Diabetologia 9 Springer-Verlag 1985

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Diabetlgia (1985) 28:330-334 Diabetlgia 9 Springer-Verlag 1985 The influence f insulin antibdy levels n the plasma prfiles and actin f subcutaneusly injected human and bvine shrt acting insulins A. J.

1 Diabetlgia (1985) 28: Diabetlgia 9 Springer-Verlag 1985 The influence f insulin antibdy levels n the plasma prfiles and actin f subcutaneusly injected human and bvine shrt acting insulins A. J. Francis, I. Hanning and K. G. M. M. Alberti Department f Medicine, The Medical Schl, Newcastle upn Tyne, UK Summary. The influence f mderate and lw insulin antibdy levels n insulin absrptin and plasma free insulin prfiles is uncertain. Tw grups f six C-peptide negative diabetic patients, ne with lw ( g/l) and ne with mderate ( ~tg/1) serum insulin antibdy levels, were studied. Subjects were given 0.3 U/kg neutral human r acid bvine sluble insulin subcutaneusly in randm rder befre breakfast n separate days. Mderate antibdy levels significantly blunted the rise in plasma free insulin that fllwed injectin f the human and bvine insulins (p < 0.05). The rise in bld glucse after breakfast was significantly greater in patients with mderate antibdy levels (p < 0.05) and mre marked fllwing the bvine than the human insulin (p < 0.05). Plasma free insulin, bld glucse and 3-hydrxybutyrate prfiles suggest that acid bvine sluble insulin has a significantly mre prtracted actin than neutral human insulin. Keywrds: Type 1 (insulin-dependent) diabetes mellitus, insulin antibdies, insulin absrptin. Insulin binding antibdies at high titre may cause insulin resistance [1, 2]. They may als prlng the actin f injected insulin but such an effect has nly been cnvincingly demnstrated during artificially induced severe insulin deficiency [3-5]. It is nt knwn whether the mre mderate levels f insulin antibdies fund in the majrity f insulin treated diabetic patients significantly affect the pharmackinetics f insulin therapy. This culd be f particular imprtance when attempting t mimic meal-time insulin secretry patterns. We have therefre examined the effect f mderate levels f insulin antibdies n the absrptin and actin f human and bvine sluble insulins. Subjects and methds Subjects Tw grups f six Type 1 (insulin-dependent) diabetic patients were studied: ne grup had serum insulin binding capacity fr prcine and bvine insulins belw 10 Ix/l, whilst the secnd grup had binding abve 10 Ix/1 (Table 1). The grups were matched fr age, duratin f diabetes, insulin dse and bdy mass index (kg/m2). Infrmed cnsent was given by patients and the study was apprved by the Newcastle upn Tyne Health Authrity Ethical Cmmittee. Prtcl T deplete residual subcutaneus insulin depts, patients were treated with shrt acting insulin alne fr 24 h befre the study, the last dse being given at hurs. At hurs n the day f study, patients were given either 0.3 U/kg highly purified human neutral sluble insulin (Human Actrapid, Nv Industri, Bagsvaerd, Denmark) r bvine acid sluble (Wellcme Medical Divisin, Crewe, UK) in, randm rder. The study was repeated with the ther insulin preparatin apprximately I week later. All injectins were given int the anterir abdminal wall 10 cm lateral t the umbilicus using a 12.7 mm needle inserted vertically t the hilt by ne physician. Patients ate their nrmal breakfast and lunch at and hurs respectively (Table 1), and remained at rest in a chair thrughut the study with the injectin site cvered by their nrmal indr clthing. Befre the study a plyethylene cannula was inserted int a frearm vein and its patency maintained by flushing with physilgical saline. Tw baseline bld samples were drawn, and bld taken every 15 min fr 2 h, and every 30 rain until 8 h. Bld fr estimatin f glucse, lactate, pyruvate, glycerl, alanine and 3-hydrxybutyrate was deprteinised immediately in perchlric acid (0.5 ml/1) and assayed by flurimetric techniques [6]. Plasma fr measurement f free insulin was separated by centrifugatin fr 2 min and antibdies precipitated immediately with plyethylene glycl [7]. Extracts were stred at -20 ~ and measured by radiimmunassay [8]. Human insulin was used as standard, and crss reactivity with bvine insulin was 100%. A serum sample was drawn n the first day f study fr measurement f insulin binding capactiy by an immunchemical methd [9], using human, prcine and bvine insulin as ligands.

2 A. J. Francis et al.: Insulin antibdies and subcutaneus insulin absrptin 331 Table l. Clinical characteristics f patients studied Case Sex Age Duratin Bdy mass Diet during study Usual Usual dse Serum insulin binding capacity n (years) diabetes index (g carbhydrate) insulin (p.g/1) (years) (kg/m 2) Breakfast Lunch Type Mrning Evening Bvine Prcine Human Grup 1 1 M SOL ISO F ACT INS F ACT MONO M ACT MONO M SOL ISO F ACT MONO Mean_ SEM _ _ _ _+0.9 Grup 2 7 M SOL ISO F SOL ISO M SOL ISO M ACT MONO M ACT MONO M SOL ISO Mean_+SEM _ All patients had undetectable serum C-peptide levels (< 0.02 nml/1) 90 rain fllwing breakfast and nrmal plasma creatinine cncentratins. SOL = Bvine acid sluble insulin purified by recrystauizatin nly; ISO = bvine isphane insulin; ACT = prcine neutral sluble insulin, highly purified; MONO = prcine lente insulin, highly purified Statistical analysis Results are expressed as mean + SEM. The areas under the free insulin and metablic curves were calculated trapezidally. Analysis was by Students paired r unpaired t-test r by Spearman's rank crrelatin unless therwise stated. T avid errrs due t cmparisn f repeated measures ver time, statistical tests were perfrmed n areas under the free insulin r metablite curves. Where a significant difference was fund differences at individual time pints have als been shwn in the figures. A cmputerised curve fitting prgram was used t describe the mean plasma free insulin prfiles. A satisfactry fit was btained using ne rising and ne falling expnential, with a lag phase befre insulin was detectable [10]. Results Insufin antibdy cncentratins Serum insulin binding capacities fr each patient are shwn in Table 1. Binding capacities were almst identical fr each f the three insulin species tested except in patient 12 wh had the highest binding capacity and whse serum shwed greater affinity fr bvine than prcine r human insulins. Effect f insulin antibdy cncentratin Mderate levels f insulin antibdies blunted the rise in plasma free insulin levels fllwing subcutaneus injectin f bth neutral human and acid bvine insulins (Fig. 1). The areas under the plasma free insulin curves were significantly greater (p < 0.05) in patients with lw antibdies fr the first and secnd hurs after human insulin and the first hur after bvine insulin. There was a significant negative crrelatin between the insulin antibdy level f each patient and the area under the plasma free insulin curve in the first hur after injectin (r = Spearman's rs = , p < 0.05) (Fig. 2). As intermediate-acting insulin had been withheld the previus evening patients were insulin deficient and bld glucse levels were high at the beginning f the study (12.1 _+ 1.3 mml/1 mderate antibdy grup, 18.7 _+ 1.4 lw antibdy grup). Hwever, the rise in bld glucse after breakfast was greater in the grup with mderate antibdy levels than in the grup with' lw antibdy levels (Fig.3), the incremental area under the bld glucse curve being significantly greater (p < 0.05) in the first hur after injectin f bvine insulin.

3 332 A.J. Francis et el.: Insulin antibdies and subcutaneus insulin absrptin 100 O Plasma free insulin (muff) 50 L$ Bld glucse (mml/i) -5 HUMAN T --10 lo0 Plasma free insulin 50 (muff) e Bld g~uese (mml/i) ~< 5 ~_~ T BOVINE,, ; 0 ~lnsulin -~-~ I I I I J J I I J Fig.l. Plasma free insulin prfiles after subcutaneus injectin f 0.3 U/kg sluble insulin in C-peptide deficient patients with lw (< 10 ~xg/1) (0---0) and mderate (9 9 insulin antibdy levels. Results fr human and bvine insulins are shwn in the upper and lwer panels respectively -10 ~ Insulin.L",,~ J- Brekfa l'uh I, I J J, J i J Fig.3. Bld glucse prfiles after subcutaneus injectin f 0.3 U/ kg sluble insulin in patients with lw (< I 0 ~g/1) ( H ) and mderate [ O) insulin antibdy levels (Fig.3), as was the rate f rise in 3-hydrxybutyrate which ccurred tward the end f the study as free insulin levels declined (Fig. 4). Area under free insulin curve 0-1h (mu h) I I I I e Insulin antibdies (tag/i) Fig.2. Relatinship between insulin binding capacity and calculated area under free insulin curve in the first hur after injectin. Results fr human (O) and bvine (O) insulin are shwn separately (r = , rs (Spearman) = , p < 0.05) Free insulin prfiles were similar in the lw and mderate antibdy grups frm 2 h after injectin until the end f the study (Fig. 1). Hwever the cmputer calculated absrptin rate suggest a slightly lnger half life in patients with mderate antibdy levels (87 versus 75 min). The rise in bld glucse cncentratin fllwing lunch was similar in the grups with lw and mderate antibdies during treatment with either insulin Effect f insulin species and preparatin The rise in plasma free insulin cncentratin frm baseline was mre rapid with neutral human than with acid bvine insulin but this was nt statistically significant. Hwever the rise in bld glucse in respnse t breakfast was significantly less fllwing the human insulin than the bvine insulin (p < 0.05 in the first hur after injectin). Plasma free insulin levels were sustained at higher levels fllwing injectin f acid bvine insulin than neutral human insulin. The area under the plasma free insulin curve was significantly greater 4-8 h after injectin f the bvine insulin than after the human insulin (p < 0.01). The calculated absrptin rate, nce steady state had been established, crrespnded t a half life f 62 min fr human and 92 rain fr bvine insulin. Bld glucse levels crrespndingly rse less after lunch with the bvine than with the human insulin (p<0.05). Bld 3-hydrxybutyrate levels remained significantly lwer between 5 and 8 h after acid bvine insulin injectin than after neutral human insulin (p < 0.05). There was n significant difference in bld lactate, pyruvate, glycerl r alanine prfiles.

A. J. Francis et al.: Insulin antibdies and subcutaneus insulin absrptin 333 0.8 Bld 3- hydrxybuwrate (mml/i) 0.4 O.B Bld 3- hydrxybutyrate {mml/i) 0.4 HUMAN ~.

4 A. J. Francis et al.: Insulin antibdies and subcutaneus insulin absrptin Bld 3- hydrxybuwrate (mml/i) 0.4 O.B Bld 3- hydrxybutyrate {mml/i) 0.4 HUMAN ~. BOVINE ~ Insulin L- ~ I, 1 ~ I, 1 O Fig.4. 3-hydrxybutyrate prfiles after subcutaneus injectin f 0.3 U/Kg sluble insulin in patients with lw (<10 9g/l) (O---O) and mderate (O O) insulin antibdy levels Discussin The results suggest that mderate levels f circulating insulin binding antibdies are assciated with a delay in the rise f plasma insulin cncentratins and the nset f actin f bth neutral human and acid bvine insulins (Figs. 1-3). Frm this study it is nt pssible t determine whether this is due t delay in the nset f absrptin frm the subcutaneus tissue r t the binding f insulin by antibdies after absrptin int the circulatin. Hwever assuming that extracellular fluid cmprises 20% f bdy weight, and that lgg cncentratin in the extravascular cmpnent is abut 60% f that in serum, ne may calculate that in ur patients with mderate antibdy levels the ttal antibdy pl culd, under maximal cnditins, bind 3-8 units f insulin and thus culd exert cnsiderable influence n the dynamics f insulin actin. In rder t mimic the nrmal meal-time insulin secretry pattern, the pre-prandial insulin injectin shuld have a rapid nset f actin, and insulin activity shuld return t base line in the pst-absrptive state. In this study we fund that differences in bld glucse fllwing breakfast persisted well beynd the bserved difference in free insulin levels. This suggests that the timing f the nset f insulin actin t meals is f critical imprtance. In this respect the chice f injectin site is imprtant as absrptin frm the abdminal wall, the site chsen fr this study, is mre rapid than frm the arm r leg [11, 12]. In additin administratin f insulin thirty minutes befre breakfast may significantly reduce the pst-prandial peak in bld glucse [13]. By adjusting the time f the pre-prandial insulin injectin it may be pssible t minimise the small difference in the nset f actin we have fund between the human and bvine insulin preparatins. It is unlikely, hwever, t cmpensate fr the mre prlnged blunting f absrptin seen in ur patients with mderate antibdy levels, as peak plasma free insulin levels were nt achieved until 2 h after injectin (Fig. 1). Several studies [3-5] have suggested that insulin antibdies may prlng insulin actin, with speculatin that they may act as a buffer gradually releasing 'free' insulin t the circulatin. We did nt find that mderate levels f antibdies, when cmpared with lw levels, significantly prlnged the actin f human r bvine insulin ver the eight hurs f study. This discrepancy is mst likely explained by the differing cnditins f study. Whilst we examined the absrptin f subcutaneusly injected sluble insulin, the earlier studies [3-5] examined the rle f antibdies during acute insulin deprivatin induced by cessatin f intravenus insulin infusin. If antibdies d play a useful rle in maintaining backgrund insulin levels this may be f greatest imprtance in the latter part f the night when free insulin levels are in decline with cnsequent deteriratin in metablic cntrl [14]. In ur study, when n intermediate acting insulin had been given the previus day, this effect was evident. The grup f patients with mderate antibdy levels had significantly lwer fasting levels f 3-hydrxybutyrate (Fig.4) and bld glucse ( versus mml/l, p < 0.05). This finding shuld hwever be interpreted with cautin. The study was designed t assess the effect f antibdies n the meal-time actin f shrt-acting insulin and it wuld be inapprpriate t extraplate these findings t vernight cntrl with lnger acting insulin preparatins. In additin mre patients (4 f 6) in the mderate antibdy grup had received acid bvine insulin the evening befre study, and we have shwn that the bvine sluble insulin had a mre prtracted actin irrespective f antibdy levels. Mderate levels f insulin binding antibdies thus markedly blunt the nset f actin f shrt acting insulins. We did nt find that insulin antibdies significantly extended the actin f subcutaneus injected shrt acting insulin but as the study was nt designed t assess ttal duratin f actin such an effect may have been missed. Acknwledgements. We are grateful t Nv Labratries Ltd and the British Diabetic Assciatin fr financial supprt. The kind help f the staff f Ward 2, Newcastle General Hspital is gratefully acknwledged.

334 A.J. Francis et al.: Insulin antibdies and subcutaneus insulin absrptin References 1. Andersn OO, Egeberg J (1977) The clinical significance f insulin antibdies. Acta Paed Scand 270:63-68 2.

5 334 A.J. Francis et al.: Insulin antibdies and subcutaneus insulin absrptin References 1. Andersn OO, Egeberg J (1977) The clinical significance f insulin antibdies. Acta Paed Scand 270: Reeves WG, Allen BR, Tattersall RB (1980) Insulin-induced lipatrphy: evidence fr an immune pathgenesis. Br Med J 280: Madsbad S, Alberti KGMM, Binder C, Burrin JM, Faber OK, Krakup T, Regeur L (1979) Rle f residual insulin secretin in prtecting against ketacidsis in insulin - dependent diabetics. Br Med J 2: Vaughan NJA, Matthews JA, Kurtz AB, Nabarr JDN (1983) The biavailability f circulating antibdy-bund insulin fllwing insulin withdrawal in Type 1 (insulin-dependent) diabetes. Diabetlgia 24: Gray RS, Cwan P, Di Mari U, Clarke BF, Duncan LJP (1983) The influence f insulin binding antibdies n bvine and bisynthetic human insulin actin in Type 1 diabetes. Diabetlgia 25: 158 (Abstract) 6. Llyd B, Burrin JM, Smyth P, Alberti KGMM (1978) Enzymatic flurimetric cntinuus-flw assays fr bld glucse, lactate, pyruvate, alanine, glycerl and 3-hydrxybutyrate. Clin Chem 24: Nakagawa S, Nakayamah H, Sassaki T, Yshin K, Yune YY, Shinzaki K, Aki S, Mashim K (1973) A simple methd fr the determinatin f free insulin levels in insulin treated patients. Diabetes 22: Seldner J, Slne D (1965) Critical variables in the radiimmunassay f serum insulin using the duble antibdy technique. Diabetes 14: Reeves WG, Kelly U (1980) An immunchemical methd fr the quantitatin f insulin antibdies. J Immunlgical Meth 34: Hme PD, Owens DR, Blain PG, Alberti KGMM (1982) A new lk at the pharmackinetics f subcutaneus injected human and prcine insulin. Diabetes 31: 58A 11. Kivist VA, Felig P (1980) Alteratins in insulin absrptin and in bld glucse assciated with varying insulin injectin sites in diabetic patients. Ann Internal Med 92: Berger M, Ciippers HJ, Hegner H, J6rgens V, Berchtld P (1982) Absrptin kinetics and bilgic effects f subcutaneusly injected insulin preparatins. Diabetes Care 5: Kinmuth AL, Baum JD (1980) Timing f pre-breakfast insulin injectin and pst-prandial metablic cntrl in diabetic children. Br Med J 280: Francis AJ, Hme PD, Hanning I, Alberti KGMM, Tunbridge WMG (1983) Intermediate acting insulin at bedtime: effect n bld glucse cncentratins befre and after breakfast. Br Med J 286: Received: 17 December 1984 and in revised frm: 8 May 1985 Dr. A.J. Francis Department f Medicine The Medical Schl Framlingtn Place Newcastle upn Tyne NE2 4HH UK

by Springer-Verlag 1977

by Springer-Verlag 1977 Diabetlgia 13, 615-61 (177) Diabetlgia @ 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,