Extending the spectrum of α-dicarbonyl compounds in vivo.

Maillard α-dicarbonyl compounds are known as central intermediates in advanced glycation end product (AGE) formation. Glucose is the primary source of energy for the human body, whereas l-threo-ascorbic acid (vitamin C) is an essential nutrient, involved in a variety of enzymatic reactions. Thus, the Maillard degradation of glucose and ascorbic acid is of major importance in vivo. To understand the complex mechanistic pathways of AGE formation, it is crucial to extend the knowledge on plasma concentrations of reactive key α-dicarbonyl compounds (e.g. 1-deoxyglucosone). With the present work, we introduce a highly sensitive LC-MS/MS multimethod for human blood plasma based on derivatization with o-phenylenediamine under acidic conditions. The impact of workup and reaction conditions, particularly of pH, was thoroughly evaluated. A comprehensive validation provided the limit of detection, limit of quantitation, coefficients of variation, and recovery rates. The method includes the α-dicarbonyls 1-deoxyglucosone, 3-deoxyglucosone, glucosone, Lederer's glucosone, dehydroascorbic acid, 2,3-diketogulonic acid, 1-deoxypentosone, 3-deoxypentosone, 3,4-dideoxypentosone, pentosone, 1-deoxythreosone, 3-deoxythreosone, threosone, methylglyoxal, glyoxal; the α-keto-carboxylic acids pyruvic acid and glyoxylic acid; and the dicarboxylic acid oxalic acid. The method was then applied to the analyses of 15 healthy subjects and 24 uremic patients undergoing hemodialysis. The comparison of the results revealed a clear shift in the product spectrum. In most cases, the plasma levels of target analytes were significantly higher. Thus, this is the first time that a complete spectrum of α-dicarbonyl compounds relevant in vivo has been established. The results provide further insights into the chemistry of AGE formation and will be helpful to find specific markers to differentiate between the various precursors of glycation.

The diagnostic value of serum concentrations of 2-(alpha-mannopyranosyl)-L-tryptophan for normal renal function.

BACKGROUND: We have previously reported that the serum concentration of 2-(alpha-mannopyranosyl)-L-tryptophan (MPT), tryptophan glycoconjugate, is a more accurate measure of renal function than that of serum creatinine concentration. The aim of the present study was to compare the diagnostic value of serum concentrations of MPT and creatinine as a measure of normal renal function. METHODS: A total of 156 subjects with serum creatinine concentration < or =1.60 mg/dL aged 0 to 88 years were recruited. Serum concentrations of MPT and creatinine, and creatinine clearance calculated by Cockcroft-Galt formula were determined. A diagnostic accuracy of serum concentrations of MPT and creatinine for normal renal function was analyzed by using receiver-operating characteristics (ROC) curves. In 82 subjects with normal renal function defined as calculated creatinine clearance > or =80 mL/min (aged 6 to 68 years), the correlations between age and/or urinary creatinine excretion, which is related to muscle mass, and serum concentrations of MPT or creatinine, were determined. RESULTS: In the ROC curve, the area under the curve (AUC) in serum MPT concentration was significantly greater than that of creatinine (0.855 versus 0.800, respectively, P < 0.001) and the cut-off levels associated with the greatest diagnostic accuracy were 90 ng/mL for serum MPT concentration and 0.70 mg/dL for serum creatinine concentration. The sensitivity, specificity, and positive and negative predictive values were 69.5%, 85.1%, 83.8%, and 71.6% for serum MPT concentration, and 53.7%, 81.1%, 75.9%, and 61.2% for serum creatinine concentration. A close correlation existed between serum creatinine concentration and age (r= 0.798, P < 0.0001) in 23 subjects aged 20 years or younger. Conversely, serum MPT concentration remained unchanged regardless of age (r=-0.135, P= 0.228). Furthermore, a close correlation existed between serum creatinine concentration and urinary creatinine excretion (r= 0.817, P < 0.0001), but not between serum MPT concentration and urinary creatinine excretion (r= 0.082, P= 0.461). CONCLUSION: The concentration of serum MPT is a more reliable diagnostic parameter than that of serum creatinine as a measure of normal renal function, and renal function can be compared in subjects independently of age and muscle mass when serum MPT concentration is measured.

Tryptophan glycoconjugate as a novel marker of renal function.

PURPOSE: Neither serum creatinine concentration nor creatinine clearance assess renal function accurately. Serum creatinine concentration is affected by muscle mass, and the creatinine clearance overestimates the glomerular filtration rate because of tubular secretion of creatinine. The present study was designed to determine whether serum concentrations of 2-(alpha-mannopyranosyl)-L-tryptophan (MPT), a tryptophan glycoconjugate, can be used as a marker of renal function. METHODS: Clearances of MPT and of inulin were compared in normal rats and in rats with cisplatin-induced acute renal failure. We also compared the clearances of MPT and of creatinine with inulin clearance in 25 patients with chronic renal disease. Serum concentrations of MPT and creatinine as a function of MPT clearance were determined in 108 patients with chronic renal disease. RESULTS: There was strong linear correlation between clearances of MPT and inulin in rats (r = 0.97) and humans (r = 0.87), indicating that renal handling of MPT is similar to that of inulin. In humans, linear regression analyses indicated that MPT was a better indicator of inulin clearance than was creatinine clearance. At the same level of renal function, serum creatinine concentrations tended to be lower in patients with less muscle mass (as indicated by a urinary creatinine excretion <1,000 mg in 24 hours) than in those who excreted >1,000 mg in 24 hours, whereas serum MPT concentrations were not affected by creatinine excretion. CONCLUSION: MPT clearance can replace inulin clearance in the clinical setting. The serum MPT concentration is an accurate measure of renal function even in patients with diminished muscle mass, and thus is a better indicator of renal function than is the serum creatinine concentration.

3-Deoxyfructose concentrations are increased in human plasma and urine in diabetes.

3-Deoxyglucosone (3-DG) is a reactive dicarbonyl sugar thought to be a key intermediate in the nonenzymatic polymerization and browning of proteins by glucose. 3-DG may be formed in vivo from fructose, fructose 3-phosphate, or Amadori adducts to protein, such as N epsilon-fructoselysine (FL), all of which are known to be elevated in body fluids or tissues in diabetes. Modification of proteins by 3-DG formed in vivo is thought to be limited by enzymatic reduction of 3-DG to less reactive species, such as 3-deoxyfructose (3-DF). In this study, we have measured 3-DF, as a metabolic fingerprint of 3-DG, in plasma and urine from a group of diabetic patients and control subjects. Plasma and urinary 3-DF concentrations were significantly increased in the diabetic compared with the control population (0.853 +/- 0.189 vs. 0.494 +/- 0.072 microM, P < 0.001, and 69.9 +/- 44.2 vs. 38.7 +/- 16.1 nmol/mg creatinine, P < 0.001, respectively). Plasma and urinary 3-DF concentrations correlated strongly with one another, with HbA1c (P < 0.005 in all cases), and with urinary FL (P < 0.02 and P = 0.005, respectively). The overall increase in 3-DF concentrations in plasma and urine in diabetes and their correlation with other indexes of glycemic control suggest that increased amounts of 3-DG are formed in the body during hyperglycemia in diabetes and then metabolized to 3-DF. These observations are consistent with a role for increased formation of the dicarbonyl sugar 3-DG in the accelerated browning of tissue proteins in diabetes.

Adenosine diphosphate ribulose in human erythrocytes: a new metabolite with membrane binding properties.

Incubation of ADPribose with yeast phosphoriboisomerase resulted in the formation of an adenylic nucleotide that was identified with ADPribulose by mass spectrometry. Synthesis of [32P]ADPribulose from [32P]NAD+ by the combined activities of commercial NAD+ glycohydrolase and phosphoriboisomerase allowed us to use it as a labeled internal standard throughout the procedure of purification from trichloroacetic acid extracts of human red blood cells. ADPribulose was purified by means of three sequential reverse phase HPLC separations and its concentration in human erythrocytes was estimated to be 0.11 +/- 0.1 microM. Unsealed erythrocyte ghosts did not transform ADPribulose, which bound to specific membrane proteins with a trichloroacetic and formic acid-resistant binding. The labeled proteins were identified as spectrin, bands 3, 4.1, 4.2 and Glyceraldehyde 3-phosphate dehydrogenase on the basis of their relative mobilities on SDS-PAGE.

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.

Effects of ketohexosemia on the ketohexose transport in the small intestine of rats.

It was observed previously (Cs aky , T.Z. and Fischer, E. (1981) Diabetes 30, 568-574), that sustained hyperglycemia enhances the intestinal transport of aldohexoses ; on the other hand, hyperfructosemia affects primarily the transport of fructose. The present study examines in detail the hyperketosemia -induced intestinal ketose transport. Intravenously infused 3-O- methylfructose produces marked 3-O- methylfructosemia without concomitant hyperglycemia; in such animals the intestinal transport of both fructose and 3-O- methylfructose increased. The hyperketosemia -induced increased ketose transport was inhibited by phloretin but only if placed on the serosal compartment. Phlorizin affects neither the basal nor the induced intestinal ketohexose transport. The enhancement of the intestinal ketohexose transport is not sodium-dependent and is not inhibited by ouabain.