by Springer-Verlag 1977

Transcription

1 Diabetlgia 13, (177) by Springer-Verlag 177 Prevalence f Residual B-Cell Functin in Insulin-Treated Diabetics Evaluated by the Plasma C-Peptide Respnse t Intravenus Giucagn C. Hendriksen, O.K. Faber 1, J. Drejer, and C. Binder Hwdre Hspital, Klampenbrg, Denmark Summary. In 83 insulin-treated diabetics the influence f the duratin f insulin treatment n the prevalence f residual insulin secretin was examined by determining the plasma C-peptide cncentratin befre and after intravenus injectin f 1 mg f glucagn. In 64 patients, plasma C-peptide cncentratin was als determined befre and after a standard meal. There was a gd crrelatin between the C-peptide respnse t glucagn and t the meal (r = 0.67; p < ) suggesting that the glucagn test will predict the B-cell respnse during everyday life. The predictive value f a psitive glucagn test was 84% and f a negative test 100%. A preserved, but reduced, B-cell functin was demnstrable in 36 f 83 patients. Residual B-cell functin was mst frequent in the patients with the shrtest duratin f diabetes. The metablic imprtance f endgenus insulin was demnstrated by the significantly lwer insulin requirement in the patients with residual B-cell functin. Key wrds: Insulin-treated diabetics, C-peptide, IV glucagn test, residual B-cell functin. Determinatin f the cncentratin f C-peptide in peripheral venus plasma yields an indirect, but specific, measure f the secretin f insulin frm the B-cells f the islets f Langerhans [1, 2, 3]. C-peptide determinatins are especially f value in subjects in whm exgenus insulin and circulating insulin antibdies interfere with quantitative measurements f endgenus insulin [1]. 1 O.K. Faber is a Research Fellw at the Umverslty f Cpenhagen It has been demnstrated that, within the first year f treatment, mst diabetics n insulin have a much reduced insulin secretin [4, 5, 6] and that, fllwing a temprary respite within the first 2-5 mnths, insulin secretin decreases with time [7]. Determinatin f the C-peptide cncentratin befre and after stimulatin with glucagn has been fund t reflect quantitatively B-ceU functin under nrmal cnditins f life in patients with insulin-dependent diabetes mellitus f shrt duratin [8]. The aim f the present wrk was, by use f the glucagn test, t examine the influence f the duratin f insulin treatment n the prevalence f residual B-cell functin. The study als includes an evaluatin f the discriminatry capacity f the test when applied t subjects with diabetes f lng duratin. Study Ppulatin and Methds The study ppulatin cmprised 83 patients with insulin-treated diabetes mellitus. Duratin f treatment was ne t 50 years (mean 12) and age at nset 3 t 50 years (mean 22) (Fig. 1). Insulin treatment was started at the time f diagnsis in all but 7 patients. This subgrup had knwn diabetes fr 1-8 years (mean 3 years) befre insulin treatment was started. At the time f study, they had been insulin-treated fr 1--4 years (mean 2 years). Their age at the time f diagnsis ranged frm 27 t 48 years (mean 38 years). The insulin dse fr the whle study ppulatin varied frm 0.07 t 0.1 units/kg/24 h (mean 0.4). All patients were within +15% f their ideal bdy weight () except ne wman (weight 77.6 kg, height 166 cm) with insulindependent diabetes since age 10. In 11 patients, the serum creatinine exceeded 120 ~tml/1 and 3 had al-

2 616 C. Hendriksen et al.: The Glucagrt/C-Peptide Test 50_ N ~, ~_ 30_ p- uj i Z 20_ uj I0_ el I ~0 ~r ~ 00 ~ ~ ~ I I I I { years INSULIN TREATMENT Fig. 1. Age at the time f diagnsis and duratin f insulin treatment in 83 patients with insulin-treated diabetes mellitus.., indicates subjects with and,, subjects withut a significant plasma C-peptide increase after 1 mg f glucagn IV buminuria. Tw thers had chrnic albuminuria, but nrmal serum creatinine. All patients gave infrmed cnsent t the investigatin. They were tested after an vemight fast. Freeflwing venus bld was sampled 10, 5 and 0 rain befre and 2, 4, 6, 8, 10, 15 and 20 rain after an IV blus f 1 mg prcine glucagn (NOV Apart frm mild nausea in sme patients 1--4 min after the injectin, n side effects were bserved during r after the administratin f glucagn. Fr each sample, 7 ml bld were transferred t a plastic tube with 700 ~tl f a trasyll-heparin mixture (Trasyll Bayer 5000 KIE/ml and Heparin Nv 5000 pu/ml, relative prprtins 10 : 1). The tubes were kept at rm temperature until after the test when they were spun at 3000 g fr 10 min at 4 ~ C. The supernatant was pipetted int plastic tubes and immediately stred at -20~ In 64 patients, venus bld was als sampled befre and ne hur after the start f a standard breakfast. The meal was cmpsed f 34% fat, 45% carbhydrate, and 21% prtein and amunted t 435 kcal (1823 kj). The glucagn test and the meal test were carried ut in randm rder with an interval f 3 t 7 days. The plasma C-peptide cncentratin was measured as described by Heding [3], emplying the antibdy M 1230 [10]. In this assay human prinsulin crss-reacts n mre than 13% n a mlar basis with C-peptide (unpublished bservatin). All samples frm a given study were analyzed in the same assay. The intra-assay cefficient f variatin in the lw part f the wrking range f the C-peptide assay was 5.4 per cent [10]. The "effective" detectin limit f the C-peptide assay was 0.06 pml/ml. This limit was defined by the upper range f values measured in 10 pancreatectmized patients. The detectin limit in the assay buffer was 0.02 pml/ml. A significant increase in the C-pepfide cncentratin was defined as an increase exceeding 16.2% (3 within-assay cefficient f variatin) f the individual fasting level prvided that ne f the values was greater r equal t 0.06 pml/ml, i. e., the "effective" detectin limit. The discriminatry capacity f the glucagn test as an indicatr f B-cell. functin during everyday life was evaluated frm the predictive values f psitive and negative tests. The predictive value f a psitive test was calculated as the number f true psitives (thse subjects wh shwed a significant C-peptide respnse t bth stimuli) divided by the number f true psitives plus the number f false psitives (thse subjects wh shwed a significant C-peptide respnse t glucagn but nt t the meal). The predictive value f a negative test was calculated in a similar way as the number f true negatives divided by the number f true negatives plus the number f false negatives. Bld glucse cncentratin was measured using a glucse-xidase methd. The significance f crrelatin was tested by the Spearman rank crrelatin cefficient. The Mann- Whitney rank sum test fr unpaired data was used fr statistical evaluatin f cmparisn between grups. Results Fasting bld glucse ranged between 3.6 and 22.1 mml/1 (mean 12.1) in the 83 patients befre the glucagn test and between 4.4 and 21.7 mml/1 (mean 13.0) in the 64 patients n the day f the meal test. The increase in glucse 20 min after glucagn ranged frm 0.6 t 5.8 mml/1 (mean 3.0) and that ne hur after the meal frm 1.4 t 10. mml/1 (mean 5.). The fasting C-peptide cncentratin in the 83 glucagn tested patients ranged frm 0 t 0.3 pml/ml. Fllwing stimulatin with glucagn, the maximum increase in C-peptide cncentratin, which ccurred 4-6 min after the blus injectin, varied between 0 and 0.35 pml/ml. A significant increase was bserved in 36 f the 83 patients. The fasting C-peptide cncentratin was psitively crrelated with the maximum increase fllwing glucagn (r = 0.7; p < ) in these patients. A significant increase in the plasma C-peptide cncentra-

3 C. Hendriksen et al.: The Glucagn/C-Peptide Test ~ e z 0.20 :~ , J 0.3 (/3 0 0 Q I 8 G..J (. 010 (Z LIJ I,-- LL 0.05 LIJ O ffl z w 0.2 0_! "~ 001 e 0 11 '2 0'3 '4 FASTING C -peptide pml/ml Fig. 2. The crrelatin between insulin dse and fasting C-peptide cncentratin. Results frm 36 insulin-treated diabetics with a significant increase in the plasma C-peptide cncentratin in respnse t glucagn (r = -0.51, p < 0.003) T ~ J, ~, 0~20, J I sn Z~ C- PEPTIDE AFTER MEAL pml/ml Fig. 3. The crrelatin between the increase in the plasma C-peptide cncentratin in respnse t 1 mg IV glucagn and in respnse t a standard meal. Results frm 32 insulin-treated diabetics with a significant increase in the plasma C-peptide cncentratin in respnse t glucagn (r = 0.67, p < ). Abscissa and rdinate pltted n a lgarithmic scale Table 1. Relatin f C-peptide respnse t IV glucagn t fasting C-peptide cncentratin. See text fr definitin f respnse Fasting C-peptide cncentratin pml/ml Number f patients => and and =< Ttal Number f patients with respnse t glucagn tin was fund in all patients with fasting C-peptide cncentratin pml/ml and in 7 patients with fasting C-peptide cncentratins < 0.08 pml/ml. There was n increase in the plasma C-peptide cncentratin in respnse t glucagn in patients with fasting C-peptide cncentratins ~< 0.03 pml/ml (Table 1). The C-peptide respnse t glucagn was significant in 31 f 3 patients (7 per cent) treated with insulin fr ne t 6 years and in 5 f 44 patients (11 per cent) treated fr 7 t 50 years (Fig. 1). In the patients with residual B-cell functin the insulin dse (mean 0.40, range units/kg/ 24 hurs) was fund t be lwer than in the patients with n B-cell functin (mean 0.55, range units/kg/24 hurs) (p < 0.001). Average fasting bld glucse cncentratin was 11.8 (range ) mml/l in patients with residual B-cell functin and 12.1 (range ) mml/1 in thse withut. The insulin dse was inversely crrelated with the fasting C-peptide cncentratin in patients with a residual B-cell functins (r = -0.51, p < 0.003) in that the patients given the highest dses had the lwest fasting C-peptide cncentratins (Fig. 2). The dse was als inversely crrelated with the increase in C-peptide cncentratin fllwing glucagn (r = -0.42; p < 0.02). There was n crrelatin between the fasting bld glucse cncentratin and the increase in the C-peptide cncentratin in respnse t glucagn. In the 64 patients in whm the glucagn as well as the meal stimulatin tests were studied the increase in the C-peptide cncentratin varied frm 0 t 0.35 pml/ml fllwing glucagn and frm 0 t 0.25 pml/ml fllwing the meal. In 27 f the 64 patients bth types f stimulatin caused a significant increase in C-pept~de cncentratin. In 5 patients there was a significant C-peptide respnse t glucagn, but nt t the meal. Of these 5 patients nly ne failed t shw significant amunts f C- peptide in the plasma. All patients wh respnded t the meal had a significant C-peptide respnse t glucagn. In 32 patients there was neither a respnse t glucagn nr t the meal. The predictive

4 618 c. Hendriksen et al.: The Glucagn/C-Peptide Test value f a psitive test was 27/(27 + 5) = 84.4% (5% cnfidence limits %) and f a negative test 32/(32 + 0) = 100% (5% cnfidence limits %). The fasting C-peptide cncentratin was crrelated with the increase ne hur after the meal in the 27 patients with a significant C-peptide respnse t the meal (r ; p < 0.001). The maximum increase in the C-peptide cncentratin fllwing glucagn stimulatin was significantly crrelated with that after meal stimulatin in the 32 patients wh shwed a significant C-peptide respnse t glucagn (r = 0.67; p < ) (Fig. 3). There was n significant difference in the average increase in plasma C-peptide in respnse t the tw tests. Discussin It has previusly been shwn that the increase in plasma C-peptide cncentratin in respnse t 1 mg f IV glucagn is a quantitative measure f the functinal B-cell secretry capacity under physilgical cnditins in patients with insulin-dependent diabetes f shrt duratin [8]. The gd crrelatin fund in the present study between the C-peptide respnse t I mg f IV glucagn and t a standard meal suggests that the glucagn test, as a measure f residual B-cell functin, is applicable als t insulintreated diabetics with a diabetes duratin f up t 50 years. Amng the 64 patients examined with bth tests, we fund n false negative results f the glucagn test. On the ther hand, the result was false psitive in 5 patients, as the plasma C-peptide cncentratin increased significantly in respnse t giucagn, but did nt shw any significant increase in respnse t the standard meal. In these patients, hwever, the magnitude f the C-peptide respnse was very small. All patients with a C-peptide increase > 0.03 pml/ml fllwing glucagn had a significant respnse t the meal (Fig. 3). In the case f prnunced B-cell failure, glucagn thus seems t exert a greater stimulus t the B-cells than the test meal. An evaluatin f the discriminatry capacity f the glucagn test shwed that the predictive value f a psitive test was 84% and that f a negative test 100%. The glucagn test, therefre, is prbably applicable t measurements f the functinal capacity f the B-cells during everyday life in diabetics n lng-term insulin treatment. Insulin-dependent diabetics were previusly held t have an irreversible and abslute endgenus insulin deficiency. The C-peptide cncentratin in plasma reprted in insulin-treated patients with diabetes f shrt duratin suggests, hwever, that residual B-cell functin is retained in the majrity f such patients [4, 5, 6]. The results frm the present study shw that residual B-cell functin is present in mst insulin-treated diabetics fr the first five years f treatment. After that time, the prevalence f residual insulin secretin shws a marked fall. Amng the diabetics with diabetes f mre than 10 years' duratin, few had any demnstrable insulin secretin (Fig. 1). In a recent study, Grajwer et al. [11] have reprted a similar prevalence f functinal B-cell survival in juvenile diabetes mellitus. It has been suggested that the fasting C-peptide cncentratin is nt nly a qualitative, but als a quantitative measure f the insulin secretin [8]. This suggestin is supprted by the present study, as the fasting C-peptide cncentratin crrelated well with the increase fllwing stimulatin. Only 2 f 43 patients with undetectable amunts f C-peptide in fasting plasma had a significant increase fllwing stimulatin with glucagn. On the ther hand, 6 patients with measurable amunts f C-peptide in fasting plasma shwed n respnse t glucagn. A pssible explanatin is an extrardinarily high r lw bld glucse level during the glucagn test. Hwever, this was nt the case. Elevated cncentratins f C-peptide have been fund in patients with reduced clearance f C- peptide wing t impaired renal functin [12]. In such patients, even an almst failing insulin secretin may be enugh fr C-peptide t be present at a lw cncentratin. Shrt-term B-cell stimulatin wuld nt be expected t induce a significant increase in plasma C-peptide cncentratin. Hwever, nne f the 6 patients withut respnse t glucagn had a reduced renal functin. Crss-reactin f human prinsulin in the C- peptide assay might als explain the missing C-peptide respnse t glucagn in patients with measurable fasting C-peptide cncentratins. Hwever, human prinsulin crss-reacts nly t a small degree in the assay emplyed. It can thus be suppsed that the patients in questin had a mdest insulin secretin, but that their B-cells were nt respnsive t glucagn. The fasting C-peptide cncentratin therefre seems t be nt nly a qualitative, but als a quantitative measure f B-cell secretry capacity under physilgic cnditins. All patients with a fasting C- peptide cncentratin abve 0.07 pml/ml will have retained sme functining B-cells, and nearly all will respnd t fd with an increased insulin secretin. Patients with a fasting C-peptide cncentratin belw 0.04 pml/ml will have an abslute B-cell

5 C. Hendriksen et al.: The Glucagn/C-Peptide Test 61 failure, whereas the B-cell functin cannt be evaluated with certainty frm the fasting C-peptide cncentratin alne in patients with fasting C-peptide cncentratins between these values (Table 1). The metablic imprtance f an endgenus insulin prductin has been demnstrated in shrtterm insulin-dependent diabetic subjects [6, 7]. This finding is supprted by the lwer insulin requirement fund in the grup with residual B-cell functin as cmpared t the grup withut any demnstrable B-cell functin, the metablic balance in the tw grups being similar as judged by the fasting bld glucse cncentratin. Als in favur f this hypthesis is the finding that, amng the patients with residual B-cell functin, the insulin requirement was lwest in the patients with the highest B-cell secretry capacity evaluated frm the fasting C-peptide cncentratin (Fig. 2). Acknwledgements. We thank Miss Bdil Mathiassen and Miss Jane Falk fr technical help. The English versin was revised by Ilna Munck. References 1. Melani, F., Rubenstein, A.H., Oyer, P., Steiner, D.F.: Identificatin f prinsulin and C-peptide in human serum by a specific immunassay. Prc. Natl. Acad. Sci. USA 67, (170) 2. Hrwitz, D.L., Starr, 3. L., Mak, M.E., Blackard, W.G., Rubenstein, A.H.: Prinsulin, insulin, and C-peptide cncentratins in human prtal and peripheral bld. J. Clin. Invest. 55, (175) 3. Heding, L.G.: Radiimmunlgical determinatin f human C-peptide in serum. Diabetlgia 11, (175) 4. Beischer, W., Heinze, E., Keller, L., Raptis, S., Kernel W., Pfeiffer, E.F.: Human C-peptide. Part II: Clinical studies. Klin. Wchenschr. 54, (176) 5. Ludvigssn, J., Heding, L G.: C-peptide in children with juvenile diabetes. Diabetlgia 12, (176) 6. Faber, O.K., Binder, C.: B-cell functin and bld glucse cntrl in insulin dependent diabetics within the first mnth f insulin treatment. Diabetlgia 13, (177) 7. Faber, O.K., Binder, C.: Plasma C-peptide during the first year f insulin dependent diabetes mellitus. Prceedings f the th IDF Cngress 177, Excerpta Medica (in press) 8. Faber, O.K., Binder, C.: C-peptide respnse t glucagn: A test fr the residual B-ceU functin in diabetes meuitus. Diabetes 26, (177). Natvig, H.: Nye Hide - vekttabeuer fr nrske kvinner g menn. Osl: Landsfreningen fr ksthld g helse Faber, O.K., Markussen, J., Naithani, V.K., Binder, C.: Prductin f antisera t synthetic benzylxycarbnyl-c-peptide f human prinsulin. Hppe Seylers Z. Physil. Chem. 357, (176) 11. Grajwer, L.A., Pildes~ R.S., Hrwitz, D.L., Rubenstein, A.H.: Cntrl f juvenile diabetes meuitus and its relatinship t endgenus insulin secretin as measured by C-peptide immunreactivity. L Pediatr. 0, (177) 12. Regeur, L., Binder, C.: The crrelatin between plasma C- peptide and kidney functin. Diabetlgia 12, 416 (176) Recetved." Aprd 20, 177, and in revised frm: July 1, 177 Dr. O. K. Faber Hvidre Hspital Emiliekildevej 1 DK-230 Klampenbrg Denmark