No abstract

To examine the mechanism of enzymatic acyl transfers from p-nitrophenyl acetate (PNPA), isotope effects were measured for the reaction of PNPA with chymotrypsin, carbonic anhydrase, papain, and Aspergillus acid protease. The isotope effects were measured at the β-deuterium (D k), carbonyl carbon (13 k), carbonyl oxygen (18 k carbonyl), leaving group phenolic oxygen (18 k lg), and leaving group nitrogen (15 k) positions. D k ranged from 0.982 ± 0.002 to 0.999 ± 0.002. 13 k ranged from 1.028 ± 0.002 to 1.036 ± 0.002. 18 k carbonyl ranged from 1.0064 ± 0.0003 to 1.007 ± 0.001. 18 k lg ranged from 1.141 ± 0.0002 to 1.330 ± 0.0007. 15 k ranged from 0.9997 ± 0.0007 to 1.0011 ± 0.0002. Uncatalyzed acyl transfer from PNPA to oxygen and sulfur nucleophiles proceeds by a concerted mechanism. All of the enzymatic reactions showed isotope effects consistent with a concerted mechanism like that seen in uncatalyzed aqueous reactions, but exhibited smaller inverse β-deuterium isotope effects than seen in the nonenzymatic aqueous reactions. This phenomenon may be explained by greater hydrogen bonding or electrostatic interaction with the ester carbonyl group in enzymatic transition states relative to nonenzymatic aqueous transition states. Quantum mechanical calculations were used to estimate the magnitude of changes in hyperconjugation and C−H bond order due to protonation of a carbonyl oxygen.

SUMMARY Although hemoglobin A1c (HbA1c) is generally considered to be an accurate index of long-term blood glucose regulation, several recent studies suggest that HbA1c may be acutely responsive to changes in blood glucose. We have examined the effects of acute changes in glucose concentration in vivo and in vitro on HbA1c. HbA1c was measured by a high-performance liquid chromatography (HPLC) method. HbA1c and plasma glucose were measured in seven diabetic patients and five control subjects before and 2 h after a standard breakfast. Only diabetic patients showed increases in HbA1c and plasma glucose values 2 h after the test meal (Δ HbA1c = 0.87 ± 0.24% and Δ glucose = 210 ± 0.33 mg/dl). The increment in HbA1c correlated significantly with the increment in plasma glucose (r = 0.73, P < 0.05). to examine the lability of these postmeal increments in HbA1c, erythrocytes from pre- and postmeal blood samples were incubated in 0.9% NaCl for 5 h at 37°C and HbA1c was re-measured. After saline incubations %HbA1c in pre- and postmeal blood samples decreased in both diabetics and controls, and from each subject time 0 and 2-h HbA1c values were nearly identical. HbA1c was then measured before and after incubations of erythrocytes from a larger group of diabetic patients (N = 55) and control subjects (N = 7). In both diabetic and control cells HbAlc decreased after saline incubations. Pre- and post-saline HbA1c values in diabetics were (means ± SEM) 10.07 ± 0.30% and 9.15 ± 0.27%, respectively; values in controls were 5.87 ± 0.11% and 5.46 ± 0.09%, respectively. The mean decrement in HbA1c was significantly greater in cells from diabetics than from controls (P < 0.001). In diabetics the HbA1c decrement correlated with both plasma glucose (r = 0.58, P < 0.001) and the preincubation HbA1c (r = 0.55, P < 0.001). After dialysis of hemolysates for 5 h at 37°C or 48–72 h at 4°C, HbA1c values showed decreases comparable to those after saline incubations of intact erythrocytes. However, decreases in HbA1c after dialysis were accompanied by increases in HbA1a+b, findings that were not observed after saline incubation. The results suggest that HbA1c exists in two chromatographically indistinguishable forms: one that represents the major portion of HbA1c in normals and diabetics, is not altered by short-term changes in plasma glucose, and can be estimated by measuring HbA1c after saline incubation of erythrocytes; and a second form that is responsive to short-term fluctuations of the blood glucose level. These labile and stable fractions may be identical to the labile Schiff-base and stable ketoamine forms of HbA1c, which have been previously described. For HbA1c to be an accurate indicator of long-term glucose control, saline incubation of erythrocytes or perhaps dialysis of hemolysates before HbA1c assay may be necessary. Otherwise the assay results will reflect recent changes in blood glucose levels.

Glycosylated hemoglobin measurement has been shown to be a potentially useful tool for both a variety of research applications and for the management of patients with diabetes mellitus. None of the methods available to quantitate glycosylated hemoglobins is ideal. We have reviewed a number of critical methodologie considerations for Chromatographie procedures including the effects of sample storage under various conditions, and the importance of removing labile components prior to analyses. We have developed a method for the colorimetrie determination of glycosylated hemoglobins that is more rapid than methods reported previously, that correlates well with results using high-performance liquid chromatogra-phy, and that can he standardized between laboratories. We have reviewed our experience using glycosylated hemoglobin in a large population of diabetic youths. We have presented a method for developing realistic goals for glucose control using glycosylated hemoglobin and for using glycosylated hemoglobin as a patient education and care reinforcement tool.