Development of an Isotope Dilution Assay for Precise Determination of Insulin, C-peptide, and Proinsulin Levels in Non-diabetic and Type II Diabetic Individuals with Comparison to Immunoassay
1997; Elsevier BV; Volume: 272; Issue: 19 Linguagem: Inglês
10.1074/jbc.272.19.12513
ISSN1083-351X
AutoresAlistair D. Kippen, Fabrice Cerini, L. Vadas, Reto Stöcklin, Lan Phuong Vu, Robin E. Offord, Keith Rose,
Tópico(s)Metabolism, Diabetes, and Cancer
ResumoWe describe the application of a stable isotope dilution assay (IDA) to determine precise insulin, C-peptide, and proinsulin levels in blood by extraction from serum and quantitation by mass spectrometry using analogues of each target protein labeled with stable isotopes. Insulin and C-peptide levels were also determined by immunoassay, which gave consistently higher results than by IDA, the relative difference being larger at low concentrations. Insulin, C-peptide, and proinsulin levels were all shown by IDA to be higher in type II diabetics than in non-diabetics, with mean values rising from 22 (± 2) to 92 (± 8), 335 (± 11) to 821 (± 24), and 6 (± 1) to 37 (± 3) pm, respectively. Interestingly, the ratio between IDA and immunoassay values for insulin levels increased from 1.3 in non-diabetics to 1.7 in type II diabetics. The ratio between proinsulin and insulin levels by IDA increased from 0.24 in non-diabetics to 0.36 in type II diabetics, whereas the ratio between C-peptide and insulin levels by IDA decreased from 17.6 to 10.7. This disproportionate change in protein levels between different types of individuals has implications for the metabolism of insulin in the diabetics studied (type II) and suggests that C-peptide levels are not always a reliable guide as to pancreatic insulin secretion. In addition, levels of the 33-residue C-peptide (partially trimmed form) were shown to be less than 10% that of the fully trimmed 31-residue C-peptide levels, and we tested IDA in a clinical context by two post-pancreatic graft studies. IDA was shown to give direct, positive identification of the target protein with unrivaled accuracy, avoiding many of the problems associated with present methodology for protein determination. We describe the application of a stable isotope dilution assay (IDA) to determine precise insulin, C-peptide, and proinsulin levels in blood by extraction from serum and quantitation by mass spectrometry using analogues of each target protein labeled with stable isotopes. Insulin and C-peptide levels were also determined by immunoassay, which gave consistently higher results than by IDA, the relative difference being larger at low concentrations. Insulin, C-peptide, and proinsulin levels were all shown by IDA to be higher in type II diabetics than in non-diabetics, with mean values rising from 22 (± 2) to 92 (± 8), 335 (± 11) to 821 (± 24), and 6 (± 1) to 37 (± 3) pm, respectively. Interestingly, the ratio between IDA and immunoassay values for insulin levels increased from 1.3 in non-diabetics to 1.7 in type II diabetics. The ratio between proinsulin and insulin levels by IDA increased from 0.24 in non-diabetics to 0.36 in type II diabetics, whereas the ratio between C-peptide and insulin levels by IDA decreased from 17.6 to 10.7. This disproportionate change in protein levels between different types of individuals has implications for the metabolism of insulin in the diabetics studied (type II) and suggests that C-peptide levels are not always a reliable guide as to pancreatic insulin secretion. In addition, levels of the 33-residue C-peptide (partially trimmed form) were shown to be less than 10% that of the fully trimmed 31-residue C-peptide levels, and we tested IDA in a clinical context by two post-pancreatic graft studies. IDA was shown to give direct, positive identification of the target protein with unrivaled accuracy, avoiding many of the problems associated with present methodology for protein determination. At present, protein levels in humans are measured by following radiolabeled material or by the use of immunological reagents. Radioiodination is the most common procedure of protein labeling and has been of great value. However, it has been recognized that chemical modification of a protein in this way can affect its biological and physicochemical behavior. Tissue dehalogenases, which are known to remove iodine from iodotyrosine, may invalidate concentration measurements, particularly of fragments of the target molecule (1Cockram C.S. Jones R.H. Boroujerdi M.A. Sonksen P.H. Diabetes. 1984; 33: 721-727Crossref PubMed Scopus (16) Google Scholar, 2Stumpo R.R. Llera A.S. Cardoso A.I. Poskus E. J. Immunol. Methods. 1994; 169: 241-249Crossref PubMed Scopus (19) Google Scholar). Tritiated proteins have been used since they escape these objections (3Davies J.G. Cerini F. Bradshaw C.G. Insulin and the Cell Membrane. Harwood Academic Publishers GmbH, Chur, Switzerland1990: 243-254Google Scholar), but the ethical issue of injecting radioactive proteins of any kind into human subjects remains under severe scrutiny. The alternative method involving the use of immunological reagents to estimate blood protein levels remains the best current approach. However, antibodies used in these measurements often cross-react with precursors of the target protein or with its smaller, degraded fragments, creating uncertainty in results (2Stumpo R.R. Llera A.S. Cardoso A.I. Poskus E. J. Immunol. Methods. 1994; 169: 241-249Crossref PubMed Scopus (19) Google Scholar, 4Temple R.C. Carrington C.A. Luzio S.D. Owens D.R. Schneider A.C. Sobey W.J. Hales C.N. Lancet. 1989; i: 293-295Abstract Scopus (348) Google Scholar, 5Temple R.C. Clark P.M. Nagi D.K. Schneider A.E. Yudkin J.S. Hales C. Clin. Endocrinol. 1990; 32: 684-693Crossref Scopus (126) Google Scholar, 6Saric T. Seitz H.J. Pavelic K. Mol. Cell. 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Although modern forms of radioimmunoassay, microparticle enzyme immunoassay (MEIA), 1The abbreviations used are: MEIA, microparticle enzyme immunoassay; CEI, chemiluminescent enzyme immunoassay; [2H14]C-peptide, [2H2]Gly7 human C-peptide analogue; [2H16]insulin, [2H8]PheB1-[2H8]ValB2human insulin analogue; ESI-MS, electrospray ionization mass spectrometry; IDA, isotope dilution assay; HPLC, high performance liquid chromatography; TPCK, l-1-tosylamido-2-phenylethyl chloromethyl ketone; IRMA, a monoclonal antibody-based two-site immunoradiometric assay. 1The abbreviations used are: MEIA, microparticle enzyme immunoassay; CEI, chemiluminescent enzyme immunoassay; [2H14]C-peptide, [2H2]Gly7 human C-peptide analogue; [2H16]insulin, [2H8]PheB1-[2H8]ValB2human insulin analogue; ESI-MS, electrospray ionization mass spectrometry; IDA, isotope dilution assay; HPLC, high performance liquid chromatography; TPCK, l-1-tosylamido-2-phenylethyl chloromethyl ketone; IRMA, a monoclonal antibody-based two-site immunoradiometric assay. and immunoradiometric assay (7Sobey W.J. Beer S.F. Carrington C.A. Clark P.M. Frank B.H. Gray I.P. Luzio S.D. Owens D.R. Schneider A.E. Siddle K. Temple R.C. Hales C.N. Biochem. J. 1989; 260: 535-541Crossref PubMed Scopus (339) Google Scholar, 8Clark P.M. Levy J.C. Cox L. Burnett M. Turner R.C. Hales C.N. Diabetologia. 1992; 35: 469-474Crossref PubMed Scopus (49) Google Scholar, 9Kjems L.L. Roder M.E. Dinesen B. Hartling S.G. Jorgensen P.N. Binder C. Clin. Chem. 1993; 39: 2146-2150Crossref PubMed Scopus (75) Google Scholar) reduce many of these cross-reactivities, the methodology still remains an indirect measure of the target molecule, where endogenous and administered exogenous protein cannot be distinguished. Also, pathological samples may contain endogenous antibodies that can interfere with conventional assays and invalidate the results obtained. Such difficulties, as shown by significant discrepancies between immunoassays performed at different centers on the same samples (10Robbins D.C. Andersen L. Bowsher R. Chance R. Dinesen B. Frank B. Gingerich R. Goldstein D. Widemeyer H.-M. Haffner S. Hales C.N. Jarett L. Polonsky K. Porte D. Skyler J. Webb G. Gallagher K. Diabetes. 1996; 45: 242-256Crossref PubMed Scopus (0) Google Scholar), have led to a feeling that measurements of insulin levels, for example, have been systematically overestimated by radioimmunoassay in type II (non-insulin-dependent) diabetic individuals, resulting in insufficient attention being paid to insulin deficiency as opposed to insulin resistance (5Temple R.C. Clark P.M. Nagi D.K. Schneider A.E. Yudkin J.S. Hales C. Clin. Endocrinol. 1990; 32: 684-693Crossref Scopus (126) Google Scholar). Indeed, many diseases, including type II diabetes and tumors such as insulinomas or certain other carcinomas, lead to a disproportional increase in the concentration of proteins such as proinsulin and its conversion intermediates that are immunologically cross-reactive with insulin (6Saric T. Seitz H.J. Pavelic K. Mol. Cell. Endocrinol. 1994; 106: 23-29Crossref PubMed Scopus (12) Google Scholar, 11Pavelic L. Pavelic K. Vuk-Pavlovic S. Cancer. 1984; 53: 2467-2471Crossref PubMed Scopus (14) Google Scholar, 12Robbins D.C. Tager H.S. Rubenstein A.H. N. Engl. J. Med. 1984; 310: 1165-1175Crossref PubMed Scopus (57) Google Scholar, 13Yudkin J.S. J. Diabetes Complications. 1993; 7: 113-123Crossref PubMed Scopus (46) Google Scholar). The uncertain clinical interpretation of results involving such important diseases remains a serious problem. A definitive measure of blood protein concentration would be most welcome in calibrating existing procedures and investigating suspect results. To be able to fully understand the fate and distribution of proteins in vivo, a technique is required that can accurately determine each of the components directly involved, allowing a distinction between endogenous and exogenous protein while avoiding the injection of radioactive material. To this aim we describe an alternative technique, referred to as isotope dilution assay (IDA), that allows precise quantitation of specific proteins at physiological concentrations in blood. In this procedure, target proteins are extracted from a 1-ml serum sample by solid-phase or immunoaffinity techniques and purified by HPLC. Their initial concentration is then determined by mass spectrometry from the ratio between the spectral peak of each target protein and that of an internal standard that has been added to the serum in known amounts. A carrier protein is also added in excess concentration, which allows easier detection of the three proteins by HPLC and helps to avoid protein loss by adsorption. The internal standards and carrier proteins are stable isotope-labeled analogues of the target proteins that exhibit slightly different mass. IDA incorporates a method known as stable isotope dilution mass spectrometry that has proved successful for the measurement of small molecules (14Dass C. Kusmierz J.J. Desiderio D.M. Biol. Mass Spectrom. 1991; 20: 130-138Crossref PubMed Scopus (45) Google Scholar, 15Desiderio D.M. Life Sci. 1992; 51: 169-176Crossref PubMed Scopus (22) Google Scholar, 16Bowers G.N.J. Fassett J.D. White E. Anal. Chem. 1993; 65: 475-479Crossref Scopus (39) Google Scholar, 17Fenselau C. Vestling M.M. Cotter R.J. Curr. Opin. Biotechnol. 1993; 4: 14-19Crossref PubMed Scopus (10) Google Scholar). However, its use for macromolecules such as proteins is less developed due to difficulty in the synthesis of the necessary internal standard and carrier analogues. Having previously demonstrated the feasibility of our approach by determination of insulin levels in serum (18Stöcklin R. Vu L. Vadas L. Cerini F. Kippen A.D. Offord R.E. Rose K. Diabetes. 1997; 46: 44-50Crossref PubMed Scopus (50) Google Scholar), we now report the development of an optimized version of this technique to measure in humans the protein pair proinsulin and insulin together with the activation-related connecting peptide (C-peptide) (13Yudkin J.S. J. Diabetes Complications. 1993; 7: 113-123Crossref PubMed Scopus (46) Google Scholar, 19Steiner D.F. Oyer P.E. Proc. Natl. Acad. Sci. U. S. A. 1967; 57: 473-478Crossref PubMed Google Scholar, 20Steiner D.F. Clark J.L. Proc. Natl. Acad. Sci. U. S. A. 1968; 60: 622-626Crossref PubMed Scopus (90) Google Scholar, 21Oyer P.E. Cho S. Peterson J.D. Steiner D.F. J. Biol. Chem. 1971; 246: 1375-1386Abstract Full Text PDF PubMed Google Scholar, 22Halban P.A. Irminger J.-C. Biochem. J. 1994; 299: 1-18Crossref PubMed Scopus (278) Google Scholar). The precursor of insulin, proinsulin, is produced by the beta cells of the pancreatic islets of Langerhans. 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Bennett P.H. Porte Jr., D. J. Clin. Endocrinol. Metab. 1990; 70: 1247-1253Crossref PubMed Scopus (180) Google Scholar, 25Linde S. Roder M.E. Hartling S.G. Binder C. Welinder B.S. J. Chromatogr. 1991; 548: 371-380Crossref PubMed Scopus (25) Google Scholar, 26Linde S. Welinder B.S. Nielsen J.H. J. Chromatogr. 1993; 614: 185-204Crossref PubMed Scopus (16) Google Scholar). However, the precise difference between levels of these proteins is difficult to evaluate since present methodology cannot yet distinguish between all of them. Increased knowledge about the secretion, mode of action, and distribution of insulin-related proteins in the body is required to understand important diseases such as diabetes. Using IDA, we investigated proinsulin, insulin, and C-peptide levels in non-diabetic non-obese, non-diabetic obese, and type II diabetic individuals and compared our results with those obtained by standard immunoassay. Carboxypeptidase B (from porcine pancreas) was obtained from Boehringer Mannheim. Trypsin (TPCK-treated, from bovine pancreas), antiserum against human insulin (developed in guinea pig), and bovine serum albumin (essentially fatty acid and globulin free) were obtained from Sigma. All other reagents (except the antiserum against human C-peptide, see "Acknowledgments") were of analytical grade and were purchased from either Fluka Chemie AG or Sigma. The proinsulin gene, in a construction containing a polyhistidine tag (27Berg H. Walter M. Mauch L. Seissler J. Northemann W. J. Immunol. Methods. 1993; 164: 221-231Crossref PubMed Scopus (15) Google Scholar), was cloned and expressed in Escherichia coli by a modified (see "Acknowledgments") standard procedures (28Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual.2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar) at a yield of 5–10 mg/liter of culture. After protein isolation using a 10-ml nickel-Sepharose fast flow column (Pharmacia Biotech Inc.), the polyhistidine tag was removed with cyanogen bromide (1.5 mg/mg of protein at pH 1), and proinsulin was purified by reverse-phase HPLC (using a Macherey Nagel 300-Å C8 column (250 × 10 mm)) with a linear gradient of acetonitrile (22–36% in 30 min) in 0.1% trifluoroacetic acid in water. Proinsulin was labeled biosynthetically with 15N by expression of the gene in a culture medium containing 15NH4Cl (28Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual.2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar, 29Muchmore D.C. Mcintosh L.P. Russell C.B. Anderson D.E. Dahlquist F.W. Methods Enzymol. 1989; 177: 44-73Crossref PubMed Scopus (473) Google Scholar), where the incorporation of 15N was calculated to be 98.8% by mass spectrometry. Furthermore, proinsulin was double-labeled with both15N and 2H by expression in15NH4Cl and 50% 2H2O medium, where the final incorporation of 2H after protein purification was calculated to be 24.3% (assuming 98.8%15N incorporation). Each proinsulin analogue was cleaved by proteolytic techniques, using trypsin (2.5 μg/mg of protein) and carboxypeptidase B (5 μg/mg of protein) at pH 8 to yield15N- and 15N2H-labeled insulin and C-peptide (31- and 33-residue forms). [2H8]PheB1-[2H8]ValB2insulin ([2H16]insulin) was prepared as described previously (30Stöcklin R. Rose K. Green B.N. Offord R.E. Protein Eng. 1994; 7: 285-289Crossref PubMed Scopus (10) Google Scholar). [2H2]Gly7 C-peptides ([2H14]C-peptides) (31- and 33-residue forms) were prepared by total peptide synthesis using standard Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry (Applied Biosystems 430A peptide synthesizer). Subsequent peptide purification was performed by reverse-phase HPLC using similar conditions as described above. The concentration of proinsulin and insulin solutions was determined from the absorbance at 280 nm (31Gill S.C. von Hippel P.H. Anal. Biochem. 1989; 182: 319-326Crossref PubMed Scopus (5010) Google Scholar), and the concentration of C-peptide solutions was determined by amino acid analysis, HPLC, and electrospray ionization mass spectrometry (ESI-MS) against a standard. Protein aliquots were prepared at a concentration of 25 μm, freeze-dried, and stored at −20 °C. To help avoid protein loss, any further protein dilution was carried out in siliconized glass tubes with a solution of bovine serum albumin (1 mg/ml). Our investigations on the C-peptide refer solely to the 31-residue form, unless the 33-residue form is specifically mentioned. After dilution with 5 ml of 0.1% trifluoroacetic acid in water, the serum sample (1 ml) was filtered through a 0.45-μm sterile filter (Millipore Corp.), and 150 IU of heparin was then added (to bind PF4, a platelet factor in serum that has a HPLC retention time similar to insulin). A known amount of each internal standard (at a concentration similar to that expected for the target protein) and each carrier (at excess concentration; 100 nm [15N]insulin, 200 nm [15N2H]C-peptide, and 100 nm [15N2H]proinsulin) were added to the serum. A Sep-Pak cartridge (Waters C18) was washed with pure acetonitrile and then equilibrated in 0.1% trifluoroacetic acid in water. The serum was passed back and forth through the cartridge three times at approximately 1 ml/min using polypropylene syringes fixed at either end. Two solutions (4 ml) consisting of 20% acetonitrile, 0.1% trifluoroacetic acid in water and 80% acetonitrile, 20% dichloromethane were then washed through the cartridge to remove unwanted proteins (32Cohen R.M. Given B.D. Licinio-Paixao J. Provow S.A. Rue P.A. Frank B.H. Root M.A. Polonsky K.S. Tager H.S. Rubenstein A.H. Metabolism. 1986; 35: 1137-1146Abstract Full Text PDF PubMed Scopus (53) Google Scholar). The target insulin proteins were co-eluted from the cartridge with 50% acetonitrile, 0.1% trifluoroacetic acid in water (2 ml), and the solution was freeze-dried. After dilution with 5 ml of 0.1m sodium phosphate buffer, pH 8, the serum sample (1 ml) was filtered through a 0.45-μm sterile filter (Millipore), and 150 IU of heparin and a known amount of each internal standard and each carrier were then added (as for solid phase). The serum was passed twice (under gravity) through an affinity column pre-equilibrated in the phosphate buffer, and the column was then washed with the buffer (6 ml). The column consisted of an Aminolink-agarose gel (3 ml, Pierce) to which both insulin antibodies (guinea pig) and C-peptide antibodies (mouse IgG, known to also bind proinsulin) were coupled at excess concentration (1 mg/ml). A Sep-Pak cartridge (Waters C18 light) was washed with pure acetonitrile, equilibrated in 0.1% trifluoroacetic acid in water, and fixed onto the outlet of the affinity column. A solution of 2 m acetic acid (6 ml) was passed down the column to elute the target proteins into the Sep-Pak cartridge. The cartridge was then removed and washed with 20% acetonitrile, 0.1% trifluoroacetic acid in water (4 ml). The target insulin proteins were eluted from the cartridge with 50% acetonitrile, 0.1% trifluoroacetic acid in water (2 ml), and the solution was freeze-dried. Following extraction by either solid phase or immunoaffinity, the samples were redissolved in 0.1% trifluoroacetic acid in water (120 μl) and loaded onto a microbore reverse-phase HPLC system (Applied Biosystems 140B, Nucleosil 300-Å C18 column (1 × 150 mm)). Each target protein extracted from the serum was then isolated and purified, together with its internal standard and carrier proteins, by elution with a linear gradient of acetonitrile (22–36% in 60 min at a flow of 40 μl/min) in 0.1% trifluoroacetic acid in water. The excess concentration of the carrier allows easy detection and purification of the three proteins (the target protein, its internal standard, and its carrier) together as a single peak by chromatography. The aliquots collected were freeze-dried. Aliquots containing each target protein, together with its respective internal standard and carrier proteins, were resuspended in 10 μl of ESI-MS solvent (50% methanol, 49% water, 1% acetic acid). The samples were then analyzed by ESI-MS carried out in positive ionization mode using either: (a) VG-Trio 2000 single quadrupole instrument (Fisons Instruments, Inc.) at a flow of 2 μl/min and a scan cycle of 10 s or (b) VG-Platform II single quadrupole instrument (Micromass) at a flow of 10 μl/min and a scan cycle of 1 s. The spectrum was scanned to cover the most intense signals of the target protein and its internal standard, whereas the large carrier signal was designed to be out of this scan range. By analysis of the ratio between the peak height of the target protein and the peak height of the internal standard (added at known concentration), the exact concentration of the target protein in the original serum sample was determined. Any loss of product during the extraction and purification procedure would not affect the ratio. Serum samples obtained from the University Hospital, Geneva, were initially analyzed by automated immunoassay. Insulin concentration was determined with a MEIA, IMx (Abbott Laboratories, Toyko, Japan), which uses mouse monoclonal antibodies against human insulin. C-peptide concentration was determined with a chemiluminescent enzyme immunoassay (CEI), Immulite (Diagnostic Product Corp., Los Angeles, CA), which uses rabbit polyclonal antibodies against C-peptide. The proinsulin concentration of these samples was not determined by immunoassay. Fresh serum samples taken from various types of individuals were obtained from the University Hospital, Geneva. The concentration of insulin (66 samples) or C-peptide (40 different samples) was measured by immunoassay. These samples represent most of the range of possible blood concentration levels: 10–1638 pm insulin and 55–6820 pm C-peptide. To compare results obtained by the two techniques, the insulin or C-peptide concentration in these samples was also measured by IDA. The amount of [2H16]insulin (the internal standard for insulin) added to each sample was varied from 10 to 1500 fmol, depending on the insulin concentration as determined by MEIA. Similarly, the amount of [2H14]C-peptide (the internal standard for C-peptide) was varied from 100 fmol to 6 pmol, depending on the C-peptide concentration as determined by CEI. The concentration of insulin, in four identical 1-ml aliquots taken from a single fresh serum sample, was determined by IDA to test the variability of results. This procedure was repeated for three different serum samples at insulin concentrations of 50, 203, and 640 pm as determined by MEIA. A similar procedure was carried out to measure by IDA the concentration of C-peptide in serum samples determined by CEI to be 210, 560, and 1080 pm. Proinsulin concentration was similarly measured by IDA of serum samples at an MEIA insulin concentration of 100, 570, and 890 pm. [2H16]Insulin and [2H14]C-peptide were added at a concentration similar to that of the target protein (as determined by immunoassay), and [15N]proinsulin (the internal standard for proinsulin) was added at one-tenth the insulin concentration. The 33-residue form of the C-peptide comprises an additional two amino acids, lysine and arginine, which remain at its C terminus after incomplete cleavage from proinsulin. This form is thought to be present in serum at a far lower concentration than the fully trimmed C-peptide consisting of 31 residues. The C-peptide concentration of 10 serum samples was determined by CEI. The 33-residue C-peptide and the 31-residue C-peptide were then individually quantitated in these samples by IDA. The amounts of internal standards added were 100 fmol of [2H14]C-peptide 33 and 150–4500 fmol of [2H14]C-peptide 31. Following a 12-h fast, fresh serum samples were obtained from three types of persons: non-diabetic non-obese (25 samples), non-diabetic obese (10 samples), and type II diabetic (20 samples). A heterogeneous population was examined from both sexes, aged 20–60 years, where obese individuals were defined as those with a body mass index above 35 kg/m2. The precise basal level of insulin, C-peptide, and proinsulin in each of these samples was investigated by IDA. The concentration of insulin (in all samples) and C-peptide (only in samples from diabetic individuals) was also measured by immunoassay. The amounts of internal standards added to each sample were 10–300 fmol of [2H16]insulin, 200–2000 fmol of [2H14]C-peptide, and 10–100 fmol of [15N]proinsulin. This patient underwent a subtotal pancreatectomy with autotransplantation of the islets of Langerhans. Following the operation, insulin and C-peptide basal levels were measured by immunoassay from blood samples (10 ml) taken every 3 h for 60 h. These 20 samples were further analyzed by IDA to determine insulin, C-peptide, and proinsulin levels during this period. After coagulation in the absence of anticoagulant, serum was separated from blood samples by centrifugation. The amounts of internal standards added to each sample were 10–30 fmol of [2H16]insulin, 500 fmol of [2H14]C-peptide, and 50 fmol of [15N]proinsulin. Following a similar operation to that described for patient 1, pancreatic reaction was monitored by an oral glucose tolerance test, where 70 g of glucose in 400 ml of water was taken orally after a 12-h fast. Insulin and C-peptide levels were measured by immunoassay from blood samples (10 ml) taken every 10 min for 50 min. For comparison, insulin, C-peptide, and proinsulin levels were determined in these samples by IDA. The same amounts of internal standards were added to each sample as in the study with patient 1. Each protein prepared comprises the human sequence and was characterized by amino acid analysis and ESI-MS (TableI). Importantly, all of these analogues prepared, both those to be used as internal standards and those that act as carrier proteins, had exactly the same retention time by HPLC as their native counterparts and differ in mass due only to isotopic labeling. Further, the carrier proteins, present in excess concentration, exhibited no spectral peaks by ESI-MS that might have obscured the measurement of their respective target protein or internal standard peaks.Table IMass data for each analogue of insulin, C-peptide, and proinsulin preparedProteinMolecular mass1-aStandard error in each measurement is ± 1.0 Da.Mass/charge of main ion1-bFrom the ion envelope formed by ESI-MS of each protein, the ion with the largest intensity was used for analysis. Standard error in each measurement is ± 0.4 m/z units.Dam/zC-peptide 31Native30201007.7 (3+)1/2H141-cAnalogue used as internal standard.30341012.31/15N2H1-dAnalogue used as carrier.31111038.0C-peptide 33Native33041102.3 (3+)2H141-cAnalogue used as internal standard.33181107.015N2H1-dAnalogue used as carrier.34081137.0InsulinNative58071162.4 (5+)2H161-cAnalogue used as internal standard.58231165.615N1-dAnalogue used as carrier.58721175.4ProinsulinNative93881174.5 (8+)15N1-cAnalogue used as internal standard.95011188.615N2H1-dAnalogue used as carrier.96571208.1Proteins comprise the human sequence, and their mass was determined by ESI-MS.1-a Standard error in each measurement is ± 1.0 Da.1-b From the ion envelope formed by ESI-MS of each protein, the ion with the largest intensity was used for analysis. Standard error in each measurement is ± 0.4 m/z units.1-c Analogue used as internal standard.1-d Analogue used as carrier. Open table in a new tab Proteins comprise the human sequence, and their mass was determined by ESI-MS. Insulin, proinsulin, and the C-peptide were efficiently isolated and purified from 1 ml of serum by both solid-phase and immunoaffinity extraction methodology, with approximately 80% recovery of the pure target protein. Both extraction procedures eliminated most of the unwanted substances in the serum (which would have overloaded the mass spectrometer) before further purification by microbore HPLC. This system keeps the working volumes small to avoid protein loss and is relatively efficient even at low protein concentration. Immunoaffinity (Fig. 1) allowed a more specific extraction of the target proteins than solid-phase methodology, which also extracted proteins of similar mass to the C-peptide (such as platelet factors) that elute at an HPLC retention time between that of the C-peptide and insulin. Following extraction and purification, an excellent signal-to-noise ratio was obtained by ESI-MS of each target protein at its physiological concentration in the presence of its internal standard, demonstrating the high sensitivity of this method (Fig.2). By extraction of a series of samples containing either negligible amounts of each targ
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