Identification and quantitation ofN-(carboxymethyl)valine adduct in hemoglobin by gas chromatography/mass spectrometry

Abstract

A sensitive, specific and reproducible method was developed for the quantitation of the hemoglobin (Hb) adduct N‐(carboxymethyl)valine (CMV). This adduct is one of various products from the Maillard reaction, involving reducing sugars and amino acids, proteins or other molecules with a free amino group. Such adducts, including N ε‐(carboxymethyl)lysine (CML), are called advanced glycation end products (AGE) and have been correlated with aging and severity of diabetes in human tissues. This method was developed to examine the CMV–Hb adduct as a possible AGE formed by reaction of Hb with glucose or other oxidation products. CMV was cleaved selectively from isolated globin using pentafluorophenyl isothiocyanate (PFPITC) in a modified Edman degradation at pH 9.5. The carboxyl group of the adduct was derivatized to its methyl ester with diazomethane. The resulting derivative, 5‐isopropyl‐1‐(methyl acetate)‐3‐pentafluorophenyl‐2‐thiohydantoin, was detected by gas chromatography/mass spectrometry with selected ion monitoring (GC/SIM/MS). Quantitation was based on the response factor of the derivative molecular ion (m/z 396) from synthesized CMV and N‐(2‐carboxyethyl)valine (molecular ion m/z 410) as internal standard. This method exhibits reproducibility and linearity in the range 0.2–100 ng CMV. The limit of quantitation (0.2 ng CMV) gave a signal‐to‐noise ratio greater than 5: 1 using a 1: 30 sample aliquot. The GC/SIM/MS method can detect CMV adduct in 5 mg globin samples with relative standard deviations less than 5%. This approach avoids tedious acid hydrolysis and interference from other amino acids. The molecular ion and other CMV derivative ion assignments from samples were confirmed by accurate mass determinations using GC/high resolution SIM/MS. Measurements from random mouse, rat and human globin samples gave mean CMV levels of about 6, 5 and 14 nmol g−1 Hb in these species, respectively. Copyright © 1999 John Wiley & Sons, Ltd.

Degradation products of proteins damaged by glycation, oxidation and nitration in clinical type 1 diabetes

“…AGEs in haemoglobin, measured previously by immunoassay of unidentified epitopes [29,30], correlated with HbA 1 c and probably detected CML [31]. Specific immunoassay of CML residues overestimated the concentration Fig.…”

“…b Schematic diagram showing a cross-section through a blood capillary lumen, illustrating the flows of nitric oxide from the endothelium into the red blood cells and formation of peroxynitrite and 3-NT residues in plasma protein and haemoglobin (Hb) ten-fold [32]. The analogous AGE N -carboxymethylvaline residues were quantified in haemoglobin by a gas chromatography-mass spectrometry assay and were approximately 0.06% of haemoglobin [31]. The concentrations of MG-H1 and 3DG-H in haemoglobin were approximately ten-fold higher than the concentrations of CML and CEL residues, and 100-and 500-fold higher than the concentrations of MOLD and pentosidine residues respectively.…”

Oxidative degradation of N ε-fructosylamine-substituted peptides in heated aqueous systems

“…However, these products were formed in relatively low quantities and thus contributed only minimally to the lack. More likely, this difference is attributed to CML, a modification that has been reported to occur on α-amino groups (Cai and Hurst 1999), but might occur to a significantly lower degree at the N α compared to the N ε position. The summed yields of CML-and unmodified lysine-containing peptide (55 %) do indeed closely resemble the yields at the amino acid level (Davidek et al 2002).…”

Inhibitory effect of fermentation byproducts on formation of advanced glycation end-products

“…Carboxymethylation of a-amino groups of N-terminal acids may also occur; e.g., N-carboxymethylvaline in haemoglobin (Cai & Hurst, 1999). CML formation is a major pathway in vivo.…”