Diabetologia 9 Springer-Verlag 1985

Transcription

1 Diabetlgia (1985) 28: Diabetlgia 9 Springer-Verlag 1985 C-peptide measurement in the differentiatin f Type 1 (insulin-dependent) and Type 2 (nn-insulin-dependent) diabetes mellitus H. L. Katzeff 1, P.J. Savage 2, B. Barclay-White,. Nagulesparan, and R H. Bennett pidemilgy and ield Studies Branch and Phenix Clinical Research Sectin, Natinal Institute f Arthritis, Diabetes and Digestive and Kidney Disease, Phenix, Arizna, USA Summary. T determine whether individual subjects with Type 1 (insulin-dependent) diabetes r Type 2 (nn-insulindependent) diabetes, wh are treated with insulin, culd be reliably distinguished, C-peptide cncentratins and urinary C- peptide excretin were measured in 10 Caucasids and 10 Pima Indians. All the subjects had develped diabetes befre 21 years f age and were receiving insulin treatment. asting C-peptide cncentratins were significantly higher in the Pima Indians ( versus nml/1 in Caucasids; p < 0.001), but there were slight verlaps in individual values. Urinary C-peptide excretin, an index f 24-h-insulin excretin, was als higher in the Pima Indian grup (27.6_ 1.85 versus pml/min in Caucasids; p <0.001) and there was n verlap in the individual values between the grups. The Pima Indians with early nset diabetes have been previusly shwn t have Type 2 diabetes, and the Caucasids with an early nset are mst likely t have Type I diabetes. These results suggest that distinctin between these tw majr types f diabetes can be made effectively by using C-peptide measurements prvided that vert renal disease is absent. This differentiatin between insulin-treated patients will be useful fr a variety f research applicatins and pssibly in making clinical management decisins. Keywrds: Type 1 diabetes, Type2 diabetes, plasma C-peptide, urinary C-peptide Pima Indian. Hetergeneity in diabetes mellitus is well recgnized [1, 2] as is reflected in the recent classificatin f the Natinal Diabetes Data Grup and Wrld Health Organizatin [3, 4]. any patients with Type 2 diabetes are treated with insulin t cntrl hyperglycaemia, and althugh they are nt prne t spntaneus ketsis, ketacidsis can ccur in Type 2 diabetes with severe intercurrent illness r ther stress. Cnversely, many patients with well cntrlled Type 1 diabetes, wh receive insulin therapy regularly, may never experience an episde f ketacidsis. Thus, in sme insulin-treated patients, unless insulin is withdrawn, it may nt be pssible t establish the type f diabetes by clinical histry alne. This is particularly true in adlescents and yung adults wh receive insulin treatment frm the time f diagnsis f diabetes, as well as in lder patients wh may have received insulin treatment fr many years. The present study was undertaken t determine whether it was pssible t discriminate between Types 1 and 2 diabetes amng subjects wh had insulin treatment frm the time f diagnsis f diabetes by examining plasma C-peptide cncentratins, fasting and in respnse t glucse and arginine, and 24 h-urinary C-peptide excretin. Present addresses: 1 Department f edicine, Crnell University edical Cllege, S ast 71 Street, New Yrk, New Yrk 10021, USA 2 Department f Internal edicine, Wayne State University, VA edical Center. Allen Park. ichigan. USA Subjects and methds Subjects Ten Caucasids and ten Pima Indians with the nset f diabetes befre age 21 years were admitted t the Clinical Research Sectin fr investigatin. The study was apprved by the Natinal Institutes f Health Clinical Research Cmmittee, and each subject gave written cnsent t participate in the study. The subjects were teenagers r yung adults, wh had been treated with insulin. They included a grup f Caucasids, wh were assumed almst certainly t have Type 1 diabetes, and Pima Indians wh, n the basis f previus evidence, were believed t have Type 2 diabetes [5]. The clinical characteristics f the individual subjects are given in Table 1. The Pima participants were either full-blded Pima Indians r a cmbinatin f Pima/Papag. The Papag tribe is clsely related t the Pima Indians and als has a high prevalence f Type 2 diabetes. Study design After a 12-h vernight fast, subjects were given their usual insulin dse and 30 min later received a 100 g carbhydrate lad (Kladex- Custm Labratries, Baltimre, aryland). Venus bld, fr determinatin f plasma glucse and C-peptide cncentratins, was drawn at -30, 0, 30, 60, 90, 120, and 180 min. On day 2 insulin was withheld and after an vernight fast, arginine mnhydrchlride (5.0 rag. kg-1. min-a) was infused intravenusly fr 40 rain. Venus bld fr measurement f glucse and C-peptide cncentratins was withdrawn frm the cntralateral frearm at- 20, - 10, 0, 5,10, 20, 30, and 40 rain. A 24-h urine cllectin fr measurement f C-peptide, creatinine and prtein was btained. Bld fr HLA typing and islet cell antibdies was als cllected.

2 H. A. Katzeff et al.: C-Peptide in Type 1 and Type 2 Diabetes 265 Table 1. Individual characteristics and C-peptide results f subjects studied Sex Age at Duratin Bdy mass Histry a HLA-B8 Islet Basal examinatin f diabetes Index f ket- antigen cell bld (years) (years) (kg/m 2) acidsis antibdies glucse (mml/l) C-peptide Basal plasma (nml/1) Urinary excretin (pml/min) Caucasids ean S NT NT NT NT Renal failure Pima Indians ean + S NT NT ~ c b ~ Renal failure d _ 1.85 d + = present; NT = Nt tested; a with bld ph ~< 7.2 r serum bicarbnate ~< 12 mml/1; b p < 0.05 ; c p < 0.01 ; d p < x_ 1,8 c 1,5 ~ 1.2 ~ c. D Z a ~ 54 ~ 45 c 36 ~~ ~ 2z - 0,3 9 ~ c1 9 c "7-- 9, -3 0,0 Caucasid Pima Caucasid Pima Subjects Indians b Subjects Indians ig. 1l. A and B Individual C-peptide levels in the nine Caucasid and nine Pima Indian subjects. The hrizntal bars represent the mean value f each grup. A asting plasma cncentratins; p<0.01. B Urinary excretin rates; p < G c -~ 50 ~ ~ 30,3 2 9 c 9----,, Duratin f dibetes (years) ig.2. Urinary C-peptide excretin rates in the nine Pima (O) and nine Caucasid (9 subjects accrding t duratin f diabetes 9 ethds Samples fr plasma glucse determinatins were cllected in tubes cntaining sdium fluride, stred at- 20 ~ and were analyzed in duplicate using the glucse xidase methd n a glucse analyzer (Beckman Instruments, Anaheim, Califrnia). Plasma fr C-peptide determinatins was stred at- 20 ~ and measured using a nn-equilibrium ethanl precipitatin radiimmunassay [6]. The sensitivity f this assay is 0.02 pml/ml with an intra- and interassay variatin f 5.8% and 9.6%, respectively. Urine fr C-peptide measurements was centrifuged at 2500 g fr 30 min t remve particulate atter and the ph adjusted t 7.0 with NaOH befre analysis [7]. HLA typing was perfrmed using standard micrlymphcyttxicity techniques fr A and B lci antigens nly [8]. Islet cell cytplasmic antibdies were measured by the methd f Bttazz et al. [9]. Statistical methds The fasting C-peptide cncentratins were taken as the mean f the - 30 and 0 rain samples fr each subject. The mean fasting plasma and urinary cncentratins f C-pepfide were cmpared using Student's t-test A isher exact test fr 2 x 2 tables was utilized t analyze the HLA and islet cell antibdy data and Pearsn crrelatin cefficients were used t examine relatinships between C-peptide cncentratins, excretin rates, besity and duratin f disease within each grup f subjects [10]. Analysis f variance with repeated measures was used t test fr differences frm baseline in respnses t ral glucse tlerance and arginine infusin tests [10]. Results The age f nset f diabetes was smewhat lwer and the duratin f diabetes slightly lnger in the Cauca- sids cmpared with the Pima Indians. All the Pima had at least ne parent with diabetes, whereas nne f the Caucasids had a parent r ther first degree rela-

3 266 H. A. Katzeff et al.: C-Peptide in Type 1 and Type 2 Diabetes 2O "" = 5 (..9 0 I I I I 1, ,-,4 d.20 jt..., 0-I co 120 I~0 Time (minutes) ig.3. ean + S bld glucse (tp panel) and plasma C-peptide (bttm panel) cncentratins befre and in respnse t a 100-g ral glucse lad in the Pima Indians (--(3--) and Caucasids ( ). ean plasma C-peptide cncentratins were higher than the basal level at 120 and 180 min (p <0.01) in the Pima Indians, but shwed n significant change in the Caucasids -6-8,3 if) m ,O , z---2 ARSlNiN NUSiON / I [ : -e e Time (minutes) ig.4. ean+s plasma C-peptide cncentratins befre and during an arginine infusin (5.0 mg. kg -1. min -1) in the Pima Indians (-- (3--) and the Caucasid (-- 9 subjects. A significant respnse ccurred in the Pima grup at 5 and 10min (p <0.01) tive with the disease. The Pima grup were mre bese than the Caucasids (p < 0.001), and their mean fasting plasma glucse levels were higher ( mml/1 in Pima and mml/1 in Caucasids). In bth grups, fur f the ten subjects had a histry f ketacidsis, althugh in the Pima the episdes had been assciated with a cmbinatin f prlnged insulin with- drawal (>/30 days) and alchl ingestin in three patients r sft tissue infectin in ne, indicating that they were nt insulin-dependent. Seven f the Caucasids were HLA-B8 psitive, whereas nne f the Pima had HLA-B8 (r BW15) (p <0.05 Table 1). ive f the six Caucasids had islet cell antibdies, whereas nne were fund in the Pima (p < 0.05). r analysis f C-peptide cncentratins and excretin, results were nt included frm ne Caucasid and ne Pima subject as they had evidence f nephrpathy with impaired creatinine clearance which has been shwn t suppress urinary C- peptide excretin [11, 12]. ean fasting plasma C-peptide levels were significantly different between the grups (Pima 0.73_ nml/1; Caucasids 0.02_ nml/1; p < 0.01), with nly ne subject in the Pima grup falling int the range bserved in the Caucasids, and vice versa (ig. 1 a). Twenty-fur hur urinary C-peptide excretin, hwever, shwed n verlapping values (ig. lb) (Pima range pml/min and Caucasids pml/min; mean_ S 27.6_+ 1.9 and pml/min, respectively; p < ). Bth fasting C-peptide and urinary C-peptide excretins were psitively crrelated with bdy mass index in the Pima Indians (r = 0.56 p < 0.05 and 0.77; p < 0.05, respectively), but nn-significant negative relatinships were fund in the Caucasids. The fasting plasma C-peptide levels shwed a crrelatin with the urinary C-peptide excretin (Pima r = 0.81 ; Caucasids 0.97; p < 0.01). Urinary excretin f C-peptide tended t be lwer with increasing duratin f diabetes in bth grups (Pimar = , p = 0.05; Caucasids- 0.58, p > 0.05; ig 2), but the levels in the Pima Indians were higher than in the Caucasid grup regardless f the duratin f diabetes. The bld glucse and C-peptide cncentratins, measured during an ral glucse tlerance test (ig. 3), shwed that while the fasting glucse levels were lwer amng the Caucasids than amng the Pima Indians, the glucse levels were similar during the secnd and third hurs f the test. The C-peptide cncentratins, hwever, remained lw and did nt change in respnse t the change in glucse cncentratin amng the Caucasids, whereas amng the Pima Indians there were increments in seven f the ten subjects, resulting in a significant increase in mean C-peptide cncentratin (p < 0.01). In respnse t arginine infusin (ig.4), mean C- peptide cncentratins in the Pima Indians were significantly higher 5 and 10 min after the start f infusin (p < 0.01), but did nt change in the Caucasids. Neither grup shwed a significant change in bld glucse in respnse t the arginine infusin. Discussin Determinatin f urinary excretin rates f C-peptide resulted in cmplete differentiatin between tw small grups f Caucasid and Pima Indian insulin-treated

4 H. A. Katzeff et al.: C-Peptide in Type 1 and Type 2 Diabetes 267 yung adult diabetic patients with Type 1 and Type 2 diabetes, respectively. easurement f the fasting serum C-peptide cncentratins als led t a substantial degree f discriminatin between the tw grups, but there was sme verlap; whereas with excretin rates the lwest level bserved in the Pima grup (8.2 pml/ min) was almst twice that f the highest seen in the Caucasid grup. Thus, fasting C-peptide cncentratins in sera, and t a greater extent urinary C-peptide measurements, appear useful t differentiate between Type 1 and Type 2 diabetes. The cnclusin that C-peptide excretin is useful in distinguishing between Type 1 and Type 2 diabetes depends n the likelihd that the Caucasids and Pima, respectively, d indeed have these different types f diabetes. The Causasids were selected because it was believed that the vast majrity f these patients, wh had n first degree relatives with the diabetes and an age f nset belw 20 years, n clinical grunds, wuld have Type 1 diabetes. In cntrast, amng the Pima Indians, even thse with diabetes with an early nset d nt appear t be insulin dependent [5]. The differences in the types f diabetes were reflected in this study in differences between the grups in degree f besity, the frequency f islet cell antibdy detectin, the frequency f a parental (r ther family) histry f diabetes, that the majrity f the Caucasids had an HLA phentype assciated with Type 1 diabetes and that in the Pima Indians ketacidsis had develped nly under stress and never spntaneusly. These findings agree with earlier studies indicating that the Pima Indians appear t have exclusively Type 2 diabetes as determined frm the absence f islet cell antibdies in thse f recent nset [13], and f their ther clinical characteristics at nset and the clinical curse f the disease [5]. That the diabetic subjects culd be partitined int tw separate grups lends additinal supprt t the cncept f different aetilgical entities in diabetes as defined in the Natinal Diabetes Data Grup and Wrld Health Organizatin classificatin [3, 4]. Prir t the availability f the C-peptide assay, bjective labratry definitin f these tw diseases in individual subjects was nt pssible in many wh had received insulin treatment, as residual insulin secretin r plasma insulin cncentratins culd nt be measured because f the presence f insulin antibdies. The determinatin f C-peptide cncentratins in urine and bld nw permits an accurate characterizatin f insulin secretin f many such patients [7, 12, 14] and facilitates the classificatin f insulin-treated subjects int the Type I and Type2 classes. This categrizatin is necessary fr studies f the genetics f the tw cnditins, as well as in ther research. Such measurements may als have clinical applicatins in determining whether r nt it is safe t withdraw insulin treatment, r in determining its necessity where distinctin is difficult n clinical grunds alne. The C-peptide results in the present study als emphasize the characteristic f insulinpenia as being pathgnmnic f Type I diabetes and add further t the evidence that diabetes in the Pima Indians, even when it has its nset at an early age, is nt characterized by insulinpenia and is f the nn-insulin-dependent type. T sme extent the differences in C-peptide cncentratins and excretin bserved between the Caucasids and Pima Indians in this study might be the result f differences in fasting bld glucse levels between the grups. The lwer fasting glucse levels in the Caucasids culd pssibly have resulted in a greater degree f suppressin f C-peptide secretin than in the Pima, in whm the levels f bth fasting glucse and C-peptide were higher. This is an imprbable explanatin fr the differences bserved, because when bld glucse levels during the ral glucse tlerance test in the tw grups became equivalent, and exceeded 10 mml/1, a cncentratin which appears t stimulate maximally C- peptide secretin [15], mean C-peptide levels became even higher in the Pima Indian grup, but remained lw and unchanged in the Caucasids. Likewise, in respnse t arginine infusin, n C-peptide respnse was seen amng the Caucasids, suggesting that the fasting level may have been reflecting maximum secretry capacity, rather than partial suppressin. Indeed, we may speculate that the greater discriminating pwer f the urinary measurement may reflect the extent t which C- peptide secretin was stimulated in Type 2 diabetes by fd intake, etc., during the cllectin perid, whereas in Type i diabetes the pancreas appeared t be refractry even t ral glucse and arginine stimulatin. The urinary C-peptide measurement appears t be a better measure than the fasting serum level fr making the distinctin between Type 1 and Type 2 diabetes as it reflects 24-'h insulin utput, except in renal failure when C-peptide excretin is reduced and serum levels may be increased [12]. Hwever, the cllectin f a 24-h urine sample can be difficult, particularly in epidemilgical studies. As there is a high crrelatin between the fasting C-peptide level and the 24-h excretin, in practice the majrity f patients can prbably be distinguished n the basis f the serum measurement alne, althugh as reprted previusly by Welbrn et al. [16], there will remain a minrity f diabetic patients where this is nt pssible. In these subjects urinary C-peptide excretin appears t be helpful, and while 24-h excretin was determined in this study, it has recently been suggested that a 4-h urine cllectin may be sufficient [17]. If renal failure is present, the fasting C-peptide level in bld may be elevated. If this is suspected the renal clearance f C-peptide can be determined, and if decreased, bth the serum and urinary cncentratins shuld be cnsidered unreliable as indices f insulin secretin. In the present study, C-peptide clearance was calculated amng the Pima Indian grup t exclude the pssibility that the higher fasting C-peptide levels culd be explained by diminished renal clearance. The results indicated that in all but ne subject, already excluded as she had renal failure n the basis f ther criteria, reduced clearance was nt respnsible fr the higher plas-

5 268 ma levels bserved. Similarly ne Caucasid was excluded because f evidence f renal failure. The use f glucse r arginine stimulatin befre C- peptide measurement did nt increase the ability t discriminate between Type 1 and Type 2 diabetes in this study althugh evidence f increased secretin in respnse t these stimuli did ccur in sme, but nt all f the Type 2 diabetic subjects. Althugh the sample size was small, and in the Caucasid grup the range f measurements narrw, there was a negative crrelatin between duratin f diabetes and C-peptide excretin in bth the Type 1 and Type 2 grups suggesting a fall in insulin secretin with increasing duratin (ig. 2). Decreases in fasting serum C- peptide levels and respnses t insulin secretggues with increasing duratin f diabetes have been reprted previusly [16, 18] and the urinary excretin data reprted here shw similar trends. These findings suggest that insulin utput falls with increasing disease duratin in bth Type 2 and Type 1 diabetes, althugh lngitudinal data are needed t validate the bservatin. In the present study, the levels f C-peptide excretin in Type 1 diabetes f less than 5 years duratin were nt measured, althugh ther reprts indicate that in Type 1 diabetes f recent nset urinary excretin f C-peptide may ften be belw 10 pml/min [19]. The levels seen in ur Type 2 diabetic patients with a duratin f 5 years r less were at least twice as high. Nevertheless, in the absence f data frm this study n Type I diabetes f less than five years duratin, the relative merits f fasting and urinary C-peptide excretin data t discriminate between Type 1 and Type 2 diabetes f shrt duratin cannt be determined. We cnclude that amng patients with early nset diabetes mellitus, wh have been treated with insulin, C-peptide measurements in fasting bld r in 24-h urine cllectins can be used t categrize subjects withut renal failure int insulin-dependent and nninsulin-dependent grups even after many years f diabetes. Applicatin f such measurements culd facilitate research int these diseases, and may smetimes help in selecting apprpriate clinical management and treatment. Acknwledgements. We wish t express ur thanks t Dr. G.. Bttazz, iddlesex Hspital, Lndn wh perfrmed the islet cell antibdy determinatins, Dr. A. H. Jhnsn f Duke University, Durham, Nrth Carlina wh did the HLA typing and rs. J. Baird fr secretarial assistance. We als wish t express ur gratitude t R Blix and Dr. A. H. Rubenstein, University f Chicag fr the C-peptide measurements and their helpful cmments n the manuscript. This wrk was supprted in part by NIH grant A t the Diabetes Research and Training Center, University f Chicag. References 1. ajans SS, lyd JC, Tattersall RB, Williamsn JR, Pek S, Taylr CI (1976) The varius faces f diabetes f the yung. Arch Int ed 136: H. A. Katzeff et al.: C-Peptide in Type I and Type 2 Diabetes 2. Rtter J, Rimin DL (1978) Hetergeneity in diabetes mellitusupdate; evidence fr further genetic hetergeneity within juvenilenset insulin-dependent diabetes mellitus. Diabetes 27: Natinal Diabetes Data Grup (1979) Classificatin and diagnsis f diabetes mellitus and ther categries f glucse intlerance. Diabetes 28: WHO xpert Cmmittee n Diabetes ellitus (1980) Secnd Reprt, Technical Reprt Series N. 640, Geneva 5. Savage PJ, Bennett PH, Senter RG, iller (1979) High prevalence f diabetes in yung Pima Indians. Diabetes 28: Kuzuya H, Blix P, Hrwitz DL, Steiner D, Rubenstein AH (1977) Determinatin f free and ttal insulin and C-peptide in insulin-treated diabetics. Diabetes 26: Hrwitz D, Rubenstein AH, Katz AI (1977) Quantitatin f human pancreatic beta-cell functin by immunassay f C-peptide in urine. Diabetes 26: Terasaki PI, Bernc A, Park D, Ozturke G, Iwaki Y (1978) icrdrp testing fr HLA-A, -B, -C, and -D antigens. Am J Clin Path 69: Bttazz G, lrin-christensen A, Dniach D (1974) Islet-cell antibdies in diabetes mellitus with autimmune plyendcrine deficiencies. Lancet 2: Snedecr GW, Cchran WC (1976) Statistical ethds, 6 ed, State University Press, Ames, Iwa 11. Kajinuma H, Tanabashi S, Ishiwata K, Kuzuya N (1979) Urinary excretin f C-peptide in relatin t renal functin. In: Baba S, Knek T, Yanihara N (eds) Pr-insulin, insulin, and C-peptide. xcerpta edica, Amsterdam-Oxfrd, pp Blix P, Bddie-Willis C, Landau RL, Rchman H, Rubenstein AH (1982) Urinary C-peptide: an indicatr f B-cell secretin under different metablic cnditins. J Clin ndcrinl etab 54: Knwler WC, Bennett PH, Bttazz G, Dniach D (1979) Islet cell antibdies and diabetes mellitus in Pima Indians. Diabetlgia 17: iestas T, Zadik A, arglis S, Kwarski AA (1981) Crrelatin f urinary excretin f C-peptide with the integrated cncentratin and secretin rate f insulin. Diabetes 30: Gerich J, Charles A, Grdsky G (1976) Regulatin f pancreatic insulin and glucagn secretin. Ann Rev Physil 38: Welbrn T, Garcia-Webb P, Busen A (1981) Basal C-peptide in the discriminatin f Type I frm Type II diabetes. Diabetes Care 4: Bantle JP, Laine DC, Hgwerf B J, Getz C (1984) The ptential usefulness f pstprandial urine C-peptide measurement in classifying diabetic patients. Diabetes Care 7: Hendriksn C, aber OK, Drejer J, Binder C (1977) The prevalence f residual beta-cell functin in insulin treated diabetics evaluated by the plasma C-peptide respnse t intravenus glucagn. Diabetlgia 13: Rappaprt B, Ulstrm RA, tzwiler DD, ife D, Hudlund B, Steffes W (1980) Urine C-peptide, B-cell functin, and insulin requirement. Am J Dis Child 134: Received: 9 August 1984 and in revised frm: 25 ebruary 1985 Dr. Peter HI Bennett Natinal Institute f Arthritis, Diabetes, and Digestive and Kidney Diseases 1550 ast Indian Schl Rad Phenix, Arizna USA