Sugars and sugar degradation products react in vivo readily with proteins (glycation) resulting in the formation of a heterogeneous group of reaction products, which are called advanced glycation end products (AGEs). AGEs notably change the structure and function of proteins so that extended protein-AGE formation is linked to complications such as nephropathy, atherosclerosis, and cataract. DNA can be glycated in vitro in a similar way as proteins, and the two diastereomers of N(2)-carboxyethyl-2'-deoxyguanosine (CEdG(A,B)) were identified as major DNA AGEs. It was postulated that DNA AGEs play an important role in aging, diabetes, and uremia. However, at the moment, sensitive methods to measure the extent and impact of DNA AGEs in vivo do not exist. In this study, we developed a monoclonal antibody, which recognized CEdG(A,B) with high affinity and specificity (MAb M-5.1.6). The I(50) value for CEdG(A,B) was 2.1 ng/mL, whereas other modified nuclueobases and AGE proteins showed negligible cross-reactivity. Unmodified 2'-deoxyguanosine was only weakly recognized with an I(50) value > 600,000 ng/mL, which is the limit of solubility. MAb M-5.1.6 was then used to measure the urinary excretion of AGE-modified nucleobases in a competitive enzyme-linked immunosorbent assay. The recovery of CEdG(A,B) from human urine was between 87.4 and 99.7% with coefficients of variations between 8.0 and 22.2%. The detection limit was 0.06 ng/mL, and the determination limit was 0.15 ng/mL with a linear range between 0.3 and 100 ng/mL. CEdG equivalents were analyzed in urine samples from 121 healthy volunteers, and concentrations between 1.2 and 117 ng CEdG equiv/mg creatinine were detected.
The reaction of folic acid with reducing sugars (nonenzymatic glycation) under conditions that can occur during food processing and preparation was studied by high-performance liquid chromatography with diode array detection. N-(p-Aminobenzoyl)-L-glutamic acid, a well-established oxidation product, was detected in the reaction mixtures. Furthermore, a new product was isolated and identified as N2-[1-(carboxyethyl)]folic acid (CEF). CEF was the main product that was formed by the nonenzymatic glycation of folic acid. For preparation, N2-[1-(carboxyethyl)]folic acid was obtained in high yields when folic acid and dihydroxyacetone (DHA), a sugar degradation product, were heated at 100 degrees C in phosphate buffer. Mixtures of folic acid and different sugars or DHA were heated under variation of reaction time and temperature, and CEF was quantified. Up to 50% of the vitamin was converted to CEF, with highest yields formed from maltose (49%) and lactose (43%).
Sugars and sugar degradation products are formed during food processing, but also endogenously in vivo. In vitro, nucleosides and DNA react readily with these carbonyl compounds during the formation of the two diastereomers of N(2)-carboxyethyl-2'-deoxyguanosine (CEdG(A,B)), leading to a loss of DNA integrity. Only little is known about DNA glycation in vivo and about the influence of nutrition on CEdG formation. In this study, we developed a sensitive method to analyze DNA glycation by HPLC. For this purpose, immunoaffinity chromatography (IAC) using a polyclonal antibody against N(2)-carboxyethylguanine (CEguanine) was coupled to HPLC-DAD. In some samples, peak identity was confirmed by LC-MS/MS. The recovery of CEguanine from the IAC columns was 52.5% +/- 3.6 (n = 4). Thus, it was possible for the first time to detect CEdG(A,B), N(2)-carboxyethylguanosine (CEG(A,B)), and CEguanine in 11 human urine samples. However, due to imprecision of IAC, valid quantification of the adducts could not be achieved. Furthermore, CEdG was also detected in the DNA of cultured human smooth muscle cells (SMCs) and bovine aorta endothelium cells (BAECs). In BAECs, CEdG(A,B) were found by HPLC-DAD and LC-MS/MS after immunoaffinity purification, whereas in SMCs DNA-advanced glycation end-products were only detected with the more sensitive LC-MS/MS method.
We determined by the ninhydrin method the plasma amino acid (AA) levels prior to, during and following, a 1-hour i.v. infusion of 1 U/kg body weight each of secretin and pancreozymin in patients with normal (n = 74) or reduced (n = 39) exocrine pancreatic function, as assessed by the duodenal aspiration test. The results of the two tests correlated significantly with each other (p less than 0.001). A maximum AA decrease of greater than or equal to 12% was observed in all patients with a normally functioning pancreas (specificity 100%), and of less than 12% in all patients with medium to high-grade impairment of pancreatic function (sensitivity 100%). Since, however, low-grade pancreas insufficiency (20-40% of the mean normal enzyme output) is recognized in fewer than one-half of the cases, the overall sensitivity of the AA-consumption test decreases to 69%. The results can, however, be improved by: 1) Calculating the mean percentage AA decrease with a limit value of 5% (sensitivity 90%); 2) determining individual AA with pancreas-specific absorption, such as serine (sensitivity 92%); 3) dropping the lower normal value of exocrine pancreatic function to 25% of the normal mean enzyme output (sensitivity 96%). Diseases that may be associated with the most common condition that causes pancreatic insufficiency--chronic pancreatitis--and which have an influence on AA metabolism, such as cirrhosis of the liver and diabetes mellitus, have no influence on the accuracy of the AA consumption test, which, considered overall, represents a competitive alternative to other tubeless tests of pancreatic function.
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