyoxal was observed when using His-His and His-Arg, although all the histidyl di-peptides used were shown to decrease the level of methylglyoxal in solution as compared to the control sample.

Reference will now be made to figure 6 of the drawings which shows the decrease in a- oxoaldehyde concentrations in heat sterilised peritoneal dialysis fluid catalysed by histidine and two substituted histidyl peptides, wherein the oc-oxoaldehydes studied were: (a.) glyoxal, (b.) methylglyoxal, (c.) hydroypyruvaldehyde (HPA), (d.) erythrosone (ES), (e.) 3-deoxyerythrosone (3-DE), (f.) 3-deoxyribosone (3-DR) and (g.) 3deoxyglucosone (3-DG).

The effect of two histidyl peptides, His-His and His-Arg, and L-histidine (His) on the concentrations of a-oxoaldehydes in a model therapeutic peritoneal dialysis fluid after thermal sterilization is shown for the aoxoaldehydes studied. The experimental dialysis fluid was a model of Baxter Healthcare produced Physioneal_ with a final concentration of of glucose of 3.86% when compartment A and B solutions are mixed. The compartment A solution prior to mixing contains 593 mM glucose (see table l). A model compartment A solution was prepared for experimental sterilization and contained: 593 mM glucose, 3.45 mM calcium chloride and 0.69 mM magnesium chloride. For the purposes of thermal processing, samples were heated to 121 C for 1 h. The a o oxoaldehyde concentrations were assayed as described in [20].

Incubations were carried out with and without 50 AM histidine and Histidyl dipeptide. a- Oxoaldehyde concentrations were assayed as described in [20]. Data are mean + SD (n = 4). Significance (t-test): *, ** and *** indicates P<0.05, P<0.01 and P<0. 00] with respect to zero test compound incubation control (Control). The a-oxoaldehyde content of the model dialysis fluid before heat sterilization is given as Control (t = 0).

These studies indicate that His-His and His-Arg have enhanced activity relative to His in lowering the concentrations of reactive a-oxoaldehydes formed during thermal sterilization of the model dialysis fluid. Overall, His-His produced the greatest decrease in the levels of the a-oxoaldehydes. It should however be noted that in the case of 3deoxyerythrosone the L-histidine gave a greater reduction in the level of the a- oxoaldehyde than either the His-His or 11is-Arg. So there may be cases where the catalyst may comprise a mixture of di, tri and/or tetra peptide used in conjunction with L- histidine.

Reference will now be made to figure 7 of the drawings which shows the decrease of a- Oxoaidehyde concentrations in heat sterilized peritoneal dialysis fluid as catalysed by His- His i.e. a dose response study for His-His.

so The dependence on concentration of His-His of the decrease in aoxoaldehyde concentrations in the model dialysis fluid during thermal sterilization was investigated.

The experimental dialysis fluid was again a model of Baxter healthcare Physioneal_ 3.86% glucose, component A with 593 mM glucose, 3.45 mM calcium chloride and 0.69 mM magnesium chloride, with sterilization by heating to 121 C for I h. The effect of I - I1M His-His on a-oxoaldehyde concentrations was studied.

Figure 7 shows the decrease of a-oxoaldehyde concentrations in heat sterilized peritoneal dialysis fluid as catalysed by His-His. Wherein the relevant a-oxoaldehyde are: (a.) glyoxal, (b.) methylglyoxal, (c.) hydroypyruvaldehyde (HPA), (d.) erythrosone (ES), (e.) 3-deoxyerythrosone (3-DE), (f.) 3-deoxyribosone (3-DR) and (g.) 3- deoxyglucosone (3- DG). Incubations were of model dialysis fluid Physioneal_ component A with 3.86% glucose, with and without I - 50 I1M His-His (in the case of 3deoxyerythrosone 1-10 M). a-oxoaldehyde concentrations were assayed as described [20]. Data are mean + SD (n = 4). Significance (-test): *, ** and *** indicates P<0.05, P<0.01 and P<0.001 with respect to zero test compound incubation control (Control). a-oxoaldehyde content of the model dialysis fluid before heat sterilization is given as Control (t = 0).

These studies showed that His-His (I - 50 M) decreased the concentrations of reactive a-oxoaldehydes present in sterilised dialysis fluid when added to the dialysis fluid prior to carrying out the heat sterilisation process. The median effective concentration IC50 values of His-His for each a-oxoaldehyde were computed by logistic regression of percentage o decrease of a-oxoaldehyde concentration on concentration of histidylhistidine, solving for IC5' Table 2 showing the computed IC50 values for the a-oxoaldehydes produced by disproportionation of glucose and these values indicate that His-His is a potent agent for decreasing the level of a-oxoaldetydes in dialysis fluids. It should further be noted that the decrease in a-oxoaldehyde concentration far exceeds the concentration levels of the His-His present, showing that the His-His is acting as a true catalyst: for example, with 50 M His-His, the concentration of 3-DG was decreased by 727 I1M, indicating that His-His engaged in at least 15 catalytic cycles in this incubation. Thus showing that His-His o peptides decrease a-oxoaldehyde levels in a model dialysis fluid sterilisation system catalytically.

Table 2 Median inhibitory concentration values of L-histidyl-histidine for the decrease of o-oxoaldehydes in model dialysis fluid sterilization o-Oxoaldehyde ICso for His-His (pM) Glyoxal 2.4 + 0.6 Methylglyoxal 6.3 + 2.6 HPA < 1 ELM Erythrosone < 1 IBM 3-Deoxyerythrosone < 1 I1M 3-Deoxyribosone 1.5 + 0.5 3-Deoxyglucosone 13.8 + 0.4 Data in Figure 7 were fitted to a logistic regression equation with regression ofpercentage decrease of o- oxoaldehyde concentration on concentration of histidyl-histidine, solving for the median inhibitory concentration ICso value. Data are mean + SD.

Commonly used abbreviations for amino acids are used throughout this document these correspond to: His - histidine, Lys - Iysine, Arg arginine and Glu - glutamic acid Reference is made in the specification to the use of L-histidine, we believe that D- histidine would work equally well as the catalyst.

It will be appreciated by those skilled in the art that various modifications may be made to the invention described herein without departing from the scope thereof.

For example, although the specific examples provided relate to situations where the so catalyst is added to a solution before thermal processing is carried out, it will be appreciated by those skilled in the art that the catalyst could be added to a system following thermal processing wherein the solution is to be stored for a prolonged period of time, such that the catalyst works at a slower rate but removes the a-oxoaldehydes present in the solution over the prolonged storage period.

It will also be appreciated by those skilled in the art the catalyst described herein can be added to solutions other than those previously described but wherein the generation of oc oxoaldehydes is known to be a problem during thermal processing or storage of the solution. .

References 1 UK Renal Registry. UK Rena] Registry Report. Ansell, D. and Feest, T. 2002.

Bristol, U.K. 2 Alloatti, S., Manes, M., Paternoster, G., Gaiter, A. M., Molino, A., and Rosati, C. (2000) Peritoneal dialysis compared with hemodialysis in the treatment of end-stage renal disease. J.Nephrol. 13, 331-342 3 Thodis, E., Passadakis, P., Vargemezis, C., and Oreopoulis, D. G. (2001) Peritoneal dialysis: Better than, equal to, or worse than hemodialysis? Data worth knowing to before choosing a dialysis modality. Peritoneal Dialysis Internat. 21, 25-35 4 Van Biesen, W., Veys, N., VanHolder, R., and Lameire, N. (2003) New concepts on peritoneal dialysis: new wine in old barrels. Artificial Organs 27, 398-405 Lindsay, R. M., Leitch, R., Heidenheim, A. P., and Kortas, C. (2003) The London Daily/Nocturnal Hemodialysis Study - Study design, morbidity, and mortality results. Amer.J.Kidney Dis. 42, S5-S12 6 Zoccali, C., Mallamaci, F., and Tripepi, G. (2003) Inflammation and atherosclerosis in end-stage renal disease. Blood Purification 21, 29-36 7 Cooker, L. A., Holmes, C., and Hoff, C. M. (2002) Biocompatibility of icodextrin.

Kidney Internat. 62, Suppl.81, S34-S45 8 Himmelfarb, J., Stenvinkel, P., Ikizler, C. A., and Hakim, R. A. (2002) The elephant in uremia: Oxidant stress as a unifying concept of cardiovascular disease in uremia.

Kidney Internat. 62, 1524-1538 9 Thornalley, P. J., Battah, S., Ahmed, N., Karachalias, N., Agalou, S., Babaei-Jadidi, R., and Dawnay, A. (2003) Quantitative screening of advanced glycation endproducts in cellular and extracellular proteins by tandem mass spectrometry.

Biochem.J. 375, 581-592 Thornalley, P. J. (1998) Cell activation by glycated proteins. AGE receptors, receptor recognition factors and functional classification of AGEs. Cell.Molec.Biol.

44, 1013-1023 ao 11 Ng, R., Argirov, O. K., Ahmed, N., Weigle, B., and Thornalley, P. J. (2002) Human serum albumin minimally modified by methylglyoxal binds to human mononuclear leukocytes via the RAGE receptor and is displaced by N-carboxymethyl-lysine and hydroimidazolone AGE epitopes. In The Maillard Reaction in Food Chemistry and Medical Science: Update for the Postgenomic Era (Horiuchi, S., Taniguchi, N., Hayase, F., Kurata, T., and Osawa, T., eds.), pp. 77-81, Elsevier, Tokyo 12 Stehouwer, C. D. A., Gall, M. A., Twisk, J. W. R., Knudsen, E., Emeis, J. J., and Parving, H.-H. (2002) Increased urinary albumin excretion, endothelial dysfunction, and chronic low-grade inflammation in type 2 diabetes. Diabetes 51, 1157-1165 13 Rabb, H., Calderon, E., Bittle, P. A., and Ramirez, G. (1996) Alterations in soluble intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 in hemodialysis patients. Amer.J.Kidney Dis. 27, 239-243 14 Boulanger, E., Wautier, M. P., Wautier, J. L., Boval, B., Panis, Y., Wernert, N., Danze, P. M., and Dequiedt, P. (2002) AGEs bind to mesothelial cells via RAGE and stimulate VCAM-1 expression. Kidney Internat. 61, -148 Kjellstrand, P., Martinson, E., Wieslander, A., Kjellstrand, K., Jeppsson, E., Svensson, E., Jarkelid, L., Linden, T., and Olsson, L. F. (2002) Degradation in peritoneal dialysis fluids may be avoided by using low pH and high glucose to concentration. Peritoneal Dialysis Internat. 21, 338-344 16 Thornalley, P. J., Yurek-George, A., and Argirov, O. K. (2000) Kinetics and mechanism of the reaction of aminoguanidine with the a- oxoaldehydes, glyoxal, methylglyoxal and 3-deoxyglucosone under physiological conditions.

Biochem.Pharmacol. 60, 55-65 17 Thornalley, P. J. and Minhas, H. S. (1999) Rapid hydrolysis and slow a, ,B- dicarbonyl cleavage of an agent proposed to cleave glucose-derived protein cross- links. Biochem.Pharmacol. 57, 303-307 ]8 Thornalley, P. J. (2003) Use of aminoguanidine (Pimagedine) to prevent the formation of advanced glycation endproducts. Arch.Biochem.Biophys. 419, 31 -40 o 19 McLellan, A. C. and Thornalley, P. J. (1992) Synthesis and chromatography of 1,2 diamino-4,5-dimethoxybenzene, 6,7-dimethoxy-2- methylquinoxaline and 6,7 dimethoxy-2,3-dimethylquinoxaline for use in a liquid chromatographic fluorimetric assay of methylglyoxal. Anal.Chhn. Acta 263, 137-142 McLellan, A. C., Phillips, S. A., and Thornalley, P. J. (1992) The assay of methylglyoxal in biological systems by derivatization with 1,2-diamino-4,5 dimethoxybenzene. Anal.Biochem. 206, 17-23 21 Agalou, S., Karachalias, N., Tucker, B., Thornalley, P. J., and Dawnay, A. (2002) Estimation of a-oxoaldehydes formed from the degradation of glycolytic intermediates and glucose fragmentation in blood plasma of human subjects with uraemia. In The Maillard Reaction in Food Chemistry and Medical Science: Update for the Postgenomic Era (Horiuchi, S., Taniguchi, N., Hayase, F., Kurata, T., and Osawa, T., eds.), p. 182, Elsevier, Tokyo '

Claims (9)

1. A process for treating a solution containing sugar and a-oxoaldehydes, comprising the step of adding a catalyst which comprises an optionally substituted histidine amino acid, such that the a-oxoaldehydes are catalytically converted to aldonic acids.

2. A process according to claim l wherein the process involves a thermal processing step which is suitable for the sterilization of the solution.

3. A process according to any preceding claim wherein the optionally substituted histidine comprises a histidine amino acid substituted with at least one further amino acid and fewer than four further amino acids

4. A process according to claim 3 wherein the optionally substituted histidine comprises a peptide comprising 2 to 4 amino acids, one of which is histidine.

5. A process according to claim 3 wherein the at least one further amino acid comprises a single amino acid chosen from the following; tyrosine, aspartic acid, histidine, arginine, glutamic acid or Iysine.

6. A process according to any preceding claim wherein the sugar comprises a reducing sugar.

7. A process according to claim 5 wherein the reducing sugar is glucose, ribose or a reducing sugar polymer.

8. A process according to any preceding claim wherein the catalyst comprises a plurality of various optionally substituted histidine amino acids.

9. A process according to any preceding claim wherein the sterilised solution is a peritoneal dialysis fluid.