|Year : 2018 | Volume
| Issue : 1 | Page : 26-29
Optimization of reagent concentration for radioiodination of rat C-peptide II in development of radioimmunoassay procedure for rats
BR Manupriya1, Shalaka Paradkar2, Lathika1, Shrikant L Patil3, HM Somashekarappa1, Bhasker K Shenoy1
1 Department of Applied Zoology and Centre for Application of Radioisotopes and Radiation Technology, Mangalore University, Mangaluru, Karnatak, India
2 Radiopharmaceuticals Production Programme, Board of Radiation and Isotope Technology, Navi Mumbai, Maharashtra, India
3 Department of Physiology, K.S. Hegde Medical Academy, Mangaluru, Karnatak, India
|Date of Submission||31-Jan-2018|
|Date of Decision||30-Mar-2018|
|Date of Acceptance||19-Apr-2018|
|Date of Web Publication||31-May-2018|
Dr. Bhasker K Shenoy
Department of Applied Zoology, Mangalore University, Mangalagangothri, Konaje, Mangalore - 574 199, Karnataka
Source of Support: None, Conflict of Interest: None
Rat C-peptide is a polypeptide molecule made up of 31 amino acids and secreted from pancreas into circulation in two isoforms I and II. Quantification of rat C-peptide II in rat serum is important as it is directly related to the diagnosis of carbohydrate metabolism abnormalities, pancreatic performance analysis, monitoring of hypoglycemia, and diabetes-related illness in rat model. The aim of the present work is to develop a tracer by chloramine-T method for radioimmunoassay (RIA) procedure and to determine the optimum amount of chloramine-T required for the preparation of stable radioiodinated product with a specific activity of around 24.97 MBq/μg, corresponding to 1 125I atom per molecule of the peptide. Tyrosylated rat C-peptide II was selected for the radioiodination procedure as rat C-peptide II does not contain either tyrosine or histidine which is mandatory for the incorporation of 125I atom to the rat C-peptide II. Tyrosylated rat C-peptide II was subjected to radioiodination by chloramine-T method with different concentrations of chloramine-T and sodium metabisulfite (MBS) to obtain a stable radiolabeled compound. Optimized reaction conditions relating to the concentration of chloramine-T (10 μg) and MBS (20 μg) yielded a stable 125I-rat C-peptide II with specific activity of 21.01 MBq/μg corresponding to 0.84 125I atoms per molecule of the peptide. Preparation of high integrity tracer of rat C-peptide II was achieved by combining one molecule of oxidant (chloramine-T) and two molecule of reductant (MBS).
Keywords: 125I-rat C-peptide II tracer, chloramine-T, radioiodination
|How to cite this article:|
Manupriya B R, Paradkar S, Lathika, Patil SL, Somashekarappa H M, Shenoy BK. Optimization of reagent concentration for radioiodination of rat C-peptide II in development of radioimmunoassay procedure for rats. Radiat Prot Environ 2018;41:26-9
|How to cite this URL:|
Manupriya B R, Paradkar S, Lathika, Patil SL, Somashekarappa H M, Shenoy BK. Optimization of reagent concentration for radioiodination of rat C-peptide II in development of radioimmunoassay procedure for rats. Radiat Prot Environ [serial online] 2018 [cited 2021 Feb 26];41:26-9. Available from: https://www.rpe.org.in/text.asp?2018/41/1/26/233643
| Introduction|| |
Preproinsulin is the precursor molecule of insulin biosynthesis. Preproinsulin is converted to proinsulin by removal of a signal peptide. Proinsulin is then cleaved into insulin and C-peptide by endopeptidases. C-peptide is a connecting peptide which plays a key role in maintaining the structure of insulin secreted along with insulin in equimolar concentration from β-cells of pancreas. Rat C-peptide is a polypeptide molecule made up of 31 amino acids (3.2 kDa). Structure of rat C-peptide I and II differ by two amino acids at positions 8 and 17. C-peptide could be an independent marker of insulin as C-peptide (33 min) has a longer half-life compared to insulin (4 min). C-peptide assay facilitates the quantification of insulin secreted by the individual. Rat is a widely used experimental model to study metabolic disorders, impairment in islet functions, and degree of insulin resistance occurring due to type II diabetes. These disorders eventually affect the level of C-peptide in the blood. Development of an indigenous method like immunoassay is necessary to estimate rat C-peptide in rat serum. The present study was planned to optimize reagent concentration required for radioiodination of rat C-peptide II by chloramine-T method.
| Materials and Methods|| |
Tyrosylated rat c-peptide II was procured from Cellmano Biotech Limited, China.125 I was provided by Board of Radiation and Isotope Technology, Navi Mumbai, India. Chloramine-T (Nchloroptoluenesulfonamide, salt trihydrate) and metabisulfite (MBS) were purchased from Sigma Chemical Company, USA. Sephadex G-50 was from Pharmacia fine chemicals company, Sweden, and Whatman 3 mm chromatography paper was from Whatmann Limited, England. Single well manual gamma counter (Electronics Corporation of India Limited, India) and a multiwell gamma counter (Stratec, Germany) instruments were used to count radioactivity.
Radioiodination procedure was designed with the aim of optimization of reagent concentrations. Chloramine-T concentration of 0.5, 2, 10, 20, 40, 80, and 100 μg was used along with 2.5, 10, 20, 40, 80, 160, and 200 μg of MBS, respectively, to optimize reagent concentrations. Reaction mixture was prepared in different combinations by adding 5 μg of tyrosylated rat C-peptide II to a mixture of 0.5 M phosphate buffer, pH - 7.4, radioactivity (111 MBq of 125 I) and chloramine-T (0.5, 2, 10, 20, 40, 80, and 100 μg), gently vortexed and incubated for 60 s at room temperature followed by the addition of 0.05 M phosphate buffer, pH - 7.4, MBS (2.5, 10, 20, 40, 80, 160, and 200 μg) and 50 μg of potassium iodide in different radioimmunoassay (RIA) tubes. After the iodination reaction, 10 μl of reaction mixture was diluted with 50 μl of potassium iodide and subjected to paper electrophoresis to determine radioiodination yield. The reaction mixture was spotted at the negative end of preequilibrated Whatmann paper strip (30 cm length) and electrophoresis unit was run for 55 min at 250 volt. At the end of the electrophoresis, paper strips were dried, cut into 1 cm and counted in a multiwell gamma counter.
The remaining reaction mixture was purified using Sephadex G-50 column and eluted the column with 0.05 M phosphate buffer, pH - 7.4. One ml of eluted fractions were collected in the RIA tubes containing 1% bovine serum albumin in 0.05 M phosphate buffer, pH-7.4. Purified fractions were vortexed and counted in single well manual gamma counter for 10 s with changed geometry. The fractions giving highest counts were pooled, and radiochemical purity (RC purity) was determined by paper electrophoresis. The equilibrated Whatman paper strips were spotted with the mixture of 10 μl 125 I-rat C-peptide II tracer and 50 μl of potassium iodide at the negative end. Electrophoresis unit was run for 55 min at 250 volt by immersing both ends of paper strips in the tank buffer (0.025 M phosphate buffer). Dried paper strips of 1 cm length were counted in the multiwell gamma counter for 1 min. The purified fractions corresponding to desired protein were dispensed into 10 ml vials and stored at −20°C for further use.
| Results|| |
Choice of reagent concentration is mainly based on the yield of radiolabeled compound. Radioiodination yield corresponds to the amount of radionuclide (125 I) incorporated into rat C-peptide II and form 125 I-rat C-peptide II compound. Radioiodination yield was calculated by the formula: radioiodination yield = (counts at the point of spotting/total counts) ×100. [Figure 1] represents the variation in the radioiodination yields with respect to chloramine-T concentration. Among all the reagent concentrations studied, the highest yield (~75%) was observed at the concentrations of 20 μg chloramine-T and 40 μg MBS. These concentrations were further selected for the optimization of amount of radioactivity. Radioiodination of rat C-peptide II with 111 MBq of radioactivity, 10 μg of chloramine T, and 20 μg of MBS concentrations resulted the radioiodinated compound with radioiodination yield of 94.61 % and specific activity of 21.01 MBq/μg corresponding to 0.84125 I atoms per molecule of the peptide. The electrophoretic pattern of reaction mixture is presented in [Figure 2]; major peak indicates the radio-labeled rat C-peptide II whereas minor peak depicts the free iodide. Sephadex G-50 column purification isolated the free iodide and furnishes a purified form of 125 I-rat C-peptide II. Similarly, electrophoretic pattern of purified compound has a single peak [Figure 3]. The graph symbolizes the total radioactivity associated with the 125 I-rat C-peptide II compound (i.e., RC purity). RC purity is calculated as RC purity = (counts at the point of spotting/total counts) ×100. RC purity was determined at specific intervals over a period of time. RC purity of the purified compound was found to be >95% at least up to 1 month from the date of radioiodination.
|Figure 1: Radioiodination yield (%) of rat C-peptide II with respect to variation in chloramine-T concentration. (a) 0.5 μg chloramine-T and 1 μg metabisulfite, (b) 2 μg chloramine-T and 4 μg metabisulfite, (c) 10 μg chloramine-T and 20 μg metabisulfite, (d) 20 μg chloramine-T and 40 μg metabisulfite, (e) 40 μg chloramine-T and 80 μg metabisulfite, (f) 80 μg chloramine-T and 160 μg metabisulfite, (g) 100 μg chloramine-T and 200 μg metabisulfite|
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|Figure 2: Electrophoretic pattern of reaction mixture of radiolabeled rat C-peptide II before purification. Major peak represents the radiolabeled rat C-peptide II and minor peak indicates free iodide|
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|Figure 3: Electrophoretic pattern of purified radiolabeled rat C-peptide II. Single peak represents the purified compound of 125I–rat C-peptide II obtained after purification of reaction mixture using Sephadex G-50 column chromatography|
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| Discussion|| |
Insulin and C-peptide are secreted to circulation in equimolar concentrations. The longer metabolic clearance rate of C-peptide encourages its detection over insulin. Preparation of a good tracer is a major criterion in the development of RIA procedure for quantification of rat C-peptide II in rat serum. In the preparation of 125 I-rat C-peptide II tracer, optimization of reagent concentrations plays a crucial role as impaired combination of chloramine-T and MBS can cause damage to the protein or may affect the radioiodination yield. The reagent concentrations of 0.5 μg of chloramine-T and 2.5 μg of MBS provided a lowest radioiodination yield (13%). This is may be due to the concentration of oxidizing agent applied was not adequate for the incorporation of radionuclide molecule into rat C-peptide II. Gradual decrease in radioiodination yield was observed above the reagent concentrations of 40 μg chloramine-T and 80 μg of MBS which imply that application of high concentration of oxidation reagent may cause damage to the protein. Lactoperoxidase method provides a gentle oxidation reaction; however, longer reaction time (~20 min), low radioiodination yield, and low-specific activity (0.185 MBq/μg) are the limitations of this method. Incorporation of iodine to protein is less in Bolton–Hunter method as Bolton–Hunter reagent (N-Succinimidyl-3-(4-hydroxyphenyl) propionate) itself undergoes hydrolysis and forms 3-(4-hydroxyphenyl) propionic acid. Therefore, chloramine-T method was preferred over other methods for radioiodination of protein.
| Conclusions|| |
The reagent concentration combinations of 10 μg chloramine-T and 20 μg MBS and 20 μg chloramine-T and 40 μg of MBS produced a radiolabeled compound with a yield of 75% and 78%, respectively. Among these, 10 μg of chloramine-T and 20 μg of MBS were selected to carry out radioiodination as this concentration combination offers a radiolabeled product with good radioiodination yield and minimum damage to the protein. In this study, it was found that two molecules of reductant are essential to hinder the action of one molecule of the oxidant.125 I-rat C-peptide II can be used for the routine preparation of tracer required for the regular production of rat C-peptide RIA kits.
Manupriya B R is grateful to University Grants Comission– Basic Scientific Research (UGC-BSR) grant, New Delhi for providing financial support to carry out this work.
Financial support and sponsorship
This study is financially supported by UGC-BSR Fellowship Scheme 2013.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3]