Jugoslov Med Biohem 2005; 24 (1) 35 UC 577,1; 61 SSN 0354-3447 Jugoslov Med Biohem 24: 35 39, 2005 Originalni nau~ni rad Original paper NSULN AND C-PEPTDE RESPONSE N HEALTHY PERSONS AND NDVDUALS WTH MPARED GLUCOSE METABOLSM DURNG ORAL GLUCOSE TOLERANCE TEST Olgica Nedi}, Jasminka Miloradovi}, Marija Ratkovi}, Romana Masnikosa NEP nstitute for the Application of Nuclear Energy, 11080 Belgrade-Zemun, Banatska 31b, Serbia and Montenegro Summary: Serial measurements of blood glucose concentration before and after giving a specific amount of glucose orally provide a standard method to evaluate glucose metabolism. Although the reference ranges for glucose concentration in various disease states that are based on impaired glucose homeostasis have been established, the reference values are not clearly defined for insulin and C-peptide concentrations. The aim of this work was to study the insulin and C-peptide response during OGTT. Healthy individuals exhibited the following profile of insulin and C-peptide levels: 15 ± 4.9 mu/l and 0.5 ± 0.19 nmol/l (0 h), 116 ± 52.8 mu/l and 2.3 ± 0.79 nmol/l (1 h) and 59 ± 26.7 mu/l and 2.0 ± 0.67 nmol/l (2 h). Persons with impaired glucose tolerance had higher C-peptide levels at 0 h, 0.6 ± 0.17 nmol/l, and significantly higher insulin and C-peptide concentrations after 1 h, 209 ± 63.8 mu/l and 3.5 ± 1.00, nmol/l and 2 h, 188 ± 48.8 mu/l and 3.6 ± 0.92 nmol/l. Diabetic patients had higher basal levels of C-peptide, 0.8 ± 0.23 nmol/l, insulin and C-peptide increased after 1 h similarly as in healthy people, but further continued to increase significantly, 181 ± 137.6 mu/l and 3.7 ± 1.49 nmol/l. Subjects that exhibited low blood glucose levels demonstrated lower insulin concentrations at all time intervals, 11 ± 2.5 mu/l (0 h), 63 ± 31.1 mu/l (1 h) and 44 ± 22.9 mu/l (2 h), but the concentration of C-peptide leveled with that of the healthy ones. The results of this work may be useful in establishing reference ranges for insulin and C-peptide concentrations for defined time intervals during OGTT, in health and disease. Key words: insulin, C-peptide, OGTT Address for correspondence: Olgica Nedi} NEP nstitute for the Application of Nuclear Energy Banatska 31b, 11080 Belgrade-Zemun Serbia and Montenegro Tel.: (+) 381 11 617 252 Fax.: (+) 381 11 618 724 e-mail: olgica@inep.co.yu ntroduction The concentration of glucose in the blood is regulated by a complex network of various biochemical processes including glycolysis, glyconeogenesis, glycogenesis, glycogenolysis and many others. These metabolic pathways are controlled and modulated by a number of hormones, in order to maintain the concentration of glucose within relatively narrow range. nsulin and, to a lesser extent, insulin-like growth factors are responsible for a decrease in blood glucose, while glucagon, cortisol, epinephrine and growth hormone have the opposite role (1). Numerous disease states that involve impaired glucose metabolism have been discovered. A disease with the highest incidence is diabetes mellitus (2). Diabetes mellitus can be regarded as a group of metabolic disorders of carbohydrate metabolism in which glucose is underutilized, producing hyperglycemia, and often followed by lifethreatening episodes that include ketoacidosis and hyperosmolar coma (1). Prolonged disease causes specific complications, many of which are related to pathological glycosylation (glycation) of various essential molecules and cellular structures. n general, structural modifications fall into two categories: (i) nonenzymatic glycation per se, which refers to the attachment of free carbohydrate to proteins in the Amadori construct, and (ii) generation of advanced glycation endproducts (AGE), which refers to a heterogeneous group of carbohydrate-modified products derived from the Amadori adduct by oxidation, polymerization and other spontaneous reactions (3). Complications,
36 Jugoslov Med Biohem 2005; 24 (1) including cardiovascular disease, neuropathy, nephropathy and others, are frequently diagnosed in patients with diabetes mellitus (4 6). The opposite, hypoglycemia, is defined as a state in which blood glucose concentration is bellow the fasting range. A rapid decrease in glucose level produces weakness, shakiness, rapid pulse, headache etc. (1). nsulin is a hormone that stimulates the uptake of glucose into cells, promotes intracellular transport and conversion into the other metabolic molecules. Proinsulin is produced from preproinsulin in pancreatic beta cells. Proinsulin is stored in secretory granules and is cleaved into insulin and C-peptide upon stimulation. A physiological role of insulin is well known, while C-peptide, being more stable than insulin in the circulation, can be regarded as a reliable monitor of insulin secretion (1). Serial measurements of blood glucose levels before and after giving a specific amount of glucose orally provide a standard method to evaluate glucose metabolism. Oral glucose tolerance test (OGTT), recommended by the World Health Organization (WHO), is performed with 75 g of glucose for non-pregnant adults and the blood samples are collected at defined time intervals: 0 h (before glucose intake), 1 h and 2 h (after glucose consumption). Variations of the method include sampling every 30 min for 2 h, as well as after 3h (1). The WHO criteria that differentiated healthy persons from diabetic were adopted in the National guide for clinical practice in Serbia (7). Metabolic response in healthy individuals exposed to OGTT should increase the concentration of blood glucose at most to 7.8 mmol/l and return to normal after 2 h. mpaired glucose tolerance results in higher levels of blood glucose and slower clearance. Although the reference ranges for glucose concentration in various disease states have been established, the reference values are not clearly defined for insulin and C-peptide concentration. The aim of this work was to study insulin and C-peptide response during OGTT and to obtain ranges for each hour of the test for healthy persons and individuals with impaired glucose metabolism. Material and Methods Eighty seven non-pregnant adult persons (18 males and 69 females) were subjected to OGTT. They were given orally 75 g of glucose in 200 ml of water after the fast blood sampling. The blood was collected after 1 h, 2 h and, upon the request of a physician, after 3 h. Each time the serum was separated by centrifugation within 45 min. Samples were assayed immediately for glucose and within a week for insulin and C-peptide. Meanwhile, sera were kept frozen at 20 C, as C-peptide is unstable in serum at higher temperatures. Glucose concentration was measured using RANDOX commercial reagent (GOD-PAP method); the reference levels according to the producer are 4.2 6.4 mmol/l. nsulin and C-peptide levels were determined by NEP commercial RA kits; the reference values are established as 5 25 mu/l for insulin and 0.3 0.7 nmol/l for C-peptide. Data were statistically analyzed using the Student t-test for the significance of differences between the groups (patients vs. healthy persons). Results According to the results obtained for the concentration of blood glucose during OGTT and clinical history, the examined persons were divided into four groups. The first group (, n = 21) was characterized as having the referent blood glucose concentration at 0 h (the lowest value was 4.0 and the highest 5.7 mmol/l), after 1 h the concentration increased (6.8 8.3 mmol/l) and returned to normal after 2 h (4.8 6.0 mmol/l). These individuals were assumed to be healthy in respect to glucose metabolism. Subjects collected as a second group (, n = 25) exhibited delayed glucose clearance and most of them were monitored for 3 h. The fasting blood glucose concentration was referent (4.0 5.7 mmol/l), then increased after 1 h (7.4 10.3 mmol/l), decreased after 2 h (6.6 8.3 mmol/l), and returned to normal after 3 h (4.0 5.5 mmol/l). Patients in group three (, n = 25) satisfied Table Blood insulin and C-peptide concentrations ( x ± SD) during OGTT in healthy persons () and individuals with impaired glucose metabolism (: impaired glucose tolerance, : diabetes mellitus, V: low blood glucose levels). Data were analyzed and compared (patients vs. healthy people) using t-test (ns: non significant) Group V nsulin (mu/l) 0h 1h 2h 15 ± 4.9 116 ± 52.8 59 ± 26.7 17 ± 4.7 209 ± 63.8 188 ± 48.8 ns p = 0.0004 p < 0.0001 18 ± 8.1 131 ± 75.1 181 ± 137.6 ns ns p = 0.0008 11 ± 2.5 63 ± 31.1 44 ± 22.9 p = 0.009 p = 0.0004 p = 0.08 C-peptide (nmol/l) 0h 1h 2h 0.5 ± 0.19 2.3 ± 0.79 2.0 ± 0.67 0.6 ± 0.17 3.5 ± 1.00 3.6 ± 0.92 p = 0.007 p = 0.002 p < 0.0001 0.8 ± 0.23 2.7 ± 1.48 3.7 ± 1.49 p = 0.0008 ns p = 0.017 0.5 ± 0.17 2.7 ± 0.93 2.0 ± 0.58 ns ns ns
Jugoslov Med Biohem 2005; 24 (1) 37 (a) nsulin concentration, mu/l (b) C-peptide concentration, nmol/l 300 250 200 150 100 50 0 3.5 3 2.5 2 1.5 1 0.5 0 nsulin response during OGTT V 0 0.5 1 1.5 2 2.5 Time intervals (h) C-peptide response during OGTT V 0 0.5 1 1.5 2 2.5 Time intervals (h) Figure 1. Typical examples of (a) insulin and (b) C-peptide responses during OGTT in healthy persons () and individuals with impaired glucose metabolism (: impaired glucose tolerance, : diabetes mellitus, V: low blood glucose levels). the OGTT criteria that classified them as diabetics, they also had other signs of diabetes mellitus confirmed biochemically and/or clinically. Their fasting blood glucose was normal to elevated (5. 4 13. 9 mmol/l), increased after 1 h (8.8 22.4 mmol/l), then moderately dropped or continued to increase after 2 h (7.5 24.0 mmol/l). Finally, subjects with low blood glucose levels were established as a fourth group (V, n = 16). At 0 h their concentration of glucose was low to normal (3.1 5.1 mmol/l), it remained almost unchanged after 1 h (4.0 5.7 mmol/l) and most often decreased to very low after 2 h (2.2 4.2 mmol/l). The concentrations of insulin and C-peptide were measured in each sample and the results (mean ± standard deviation) are presented in Table. Although there were significant interindividual variations, which can be seen from the SD values, specific patterns of insulin and C-peptide alterations during OGTT emerged. Figures 1a and 1b present typical insulin and C-peptide responses in four groups of people during OGTT. Healthy individuals exhibited increase in insulin and C-peptide levels 1 h after the stimulation and they decreased after 2 h. Persons with delayed glucose clearance had slightly higher C-peptide levels at 0 h and significantly higher insulin and C-peptide concentrations compared to healthy subjects after 1 h and 2 h. Concentrations of insulin and C-peptide remained very high after 2 h and dropped after 3 h (data not shown). Diabetic patients had higher than normal basal levels of C-peptide. After 1 h insulin and C-peptide increased, on average, as in healthy people but further continued to increase significantly. After 2 h the concentration of insulin and C-peptide in groups and were within the same range. Subjects with low blood glucose levels demonstrated lower insulin concentrations at all time intervals, but the concentration of C-peptide leveled with that of the healthy ones. Discussion The results for insulin concentration during OGTT in healthy persons are in accordance with the data reported in Tietz Textbook of Clinical Chemistry, 0 h: 6 21 mu/l, 1 h: 20 120 mu/l and 2 h: 18 56 mu/l (8) and Human Clinical Endocrinology, 0 h: 20 ± 2 mu/l, 1 h: 75 ± 10 mu/l and 2 h: 45 ± 6 mu/l (9). n obese persons both basal and stimulated levels may be high, 0 h: 80 ± 5 mu/l, 1 h: 130 ± 7 mu/l and 2 h: 80 ± 5 mu/l (9). mpaired glucose tolerance was diagnosed in individuals who had fasting blood glucose levels less than those required for a diagnosis of diabetes mellitus, but had a glucose response during OGTT between normal and diabetic (1). nsulin and C-peptide response, on the other hand, did not follow that pattern. The initial response after 1 h was, on average, more intense than in diabetics, suggesting greater pancreatic sensitivity to glucose stimulation and hormonal secretion which, unfortunately, was less successful in decreasing the blood glucose. The state of relative insulin resistance at cellular level is most often responsible for this insensitivity (10). Mathematical analysis of the relationship between insulin sensitivity and secretion has revealed a hyperbolic function, such that the product of two variables is constant (11). Measurements of insulin sensitivity provide clinicians and clinical researchers with valuable tool to evaluate the efficiency of both current and potentially useful therapeutic preparations. Although several methods had been developed and validated to evaluate insulin sensitivity, none of these methods can be universally used in all patients (12). Evenmore, a method suitable for clinical or basic research may not necessarily be a practical method for use in regular practice or for epidemiological studies. nsulin activity may be also influ-
38 Jugoslov Med Biohem 2005; 24 (1) enced by the presence of anti-insulin or anti-insulin receptor antibodies (13, 14). Two hours after the glucose intake, insulin/c-peptide status in both groups of patients ( and ), on average, became the same. These results should be, however, interpreted with caution due to very high standard deviation values in group. All examined diabetic persons had normal to high insulin levels upon stimulation, which classified them as insulin-independent cases of diabetes mellitus (1). Subtyping of these patients would, possibly, narrow insulin and C-peptide ranges for each subgroup. Blood glucose concentration limits are hard to define for hypoglycemia, as glucose levels are often bellow fasting range and transient decline may occur (1). OGTT is not a test regularly recommended for diagnosis of hypoglycemia. Blood insulin concentration in persons with low blood glucose levels was found to be significantly lower than in healthy people, both after fasting and during OGTT. C-peptide levels, on the other hand, were within reference range at all sampling intervals. These data suggested that pancreatic stimulation was preserved, but secreted insulin was either cleared from the circulation faster than normal or it was present in a form that was not detected by the applied analytical method (immune complexes?). Although OGTT and blood glucose determinations are routinely performed for diagnosis of diabetes mellitus and impaired glucose tolerance (15, 16), parallel measurement of insulin and C-peptide levels, in all cases of impaired glucose metabolism, certainly helps in diagnosis and management of these disorders. The results of this work may be useful in establishing reference ranges for insulin and C-peptide concentrations for defined time intervals during OGTT, in health and disease. PROMENA KONCENTRACJE NSULNA C-PEPTDA KOD ZDRAVH LJUD OSOBA SA POREME]ENM METABOLZMOM GLUKOZE TOKOM ORALNOG TESTA TOLERANCJE GLUKOZE Olgica Nedi}, Jasminka Miloradovi}, Marija Ratkovi}, Romana Masnikosa NEP-nstitut za primenu nuklearne energije, 11080 Beograd-Zemun, Banatska 31b, Srbija i Crna Gora Kratak sadr`aj: Metabolizam glukoze se uobi~ajeno procenjuje serijskim odre ivanjem koncentracije glukoze u krvi pre i posle oralnog uno{enja odre ene koli~ine glukoze. ako je referentni opseg za koncentraciju glukoze utvr en kod razli~itih stadijuma bolesti u ~ijoj osnovi je poreme}ena homeostaza glukoze, referentne vrednosti za koncentraciju insulina i C-peptida nisu jasno definisane. Cilj ovog rada je bio da ispita promenu koncentracije insulina i C-peptida tokom OGTT. Zdrave osobe su ispoljile slede}i profil promene koncentracije insulina i C-peptida: 15 ± 4,9 mu/l i 0,5 ± 0,19 nmol/l (0 h), 116 ± 52,8 mu/l i 2,3 ± 0,79 nmol/l (1 h) i 59 ± 26,7 mu/l i 2,0 ± 0,67 nmol/l (2 h). Osobe sa smanjenom tolerancijom glukoze su imale vi{i nulti nivo C-peptida, 0,6 ± 0,17 nmol/l i zna~ajno vi{e koncentracije insulina i C-peptida posle 1 h, 209 ± 63,8 mu/l i 3,5 ± 1,00 nmol/l i 2 h, 188 ± 48,8 mu/l i 3,6 ± 0,92 nmol/l. Dijabeti~arima je izmeren vi{i bazalni nivo C-peptida, 0,8 ± 0,23 nmol/l, posle 1 h koncentracije insulina i C-peptida su pove}ane sli~no kao kod zdravih ljudi, ali su nastavile i dalje da zna~ajno rastu, 181 ± 137,6 mu/l i 3,7 ± 1,49 nmol/l. Kod osoba sa niskom koncentracijom glukoze u krvi utvr ena je niska koncentracija insulina u svim testiranim vremenskim intervalima, 11 ± 2,5 mu/l (0 h), 63 ± 31,1 mu/l (1 h) i 44 ± 22,9 mu/l (2 h), dok je nivo C-peptida bio sli~an kao kod zdravih. Rezultati ovog rada bi mogli biti korisni za odre ivanje referentnih opsega za koncentraciju insulina i C-peptida, za utvr ene vremenske intervale tokom OGTT, u zdravom stanju i tokom bolesti. Klju~ne re~i: insulin, C-peptid, OGTT
Jugoslov Med Biohem 2005; 24 (1) 39 References 1. Sacks DB. Carbohydrates. n: Burtis CA, Ashwood ER. eds. Tietz Textbook of Clinical Chemistry. Philadelphia: Saunders Company, 1994: 928 1001. 2. Majki}-Singh N. nstructions and recommendations for use of the laboratory methods in diabetes mellitus diagnosis and monitoring. Jugoslov Med Biohem 2003; 22 (Suppl 1): 1 4. 3. Cohen MP. ntervention strategies to prevent pathogenic effect of glycated albumin. Arch Biochem Biophys 2003; 419: 25 30. 4. Podwall D, Gooch C. Diabetic neuropathy: clinical features, etiology and therapy. Curr Neurol Neurosci Rep 2004; 4: 55 61. 5. Khamaisi M, Wexler D, Skrha J, Strojek K, Raz, Milicevic Z. Cardiovascular disease in type 2 diabetes: epidemiology, risk factors and therapeutic modalities. sr Med Assoc J 2003; 5: 801 6. 6. Jerums G, Panagiotopoulos S, Forbes J, Osticka T, Cooper M. Evolving concepts in advanced glycation, diabetic nephropathy and diabetic vascular disease. Arch Biochem Biophys 2003; 419: 55 62. 7. Zamlakar M, Bajovi} Lj, Stefanovi} M. Definicija, dijagnoza i podela dijabetesa. n: Nacionalni vodi~ klini~ke prakse, diabetes mellitus. Ministarstvo zdravlja Republike Srbije, 2002: 3 10. 8. Painter PC, Cope JY, Smith JL. Appendix Reference ntervals. n: Burtis CA, Ashwood ER. eds. Tietz Textbook of Clinical Chemistry. Philadelphia: Saunders Company, 1994: 2161 217. 9. Slijep~evi} D, Vujovi} S, Nestorovi} Z. Humana klini~ka endokrinologija. Beograd: Obele`ja, 2002: 829 901. 10. Pirola L, Johnson AM, Van Obberghen E. Modulators of insulin action and their role in insulin resistance. nt J Obes Relat Metab Disord 2003; 27 (Suppl 3): 61 4. 11. Ahren B, Pacini G. mportance of quantifying insulin secretion in relation to insulin sensitivity to accurately asses beta cell function in clinical studies. Eur J Endocrinol 2004; 150: 97 104. 12. Monzillo LU, Hamdy O. Evaluation of insulin sensitivity in clinical practice and in research settings. Nutr Rev 2003; 61: 397 412. 13. Eisenbarth GS. nsulin autoimmunity: immunogenetics/immunopathogenesis of type 1A diabetes. Ann N Y Acad Sci 2003; 1005: 109 18. 14. Yamasaki H, Yamaguchi Y, Fujita N, Kato C, Kuwahara H, Yamauchi MD et al. Anti-insulin receptor autoantibodies in a patient with type B insulin resistance and fasting hypoglycemia. Acta Diabetol 2000; 37: 189 96. 15. Luyckx FH, Scheen AJ. How explore the controversy concerning the role of oral glucose tolerance test in clinical practice. Rev Med Liege 2003; 58: 701 5. 16. gnjatovi} S. Diagnosis of diabetes mellitus. Jugoslov Med Biohem 2003; 22 (Suppl 1): 13 19. Received: April 2, 2004 Accepted: August 5, 2004