Diabetologia 9 Springer-Verlag 1984

Transcription

1 Diabetlgia (1984) 26: Diabetlgia 9 Springer-Verlag 1984 Ketne bdies as markers fr Type 1 (insulin-dependent) diabetes and their value in the mnitring f diabetic cntrl Y. Haran, K. Ksugi, T. Hysu, M. Suzuki, H. Hidaka, A. Kashiwagi, S. Un I and Y. Shigeta The Third Department f Medicine, Shiga University f Medical Science, Ohtsu, Shiga, and ~ Sanwa Kagaku Kenkynsh, Nagya, Japan Summary. Serum levels f acetacetate, 3-hydrxybutyrate and the 3-hydrxybutyrate/acetacetate rati were determined in Type 1 (insulin-dependent) and Type 2 (nn-insulindependent) diabetic patients by a new sensitive methd. ffrts were made t differentiate Type 1 and Type 2 diabetes by serum levels f ketne bdies and t determine whether their measurement is a useful way f mnitring diabetic cntrl. In Type 2 diabetes, serum levels f ttal ketne bdies did nt exceed 2. mml/l even if the patients were untreated r prly cntrlled. In Type 1 diabetic subjects, treated with nce r twice daily injectins f insulin, mrning serum levels f acetacetate, 3-hydrxybutyrate and ttal ketne bdies were significantly elevated by fur-, ten- and sevenfld, respectively. In Type 2 diabetic subjects treated with diet r sulphnylu- reas, serum levels f 3-hydrxybutyrate were highest befre breakfast, next highest befre dinner and decreased after each meal. The changes were rughly inversely prprtinal t serum insulin levels. In additin, insulin treatment nrmalized fasting serum levels f ketne bdies better than diet r sulphnylurea treatment. Acetacetate was als significantly increased in bth types f diabetes t a lesser extent, but n apparent diurnal rhythm was bserved. Determinatin f serum levels f ketne bdies is useful fr the diagnsis f Type 1 diabetes (thse with ttal ketne bdies > 2 mml/1) and fr detecting insufficient insulin therapy. Key wrds: Ketne bdies, acetacetate, 3-hydrxybutyrate, Types 1 and 2 diabetes, ketsis. Prneness t ketsis is an imprtant measure fr differentiating Type 1 (insulin-dependent) and Type 2 (nninsulin-dependent) diabetes [1, 2]. Rutine tests fr ketne bdies (nitrprusside reactin) nly detect acetacetate (AcAc), and acetne t a lesser degree, but nt 3-hydrxybutyric acid (3-OHBA), the majr ketne bdy which is increased in prly cntrlled diabetes [31. Ketne bdies are increased als in the sera f healthy diabetic subjects [4, 5]. Therefre, quantificatin f serum levels may be clinically imprtant fr assessing cntrl in bth types f diabetes. We have develped a sensitive and simplified methd fr the determinatin f AcAc and 3-OHBA. With this methd, we have tried t assess whether quantificatin f serum levels f ketne bdies is useful (a) t differentiate Type 1 frm Type 2 diabetes, and (b) t mnitr diabetic cntrl. Subjects and methds Subjects The 91 nrmal subjects were aged between 18 and 65 years. The diabetic subjects were classified int Type 1 and Type 2 diabetes accrd- ing t the Natinal Diabetes Data Grup (t) and WHO xpert Cmmitee [2]. Overnight fasting bld was btained frm 117 treated Type t diabetic subjects, aged 7-2 years. All had urinary C-peptide reactivity excretin < 1 ag/day. Haemglbin A1 levels were elevated (mean+sm, %, n=53; upper limit f ur nrmal range: 8%). All had been treated with intermediate-type insulin (mean + SM, U) befre breakfast. ighteen received additinal intermediate-type insulin ( U) befre dinner. Twentytw subjects received shrt-acting insulin (6.7_+.9 U) cncmitant with the mrning intermediate-type insulin. Furteen adult-nset Type I diabetic patients, including fur with ketacidsis, were als included t study the relatinship between fasting plasma glucse and serum ketne bdies levels. The ther t subjects were classified as having Type 1 diabetes n the basis f ketsis-prneness, episde f ketsis and rapid prgressin t insulin dependency. At least 26 Type 2 diabetic subjects treated r untreated (28 subjects) were studied. They had a urinary C-peptide reactivity excretin> 2 Ixg/day (up t a maximum f 8 ~tg/day). One-third had nrmal glycsylated haemglbin (HbA levels ( %, mean SM, n = 91). Thse wh received insulin fr diabetic cntrl had previusly been n diet r sulphnylurea treatment. They were classified int thse n diet, sulphnylureas and insulin, and further divided accrding t the degree f fasting hyperglycaemia. In mst subjects, ketne bdies were determined repeatedly: using a representative stabilized value after 2-4 weeks treatment. Only ne value was used fr each subject except in severe and untreated subjects whse initial values were als used. Newly diagnsed untreated subjects were analysed with the diet grup, since their ttal calrie intake per day and fd cmpsitin were similar. The Type 2 diabetic subjects in this study were mstly

2 344 Y. Haran et al.: Determinatin f ketne bdies in diabetic subjects Table 1. Serum ketne bdies levels in juvenile Type 1 diabetic subjects accrding t fasting plasma glucse levels Fasting N. Acet- 3-Hydrxy- Ttal 3-OHBA/AcAc plasma f acetate butyrate (Ixml/1) rati glucse subject (ttml/1) (~tml/1) (mml/1) Nrmal subjects _ Type 1 diabetic subjects < "q- 11 b b 295 _+ 39 a 1.6 _+.2 a b b b a _t3 b b 465_+53 b > _13 b b b 2.2 b Ttal _ 7 b b b 2. -I"-.1 b Results expressed as mean + SM a p <.2, b p <.1 versus nrmal subjects nn-bese (mean_+sm percentage ideal weight, 11_+.2%). Thse wh had been admitted t hspital with ketacidsis were excluded. Determinatin f ketne bdies Bld was drawn befre breakfast unless therwise stated. N difference in ketne bdy cncentratin was nted fr either heparinized plasma r serum. Ketne bdies were stable fr a few hurs at rm temperature while the bld cltted. Ketne bdies were als unchanged when stred fr 12 h as whle bld in a refrigeratr (4 ~ Usually ketne bdies were determined n the day f sampling. In sme samples, they were determined within 1 week after strage at - 7 ~ (acetacetate was fund t be stable fr up t 2-3 weeks at -7 ~ 3-Hydrxybutyrate did nt change during strage fr this perid even at - 2 ~ Acetacetate was determined by a newly-devised technique [6], using a mdificatin f Salway's methd [7]. In the present methd, p- nitrphenyl-diaznium flurbrate, which is mre stable than the riginal diaznium salt, was used. In additin, the reactin prduct was mre stable and quantitative. 3-OHBA cncentratin was calculated frm the difference between the ttal ketne bdy and AcAc levels. Ttal cncentratin was determined in sera after enzymatic cnversin f 3-OHBA t AcAc. The mrlar extinctin cefficient f azcmpund at 645nm is , which is five times greater than NADH. A gd crrelatin was nted between the present and the enzymatic methd (r = , n = 2). Glucse [8], insulin [9], glucagn [1], grwth hrmne, and lipid levels were determined as described previusly [11]. HbAa was determined by the methd f Trivelli et al. [12] and urinary C-peptide reactivity was determined as described by Kuzuya et al. [13]. Chemicals and reagents Lithium acetacetate was btained frm Sigma, St. Luis, Missuri, USA. p-nitrphenyl diaznium flurbrate was synthesized [14], but is nw available frm Sanwa Kagaku Kenkyush, Nagya, Japan. 3-Hydrxybutyrate was purified frm pseudmnas lemignei with specific activity ver 4 U/mg prtein, which is als available frm Sanwa Kagaku Kenkyush. Statistical methds The Student's t-test was used t analyse the significance f the difference. Paired t-test was used t evaluate pst-prandial changes f bld glucse, serum insulin and ketne bdies (Fig. 4). Crrelatin cefficient (r) and prbability f rejectin f null hypthesis (p) are shwn. quatin f linear regressin was als calculated fr the relatinships between bld glucse and ketne bdies levels. Results The serum levels f ketne bdies in juvenile Type 1 diabetic patients, accrding t fasting plasma glucse levels are shwn in Table 1. The Type 1 diabetic subjects were split int fur grups accrding t fasting plasma glucse levels. Serum levels f AcAc, 3-OHBA and ttal ketne bdies were all elevated by five-, furteen- and ninefld in the diabetic subjects with fasting plasma glucse >11.1 mml/1 (Table 1). The degree f elevatin, especially fr 3-OHBA, was rughly prprtinal t the fasting plasma glucse levels, and therefre the 3-OHBA/AcAc rati was significantly increased (p <.1). ven in thse patients with fasting plasma glucse ~< 5.6 mml/1, AcAc, 3-OHBA and ttal ketne bdies were increased by 2.7-, 5.4- and furfld, respectively. Thus, in spite f insulin treatment, mst f the Type I diabetic subjects exhibited ketnaemia. Relatinship between fasting plasma glucse and serum ttal ketne bdies levels in Type 1 diabetes A crrelatin was nted between fasting plasma glucse and serum levels f ttal ketne bdies (r =.47, p <.1, Fig. 1). Seven subjects, whse ttal ketne bdies were > 1.8 mml/1, were adult Type I diabetic subjects. Amng them, fur, whse ttal ketne bdies exceeded 3. mml/1, were untreated r withut insulin fr a few days and were ketacidtic n admissin. The 3-OH- BA/AcAc rati ranged frm 1.5 t 5.8 in these subjects. The majr increase f serum ketne bdies was attributable t 3-OHBA, with AcAc increased t a lesser degree. The remaining subjects were treated with insulin (Fig.l). In Type I diabetes, ketne bdies increased accrding t the degree f hyperglycaemia. Serum ketne bdies levels and 3-OHBA/AcAc rati in Type 2 diabetes in relatin t graded fasting hyperglycaemia In prly-cntrlled Type 2 patients, whse plasma glucse exceeded 11.1 mml/1, serum levels f AcAc,

3 Y. Haran eta].: Determinatin f ketne bdies in diabetic subjects (28,s5.5) A 35- R O 3- _= ,,~ 1. 5" ".t ".. "" r 2". " t_.o, :.'..$$ 9 g..~ 4#~.. %.~.",~ ~9 (3;,33.7) I! f I J'J' Serum levels f ttal ketne bdies (pml / I ) (765;,37.2) (168,34.9) Fig. 1. Relatinship between fasting plasma glucse and serum ttal ketne bdies levels in 131 Type 1 diabetic patients. The vertical line at 2 Fml/1 indicates the cut ff pint fr prneness t ketsis. Figures in parentheses indicate actual values f ttal ketne bdies and glucse. (Crrelatin cefficient, r =.47, p <.1, regressin equatin, Y= 4.X + 1.6) A B C D / 3. 6t 2. 4 i Nrmal Diet Sulphnyl Insulin Diet Sulphnyl Insulin Diet Sulphnyl Insulin Diet Sulphnyl Insulin Subjects ( 8 ) -urea ( 16 ) ( 22 ) -urea ( 25 ) ( 27 )-urea ( 18 ) ( 44 ) -urea ( 15 ) (91) (13) (32) (14) (4) Fig. 2. A-D Serum levels f individual ketne bdies in relatin t fasting plasma glucse levels in nrmal subjects and Type 2 diabetic patients treated with diet, sutphnylureas r insulin (numbers f patients given in parentheses). A Prly cntrlled diabetic patients (fasting plasma glucse > 11.1 mml/1); B fairly well cntrlled diabetic patients (11.1 _-> fasting plasma glucse > 7.8 mml/1); C well cntrlled diabetic patients (7.8 > fasting plasma glucse > 6.1 mml/1); D strictly cntrlled diabetic patients (fasting plasma glucse < 6.1 mml/1). Bars indicate mean + SM; r-q AcAc; ~ 3-OHBA; mm ttal ketne bdies; ~ 3-OHBA/AcAc rati. * p <.5; ** p <.1 versus nrmal subjects 3-OHBA and the 3-OHBA/AcAc rati were all significantly elevated by tw- t tenfld, whether n diet, sulphnylureas r insulin treatment (p <.1; Fig.2A). Since the elevatin f 3-OHBA was greater than the AcAc rise, the 3-OHBA/AcAc rati was increased. Mean fasting plasma glucse levels did nt differ except in the diet grup whse fasting plasma glucse was 2.8 mml/1 higher than the ther tw grups. In Type 2 diabetic subjects, whse plasma glucse ranged frm 7.8 t 11.1 mml/l, serum levels findivid- ual ketne bdies increased tw- t fivefld in thse n diet, sulphnylureas r insulin treatment (Fig. 2B). The rati was always greater than in cntrl subjects. In thse whse fasting plasma glucse was between 6.1 and 7.8 ram9 serum levels f AcAc increased twfld, and 3-OHBA by tw- t fivefld (Fig.2C). In strictly-cntrlled diabetic subjects (fasting plasma glucse ~<6.amml/1), nly individual serum ketne bdies were nrmal in the insulin-treated grup: this was nt the case in the sulphnylurea- r diet-treated

4 346 Y. Haran et al.: Determinatin f ketne bdies in diabetic subjects (9 u~ ~ r,, LL 2" 15' 1 5 % 8 ~ ~%% d~%~ ~. B O ~ c % ~ ~ ~ %~ I I I Fig. 3. The relatinship between fasting plasma glucse and serum ttal ketne bdies levels in 281 Type 2 diabetic patients with r withut treatment (r =.45, p <.1, Y =.7X + 6.4) Serum levels f ttal ketne bdies (juml / I ) A c 3-2O 1 3 A // '"',% 21 ~~T;,,.,' '" '""" """'-,,, "'"'"" / -15 I1) -lo _= 5 h- O 8; 1': 12.] 14; 16'.' 18': I" t' 4' Breakfast Lunch Dinner 3 B _~ "..Q 1"... ============================ 8: 1: 12: 14: 16: 18: f f 4 ~ Breakfast f Lunch Dinner Lente Insulin ( 16-38U) :_:] 2: 22~ 3 5 O. Fig.4 A and B. The diurnal changes f serum ketne bdies levels in Type2 diabetic patients n diet, sulphnylureas r insulin treatment. /x --- A : serum insulin; H : bld glucse; O ---O : 3-OHBA and : AcAc. Bars indicate mean + SM. A Type 2 diabetic subjects (n = 17) treated by diet r sulphnylureas, BType 2 diabetic subjects ( n = 19) treated with insulin. * p <.5, ** p <.25 versus pre-prandial value

5 Y. Haran et al.: Determinatin f ketne bdies in diabetic subjects 347 grups (Fig. 2D). Fasting plasma glucse levels were nt significantly different between the three grups. Insulin treatment seemed t be mst efficient in nrmalizing serum ketne bdies levels in Type 2 diabetes. Relatinship between fasting plasma glucse and serum ttal ketne bdies levels in Type 2 diabetic subjects with r withut treatment Serum levels f ttal ketne bdies were pltted against fasting plasma glucse levels in Type 2 diabetic subjects with r withut treatment (Fig. 3). A crrelatin was bserved between fasting glucse and serum levels f ttal ketne bdies (r =.45, p <.1 ; Fig. 3). ven in thse with untreated diabetes, serum levels f ttal ketne bdies were < 2. mml/1. Mild ketnaemia, bserved in subjects with plasma glucse < 1 mml/1, was mst prbably attributable t calrie restrictin. Diurnal changes f serum levels f ketne bdies in Type 2 diabetic subjects n diet, sulphnylureas r insulin treatment In diabetic subjects n diet r sulphnylurea treatment, nearly mirrr image diurnal changes were nted between serum levels f 3-OHBA and glucse r insulin. 3-OHBA levels were highest befre breakfast r dinner when bld glucse and insulin reached their nadir, and decreased significantly after meals (Fig. 4A). Diurnal changes f AcAc were nt bserved but were slightly high befre breakfast. In the insulin-treated subjects, 3-OHBA was highest befre breakfast with a gradual decrease in the evening (Fig. 4B). In this grup, the pstprandial reductin f ketne bdies was nt bserved in spite f hyperglycaemia after meals. This was thught t be attributable t lack f endgenus insulin secretin. Therefre, insulin seems t play a majr rle in the diurnal changes f serum 3-OHBA levels. Discussin Ketnaemia is ne f the cmmnest metablic abnrmalities in diabetes [4, 5]. In all Type 2 diabetic subjects studied here, ttal ketne bdies were belw 2. mml/1 even if the patients were untreated r prly cntrlled. We have nted a transient ketnaemia in Type 2 diabetes n an extremely lw calrie diet (8 calries/day). Therefre certain cnditins, such as extreme diet, exercise, stress r infectin, might cause ketnaemia. Otherwise, ketne bdies seem rarely t exceed 2. mml/1 in Type2 diabetic subjects. In Type 1 diabetes, even in thse wh were treated with insulin, serum ketne bdies were elevated several fld, and in decmpensated state up t 2 mml/1. Since the majr ketne bdy which increases in bld is 3-OHBA, rutine urine tests with the nitrprusside reactin are inadequate t evaluate ketnaemia. At a recent summer camp held in Osaka, ut f 47 juvenile Type I diabetic subjects, nly eight (5%) exhibited ketnuria, while serum 3-OHBA levels were elevated in 25 (>.2 mml/1; 53%). In additin, serum 3-OHBA was increased abve 2 mml/1 in fur subjects wh were nt ketnuric by rutine tests [15]. The degree f ketnaemia and the increased 3-OH- BA/AcAc rati were rughly parallel t fasting plasma glucse levels. A significant crrelatin was bserved between ttal ketne bdies and the 3-OHBA/AcAc rati (r =.5, p <.1, n = 2 fr Type 2, and r =.69, p <.1, n = 82 fr Type 1 diabetic subjects). The preferential elevatin f 3-OHBA in sera and the early reductin after insulin therapy have als been nted by ther investigatrs in a limited number f subjects [3, 5]. Hwever, the present study has demnstrated clearly the preferential elevatin f 3-OHBA with insulinpenia in the bth types f diabetes. Ketnaemia was mst prminent in the mrning fllwed by anther peak befre dinner. Pst-prandially, 3-OHBA decreased in thse wh had an endgenus insulin respnse, but nt in thse n insulin treatment (single dse f intermediate-type insulin). This indicates that hyperglycaemia per se cannt suppress ketnaemia. Insulin seems t be the mst imprtant factr fr the diurnal changes f 3-OHBA. In nrmal subjects, Wildenhff nted a peak rise f ketne bdies at midnight fllwed by a peak befre dinner r breakfast [t6]. In additin, grwth hrmne was nt attributed as a trigger fr the rise f ketne bdies [17]. In juvenile Type 1 diabetic patients, serum glucagn levels were previusly fund t be elevated befre breakfast [15]. Glucagn and crtisl may play a rle in accelerating mrning ketnaemia, especially fr 3-OHBA [15, 18]. A mrning rise f these insulin-antagnistic hrmnes may be attributable t insufficient levels f insulin in the early mrning. In Type 2 diabetes thse with fasting plasma glucse belw 6.1 mml/1 exhibited nrmal individual ketne bdies in the insulin-treated grup, but nt in the dietary r sulphnylurea-treated grups. This indicates that in subjects n diet r sulphnylureas, nrmglycaemia was attained at the expense f increased fat mbilizatin and xidatin. Insulin seems t be mst efficient in nrmalizing these metablic derangements. Serum ketne bdies levels, therefre, serve as a metablic marker t mnitr the derangement f fuel metablism, which is characterized by increased fatty acid xidatin. Dissciated respnses f serum glucse and ketne bdies after insulin treatment is mre prminent in Type I diabetes. ven in thse whse fasting plasma glucse fell belw 5.6 mml/1, a marked elevatin f ketne bdies was nted. When using a typical insulin regimen (single r twice daily injectin f intermediate insulin) in juvenile Type I diabetes, the mrning level f serum insulin seemed t be insufficient. In Type 2 diabetes, residual pancreatic B-cell functin may supply sufficient basal levels f insulin. High mrning levels f

6 348 Y. Haran et al.: Determinatin f ketne bdies in diabetic subjects ketne bdies may reflect mrning insulinpenia [19]. Therefre, determinatin f ketne bdies may be a useful metablic marker t estimate adequacy f insulin dses. Sme investigatrs have already used 3-OHBA, determined by the enzymatic methd as a metablic marker in Type 1 diabetes [18, 2]. Hwever, their nrmal ranges were higher than urs. Since haemglbin At was increased in mst f ur Type 1 diabetic subjects, residual metablic derangement may have cntributed t the bserved ketnaemia in nrmglycaemic subjects. Hepatic esterificatin f nn-esterified fatty acid may be diminished in these subjects due t insufficient insulin actin, cntributing t the verprductin f ketne bdies. Nn-esterified fatty acid may als serve as a metablic marker, but it fluctuates little (.2-2 mml/1) cmpared with ketne bdies (.5-2 mml/1). There was better crrelatin between nn-esterified fatty acid and ttal ketne bdies in Type 1 (r =.7, p <.1, n = 44) cmpared with Type2 diabetes (r=.33, p<.1, n = 221). Ketnaemia als reflects the ketgenic activity f the liver, which is accelerated by glucagn [21], with enhanced liplysis in adipse tissue. Pst-prandial serum levels f ketne bdies (2-3 h) were less remarkable in bth types f diabetes. Althugh values were still greater than nrmal, detectin f such lw levels f ketne bdies (< 1 lzml/1) requires a sensitive and accurate assay. Althugh it is best t determine ttal ketne bdies, 3-OHBA increases mst in diabetic subjects, and this may be useful as a rutine test. In additin, the 3-OHBA/AcAc rati may reflect altered tissue redx ptential and give sme insight int deranged tissue metablism. Further studies are necessary t elucidate the clinical implicatin f the increased 3-OHBA/AcAc rati. Determinatin f ketne bdies, especially befre breakfast, may serve as a metablic marker f insulinpenia and subtle derangement f fatty acid and intermediary metablism. Acknwledgements. We are grateful t Drs. M. Hshi f Kseinenkin Hspital, K. Izumi f Osaka Natinal Hspital and G. Ishiki f Osaka City University fr enabling us t perfrm the clinical study at the summer camp spnsred by the Osaka Diabetes Assciatin. References 1. Natinal Diabetes Data Grup (1979) Classificatin and diagnsis f diabetes mellitus and ther categries f glucse intlerance. Diabetes 28: WHO xpert Cmmittee n Diabetes Mellitus (198). Secnd reprt, Technical Reprt Series 646, Geneva 3. KeisbergRA (1978) Diabetic ketacidsis: new cncepts and trends in pathgenesis and treatment. Ann Int Med 88: WerkJr, Knwles HC (196) The bld ketne and plasma free fatty acids cncentratin in diabetic and nrmal subjects. Diabetes 1: MacGillivray MH, Li PK, Lee JT, Mills B J, Vrhess ML, Putman TI, Schaefer PA (1981) levated plasma fl-hydrxybutyrate cncentratins withut ketnuria in healthy insulin-dependent diabetic patients. J Clin ndcrinl Metab 52: Haran Y, Ksugi K, Hysu T, Un S, Iehikawa Y, Shigeta Y (1983) Sensitive and simplified methd fr the differential deterruinatin f serum levels f ketne bdies. Clinica Chim Acta 134: Salway JG (1969) The simultaneus determinatin f acetacetate and glucse in capillary bld. Clin Chim Acta 25: Hffman WS (1973) A rapid phtelectric methd fr the determinatin f glucse in bld and urine. J Bil Chem 12: Yalw RS, Glick SM, Rth J, Bersn SA (1965) Plasma insulin and grwth hrmne levels in besity and diabetes. Ann NY Acad Sci 131 : Imagawa K, Nishin T, Shin T, Uegata S, yanaihara C, Yanaihara N (1979) Prductin f anti-glucagn sera with a C-terminal fragment f pancreatic glucagn. ndcrinl Japn 26: Haran T, Ohgaku S, Ksugi K, Yasuda H, Nakan T, Kbayashi M, Hidaka H, Izumi K, Kashiwagi A, Shigeta Y (1893) Clinical significance f altered insulin sensitivity in diabetes mellitus assessed by glucse, insulin and smatstatin infusin. J Clin ndcrinl Metab 52: Trivelli LA, Ranney HM, Lai HT (1971) Hemgllin cmpnents in patients with diabetes mellitus. New ngl J Med 284: Kuzuya T, MastudaA, Sakamt Y (1978) C-peptide immunreactivity in urine. Diabetes 27 (Suppl 1): SterkeyB (1943) P-dinitrbenzene. Organic Synthesis. Cll 2: HaranY, KjimaH, Suzuki M, KashiwagiA, HysuT, Hidaka H, Hshi M, Izumi K, Shigata Y (1984) Serum levels f ketne bdies as metablic markers mnitring diabetic cntrl in Type 1 diabetic subjects. Med J Osaka Univ (in press) 16. Wildenhff K (1972) Diurnal variatins in the cncentratins f bld acetacetate, 3-hydrxybutyrate and glucse in nrmal persns. Acta Med Scand 191: WildenhffK, HhansenJP, KarstftH, YdeH, SrensenS (1974) Diurnal variatins in the cncentratins f bld acetacetate and 3-hydrxybutyrate. Acta Med Scand 195: MacGillivray MH, Vrhess ML, Putnam TI, Li PK, Schaefer PA, Bruck (1982) Hrmne and metablic prfiles in children and adlescent with Type I diabetes mellitus. Diabetes Care 5 (Suppl 1) Haran Y, Hidaka H, Suzuki M, Kjima H, Shigeta Y (1984) Imprtance f determining serum levels f 3-hydrxybutyrate as a metablic marker f insulinpenia. In: Sakamt N (ed) Secnd Japan-Krea sympsium n diabetes meuitus. xcerpta Medica, Amsterdam (in press) 2. Dahlquist G, Blm L, Blme P, Hagenfeldt L, Linggren F, Derssn B, Thalme B, Therell M, Westin S (1982) Metablic cntrl in 131 juvenile-nset diabetic patients as measured by HBAlc: relatin t age, duratin, C-peptide, insulin dse, and ne r tw insulin injectins. Diabetes Care 5: Haran Y, Ksugi K, Kashiwagi A, Akan T, Hidaka H, Sbigeta Y (1982) Regulatry mechanism f ketgenesis by glucagn and insulin in islated and cultured hepatcytes. J Bichem (Tky) 91 : Received: 14 Octber 1982 and in revised frm: 6 March 1984 Dr. Yutaka Haran Third Department f Medicine Shiga University f Medical Science Seta, Ohtsu Shiga Japan