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Hyperglycemia may determine fibrinopeptide A plasma level increase in humans
Hyperglycemia
May Determine Fibrinopeptide
A Plasma Level Increase in Humans
Antonio Ceriello, Dario Giugliano, Antonio Quatraro, Patrizia Della Russo, Egidio Marchi, and Roberto Torella The effects of hyperglycemia on plasma fibrinopeptide A (FPA) levels in normal subjects are reported. An increase of FPA concentration parallel to sustained hyperglycemia was observed; when the glycemia returned to basal values, FPA showed values in normal renge. Heparin infusion was able to significantly decrease the hyperglycemia-induced augment of FPA levels. Isovolumic-isotonic NaCl solution infusion produced a slight (NS) increase in FPA levels; however, mild hyperglycemia, achieved by glucagon. was also able to produce a significant increase in FPA concentration. These data demonstrate the direct role of hyperglycemia in conditioning FPA level, and suggest that hyperglycemia, by itself, is a sufficient stimulus to produce thrombin activation in humans. @ 1989 by W.8. Saunders Company.
S
EVERAL STUDIES of the fluid phase of coagulation suggest that diabetes is associated with a hypercoagulable state.‘.2 However, the existence of a direct link between abnormal glucose levels and the increased activity of coagulation system is still unclear.‘T2 Fibrinopeptide A (FPA) is the amino-terminal peptide that is cleaved from A-alpha-chain of fibrinogen by the action of thrombin.’ This reaction is the initial step in conversion of fibrinogen to fibrin, and the measurement of FPA is a sensitive test of in vivo thrombin activity.4 This study describes the effects of induced hyperglycemia on FPA plasma levels in healthy subjects. MATERIALS AND METHODS
Five young normal male subjects were recruited from the medical student body of the University of Naples. Ages ranged between 21 and 26 years, and body weight (68 to 75 kg) was within 15% of their ideal body weight (Metropolitan Life Insurance tables, 1959). They had no family history of diabetes and showed a normal oral glucose tolerance test, according to National Diabetes Data Group criteria.’ The subjects consumed weight-maintaining diets with at least 250 g carbohydrate/d for three days before the study. This experiment was approved by the review board for human experiments of our institute, and all subjects gave informed consent to participate in the study after a detailed explanation of its experimental nature. Study Protocol Four studies were performed in each subject in randomized order on different days. In the morning in the postabsorptive state and after a 12 to 14 hour fast, an intravenous cannula was introduced into an antecubital vein of each subject and kept patent with slow infusion of 0.9% NaCl. Venous samples for laboratory analysis were obtained by repeated venipuncture from one cannula; another cannula was used for administration of glucose, heparin + glucose, glucagon, or isovolumic-isotonic NaCl solution. After the subjects had rested for 60 minutes, two basal samples were obtained (-30 and 0 min) before the start of the study. In a first study, hyperglycemia was induced according to Cerasi and Luft6 Each subject received a pulse of 0.5 g/kg gIucose injected in less than two minutes; glucose (0.33 g - kg-’ . min-‘) was then infused via a peristaltic pump for one hour. This study was repeated From Cattedra di Diabetologia e Dietoterapia, I Facolth di Medicina. Universith di Napoli. Napoli, Centro di Diabetologia, Casa di Cura S Rita, Taranto, and AIfa Ricerche, Bologna, Italy. Address reprint requests to Antonio Ceriello, MD, Viale Lincoln 4, Pare0 Mecos. I-81 100 Caserta. Italy. Q 1989 by W.B. Saunders Company. 0026-0495/s9/3812_ooa3%3.00/0
1162
in each subject, with the sole exception that heparin (5,000 U as IV bolus, followed by an IV infusion at the rate of 750 U/h) accompanied the hyperglycemic infusion. In a third protocol, each subject received an equivalent infusion of an isovolumic-isotonic NaCl solution. In the last study, mild hyperglycemia was achieved by the IV administration of 1 mg glucagon. Blood samples in each protocol for glycemia and FPA concentration were collected every ten minutes in the first hour and at 90,120, and 180 minutes. Analytic and Statistical Methods Plasma glucose was measured with a Beckman Analyzer. FPA estimation was performed by radioimmunoassay, using a commercially obtained kit (Mallinkrodt, St Louis). Samples were drawn directly into vacutainer containing the anticoagulant diluent supplied with the kit. FPA measurement was performed in triplicate, and the intraassay and interassay coefficients of variation for this method were 5.5% and 6.7% respectively. For each time point of the study, the ratio of the mean basal normoglycemic period hematocrit to the observed hematocrit ranged between 1.04 and 1.06. This factor was used to correct the dilutional error introduced by blood volume expansion. Statistical significance was determined by ANOVA. Data are reported as mean + SE. RESULTS
Time zero glycemia and FPA concentration were similar in the four tests (Fig 1). All subjects had sustantial blood glucose elevations in the hyperglycemic range as the results of glucose infusion. No significant difference between mean glycemic area (from 0 to 180 min) existed in these two experiments (13.5 t 1.2 v 13.7 f 1.1 mmol/L). An increase of plasma FPA level v fasting basal values concomitant with hyperglycemia was observed at 10 to 90 min (P < .OOl). When the glycemia returned to basal values, FPA concomitantly showed values in basal range. Heparin infusion reduces significantly FPA increase during induced hyperglycemia (P < .Ol) (Fig 1). Glucagon produced a short and mild hyperglycemia that was accompanied by a significant increase in FPA levels (P -C .05) (Fig 1). Isovolumic-isotonic infusion produced a slight but not significant rise in FPA concentration (Fig 1). DISCUSSION
Recent studies suggest that abnormalities of the hemostatic system are present in diabetes mellitus.‘B2However, it is not yet clear whether the reported coagulation abnormalities are causally related or are related to abnormal glucose levels.‘s2 FPA level is considered a good index of thrombin
Metabolism,Vol36, No 12 (December), 1989: pp
1162-l 163
1163
subjects with vascular complications, whereas patients with no vascular complications showed only a trend toward elevated FPA in response to glucose challenge.’ These results suggested that vascular complications might be considered as contributory but not the only factor that produces an increase of thrombin activity in diabetes.’ Our results in healthy normal subjects do not question that vascular complications may contribute to enhanced FPA formation, but at the same time they clearly show that hyperglycemia may be a sufficient stimulus to produce an increase of FPA level in plasma. Reduction of FPA levels in diabetes may be achieved by addition of heparin to hyperglycemic glucose infusion, suggesting a mechanism based on augmented thrombin activity.’ The capacity of heparin to reduce hyperglycemia-stimulated increase of FPA in our study supports this hypothesis.
4r
Blood volume expansion can participate at the augmented FPA formation in the sustained hyperglycemic experiments; however, the demonstration that a mild increase of glycemia, achieved by glucagon administration, is also able to stimulate FPA formation, stresses the direct role of abnormal glucose
MINUTES Fig 1. IO). Glycemia and FPA variations during glucose infusion. (0). Glucos@ + hapwin infusion: (A), glucagon: (HI. isoosmoticisovolumic infusion.
formation4 Increased FPA levels more related to abnormal glucose levels than to long-term control have been reported in diabetes.’ However, an increase of FPA levels in response to glucose challenge have been reported in type I diabetic
level in conditioning by itself coagulation hyperactivity in humans. Glucose has a toxic action for endothelial cells of arterial walls,’ and seems to condition a hyperglycemia-mediated enhancement of procoagulant activity of endothelial cells.’ These mechanisms may explain the hyperglycemia-stimulated thrombin activation. Moreover, the existence of hyperglycemia-dependent reduction of antithrombin III biologic activity in both diabetic” and normal” subjects suggests one additional explanation for the enhanced FPA formation. In conclusion, this study shows the existence of a direct link between hyperglycemia and coagulation hyperactivity in humans.
REFERENCES 1. Jones RL, Peterson CH: The fluid phase of coagulation and the accelerated atherosclerosis of diabetes mellitus. Diabetes 30:33-38, 1981 (suppl2) 2. Ostermann H, van de Loo J: Factors of the hemostatic system in diabetic patients. A survey of controlled study. Haemostasis 16:386-416,1986 3. Nossel HL, Younger LR, Wilner GD, et al: Radioimmunoassay of human fibrinopeptide A. Proc Nat1 Acad Sci USA 68:23502353,197l 4. Nossel HL, Yudelman J, Canfield RE, et al: Measurement of fibrinopeptide A in human blood. J Clin Invest 54:43-53,1974 5. National Diabetes Data Group: Classification and diagnosis of diabetes mellitus and other categories of glucose intolerance. Diabetes 28:1039-1057, 1979 6. Cerasi E, Luft R: “What is inherited-What is added?” Hypothesis for the pathogenesis of diabetes mellitus. Diabetes 16:615-627,1967
7. Jones RL: Fibrinopeptide A in diabetes mellitus: Relation to levels of blood glucose, fibrinogen disappearance, and hemodynamic changes. Diabetes 34:836-841,198s 8. Stout RW: Insulin and atheroma. An update. Lancet 2:10771078,1987 9. Schemthaner A, Vukovich TC, Knob1 PN, et al: Reduction of hypercoagulability and endothelial cell dysfunction in nonretinopathic IDD patients by near normoglycaemic control. Diabetologia 31:540A, 1988 10. Ceriello A, Giugliano D, Quatraro A, et al: Heparin preserves antithrombin III biological activity from hyperglycemia-induced alterations in insulin-dependent diabetics. Haemostasis 16:458-464, 1986 11. Ceriello A, Giugliano D, Quatraro A, et al: Inducedhyperglycemia alters antithrombin III activity but not its plasma concentration in healthy normal subjects. Diabetes 36:320-323, 1987
May Determine Fibrinopeptide
A Plasma Level Increase in Humans
Antonio Ceriello, Dario Giugliano, Antonio Quatraro, Patrizia Della Russo, Egidio Marchi, and Roberto Torella The effects of hyperglycemia on plasma fibrinopeptide A (FPA) levels in normal subjects are reported. An increase of FPA concentration parallel to sustained hyperglycemia was observed; when the glycemia returned to basal values, FPA showed values in normal renge. Heparin infusion was able to significantly decrease the hyperglycemia-induced augment of FPA levels. Isovolumic-isotonic NaCl solution infusion produced a slight (NS) increase in FPA levels; however, mild hyperglycemia, achieved by glucagon. was also able to produce a significant increase in FPA concentration. These data demonstrate the direct role of hyperglycemia in conditioning FPA level, and suggest that hyperglycemia, by itself, is a sufficient stimulus to produce thrombin activation in humans. @ 1989 by W.8. Saunders Company.
S
EVERAL STUDIES of the fluid phase of coagulation suggest that diabetes is associated with a hypercoagulable state.‘.2 However, the existence of a direct link between abnormal glucose levels and the increased activity of coagulation system is still unclear.‘T2 Fibrinopeptide A (FPA) is the amino-terminal peptide that is cleaved from A-alpha-chain of fibrinogen by the action of thrombin.’ This reaction is the initial step in conversion of fibrinogen to fibrin, and the measurement of FPA is a sensitive test of in vivo thrombin activity.4 This study describes the effects of induced hyperglycemia on FPA plasma levels in healthy subjects. MATERIALS AND METHODS
Five young normal male subjects were recruited from the medical student body of the University of Naples. Ages ranged between 21 and 26 years, and body weight (68 to 75 kg) was within 15% of their ideal body weight (Metropolitan Life Insurance tables, 1959). They had no family history of diabetes and showed a normal oral glucose tolerance test, according to National Diabetes Data Group criteria.’ The subjects consumed weight-maintaining diets with at least 250 g carbohydrate/d for three days before the study. This experiment was approved by the review board for human experiments of our institute, and all subjects gave informed consent to participate in the study after a detailed explanation of its experimental nature. Study Protocol Four studies were performed in each subject in randomized order on different days. In the morning in the postabsorptive state and after a 12 to 14 hour fast, an intravenous cannula was introduced into an antecubital vein of each subject and kept patent with slow infusion of 0.9% NaCl. Venous samples for laboratory analysis were obtained by repeated venipuncture from one cannula; another cannula was used for administration of glucose, heparin + glucose, glucagon, or isovolumic-isotonic NaCl solution. After the subjects had rested for 60 minutes, two basal samples were obtained (-30 and 0 min) before the start of the study. In a first study, hyperglycemia was induced according to Cerasi and Luft6 Each subject received a pulse of 0.5 g/kg gIucose injected in less than two minutes; glucose (0.33 g - kg-’ . min-‘) was then infused via a peristaltic pump for one hour. This study was repeated From Cattedra di Diabetologia e Dietoterapia, I Facolth di Medicina. Universith di Napoli. Napoli, Centro di Diabetologia, Casa di Cura S Rita, Taranto, and AIfa Ricerche, Bologna, Italy. Address reprint requests to Antonio Ceriello, MD, Viale Lincoln 4, Pare0 Mecos. I-81 100 Caserta. Italy. Q 1989 by W.B. Saunders Company. 0026-0495/s9/3812_ooa3%3.00/0
1162
in each subject, with the sole exception that heparin (5,000 U as IV bolus, followed by an IV infusion at the rate of 750 U/h) accompanied the hyperglycemic infusion. In a third protocol, each subject received an equivalent infusion of an isovolumic-isotonic NaCl solution. In the last study, mild hyperglycemia was achieved by the IV administration of 1 mg glucagon. Blood samples in each protocol for glycemia and FPA concentration were collected every ten minutes in the first hour and at 90,120, and 180 minutes. Analytic and Statistical Methods Plasma glucose was measured with a Beckman Analyzer. FPA estimation was performed by radioimmunoassay, using a commercially obtained kit (Mallinkrodt, St Louis). Samples were drawn directly into vacutainer containing the anticoagulant diluent supplied with the kit. FPA measurement was performed in triplicate, and the intraassay and interassay coefficients of variation for this method were 5.5% and 6.7% respectively. For each time point of the study, the ratio of the mean basal normoglycemic period hematocrit to the observed hematocrit ranged between 1.04 and 1.06. This factor was used to correct the dilutional error introduced by blood volume expansion. Statistical significance was determined by ANOVA. Data are reported as mean + SE. RESULTS
Time zero glycemia and FPA concentration were similar in the four tests (Fig 1). All subjects had sustantial blood glucose elevations in the hyperglycemic range as the results of glucose infusion. No significant difference between mean glycemic area (from 0 to 180 min) existed in these two experiments (13.5 t 1.2 v 13.7 f 1.1 mmol/L). An increase of plasma FPA level v fasting basal values concomitant with hyperglycemia was observed at 10 to 90 min (P < .OOl). When the glycemia returned to basal values, FPA concomitantly showed values in basal range. Heparin infusion reduces significantly FPA increase during induced hyperglycemia (P < .Ol) (Fig 1). Glucagon produced a short and mild hyperglycemia that was accompanied by a significant increase in FPA levels (P -C .05) (Fig 1). Isovolumic-isotonic infusion produced a slight but not significant rise in FPA concentration (Fig 1). DISCUSSION
Recent studies suggest that abnormalities of the hemostatic system are present in diabetes mellitus.‘B2However, it is not yet clear whether the reported coagulation abnormalities are causally related or are related to abnormal glucose levels.‘s2 FPA level is considered a good index of thrombin
Metabolism,Vol36, No 12 (December), 1989: pp
1162-l 163
1163
subjects with vascular complications, whereas patients with no vascular complications showed only a trend toward elevated FPA in response to glucose challenge.’ These results suggested that vascular complications might be considered as contributory but not the only factor that produces an increase of thrombin activity in diabetes.’ Our results in healthy normal subjects do not question that vascular complications may contribute to enhanced FPA formation, but at the same time they clearly show that hyperglycemia may be a sufficient stimulus to produce an increase of FPA level in plasma. Reduction of FPA levels in diabetes may be achieved by addition of heparin to hyperglycemic glucose infusion, suggesting a mechanism based on augmented thrombin activity.’ The capacity of heparin to reduce hyperglycemia-stimulated increase of FPA in our study supports this hypothesis.
4r
Blood volume expansion can participate at the augmented FPA formation in the sustained hyperglycemic experiments; however, the demonstration that a mild increase of glycemia, achieved by glucagon administration, is also able to stimulate FPA formation, stresses the direct role of abnormal glucose
MINUTES Fig 1. IO). Glycemia and FPA variations during glucose infusion. (0). Glucos@ + hapwin infusion: (A), glucagon: (HI. isoosmoticisovolumic infusion.
formation4 Increased FPA levels more related to abnormal glucose levels than to long-term control have been reported in diabetes.’ However, an increase of FPA levels in response to glucose challenge have been reported in type I diabetic
level in conditioning by itself coagulation hyperactivity in humans. Glucose has a toxic action for endothelial cells of arterial walls,’ and seems to condition a hyperglycemia-mediated enhancement of procoagulant activity of endothelial cells.’ These mechanisms may explain the hyperglycemia-stimulated thrombin activation. Moreover, the existence of hyperglycemia-dependent reduction of antithrombin III biologic activity in both diabetic” and normal” subjects suggests one additional explanation for the enhanced FPA formation. In conclusion, this study shows the existence of a direct link between hyperglycemia and coagulation hyperactivity in humans.
REFERENCES 1. Jones RL, Peterson CH: The fluid phase of coagulation and the accelerated atherosclerosis of diabetes mellitus. Diabetes 30:33-38, 1981 (suppl2) 2. Ostermann H, van de Loo J: Factors of the hemostatic system in diabetic patients. A survey of controlled study. Haemostasis 16:386-416,1986 3. Nossel HL, Younger LR, Wilner GD, et al: Radioimmunoassay of human fibrinopeptide A. Proc Nat1 Acad Sci USA 68:23502353,197l 4. Nossel HL, Yudelman J, Canfield RE, et al: Measurement of fibrinopeptide A in human blood. J Clin Invest 54:43-53,1974 5. National Diabetes Data Group: Classification and diagnosis of diabetes mellitus and other categories of glucose intolerance. Diabetes 28:1039-1057, 1979 6. Cerasi E, Luft R: “What is inherited-What is added?” Hypothesis for the pathogenesis of diabetes mellitus. Diabetes 16:615-627,1967
7. Jones RL: Fibrinopeptide A in diabetes mellitus: Relation to levels of blood glucose, fibrinogen disappearance, and hemodynamic changes. Diabetes 34:836-841,198s 8. Stout RW: Insulin and atheroma. An update. Lancet 2:10771078,1987 9. Schemthaner A, Vukovich TC, Knob1 PN, et al: Reduction of hypercoagulability and endothelial cell dysfunction in nonretinopathic IDD patients by near normoglycaemic control. Diabetologia 31:540A, 1988 10. Ceriello A, Giugliano D, Quatraro A, et al: Heparin preserves antithrombin III biological activity from hyperglycemia-induced alterations in insulin-dependent diabetics. Haemostasis 16:458-464, 1986 11. Ceriello A, Giugliano D, Quatraro A, et al: Inducedhyperglycemia alters antithrombin III activity but not its plasma concentration in healthy normal subjects. Diabetes 36:320-323, 1987