C-peptide assay

The most definitive laboratory test to distinguish type 1 from type 2 diabetes is the C-peptide assay, which is a measure of endogenous insulin production. With type 2 diabetes, proinsulin can be split into insulin and C-peptide lack of C-peptide indicates type 1 diabetes. The presence of anti-islet antibodies (to glutamic acid decarboxylase, insulinoma associated peptide-2 or insulin) or absence of insulin resistance (determined by a glucose tolerance test) is also suggestive of type 1. 

Measurement of C-peptide has a number of advantages over insulin measurement. Because hepatic metabolism is negligible, C-peptide concentrations are better indicators of P-ceU ffinction than is peripheral insulin concentration. Furthermore, C-peptide assays do not measure exogenous insulin and do not cross-react with insulin antibodies, which interfere with the insulin immunoassay. 

The measurement of C-peptide provides a fully validated means of quantifying endogenous insulin secretion, preventing influence of exogenous insulin or insulin antibody. However, most C-peptide assay kits cannot differentiate C-peptide from proinsulin and proinsulin conversion products. The influence of proinsulin may be significant in cases where serum proinsulin is elevated, as in Type 2 diabetes, familial hyperproinsulinemia and in patients with proinsulin antibody. 

All evaluations of this C-peptide assay performance were performed by LUMIPULSE FORTE. The method used on LUMIPULSE FORTE was as follows 20 pL of serum or standard was mixed with 250 pL of antibody-coated ferrite particle suspension and the mixture was incubated for 37 °C, 10 min. After B/F separation, 250 pL of Enzyme-labeled antibody was added to the ferrite particle suspension and the mixture was incubated for 37 °C, 10 min. After the second B/F separation, 200 pL of AMPPD solution was added. During the washing steps, the ferrite particles were magnetically separated from the bulk solution on the wall of the cartridge. After the enzyme reaction had proceeded for 5 min, the chemiluminescent light emission was measured for 2 s. 

Figure 1. Precision profile for C-peptide assay Broken lines indicate functional sensitivity (CV=10%) of the assay...

We showed reactivity to C-peptide, proinsulin and insulin of the C-peptide assay (Fig. 2). Cross-reactivity to proinsulin and insulin was <1.7% (proinsulin) and <0.03% (insulin) of C-peptide on a molar basis. 

The use of a monoclonal antibody that recognizes the N-terminal of the C-peptide molecule made it possible to realize a low cross-reactivity to proinsulin. Our results indicate that the C-peptide assay on the LUMIPULSE system shows good specificity, sensitivity, precision and linearity. Accordingly, the C-peptide assay enables for the accurate measurement of C-peptide at low concentrations. Especially, it is useful for the presumption of residual P-cell function of diabetic patients who are nearly depleted of insulin secretion. 

Fia. 27. Effects of specific salts and of ionic strength on the C > p activity of RNase. (a) The activity is plotted as that observed relative to the value at low ionic strength (0.1 If). All C>p assays were carried out at pH 7. Insert Substrates, (0—0) RNA (pH 5.0) ( — ) C>p, salt, (NH,)2S04. (b) Effect of ammonium sulfate on the activity of various enzyme preparations (O) RNase-A, ( ) RNase-A (dimer), (A) RNase-S, (A) S-protein incubated with substrate and then activated with S-peptide. [Reproduced from Winstead and Wold (510).]... 

With this assay, basal insulin values of 5-15 pU/mL (30-90 pmol/L) are found in normal humans, with a peak rise to 60-90 U/mL (360-540 pmol/L) during meals. Similar assays for measuring all of the known hormones of the endocrine pancreas (including C-peptide and proinsulin) have been developed. 

Insulin [IN suh lin] is a small protein consisting of two polypeptide chains that are connected by disulfide bonds. It is synthesized as a precursor protein (pro-insulin) that undergoes proteolytic cleavage to form insulin and peptide C, both of which are secreted by the p-cells of the pancreas.4 [Note Normal individuals secrete less pro-insulin than insulin, whereas NIDDM patients secrete high levels of the prohormone. Since radioimmunoassays do not distinguish between the two insulin types, NIDDM patients may have lower levels of the active hormone than the assay indicates. Thus measurement of circulating C peptide provides a better index of insulin levels.]... 

Indirect Two-Site Immunoradiometric Assay of Human Proinsulin. The method used is that described by Rainbow et al. Plastic tubes coated with purified guinea pig anti-insulin antibodies are prepared as described above 200-jul samples containing human proinsulin are added to these coated tubes and incubated at 4° for 24 hr. After removal of the sample, tubes are washed twice with 400 /tl of NIGP buffer. Rabbit antibody to human C-peptide is diluted to 1/1000 in 50 mM sodium phosphate buffer, pH 7.4, containing 150 mM sodium chloride, 10 g of bovine serum albumin per liter, and 100 mg of guinea pig IgG per liter 200 /til are added to each tube. After a further 24 hr of incubation at 4 the tubes are washed twice as previously and 200 /u,l of I-labeled sheep anti-rabbit IgG (10,000 cpm) are added in the same buffer as that used for diluting the C-peptide antiserum. After a final 24 hr of incubation and two further washes as above, the tubes are counted. 

Box 25-1 lists the clinical conditions in which hormones that regulate glucose, namely insulin, proinsulin, C-peptide, and glucagon, have been measured. Although there is interest in the possible clinical value of measurement of the concentrations of insulin and its precursors, the assays are useful primarily for research purposes. There is no role for routine testing for insulin, proinsulin, or C-peptide in patients with diabetes mellitus. It must be emphasized that the diagnostic criteria for diabetes mellitus do not include measurements of hormones, which remain predominantly research tools. 

BOX 25- i Clinical Utility of insulin, Proinsulin, C-Peptide, and Glucagon Assays... 

Accurate measurement of proinsulin has been difhcult for several reasons the blood concentrations are low antibody production is difficult most antisera cross-react with insulin and C-peptide, which are present in much higher concentrations the assays measure intermediate cleavage forms of proinsulin and reference preparations of pure proinsulin are not readily available. However, a more sensitive nonequiUb-rium RIA method for measuring proinsiilin was developed by adsorbing the initial antiserum with biosynthetic human C-peptide coupled to agarose to eliminate cross-reactivity with C-peptide.An enzyme-linked immunosorbent assay (ELISA) has been described that employs an antibody to C-peptide as the coating antibody and antiinsulin antibody for detection. The detection limit is 0.25 pmol/L. ... 

Antisera raised against insulin show some cross-reactivity with proinsuUn but not with C-peptide. Specificity is not a problem in healthy individuals because the low proinsulin concentrations do not appreciably affect the absolute values of insulin. In certain situations (e.g., islet cell tmnors and diabetic individuals), proinsulin is present at higher concentrations and direct assay of plasma may falsely overestimate the true insulin concentration. Because proinsulin has very low activity, incorrect conclusions regarding the availability of biologically active insulin may be reached in patients with diabetes. The magnitude of the error depends on the concentration of proinsulin and the extent of cross-reactivity of the... 

A stable isotope dilution assay using mass spectrometry to measure insulin, proinsulin, and C-peptide has been developed. The difference in mass among the three analytes allows specific measurement of each protein. Comparison of patient samples revealed that most, but not all, results were higher by immunoassay than mass spectrometry. Thus immunoassays may overestimate insulin, particularly at low concentrations. The high protein concentration in the serum requires extraction of proteins (e.g., by immunoaffinity) and purification by high-performance liquid chromatography (HPLC) before quantification by mass spectrometry. This method is not suitable for routine laboratory analysis. 

The laboratory diagnosis of diabetes is made exclusively by the demonstration of hyperglycemia. Other assays, such as the OGTT, contribute to the classification and characterization. Although other tests (e.g., C-peptide and insulin analysis) have been proposed to assist in the diagnosis and classification of the disease, these do not at present have a role outside of research studies. ... 

EQppen AD, Cerini F, Vadas L, Stocklin R, Vu L, Offord RE, et al. 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. 

Zn + is released when insulin is secreted. Conversion of proinsulin to insulin in the secretory granule is not complete and some proinsulin is also released upon secretion of insulin. Proinsulin has less than 5% of the biological activity of insulin. The C-peptide has no physiological function but assay of C-peptide helps distinguish between endogenous and exogenous sourees of insulin. 

Fig. 3. Quantification of Bcl-2 family activities with the long-format cytochrome c release assay. Results are the average of duplicate measurements in (A) and are averages of triplicate measurements +/-SEM in (B-D) (many error bars in panels [B-D] are obscured by the symbols). In panels (A-D) open circles indicate cytochrome c detected when Triton X-100 was added to mitochondria and open diamonds indicate cytochrome c detected when no Bcl-2 family proteins were added to mitochondria. All incubations of mitochondria except those in (B) were for 30 min. (A) Recombinant human Bid (solid circles) and caspase-8-cleaved human Bid (squares) induce release of cytochrome c from isolated mouse liver mitochondria in a dose- dependent manner. (B) Kinetics of 52 nM (solid squares) and 5.2 nM (solid circles) human cleaved Bid-induced cytochrome c release from isolated mouse liver mitochondria. (C) Bcl-xL inhibition of caspase-8-cleaved human Bid and cleaved mouse Bid induced cytochrome c release. Mitochondria were incubated with 52 nM cleaved human Bid without (solid diamond) or with ( solid squares) the indicated concentrations of mouse Bcl-xL. Mitochondria were also incubated with 17 nM cleaved mouse Bid without (open square) or with (solid circles) the indicated concentrations of mouse Bcl-xL. (D) A synthetic peptide (GQVGRQLAIIGDDINR) corresponding to the amino acid 72-87 BH3 region of Bak prevents Bcl-xL from inhibiting caspase-8-cleaved mouse Bid induction of cytochrome c release. Mitochondria were incubated with 17 nM caspase-8-cleaved mouse Bid without (triangle) or with (open square) 155 nM mouse Bcl-xL and the indicated concentrations of Bak-BH3 (solid circle) or the corresponding Bak peptide (GQVGRQAAIIGDDINR) with a L to A substitution (solid squares).

To solve the problem, we have developed a highly specific and sensitive assay for C-peptide in serum and plasma using specific monoclonal antibody (MoAb) to the N-terminal of the C-peptide molecule. In this report, we describe the assay performance of C-peptide on the LUMIPULSE system. The system is a fully automated chemiluminescent enzyme immunoassay (CLEIA) system that uses AMPPD as a substrate for alkaline phosphatase and ferrite micro-particles as a solid phase. ... 

The assay format for C-peptide was based on the two-step method and the two-site immunometric principle using two MoAbs, which recognize different epitopes. 

We developed a simultaneous bioluminescent assay of acetate kinase (AK) and pyruvate phosphate dikinase (PPDK). In the method, the detection limits (blank + 3SD) of AK and PPDK were 1.03x10 and 2.05x10 mol/assay, respectively. Previously, we successfully applied a tandem bioluminescent enzyme immunoassay (BL-EIA) for simultaneous measurement of insulin and c-peptide. In this study, we also applied the method to tandem BL-EIA for Angiotensin I and Endothelin-1, which are hypertension related peptides. The tandem BL-EIA used a competitive immuno-reaction for Angiotensin I and sandwich inununo-reaction for Endothelin-1. 

Table 12. Superoxide dismutase activity of different Cui+-amino acid chelates. All the amino acids and peptides used were in the L-form. The cytochrome-c reductase assay was performed as given in Table 11 (179)...

Adrenal angiography Adrenal venography Adrenocorticotropic hormone stimulation test Aldosterone assay, blood Androstenedione Antidiuretic hormone Antithyroglobulin antibody Antithyroid microsomal antibody Blood glucose Calcitonin Calcium Catecholamines Chromosome karyotype Computed tomography of adrenals Cortisol blood urine C-peptide... 

Plasma C-peptide. Insulin secretion in insulin-treated diabetics cannot be assessed by the measurement of plasma insulin since the insulin given therapeutically will also be measured in the assay. However, insulin and its associated connecting-peptide (or C-peptide) are secreted by the islet cells in equimolar amounts (Fig. 2)... 

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