INSULIN RESPONSE IN GLUCOSE-TOLERANCE TESTS

Transcription

1 THE AMERICAN JOURNAL OF CLINICAL PATHOLOGY Copyright 1967 by The Williams & Wilkins Co. Vol. 47, No. 0 Printed in U.S.A. INSULIN RESPONSE IN GLUCOSE-TOLERANCE TESTS STANLEY BURROWS, M.D. Department of Pathology, The Cooper Hospital, Camden, New Jersey The development of immuno-assay technics for the measurement of plasma insulin in recent years has led to many reports dealing with levels of plasma insulin in normal individuals and in various pathologic conditions, particularly diabetes mellitus. All materials for the accurate quantitation of insulin are now readily available from commercial sources, making it a practical procedure for a general clinical laboratory. We decided to determine plasma insulin levels on patients undergoing glucose tolerance tests to determine if any additional data of diagnostic significance could be obtained that would not otherwise be available. METHOD A standard -Gm. oral glucose tolerance test was performed on 24 adult patients after an overnight fast. There was no selection of patients with the exception that none had received insulin. Venous blood was drawn in the fasting state, at 3^, 1, 2, and 3 hr. after the ingestion of glucose. Serum glucose concentrations were determined by a ferricyanide method on the AutoAnalyzer.* Serum insulin was measured by the immuno-assay method of Yalow and Berson. 31 The method depends upon the competition between native insulin and a relatively small amount of tracer insulin tagged with I 131 for a limited amount of anti-insulin antibody, with subsequent separation of antibodybound insulin from remaining free insulin by chromatoelectrophoresis. Guinea pig serum containing antibodies against bovine insulinf was used in a 1:4000 Received, August 25, Presented at the annual meeting of the American Society of Clinical Pathologists, Washington, D. C, September 16 to 24, I960. This investigation was supported by the James Picker Foundation. * Tcchnicon Instruments Corporation, Ardsley (Chauncey), New York. f Hyland Laboratories, Los Angeles, California. 709 dilution. Pork insulin labeled with I 131 of high specific activity} (greater than mc. per mg.) was used as tracer insulin. Crystalline human insulin, kindly provided by Dr. Root of the Lilly Research Laboratories, was used hi the preparation of insulin standards. All standards and serum specimens were analyzed in quadruplicate, and a complete set of standards was analyzed with each group of sera. Chromatoelectrophoresis was performed at room temperature on Whatman 3MC paper. The strips were subsequently cut into two portions, representing the free and bound insulin, and their radioactivity was measured in a well counter. Bound to free ratios were calculated for all specimens, the insulin concentrations of the sera being determined by comparison with standards. RESULTS The 24 subjects were divided into three groups on the basis of then- blood glucose levels during the glucose-tolerance test. Of the 24 patients, 12 (50%) showed normal glucose tolerance, with a fasting and 3-hr. blood glucose level of less than mg. per ml. and a peak of less than 160 mg. per ml. Eight of the 24 patients (33%) exhibited diabetic responses, with a fasting or 3-hr. blood glucose concentration of over 110 mg. per ml. or a peak of over 170 mg. per ml. Four of the 24 patients (17 %) fell in a borderline zone, with a fasting or 3-hr. blood glucose concentration of to 110 mg. per ml. or a peak of 160 to 170 mg. per ml. As a group, the diabetic patients exhibited a greater and more persistent rise in blood insulin levels in response to the glucose load than did the normal group (Fig. 1). The borderline group showed an abnormally persistent elevation of blood insulin in relation to the normal group. Abbott Laboratories, North Chicago, Illinois.

2 710 BURROWS Vol. 47 S B 3 ~ 150_ TIME (hours) NORMAL GROUP BORDERLINE GROUP DIABETIC GROUP FIG. 1. Plasma insulin response to glucose load A tabulation (Table 1) of the ranges of blood insulin levels in the three groups emphasizes that, although the mean levels show definite patterns, the levels in individual patients were variable, with considerable overlap between groups, which casts doubt upon the interpretation of the insulin responses in any single patient. DISCUSSION All immuno-assay methods for measuring plasma insulin make use of the competition between native insulin and radioactive tagged tracer insulin for a limited quantity of guinea pig anti-insulin antibody. The various methods differ in the means of separation of the antibody-bound insulin from the remaining free insulin; they have included chromatoelectrophoresis, 31 sodium sulfite precipitation, 8 immunoprecipitation with rabbit serum containing antibodies against guinea pig serum, 7 ' 12 ' 20 and resin adsorption. 18 All methods should give comparable results if similar reference standards of insulin are used. 25 In our limited experience we found that chromatoelectrophoresis offered greater dependability than immunoprecipitation; we lost several batches of determinations by the latter procedure without apparent cause. The insulin response to a glucose load shows considerable variation in normal persons (Table 2). Welborn and associates 23 noted that each individual tends to show a high, medium, or low insulin level throughout a glucose-tolerance test, with a tendency for slightly higher levels of blood glucose in the high insulin secretors and lower glucose levels in low insulin secretors. They suggested that the wide range of insulin levels in a presumably normal group may merely reflect biologic variation. The very wide range of insulin levels in normal individuals must be appreciated in the interpretation of high or low plasma insulin concentrations in pathologic conditions. Yalow and Berson 30 have cautioned that, as a result of sporadic sampling instead of continual monitoring of blood levels, the actual peak of insulin concentration in response to a glucose load may be missed. There is general agreement 3 c that plasma insulin levels are low or absent in severe juvenile diabetes mellitus, with little if any response to glucose stimulation. Patients with diabetes secondary to chronic pancreatitis also show negligible concentrations of plasma insulin. 3 However, the plasma TABLE 1 MEAN INSULIN LEVELS DURING IOO-GM. ORAL GLUCOSE-TOLERANCE TEST* Group Fasting MHr.t 1 Hr.f 2Hr.t 3Hr.f Normal Borderline Diabetic 13 (0-30) 14 (0-25) 16 (0-45) 53 (15-200) 61 (15-155) 90 (5-300) microunits/ml. 90 (10-240) 89 (20-175) 147 (5-455) 58 (10-320) (60-145) 95 (0-200) 25 (0-155) 06 (0-155) 09 (0-200) * The ranges of the results obtained are listed in parentheses. t Times given represent lengths of time after administration of glucose.

3 June 1967 INSULIN RESPONSE IN GLUCOSE-TOLERANCE TESTS 711 TABLE 2 NORMAL INSULIN LEVELS IN GLUCOSE-TOLERANCE TESTS No. of Cases Glucose Dose 0 min. Range of Insulin Concentration 30 min. 60 min. 120 min. 150 min. 180 min. Hales and Randle 11 Hales and Randle 11 Yulow and Berson 31 Meade and Klitgaard 18 Wei born et al. a Nikkila el al." Ehrlich and Bambers 6 Wong el al.*> Burrows t Cm /kg. 1/kg * * * * * * microunits/ml * (40-500) 64* * 47* * Average, f Children. % 90 min. after glucose. insulin levels in diabetes mellitus of maturity-onset type and the prediabetic state, and their relationship to the metabolic abnormalities observed, are still disputable. In patients with diabetes mellitus of maturity-onset type, the fasting plasma insulin concentration may 2,10,13,26,29 or may not be elevated. Hales and Randle 11 noted a correlation between the fasting glucose and insulin concentrations in diabetic patients. The fasting plasma insulin level was normal if the fasting blood glucose concentration was normal; they observed increasing levels of fasting plasma insulin with increasing elevations of the fasting blood glucose. A small group of diabetic patients with severe ketonuria and advanced hyperglycemia had plasma insulin concentrations similar to those of fasting normal persons. In response to a glucose load, maturityonset diabetic patients show an excessive plasma insulin response with a delayed return to normal, 2 ' " 24,32 ' 33 except that diabetic patients with ketosis may show an absolute deficiency of plasma insulin, with a poor and delayed rise in plasma insulin concentration. 10, J1 The abnormally high and sustained response of plasma insulin of the diabetic patient generally parallels the elevated plasma glucose responses 2 and suggests a diminished effectiveness of the insulin, with decreased ability of the tissues to respond to insulin. 3 n ' Karam and colleagues 14 " 16 and Young and Jenkinson 34 have recently noted that the excessive insulin response of maturity-onset diabetic persons correlates more with obesity than with the diabetic state. They observed that non-obese diabetics exhibited normal or low insulin responses to glucose and that obese nondiabetic subjects showed excessive insulin responses to glucose. Yalow and coworkers 33 have also observed hyperinsulinism related to obesity, but they believe that they can separate the effects of obesity from the abnormalities observed in diabetes, the peak insulin concentrations occurring later in diabetes and the highest insulin concentrations being noted when obesity and diabetes coexist. Sussman 27 emphasizes the confusion of the situation by noting that the metabolic abnormalities seen in obesity and diabetes are similar, and that obese subjects may have latent or overt chemical diabetes mellitus. Plasma insulin levels have been evaluated in "prediabetic" subjects, who by family history are likely to develop diabetes, although their glucose-tolerance tests are within normal limits at the time of study. Results have been conflicting, with some noting elevated plasma insulin concentrations, and others 9 28 finding essentially normal plasma insulin responses to a glucose stimulus. Hyperinsulinism has also been reported in islet-cell tumors, 3 ' 30 acromegaly, 16 essential hypertension and peripheral vascular dis-

4 712 BURROWS Vol. 47 ease, 4 myocardial infarction, old age, 5,19 thyrotoxicosis, 31 decompensated cirrhosis, 31 leucine-induced hypoglycemia, 31 and after administration of growth hormone 17 or adrenal corticoids The large group of hyperinsulin states adds even more difficulty to the interpretation of plasma insulin levels in any single subject. In conclusion, the present study agrees with others that maturity-onset diabetic and borderline patients, as defined by blood glucose response to a glucose load, tend to exhibit an abnormally high and prolonged plasma insulin response to a glucose stimulus. However, the range of plasma insulin levels in both normal and diabetic persons is so great and may be subject to so many other factors that it is of doubtful significance in the evaluation of the diabetic or prediabetic state in any single person. Although plasma insulin assays can easily be performed, the results may not offer any additional information for the diagnosis of diabetes or prediabetes that is not available from the blood glucose levels alone. SUMMARY Plasma insulin concentrations in response to a -Gm. oral glucose tolerance test were measured in an unselected group of 24 patients. Diabetic and borderline patients had a tendency to exhibit abnormally high and prolonged insulin responses to the glucose stimulus; however, the range of variation in normal and diabetic subjects was so great that the determination of plasma insulin concentrations is of doubtful value in the diagnosis of the diabetic or prediabetic state. REFERENCES 1. Berger, S., Downey, J. L., Traisman, H. S., and Metz, R.: Mechanism of the cortisonemodified glucose tolerance test. New England J. Med. 374: , Berger, S., and Vongaraya, N.: Insulin response to ingested protein in diabetes. Diabetes, 15: , Berson, S. A., and Yalow, R. S.: Immunoassay of plasma insulin. In Ciba Foundation Colloquia of Endocrinology, Vol. 14, Immunoassay of Hormones. Boston: Little, Brown & Company, 1962, pp Breckenridge, A., Rubinstein, A. IT., Dollery, C. T., and Fraser, T. R.: Serum-insulin in essential hypertension and in peripheral vascular disease. Lancet, i: , Crockford, P. M., Harbeck, R. J., and Williams, R. H.: Influence of age on intravenous glucose tolerance and serum immunoreactive insulin. Lancet, i: , Ehrlich, R. M., and Bambers, G.: Immunologic assay of insulin in plasma of children. Diabetes, 13: , Goetz, F. C, Greenberg, B. Z., Ells, J., and Meinert, C.: A simple immunoassay for insulin: application to human and dog plasma. J. Clin. Endocrinol., 28: , Grodsky, G. M., and Forsham, P. H.: An immunochemical assay of total extractable insulin in man. J. Clin. Invest., 39: , Grodsky, G. M., Karam, J. H., Pavlatos, F. C, and Forsham, P. H.: Serum-insulin response to glucose in prediabetic subjects. Lancet, i: , Hales, C. N.: Plasma insulin in diabetes. In Ciba Foundation Colloquia on Endocrinology, Vol. 15, Aetiology of Diabetes Mellitus and its Complications. Boston: Little, Brown & Company, 1964, pp Hales, C. N., and Randle, P. J.: Effects of lowcarbohydrate diet and diabetes mellitus on plasma concentrations of glucose, nonesterified fatty acid, and insulin during oral glucose-tolerance tests. Lancet, i: , Hales, C. N., and Randle, P. J.: Immunoassay of insulin with insulin-antibody precipitate. Biochem. J., 88: , Hales, C. N., Walker, J. B., Garland, P. B. and Randle, P. J.: Fasting plasma concentrations of insulin, non-esterified fatty acids, glycerol, and glucose in the early detection of diabetes mellitus. Lancet, i: 65-67, Karam, J. H., Grodsky, G. M., and Forsham, P. H.: Excessive insulin response to glucose in obese subjects as measured by immunochemical assay. J. Am. Diet. A., IS: , Karam, J. H., Grodsky, G. M., and Forsham, P. H.: The relationship of obesity and growth hormone to serum insulin levels. Ann. New York Acad. Sc, 131: , Karam, J. I-I., Grodsky, G. M., Pavlatos, F. C, and Forsham, P. H.: Critical factors in excessive serum-insulin response to glucose. Lancet, i: , Luft, R., and Cerasi, E.: Effect of human growth hormone on insulin production in panhypopituitarism. Lancet ii: , Meade, R. C, and Klitgaard, H. M.: A simplified method for immuno-assay of human serum insulin. J. Nuclear Med., 3: , Metz, R., Surmaczynska, B., Berger, S., and Sobel, G.: Glucose tolerance, plasma insulin and free fatty acids in elderly subjects. Ann. Int. Med., 64: , Morgan, C. R., and Lazarow, A.: Immunoassay of insulin using a two-antibody system. Proc. Soc. Exp. Biol. & Med., 110: 29-32, Nikkila, E. A., Miettinen, T. A., Vesenne, M., and Pelkonen, R.: Plasma-insulin in coronary heart-disease. Lancet, ii: 50S-511, 1965.

5 June 1967 INSULIN RESPONSE IN GLUCOSE-TOLERANCE TESTS Perley, M., and Kipnis, D. M.: Effect of glucocorticoids on plasma insulin. New England J. Med., 274: , Peters, N., and Bales, C. N.: Plasma-insulin concentrations after myocardial infarction. Lancet, i: , Power, L., Reyes-Leal, B., and Conn, J. W.: Serum insulin-like activity in genetic and experimental diabetes mellitus. Metabolism, 18: , Samols, E., and Bilkus, D.: A comparison of insulin immunoassays. Proc. Soc. Exp. Biol. &Med., 115: 79-84, Steinko, J., Soeldner, J. S., Camerini-Davalos, R. A., and Renold, A. E.: Studies on serum insulin-like activity in prediabetes and early overt diabetes. Diabetes, 12: , Sussman, K. E.: Effect of prolonged fasting on glucose and insulin metabolism in exogenous obesity. Arch. Int. Med., 117: , Welborn, T. A., Rubenstein, A. II., Haslam, R., and Fraser, 11.: Normal insulin response to glucose. Lancet, i: , Wong, G. S., Cutler, J. M., and Little, J. A.: Blood glucose, insulin and lipid levels in normal, prediabetic, borderline diabetic and diabetic subjects. Canad. M. A. J. 94: 676, Yalow, R. S., and Berson, S. A.: Dynamics of insulin secretion in hypoglycemia. Diabetes, 14: , Yalow, R. S., and Berson, S. A.: Immunoassay of endogenous plasma insulin in man. J. Clin. Invest., 89: , Yalow, R. S., and Berson, S. A.: Plasma insulin concentrations in nondiabetic and early diabetic subjects: determinations by a new sensitive immunoassay technic. Diabetes, 9: , Yalow, R. S., Click, S. M., Roth, J., and Berson, S. A.: Plasma insulin and growth hormone levels in obesity and diabetes. Ann. New York Acad. Sc, 131: , Young, J. D., and Jenkinson, I. S.: Scruminsulin and glucagon during glucose-tolerance test. Lancet, i: 1321, 1966.