Boronate Affinity Column Research Articles

Fewer Exposed Lysine Residues May Explain Relative Resistance of Chicken Serum Albumin to In Vitro Protein Glycation in Comparison to Bovine Serum Albumin.

Protein glycation and formation of advanced glycation end products is associated with several diseases resulting from high blood glucose concentrations. Plasma albumin is directly exposed to circulating glucose concentrations and is therefore at greater risk of glycation than hemoglobin. As plasma glucose concentrations in birds are 1.5-2 times higher than mammals of similar mass, avian albumin may be particularly at risk of glycation. Thus, the goal of the present study was to compare the in vitro formation of glycated albumin in chicken serum albumin (CSA) and bovine serum albumin (BSA) exposed to a range of glucose concentrations over a 16-week period. The level of glycation for CSA and BSA was quantified using boronate affinity columns to separate glycated albumin from non-glycated albumin and calculating the difference in protein concentration of each sample. The results indicate that CSA is glycated to a lesser degree than BSA when the albumins are exposed to increasing concentrations of glucose (38.8-500mM). This was most apparent at week sixteen (500mM glucose) where BSA expressed a higher degree of glycation (37.8 ± 0.76%) compared to CSA (19.7 ± 1.06%, P < 0.05). Additionally, percent glycation at week sixteen was significantly higher than the glucose-free solutions for both BSA and CSA, indicating that glycation is glucose-dependent. Analyses of the protein structures suggest that the relative resistance of CSA to glycation may be due to fewer lysine residues and variations in protein folding that shield more lysine residues from the plasma. Moreover, comparisons of reconstructed ancestral albumin sequences show that the ancestor of birds had 6-8 fewer lysine residues compared to that of mammals.

Characterization of unstable hemoglobin A1c complexes by dynamic capillary isoelectric focusing

Glucose spontaneously reacts with hemoglobin amino groups to produce unstable Schiff base complexes that can dissociate or rearrange to form stable Amadori products. We used dynamic capillary isoelectric focusing and boronate affinity chromatography to assess the formation and dissociation of unstable hemoglobin complexes in vitro. Formation was studied by incubating erythrocytes at 37°C for up to 24h in phosphate-buffered saline (PBS) supplemented with 0 to 55.6mmol/L glucose. Dissociation was studied by incubating glucose-loaded erythrocytes in PBS without glucose. Dynamic capillary isoelectric focusing separated hemoglobin A1c into two subfractions identified as A1c1 and A1c2. The A1c1 subfraction contained both stable and unstable hemoglobin complexes. The A1c2 subfraction contained only unstable hemoglobin complexes. Both subfractions quantitatively increased in the presence of glucose and decreased in its absence. Rates of increase and decrease were faster and time to equilibrium was shorter for A1c2 (∼4h) compared with A1c1 (∼20h). Unstable hemoglobin complexes did not bind to boronate affinity columns but instead eluted intact in A1c1 and A1c2 subfractions from nonglycated affinity fractions. Cyanoborohydride reduction confirmed the presence of Schiff base complexes. Evidence of multiple unstable hemoglobin complexes with different rates of glycation suggests that new models are needed to describe nonenzymatic hemoglobin glycation.

A methodology for simultaneous fluorogenic derivatization and boronate affinity enrichment of 3-nitrotyrosine-containing peptides

We synthesized and characterized a new tagging reagent, (3 R,4 S)-1-(4-(aminomethyl)phenylsulfonyl)pyrrolidine-3,4-diol (APPD), for the selective fluorogenic derivatization of 3-nitrotyrosine (3-NT) residues in peptides (after reduction to 3-aminotyrosine) and affinity enrichment. The synthetic 3-NT-containing peptide, FSAY(3-NO 2)LER, was employed as a model for method validation. Furthermore, this derivatization protocol was successfully tested for analysis of 3-NT-containing proteins exposed to peroxynitrite in the total protein lysate of cultured C2C12 cells. The quantitation of 3-NT content in samples was achieved through either fluorescence spectrometry or boronate affinity chromatography with detection by specific fluorescence (excitation and emission wavelengths of 360 and 510 nm, respectively); the respective limits of detection were 95 and 68 nM (19 and 13 pmol total amount) of 3-NT. Importantly, the derivatized peptides show a strong retention on a synthetic boronate affinity column, containing sulfonamide-phenylboronic acid, under mild chromatographic conditions, affording a route to separate the derivatized peptides from large amounts (milligrams) of nonderivatized peptides and to enrich them for fluorescent detection and mass spectrometry (MS) identification. Tandem MS analysis identified chemical structures of peptide 3-NT fluorescent derivatives and revealed that the fluorescent derivatives undergo efficient backbone fragmentations, permitting sequence-specific identification of protein nitration at low concentrations of 3-NT in complex protein mixtures.

The analysis of avian plasma for glycated serum albumin

Plasma glucose concentrations (Pglu) in humans are regulated at 80–100mg%. If levels reach 150mg% for 4–6 weeks, the symptoms of Type 2 diabetes appear due to the glycation of serum albumin and the subsequent formation of advanced glycation end‐products (AGEs). AGEs bind to vascular tissues leading to the pathologies associated with Type 2 diabetes. In contrast to humans, the Pglu of birds range from 350–750mg% and from what is observed, birds do not suffer the pathologies of Type 2 diabetes. To gain an understanding of this discrepancy, in vitro experiments were conducted to analyze the reaction between glucose and avian serum albumin (ASA) and bovine serum albumin (BSA) which was used as a control. Glycated ASA and BSA were separated using boronate affinity columns and percent glycation was quantified using the Bradford protein assay. Results indicate that ASA is glycated to a lesser degree than BSA when exposed to varying concentrations of glucose. After 16 weeks of in vitro incubation with 500mM glucose, 37.8 ± 0.76% of BSA was glycated compared to 19.7 ± 1.06% for CSA, a highly significant difference (P&lt;0.05). Additionally, glycation appeared to be glucose dependent, where the percent glycation at week 16 was significantly higher than the control solutions for both BSA (P&lt;0.001) and CSA (P&lt;0.05, P&lt;0.001 and P&lt;0.001 respectively for the glucose concentrations of 38.8mM, 100mM and 500mM).

Glycation Isotopic Labeling with 13C-Reducing Sugars for Quantitative Analysis of Glycated Proteins in Human Plasma

Non-enzymatic glycation of proteins is a post-translational modification produced by a reaction between reducing sugars and amino groups located in lysine and arginine residues or in the N-terminal position. This modification plays a relevant role in medicine and food industry. In the clinical field, this undesired role is directly linked to blood glucose concentration and therefore to pathological conditions derived from hyperglycemia (>11 mm glucose) such as diabetes mellitus or renal failure. An approach for qualitative and quantitative analysis of glycated proteins is here proposed to achieve the three information levels for their complete characterization. These are: 1) identification of glycated proteins, 2) elucidation of sugar attachment sites, and 3) quantitative analysis to compare glycemic states. Qualitative analysis was carried out by tandem mass spectrometry after endoproteinase Glu-C digestion and boronate affinity chromatography for isolation of glycated peptides. For this purpose, two MS operational modes were used: higher energy collisional dissociation-MS2 and CID-MS3 by neutral loss scan monitoring of two selective neutral losses (162.05 and 84.04 Da for the glucose cleavage and an intermediate rearrangement of the glucose moiety). On the other hand, quantitative analysis was based on labeling of proteins with [(13)C(6)]glucose incubation to evaluate the native glycated proteins labeled with [(12)C(6)]glucose. As glycation is chemoselective, it is exclusively occurring in potential targets for in vivo modifications. This approach, named glycation isotopic labeling, enabled differentiation of glycated peptides labeled with both isotopic forms resulting from enzymatic digestion by mass spectrometry (6-Da mass shift/glycation site). The strategy was then applied to a reference plasma sample, revealing the detection of 50 glycated proteins and 161 sugar attachment positions with identification of preferential glycation sites for each protein. A predictive approach was also tested to detect potential glycation sites under high glucose concentration.

Development of an On-Line SPE-LC–ESI-MS Method for Urinary Nucleosides: Hyphenation of Aprotic Boronic Acid Chromatography with Hydrophilic Interaction LC–ESI-MS

The development of an on-line automated SPE-HPLC--ESI-MS method is described for targeted metabolomic analysis of urinary modified nucleoside levels. The setup comprises a boronate affinity column as a trapping device, a hydrophilic interaction chromatography (HILIC) separation and information-dependent MS detection modes. The system was optimized using standards and tested on biological samples, detecting a number of modified nucleosides. Other urinary biomarkers could also be analyzed by the system developed: for example, the urinary nucleobases were also available for analysis. A simultaneous creatinine-monitoring experiment was also demonstrated to be viable when utilizing the method, which is of benefit as creatinine is a urinary normalizing factor.

Electrochemical detection of HbA 1c, a maker for diabetes, using a flow immunoassay system

An on-chip electrochemical flow immunoassay system for the detection of hemoglobin A 1c (HbA 1c) was developed using anti-human hemoglobin (Hb) IgG labeled with ferrocene monocarboxylic acid (Fc–COOH) and boronate-affinity chromatography. An on-chip column packed with boronate-activated agarose beads was used for the separation of HbA 1c from both non-glycated Hb and free antibody. Anti-human Hb IgG conjugated to Fc–COOH (Fc–IgG) was used for the electrochemical detection of HbA 1c. The assay procedure included immunoreactions with Fc–IgG and HbA 1c, separation of immunocomplexes by boronate affinity, and electrochemical detection of Fc–IgG–HbA 1c immunocomplexes. The immunoreaction mixtures were injected onto a boronate-affinity column. HbA 1c–antibody complexes were then trapped onto the column by the affinity of HbA 1c to boronic acid. Subsequently, elution buffer containing sorbitol was applied to elute HbA 1c–antibody complexes and a current was detected by applying 600 mV versus Ag/AgCl. The elution signal was an estimation of the HbA 1c amount. A linear correlation between the increase of current and HbA 1c concentration was obtained up to an HbA 1c concentration of 500 μg/ml. The HbA 1c flow immunoassay was successfully achieved using hemolysates. This electrochemical flow immunoassay system enabled us to construct a novel point-of-care testing device for the monitoring of glycated proteins including HbA 1c.

Effects of oxidative modifications induced by the glycation of bovine serum albumin on its structure and on cultured adipose cells

Non-enzymatic glycosylation (glycation) and oxidative damages represent major research areas insofar as such modifications of proteins are frequently observed in numerous states of disease. Albumin undergoes structural and functional alterations, caused by increased glycosylation during non insulin-dependent diabetes mellitus, which is closely linked with the early occurrence of vascular complications. In this work, we first characterized structural modifications induced by the glycation of bovine serum albumin (BSA). A pathophysiological effect of glycated BSA was identified in primary cultures of human adipocytes as it induces an accumulation of oxidatively modified proteins in these cells. BSA was incubated in the presence or absence of physiological, pathological or supra-physiological concentrations of glucose at 37 °C for 7 weeks. Enhanced BSA glycation percentages were determined using boronate affinity columns. The occurrence of oxidative modifications was found to be enhanced in glycated BSA, after determination of the free thiol groups content, electrophoretic migration and infrared spectrometry spectra. An accumulation of carbonyl-modified proteins and an increased release of isoprostane were observed in cell media following the exposure of adipocytes to glycated albumin. These results provide a new possible mechanism for enhanced oxidative damages in diabetes.

Stationary phase-based two-dimensional chromatography combining both covalent and noncovalent interactions on a single HPLC column

A new type of 2-D separation material was synthesized and studied. The material is suitable for 2-D chromatography utilizing both covalent and noncovalent interactions. The first dimension is boronate affinity chromatography, and the second dimension is RP chromatography (or vice versa). The polymeric media were prepared using p-vinylphenylboronic acid as the functional monomer. This monomer was selected due to the presence of the boronic acid group for the cis-diol/boronate interaction in boronate chromatography. Two crosslinkers were evaluated, namely ethylene glycol dimethacrylate and divinylbenzene. The crosslinker content was varied to maximize the polymer strength and the RP performance of the packed column. Several parameters were evaluated to define the optimum for polymer strength and column performance including crosslinker, porogen, initiator, and column-packing parameters. The polymer-based HPLC columns were successful in separating phenol, catechol, dimethylphthalate, and hydroquinone under RP conditions, and thus can be used as an RP HPLC column. The columns were also successful in separating catechol and adenosine under boronate chromatography conditions, and thus can be used as a boronate affinity column. Moreover, the two types of chromatography can be performed consecutively on the same column during one complete chromatographic run, making it a 2-D chromatography. Under these 2-D conditions, the catechol was separated from a mixture of phenol, catechol, dimethylphthalate, and hydroquinone; the adenosine ribonucleoside was separated from a mixture of adenosine ribonucleoside, adenosine deoxyribonucleoside, and uridine deoxyribonucleoside. This type of single-column 2-D HPLC eliminates the requirement of a complex and expensive multidimensional HPLC instrument and provides increased peak capacity for separation.

Early Glycation Products Produce Pentosidine Cross-links on Native Proteins: NOVEL MECHANISM OF PENTOSIDINE FORMATION AND PROPAGATION OF GLYCATION

Bovine lens alpha-crystallin was immobilized on EAH-Sepharose gel and glycated using d-ribose. Incubation with 500 and 100 mm d-ribose for 2 and 15 days produced short-term glycated (STGP gel) and long-term glycated proteins (LTGP gel). Both STGP and LTGP gels produced oxygen free radicals. Hydroxyl radical production was twice that in STGP gel compared with the LTGP gel. Incubation with the glycated gels produced pentosidine in a mixture of N-alpha-acetylarginine + N-alpha-acetyllysine, bovine lens proteins (BLP), and lysozyme; the amounts measured with STGP gel were higher than those with LTGP gel. Reactive oxygen species scavengers decreased the formation of pentosidine. Pentosidine was also formed in BLP when incubated with water-insoluble proteins extracted from aged or brunescent human lenses. Early glycated proteins from aged or diabetic lenses were bound to a boronate affinity column, the protein-containing gel was incubated with BLP, and pentosidine was measured in the incubation mixtures. With this method we found that diabetic lens proteins produced more pentosidine on BLP than did aged lens proteins. Further investigation indicates that two and three carbon carbohydrates possibly formed from oxidative cleavage of early glycation products are involved in pentosidine formation. Based on our findings, we propose a novel pathway for pentosidine formation on native proteins from glycated proteins.

Spontaneous Generation of Superoxide Anion by Human Lens Proteins and by Calf Lens Proteins Ascorbylated In Vitro

The proteins isolated from aged human lenses and brunescent cataracts exhibit extensive disulfide bond formation. Diabetic rat lenses similarly contain disulfide-bonded protein aggregates. These observations are consistent with the known link between diabetes, glycation and oxidative damage, and suggest a role for reactive oxygen species (ROS) in this process. To assess whether the glycation-related modifications in human lens proteins spontaneously generate ROS, superoxide anion formation was measured using both cataractous lens proteins and calf lens proteins glycated in vitro with ascorbic acid (ascorbylated).The water-insoluble fraction from aged normal human lenses generated 0.3–0.6 nmol superoxide h−1mg protein−1, whereas the activity increased to 0.5–1.8 nmol h−1mg protein−1with the WI fraction from brunescent cataracts, and 2.3 nmol h−1mg protein−1with calf lens proteins ascorbylated for 4 weeks in vitro. The activity in the human lens proteins was observed in both the water-soluble and water-insoluble fractions, and was completely dependent upon the presence of oxygen. The pH optimum curve for superoxide formation increased from pH 6.5 to 10 with both the cataract and ascorbylated proteins. The superoxide-generating activity in human lens was completely bound to a boronate affinity column, but only partially bound with the ascorbylated proteins.The superoxide anion produced by a 5 m m solution of purifiedNϵ -fructosyl–lysine was barely detectable, and therefore, could not account for the superoxide formed by any of the lens protein preparations. Also, superoxide formation increased 10-fold at pH 8.8 with fructosyl–lysine, but only 1.3–1.8-fold with human lens proteins. The addition of copper-stimulated superoxide formation with glycated bovine serum albumin, but no stimulation was seen with cataractous proteins. Assays of specific compounds showed that catechol, hydroquinone, 3-OH kynurenine and 3-OH anthranylic acid exhibited the greatest activity for superoxide generation, but had a very short halflife. 2,3-Dihydroxypyridine and 4,5 dihydroxynaphthalene were one and two orders of magnitude less reactive.In long-term incubations at 37°, cataractous proteins retained the potential to produce superoxide anion, losing only half of the initial activity after 6–7 days. Therefore, the water-insoluble fraction from aged human lenses and dark brown cataracts are potentially capable of generating >100 nmol mg protein−1and >170 nmol mg protein−1of superoxide anion respectively, likely due to the presence of advanced glycation endproducts in human lens proteins. This spontaneous generation of superoxide anion in vivo could account for a major portion of the oxidation of sulfur amino acids seen during aging and cataract formation.

Human erythrocyte bisphosphoglycerate mutase: inactivation by glycation in vivo and in vitro.

2,3-Bisphosphoglycerate mutase (BPGM) [EC 5.4.2.4] is a multifunctional enzyme that catalyzes both the synthesis and the degradation of 2,3-diphosphoglycerate (2,3-DPG) and contains three types of activities in that it functions as a 2,3-DPG synthetase, a phosphoglycerate mutase and a 2,3-DPG phosphatase. In humans, BPGM occurs only in erythrocytes and plays a pivotal role in the dissociation of oxygen from hemoglobin via 2,3-DPG. The present study shows that the specific activity of BPGM in erythrocytes of diabetic patients is decreased, compared to normal controls as judged by 2,3-DPG synthetase activity and immunoreactive contents. To understand the mechanism by which the enzyme is inactivated, the enzyme was purified from pooled erythrocytes from diabetic patients and subjected to a boronate affinity column. The flow through fraction was active while the bound fraction was completely inactive. The bound fraction was reactive to an anti-hexitollysine antibody, indicating that the enzyme had undergone glycation and inactivation. The primary glycated site of the enzyme was found to be Lys158 as judged by amino acid sequencing and the reactivity with an anti-hexitollysine IgG, after reverse-phase HPLC of the lysyl-endopeptidase-digested peptides. Extensive glycation of recombinant BPGM in vitro indicated that the glycation sites were Lys2, Lys4, Lys17, Lys42, Lys158, and Lys196. From these results, the loss of enzymatic activity appears to be due to the glycation of Lys158 which may be located in the vicinity of the substrate binding site.

Determination of glycated albumin by enzyme-linked boronate immunoassay (ELBIA)

A new affinity method for quantification of glycated albumin by an enzyme-linked boronate-immunoassay (ELBIA) has been established, based on the interaction between boronic acids and the cis-diols of glycated human serum albumin (HSA) trapped by anti-HSA antibody. To evaluate the ELBIA, we first examined the accuracy of the conventional boronate affinity chromatographic (BAC) method. In the BAC method, 8.1-18.9% of nonglycated albumin calibrator nonspecifically bound to the boronate affinity column, values that were regarded as the column blank. In the modified BAC method, therefore, we substracted the column blank value from the measured glycated albumin value to obtain the true value. Because glycated albumin values measured by ELBIA were exactly the same as reported by the modified BAC method, we suggest that the ELBIA results reflect the real status of albumin glycation. We have also developed a fully automated ELBIA system, allowing multiple, rapid, and precise measurements of glycated albumin.

Glycation and inactivation of aspartate aminotransferase in diabetic rat tissues.

Glycated cytosolic aspartate aminotransferase was detected in the liver and kidney of streptozotocin diabetic rats using a boronate affinity column for adjacent cis-hydroxyl groups and an immunoblotting technique. The enzymatic activity and amount of immunoreactive substance were determined in the liver, kidney, and erythrocytes of diabetic and control rats. The ratio of enzymatic activity to the amount of enzyme was lower in diabetic rat tissue than in that in the control rats. It has been suggested that there is an inactive aspartate aminotransferase molecule in the tissues of diabetic rats. We therefore suggest that the cytosolic aspartate aminotransferase was inactivated in the diabetic rat tissues by a glycation reaction, accompanied by an impairment in glucose utilization.

Glycohemoglobin measured by automated affinity HPLC correlates with both short-term and long-term antecedent glycemia

We evaluated glycohemoglobin (GHb) and glycated plasma protein (GPP) by automated affinity HPLC for their ability to monitor both short-term and long-term antecedent glycemia in 70 diabetic subjects. We placed 30 subjects on an intervention protocol in which insulin and (or) dietary changes were made twice weekly to acutely decrease glycemia. We monitored 40 subjects at 6-week intervals; changes in the clinical regimen were made at that time only. Despite weekly changes in mean blood glucose in the subjects who received more intensive intervention. GHb concentrations correlated significantly with the weekly (r = 0.66, P < 0.001), 2-week (r = 0.70, P < 0.001), 3-week (r = 0.72, P < 0.001), and 6-week (r = 0.83, P < 0.001) mean glucose concentrations. GPP correlated significantly with measured glycated albumin determined by boronate affinity columns (r = 0.83, P < .001) and correlated best with the 1-week (r = 0.66, P < 0.001), 2-week (r = 0.64, P < .001) and 3-week (r = 0.60, P < 0.001) mean antecedent glucose concentration. Thus, GHb, traditionally considered a marker for only long-term diabetic control, correlated significantly with both short-term and long-term antecedent glycemia.

The glycation and cross-linking of isolated lens crystallins by ascorbic acid

Individual lens crystallins were isolated from calf lens extracts and incubated in the presence of ascorbic acid for 3 weeks under aerobic conditions. Both α-crystallin and β H-crystallin rapidly cross-linked to form high molecular weight proteins, which did not enter the resolving gel on SDS-PAGE. Beta l -crystallin was somewhat less reactive, but γ-crystallin showed little or no crosslinking. Gamma-crystallin, however, was almost equivalent to the other crystallins as a substrate for glycation. This was measured by: ( a) the binding of protein to a boronate affinity column; ( b) the incorporation of 3H from NaB 3H 4 into protein; ( c) amino acid analysis of the modified proteins to estimate the extent of lysine modification; and ( d) the incorporation of [1- 14C]ASA into individual crystallins. When the separated crystallins were combined with [ 125I]γ-crystallin and incubated with ascorbic acid, radioactivity was readily incorporated into the cross-linked products with other crystallins, but again not with γ-crystallin itself. Gel filtration chromatography of a mixture of [ 125I]γ-crystallin and α-crystallin showed the formation of a complex between γ- and α-crystallins. These data suggest that all crystallins are glycated, but that cross-linking occurs preferentially between proteins, which are already bound together non-covalently.

New ligands for boronte affinity chromatography : Synthesis and properties a

In order for a boronate ligand to be useful in affinity chromatography for the purification of biomolecules, it must be able to form a stable complex in an environment (pH) in which the affinity molecule is stable. A major limitation of the widely used ligand, 3-aminophenylboronate, is its high ionization constant (p K a 8375). to make this complex under more faborable pH conditions, different methods have been explored here in order to introduce an electron-withdrawing (nitro) group in the phenyl ring. Reagents and procedures for the preparation of ortho-, meta- and para-nitro derivatives of succinamidophenhylboronic acid using nitronium trifluromethanesulfonate are described. Preferential substitution of the nitro functinality into the ortho position of the boronic acid is exploited by selective use of acetic anhydrided for the reaction medium. This method yields mostly an ortho-nitro derivative (; K a 7.4) under selected reaction conditions. The ionization and solute—ligand interaction of several phenylboronates are studied in solution by using 11B NMR and spectrophotometric methods. The results indicate the presence of specific chemical shifts for the neutral (δ 30), the boronate anions (δ 30, and the cis-diol-complex boronate species (δ 7.5). In the presence of a cis-diol derivative, the complex formation is favored over anionization of neutral species. Moreover, the complex is formed approximately on pH unit below the ionization constant of the ligand and is stable, i.e. fails to break down in boronate anion, even when the solution pH is raised appreciably. Two boronate affinity column matrices were examined for their binding capacity and apparent dissociation constant. The results clearly indicate that the formation and also the breakdown of the complex are greatly enhanced because of the presence of the elecgtron-withdrawing group in the boronate ligand. The results further demonstrate that small structural differences in affinity moleculew have significant differences on their binding capacities. A comparison of binding betwen alkyl- cis-diols and aryl cis-diols to different boronate matrices indicates taht the aryl affinity molecules not only form a complex but do so very effectively. The significance of this work lies in the demonstration that the best environment for the ligand—solute interaction can be established by carrying out studies in solution, without prior immobilization of the ligand. The results from in-solution studies and those from the affinity columns are in very good agreement. The new nitrophenylboronate matrix offers enhanced binding of most affinity molecules over those examined with the phenylboronate matrix. In addition, the new matrix offers chromatographic separations of alkali-unstable biomolecules.

Glycation of lens proteins by the oxidation products of ascorbic acid

Bovine lens water-soluble proteins were incubated with [I-14C]ascorbic acid (ASA) for 6 days, and the incorporation into protein was measured at daily intervals. Aliquots were also withdrawn to determine the distribution of label among the various ASA oxidation products. A linear incorporation into protein was observed in the presence of NaCNBH 3, however, little or no incorporation was seen in its absence. TLC analysis showed a complete loss of ASA by day 3, whereas both dehydroascorbate (DHA) and diketogulonic acid (DKG) remained constant for 6 days, consistent with the linear incorporation into protein. The amino acid composition of the proteins glycated in the presence of NaCNBH 3 was identical to controls except for a 70% reduction in lysine residues and a corresponding increase in an unknown product which eluted slightly earlier than methionine. In the absence of NaCNBH 3 lysine decreased linearly to 20% with an additional decrease in arginine and histidine at later times concurrent with protein crosslinking. DHA and DKG were prepared and incubated directly with lens proteins for an 8 day period. Both compounds glycated lens protein as evidenced by an increased binding to a boronate affinity column. SDS-PAGE showed that both compounds were also capable of causing protein crosslinking. DHA is apparently capable of reacting directly with protein since glycation was observed with the ASA analog, reductic acid, which can be oxidized to dehydroreductic acid, but which cannot be hydrolyzed to an open chain structure. DHA also produced a lysine adduct which was not obtained with DKG, supporting the idea that both species have glycating ability.

Ascorbic acid-induced crosslinking of lens proteins: evidence supporting a Maillard reaction

The incubation of calf lens extracts with 20 mM ascorbic acid under sterile conditions for 8 weeks caused extensive protein crosslinking, which was not observed with either 20 mM sorbitol or 20 mM glucose. While no precipitation was observed, ascorbic acid did induce the formation of high-molecular-weight protein aggregates as determined by Agarose A-5m chromatography. Proteins modified by ascorbic acid bound strongly to a boronate affinity column, however, crosslinked proteins were present mainly in the unbound fraction. These observations suggest that the cis-diol groups of ascorbic acid were present in the primary adduct, but were either lost during the crosslinking reaction or sterically hindered from binding to the column matrix. The amino acid composition of the ascorbic acid-modified proteins was identical to controls except for a 15% decrease in lysine. Amino acid analysis after borohydride reduction, however, showed a 25% decrease in lysine, a 7% decrease in arginine and an additional peak which eluted between phenylalanine and histidine. Extensive browning occurred during the ascorbic acid-modification reaction. This resulted in protein-bound chromophores with a broad absorption spectrum from 300 to 400 nm, and protein-bound fluorophores with excitation/emission maxima of 350/450 nm. A 4 week incubation of dialyzed crude lens extract with [1- 14C]ascorbic acid showed increased incorporation for 2 weeks, followed by a decrease over the next 2 weeks as crosslinking was initiated. The addition of cyanoborohydride to the reaction mixture completely inhibited crosslinking and increased [1- 14C]ascorbic acid incorporation to a plateau value of 180 nmol per mg protein. Amino acid analysis showed a 50% loss of lysine, an 8% decrease in arginine and the presence of a new peak which eluted slightly earlier than methionine. These data are consistent with the non-enzymatic glycation of lens proteins by either ascorbic acid or an oxidation product of ascorbic acid via a Maillard-type reaction.

ALLOPURINOL (AP) INDUCED OROTIDINURIA (ODNU): A TEST OF HETEROZYGOSITY FOR ORNITHINE TRANS-CARBAHYLASE (OTC) DEFICIENCY

In addition to its effects on purine metabolism, AP, via its metabolite, oxipurinol ribonucleotide, inhibits pyrimidine biosynthesis (PB) at the level of orotidine mono-phosphate decarboxylase. This inhibition results in increased ODNU. Women heterozygous at the OTC locus have 2 populations of hepatocytes; OTC normal and OTC deficient. We hypothesize that, as a consequence of carbamyl phosphate accumulation in the latter cells, PB is chronically increased. Therefore, inhibition of PB by AP should result in an even greater increase in ODNU in OTCH as compared to controls. The AP test includes administration of 300mg of AP followed by four pooled consecutive 6 hr urine collections. Urinary orotidine was measured by reverse phase HPLC after isolation of nucleosides on a boronate affinity column. We studied 11 control women (C) and 12 obligate OTC heterozygotes (OTCH). The mean (±SEM) peak urine orotidine (umol/mmol creatinine) of C and OTCH was respectively 7.36±0.46 (range 5.7-10.1) and 39.7±7.3. One OTCH fell in the normal range (7.71); other OTCH values ranged from 17.9 to 97.9. Comparison of these results with a protein tolerance test (PTT) administered to the same subjects, shows that the diagnostic sensitivity of the 2 tests are the same and that the same OTCH was negative in both. We conclude that PB is chronically increased in OTCH; and that the AP test identifies OTCH as well as the PTT, eliminates the risk of hyperammonemia, and is more convenient and acceptable.