Milton S. Feather

Detection of 3-deoxyfructose and 3-deoxyglucosone in human urine and plasma: Evidence for intermediate stages of the maillard reaction in vivo☆

3-Deoxyglucose (3-deoxy-D-erythro-hexos-2-ulose) (3-DG) is a reactive dicarbonyl intermediate involved in the polymerization and browning of proteins by glucose in vitro. Damage to protein by formation of 3-DG in vivo is thought to be limited by enzymes which convert 3-DG to less reactive species, such as 3-deoxyfructose (3-DF). We have developed a sensitive and specific assay for measuring 3-DG and 3-DF in human urine and plasma. In this assay, 3-DG and 3-DF are reduced to 3-deoxy-hexitols (3-DH), using either NaBH4 or NaBD4, and then analyzed by selected ion monitoring gas chromatography-mass spectrometry. Based on comparative analysis of samples reduced with NaBD4 versus NaBD4, 3-DH in urine was derived exclusively (greater than 99%) from 3-DF, while 3-DG accounted for approximately 15% of 3-DH in plasma. The concentrations of 3-DH in fasting human urine and plasma were 5.3 +/- 1.5 micrograms/mg creatinine (n = 18) and 7.2 +/- 1.7 micrograms/dl (n = 18), respectively. The concentrations of 3-DG and 3-DF in plasma (n = 7) were 1.0 +/- 0.2 and 6.7 +/- 1.6 micrograms/dl, respectively. These results suggest that several milligrams of 3-DG are formed in the body per day and detoxified by reduction to 3-DF and support the role of 3-DG as an intermediate in the browning of protein via the Maillard reaction in vivo.

The rapid, quantitative determination of neutral sugars (as aldononitrile acetates) and amino sugars (as O-methyloxime acetates) in glycoproteins by gas--liquid chromatography.

Abstract O -Methyloximes have been prepared from 2-amino-2-deoxy- d -glucose, - d -mannose, and - d -galactose. The acetates of these derivatives yield stable compounds which are readily separated quantitatively by gas-liquid chromatography on a number of polar phases. The above compounds along with the aldononitrile acetates of neutral sugars can be easily separated from one another on a single column in one chromatographic run. The procedures developed were tested on a number of glycoproteins of known composition as reported by other workers utilizing more classical methodologies, resulting in excellent agreement in terms of sugar composition. An improved method is also described for converting neutral sugars to oximes which can be either converted to the trimethylsilyl derivatives or, upon acetylation, derivatized to aldononitrile acetates.

The precipitation and cross-linking of lens crystallins by ascorbic acid.

Bovine lens beta-crystallin was incubated with increasing concentrations of sugars and sugar derivatives for a period of 2 weeks in the dark at 37 degrees C. Marked protein precipitation and a browning reaction was observed with both ascorbic acid (ASA) and dehydroascorbic acid (DHA), but little or no reaction was seen with several other sugars and sugar analogs. Similar incubations were carried out with 20 mM ASA, 20 mM DHA and 20 mM glucose, but with increasing amounts of the individual crystallins. Glucose was capable of precipitating gamma-crystallin in the presence of air, but this reaction was decreased if dithiothreitol and a chelating agent were added prior to incubation. ASA and DHA produced precipitation and browning with gamma- and beta-crystallin, but not with alpha-crystallin or lens soluble proteins. Similar reactivities were observed both in air and under reducing conditions. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of these reaction mixtures showed little or no cross-linking with any of the lens proteins by glucose. ASA and DHA caused detectable dimer formation with gamma-crystallin, but produced the formation of dimers as well as highly polymerized proteins at the top of the gel with all the other crystallins and with lens soluble proteins. A time-course experiment with alpha-crystallin in the presence of air showed no cross-linking with 100 mM glucose over a 6-week period; however, 10 mM ASA caused definite cross-linking at 2 weeks, and at 6 weeks a dark smear of protein was visible throughout the gel. ASA was still capable of inducing cross-linking under low oxygen conditions but the protein smearing was markedly diminished. Further, the cross-linking pattern was similar to that seen in the water-insoluble fraction from older human lenses and cataracts. This reaction may be significant in vivo because cross-linking was observed under low-oxygen conditions with as little as 2 mM ASA, which is the level of ASA normally present in human lenses.

An improved preparation of 3-deoxy-d-erythro-hexos-2-ulose via the bis(benzoylhydrazone) and some related constitutional studies☆

Abstract Sugar osazones and glycosuloses rapidly and quantitatively react with hydroxylamine to produce oximes that give trimethylsilyl derivatives suitable for g.l.c. and mass spectral analysis. The reaction of d -glucose with benzoylhydrazine to give the bishydrazone of 3-deoxy- d - erythro -hexos-2-ulose ( 1 ) [H. El Khadem et al., Carbohydr. Res. , 22 (1973) 381-89] was re-investigated, together with the conversion of this compound to the hexosulose. Although by-products are produced in the reaction, including the bis(benzoylhydrazone) (osazone) of d -glucose, the major product is the monohydrate of the bis(benzoylhydrazone) of 1 (colorless). The anhydrous (yellow) form can be prepared from the monohydrate by crystallization from absolute ethanol and has quite different physical properties. Improvements of the original preparation are described that allow the preparation of the bishydrazone and its subsequent conversion to 1 via transhydrazonation in 44% overall yield, and with no detectable contamination by d -glucose or d -glucosone. Evidence is presented that the previously reported cyclic form of the bis(benzoylhydrazone) of d -glucose is the bis(benzoylhydrazone) (monohydrate) of 1 .

The preparation and characterization of some Amadori compounds (1-amino-1-deoxy-d-fructose derivatives) derived from a series of aliphatic ω-amino acids☆

Abstract Amadori compounds (1-amino-1-deoxy- d -fructose derivatives) were prepared by reacting d -glucose with a series of aliphatic amino acids. These include Amadori compounds derived from glycine (1), β-alanine (2), γ-amino butyric acid (3), δ-aminovaleric acid (4), ϵ-aminocaproic acid (5) and Nα-formyl- l -lysine (6). In the FAB mass spectra, molecular-ion clusters as well as fragment ions corresponding to loss of water or CO2 molecules were observed. The 13C NMR spectra indicate that all the compounds are conformationally unstable, but that the predominant from present in solution (D2O) is the β-pyranose form. The 1H NMR spectra of 1 and 2 indicate a slow rotation around the C-1—C-2 bond, possibly as a result of an intramolecular hydrogen bond involving the carboxyl group. The pKas of all compounds were measured by pH-potentiometric titration in 0.2 M KNO3 solution at 25°C. All compounds showed a decrease in the basicity of their amino groups (in the order of ≈ 1.5 of the Ka value), and 1 and 2 showed a decrease in the basicity of their carboxyl groups (in the order of ≈ 0.2) in comparison with that of parent amino acids.

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 NaCNBH3, 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 NaCNBH3 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 NaCNBH3 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.

The reaction of some dicarbonyl sugars with aminoguanidine

The reactions of aminoguanidine (guanylhydrazine) with 3-deoxy-D-erythro-hexos-2-ulose (1a), 3-deoxy-D-glycero-pentose-2-ulose (1b), D-erythro-hexos-2-ulose (1c), and D-glycero-pentose-2-ulose (1d) were examined at 37 degrees at a solution pH of 7.0 (phosphate buffer). For 1a and 1b, two major products were observed and shown respectively to be the 5- and 6-substituted 3-amino-1,2,4-triazine derivatives. The ratios of the products were independent of the amount of aminoguanidine present or the order of mixing the reagents prior to the experiments. For 1c and 1d, only the 5-substituted triazine derivatives were formed. No evidence for hydrazone or bishydrazone formation was observed.

The characterization of a chitin-associated D-glucan from the cell walls of Aspergillus niger

Abstract Exhaustive extraction of the cell walls of Aspergillus niger with 10% NaOH solution leaves an alkali-resistant residue containing chitin and glucan as the major components. The glucan in this residue comprises 58.7% of the total cell wall glucan and was characterized by permethylation, and identification of the resulting O- methyl- D -glucoses obtained after hydrolysis by gas-liquid chromagtography and mass spectrometry of the derived partially acetylated, partially methylated, [1- 2 H]alditols. The glucan was separated from the chitin by acetylation of the alkali-resistance material, a procedure which separates a large portion of the total glucan as a chloroformsoluble acetate, abd by treatment of the alkali-insoluble residue with nitrous acid, a procedure which was found to render the complex soluble in dimethylsulfoxide and amenable, therefore, to permethylation. The data collected suggests that the preparation is an essentially linear glucan containing 85–95% 1 → 3 linkages and 10–15% 1 → 4 linkages. An analysis of the glycosidic linkages using NMR spectroscopy indicate that both α and β linkages are present in the ratio of 4:1. An identical glucan appears to be present in the cell walls of Penicillium chrysogenum as well as the spore cell walls of both organisms, as evidenced by methylation studies.

Evidence for a C-2→C-1 intramolecular hydrogen-transfer during the acid-catalyzed isomerization of D-glucose to D-fructose ag]☆☆☆

Abstract In mechanistic studies by isotope-exchange tecniques of the conversion of D -fructose and D -glucose into 2-(hydroxyacetyl)furan, it was shown that both sugars are converted in acidified, tritiated water into the furan containing essentially no carbon-bound tritium. As the hydroxymethyl carbon atom of the furan corresponds to C-1 of the hexose, this result suggests that one of the hydrogen atoms in this group, when it is produced from D -glucose, must arise intramolecularly. This hypothesis was verified by synthesizing D -glucose-2- 3 H and converting it into the furan in acidified water. The 2-(hydroxyacetyl)furan obtained was labeled exclusively on the hydroxymethyl carbon atom, thus showing that intramolecular hydrogen-transfer occurs, during the conversion, from C-2 of D -glucose to the carbon atom corresponding to C-1. The specific activities of the product and reactant permitted calculation of the tritium isotope-effect ( k h /k t 4.4) for the reaction. The precise step for the transfer from C-2 of the aldose to the carbon atom corresponding to C-1 was found to be during the isomerization of D -glucose to D -fructose, as evidenced by the conversion of D -glucose- 2 - 3 H into D -fructose- 1 - 3 H in acidified water.

Inhibition of colony formation in agarose of metastatic human breast carcinoma and melanoma cells by synthetic glycoamine analogs

We studied the influence of 10 synthetic glycoamine analogs on colony formation in 0.3 and 0.9% agarose by metastatic human breast carcinoma (MDA-MB-435) and melanoma (TXM-13) cells. Nine synthetic analogs significantly inhibited the colony formation in 0.9% agarose of MDA-MB-435 human breast carcinoma cells; five compounds caused a 73–83% reduction of colony formation. Seven synthetic glycoamines caused a significant inhibition of colony formation in 0.9% agarose by TXM-13 melanoma cells with the inhibitory effect ranging from 71 to 87%. The 50% inhibition (I50) doses and relative activity rank of the compounds were similar for both breast carcinoma and melanoma cell lines. The murine B16 melanoma cell aggregation assay was employed to elucidate the potential mechanism(s) of the inhibitory activity of synthetic glycoamines. The relative activity ranks of the compounds based on the independently determined I50 doses for both cell aggregation and clonogenic growth assays were very similar for the four most active synthetic analogs and clearly indicated the importance of hydrophobic amino acid in mediating the bioactivity of synthetic glycoamines. In both experimental systems (clonogenic growth in agarose and cell aggregation assay) the leading compound was N-(1-deoxy-d-fructos-1-y1)-d-leucine (Fru-d-Leu) and the least active analog was N-(1-deoxy-d-fructos-1-yl)-glycine (Fru-Gly). These results show that synthetic glycoamines may act by competing for specific carbohydrate-lectin interactions, particularly those involving β-galactoside-specific lectins expressed on metastatic cells.