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Manuscript Post Print Page 5 of 28 Analytical & Bioanalytical Chemistry 1 2 3 IMMUNOASSAY QUANTIFICATION OF HUMAN INSULIN ADDED TO 4 TERNARY PARENTERAL NUTRITION CONTAINERS: COMPARISON OF TWO 5 METHODS 6 7 Authors: Hélose Henry 1,2 , Damien Lannoy 1,2 , Nicolas Simon 1,2 , David Seguy 3, , Mich"le 8 5 1 1,2 1 1,2 9 D#Herbome% , Christine (arthélémy , (ertrand Décaudin , Thierry Dine , Pascal Odou 10 11 12 1. -niv. Lille, .A 7305 1 23ITA 1 2roupe de 3echerche sur les formes In7ectables et les Technologies 13 Associées, 8159000 Lille, 8rance 14 2. Pharmacy Institute, CH- Lille, 8159000 Lille, 8rance 15 3. -niversity of Lille, -995 ; LI3IC ; Lille Inflammation 3esearch International Center, 8159000 Lille, 8rance 16 . -niversity Hospital of Lille, Department of Nutrition, 8159000 Lille, 8rance 5. Immunoanalysis center, (iology and Pathology Center, CH- Lille, 8159000 Lille, 8rance 17 18 19 For Peer Review 20 21 22 Corresponding author: 23 24 Dr. Damien Lannoy 25 26 8aculté des Sciences Pharmaceutiques et (iologiques, .A 7305 ; 23ITA Laboratoire de 27 28 29 (iopharmacie, Pharmacie 2alénique et Hospitali"re, 3, rue du Professeur Laguesse ; (.P. 83, 30 31 59000 Lille Cedex, 8rance. 32 33 Tel: ?33 3 20 90 0 29 34 35 8ax: ?33 (0A3 20 95 90 09 36 37 38 .1mail address: damien.lannoyBuniv1lille2.fr. 39 40 41 42 43 44 AcCnowledgments: the authors especially want to thanC Mrs Alexandra Tavernier for her 45 46 extensive revision of the final manuscript. 47 48 49 50 51 52 53 54 55 56 57 58 59 60 1 Analytical & Bioanalytical Chemistry Page 6 of 28 1 2 3 ABSTRACT 4 5 Background : Adding insulin directly into infusion bags seems to be a useful method for 6 7 controlling hyperglycemia in patients under ternary parenteral nutrition (TPNA. Its efficacy is 8 9 10 assessed by glycemic monitoring but few data are available on insulin stability in this 11 12 situation. Among the various methods for quantifying insulin levels in human serum, the 13 14 immunoassay ones seemed potentially appropriate for a TPN admixture containing high lipid 15 16 concentrations. Ee sought to identify and validate which of two immunoassay methods was 17 18 19 the better to quantifyFor human insulin Peer and consequent Reviewly be adapted to studying its stability in a 20 21 TPN admixture. 22 23 M thods : Two immunoassay methods to quantify recombinant human insulin were assessed 24 25 in industrial TPN: an immunoradiometric assay (I3MAA and an 26 27 28 immunoelectrochemiluminometric assay (I.CMAA. Falidation trials for both methods were 29 30 based on the accuracy profile method. 31 32 R sults : Interference with immunometric assays due to the high lipidic content of TPN was 33 34 eliminated through an improved preparation protocol using a bovine serum albumin ((SAA 35 36 diluted in phosphate buffer saline (P(SA. The relative total error of I.CMA varied from 1.7 37 38 39 to .52G while it varied from ; 0.32 to 8.37G with I3MA. Only I.CMA provided an 40 41 accuracy profile with a 95G confidence interval of calculated1tolerance limits falling between 42 43 the chosen acceptance limits (i.e. total error H10GA. 44 45 Conclusions : I.CMA combined with a (SA dilution is a simple and semi1automatic method 46 47 48 that provides an accurate quantification of human insulin in a TPN admixture without any 49 50 interference from lipids. 51 52 53 54 K ywords: human insulin, parenteral nutrition solutions, immunoassay, validation, accuracy 55 56 57 profile 58 59 60 2 Page 7 of 28 Analytical & Bioanalytical Chemistry 1 2 3 4 5 INTRODUCTION 6 7 The aim of parenteral nutrition therapy is to supplement or cover nutritional requirements via 8 9 10 the intravenous route when both oral and enteral intaCes are insufficient or contraindicated. 11 12 Ternary parenteral nutrition (TPNA corresponds to an admixture providing macronutrients 13 14 (glucose, amino1acids, lipidsA and micronutrients (electrolytes, vitamins and trace elementsA. 15 16 Considering the amount of glucose infused, hyperglycemia is very common during TPN in 17 18 19 elderly and home parenteralFor nutritionPeer patients, Reviewand concerns most patients in intensive care 20 21 units I1,2J. Hyperglycemia in both non1critically and critically ill patients receiving TPN is 22 23 Cnown to be responsible for an increase in the risC of hospital mortality I3J. Such patients 24 25 may require exogenous insulin in their TPN, but in spite of the guidelines on safe practices for 26 27 28 parenteral nutrition provided by the American Society of Parenteral and .nteral Nutrition I J, 29 30 few data concerning the stability of insulin in TPN are available I5,0J. 31 32 Insulin concentration is currently measured by separative methods, such as high performance 33 34 liquid chromatography coupled with -F detection I7;9J or si%e exclusion chromatography 35 36 I10J, which are not adapted to the complex TPN medium. Mass spectrometry1based methods, 37 38 39 more sensitive but more expensive, require a complicated sample preparation (i.e. 40 125 41 immunoextractionA and dedicated equipment I11J. 3adiolabeled insulin ( I1insulinA I12J is 42 43 useful for studying adsorption on containers, but cannot indicate any modification in 44 45 biological activity, or offer any assessment of either structural or conformational changes in 46 47 48 insulin (from primary to quaternary structureA. The presence of lipids hinders the accurate 49 50 measurement of insulin concentration in all1in1one TPN admixtures regardless of the 51 52 quantification method I13J. This means that other assays, capable of measuring the amount of 53 54 insulin in TPN admixtures precisely, have to be carried out. Immunometric assays are 55 56 routinely used today to quantify human insulin and its analogues in serum I1 ;10J. The 57 58 59 60 3 Analytical & Bioanalytical Chemistry Page 8 of 28 1 2 3 Immuno3adioMetric Assay (I3MAA I17J and Immuno.lectroChemiluminoMetric Assay 4 5 (I.CMAA I18J have shown a high level of specificity for human insulin because of their 6 7 mechanism involving anti1insulin antibodies as well as their high sensitivity. 8 9 10 The aim of our study was to determine the ability of such techniques to quantify insulin at low 11 12 concentrations in a TPN admixture, to validate them and maCe use of one in a stability study 13 14 of insulin in a TPN admixture. 15 16 17 18 19 MATERIALS ANDFor METHODS Peer Review 20 21 Drugs, chemicals and reagents 22 23 Olimel TM N7. (1.5 L bags, (axter, Deerfield, -SAA is the mixture used as TPN (containing 24 25 glucose, amino acid solution, with other electrolytes and lipid emulsionA. Decan TM (Aguettant, 26 27 TM 28 Lyon, 8ranceA and Cernevit ((axter, Deerfield, -SAA were used as additives to TPN. 29 TM 30 -muline rapid (100 I-/mL human insulinA was purchased from .li1Lilly (Suresnes, 31 32 8ranceA. Isotonic saline serum (0.9G NaClA was purchased from (axter. 33 34 Sodium dihydrogen phosphate monohydrate (NaH 2PO , H 2OA and sodium phosphate dibasic 35 36 dodecahydrate (Na HPO , 12 H OA, designed for phosphate buffer saline (P(SA preparations, 37 2 2 38 39 were purchased from Cooper (Melun, 8ranceA. Hydrochloric acid (HCl 25GA and 40 41 tris(hydroxymethylAaminomethane (T3ISA powder were purchased from MercC Millipore 42 43 (Molsheim, 8ranceA. (ovine serum albumin ((SAA F1fraction was purchased from 44 45 .uromedex (Souffelmeyersheim, 8ranceA. Diluted human serum albumin (dHSA, Fialebex TM 46 47 48 0 mg/mLA was purchased from L8( (Lille, 8ranceA. Human gamma globulins (H2(OA were 49 50 purchased from Sigma Aldrich (Saint Louis, -SAA. 51 52 53 54 55 56 57 58 59 60 Page 9 of 28 Analytical & Bioanalytical Chemistry 1 2 3 Sample preparation 4 5 Cernevit TM was reconstituted with 5 mL of 0.9G NaCl and gently shaCen to obtain a 6 7 homogenous solution. Decan TM was then directly introduced into the TPN bag. Seals were 8 9 10 finally broCen by rolling the bag onto itself to mix all nutrients of the TPN admixture. After 11 12 mixing, this worC medium corresponded to the TPN admixture (emulsion enriched with 13 14 vitamins and trace elementsA. It could be stored at 5H3LC up to 2 hours and used for other 15 16 assays. 17 18 19 All assays were Forperformed atPeer a 20,000 MI-/mL Review (i.e. 20 I-/LA insulin concentration, 20 21 corresponding to the usual concentration in medical wards when insulin is added to TPN. 22 23 .very step was carried out with calibrated laboratory materials. Samples were taCen from 24 25 flasCs immediately after preparation without any intermediate transfer step into another 26 27 28 container. 29 30 31 32 Instrumentation 33 34 Immunoreactive insulin concentration was determined by I3MA with the (i1insulin I3MA TM 35 36 set (Cisbio international, Paris, 8ranceA or by I.CMA with the Insulin .lecsys TM assay 37 38 39 (3oche diagnostics, Meylan, 8ranceA. As calibration and measuring ranges were respectively 40 TM TM 41 1 to 500 MI-/ml for (i1insulin I3MA and 0.2 to 1000 MI-/ml for Insulin .lecsys , a two1 42 43 step 1/200 dilution of all samples was performed. 8inal samples were analy%ed on two 44 45 different instruments: radioactivity was determined on a Ei%ard 1 70 2amma coulter 46 47 48 (Cis(io, Saclay, 8ranceA, while I.CMA used an e001 Cobas (3oche diagnostics, Mannheim, 49 50 2ermanyA. The latter apparatus was calibrated with a Calset insulin calibrating Cit and 51 52 controlled with Precicontrol multimarCer solutions both purchased from the same 53 54 manufacturer (3oche, Mannheim, 2ermanyA. All assays were performed in glass containers 55 56 (volumetric flasCs or tubesA to avoid container;content interactions.
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