Proinsulin in Human Serum: Problems in Measurement and Interpretation

Gray, I. P.; Siddle, Kenneth; Docherty, Kevin; Frank, Bruce H.; Hales, C. N.

doi:10.1111/j.1365-2265.1984.tb00134.x

Cited by 27 publications

(16 citation statements)

References 10 publications

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“…Proinsulin levels determined with this antiserum, therefore, represent not only intact proinsulin but also conversion intermediates of proinsulin to an unknown extent. The possibility of cleavage of circulating proinsulin is small [16], but there is evidence that considerable amounts of proinsulin intermediates exist in the blood [17]. Similarly, insulin assay values may include intact proinsulin and its derivatives by crossreaction.…”

Section: Discussionmentioning

confidence: 98%

Serum proinsulin levels at fasting and after oral glucose load in patients with Type 2 (non-insulin-dependent) diabetes mellitus

et al. 1988

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A simple and sensitive human proinsulin radioimmunoassay system was developed using guinea pig anti-proinsulin serum, which cross-reacted neither with human insulin nor C-peptide. The recognition site of the antiserum seems to be located near the junction between the B chain and C-peptide. With this assay system, we studied the serum proinsulin concentration at fasting and after an oral 100 g glucose load in 25 healthy subjects, 21 subjects with impaired glucose tolerance and 40 patients with Type 2 (non-insulin-dependent) diabetes mellitus. At fasting, serum proinsulin was 5.8 +/- 3.3 pmol/l in normal subjects as compared to 9.5 +/- 6.9 pmol/l (p less than 0.05) in subjects with impaired glucose tolerance and 12.6 +/- 7.5 pmol/l (p less than 0.001) in diabetic patients. The molar ratio of proinsulin to insulin was also increased in subjects with impaired glucose tolerance or diabetes compared to control subjects. After a 100 g oral glucose load, serum proinsulin increased more slowly than insulin. The proinsulin response after an oral glucose load was augmented in subjects with impaired glucose tolerance and diabetes, while the insulin response decreased with the elevation of fasting plasma glucose. Diabetic patients with high fasting plasma glucose had a very poor insulin response, but the proinsulin response was similar to control subjects. There was a linear correlation between summed proinsulin values and summed insulin values, but the slope of the regression line was steeper in diabetic patients than in control subjects. There was a relative increase in serum proinsulin both in subjects with impaired glucose tolerance and diabetic patients.(ABSTRACT TRUNCATED AT 250 WORDS)

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Section: Discussionmentioning

confidence: 98%

Serum proinsulin levels at fasting and after oral glucose load in patients with Type 2 (non-insulin-dependent) diabetes mellitus

et al. 1988

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“…Identical response curves were obtained whether standards were added in veronal buffer or in serum from which endogenous proinsulin had been removed by prior incubation with immobilized rabbit polyclonal anti-insulin immunoglobulin..Ŝ ubjects. Fasting proinsulin levels and the response to a 75-g glucose load were determined in nine normal, nonobese, healthy subjects (7 men, 2 women) with an age range of 20-24 yr. Levels were determined according to the methods described above (monoclonal assay) and a previously described method (indirect assay) (2,15).…”

Section: Methodsmentioning

confidence: 99%

“…1). We have reported that an indirect two-site immunoradiometric assay that employs polyclonal antisera directed against insulin and C-pepide appears only to measure partially hydrolyzed proinsulins and not intact proinsulin (2). Proinsulin concentrations in human serum measured with this and other methods (4,5) are similar, suggesting that circulating human proinsulin immunoreactivity may be predominantly if not entirely due to hydrolyzed proinsulins.…”

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confidence: 85%

Characterization and Use in Immunoradiometric Assay of Monoclonal Antibodies Directed Against Human Proinsulin

et al. 1987

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Proinsulin in human serum is heterogenous. Existing assay methods do not distinguish between the various forms intermediate on the pathway of processing from proinsulin to insulin. We report the production of mouse monoclonal antibodies against human proinsulin with biosynthetic human proinsulin (produced by recombinant DNA technology) as immunogen. Four monoclonal antibody-producing cell lines were obtained from two separate fusions. Two of the antibodies had affinity constants for intact proinsulin of 3.1 X 10(10) and 5.6 X 10(10) L/mol and bound all forms of proinsulin and insulin. Cross-reactivity with intact proinsulin was greater than with 65-66 and 32-33 split proinsulins and des 64-65 and des 31-32 proinsulins. The third antibody had an affinity constant for intact proinsulin of 6.3 X 10(10) L/mol and reacted only with intact proinsulin, 65-66 split proinsulin, and des 64-65 proinsulin; there was no reaction with 32-33 split proinsulin, des 31-32 proinsulin, or C-peptide. The fourth antibody reacted only with intact proinsulin and had an affinity constant of 4.1 X 10(8) L/mol. We report the use of two antibodies in a sensitive two-site immunoradiometric assay for intact proinsulin. Insulin, C-peptide, and 32-33 split proinsulin did not react in the assay up to a concentration of 100 pM. The 65-66 split proinsulin and des 64-65 proinsulin reacted with a potency of approximately 55% relative to intact proinsulin. Serum proinsulin concentrations measured with this assay were compared with those determined by an alternative method that detects only split proinsulins.(ABSTRACT TRUNCATED AT 250 WORDS)

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“…The measurement of proinsulin is complicated by the presence of proinsulin conversion intermediates in the circulation (11,12,36,37). We characterized several cleaved derivatives of proinsulin for cross-reactivity with antibody 168AB (Table 1).…”

Section: Discussionmentioning

confidence: 99%

“…First, proinsulin is present in low concentration in serum (8)(9)(10), and proinsulin conversion intermediates are present in fluids and tissues (11,12). Second, other potentially cross-reactive peptides, including insulin and C-peptide, can interfere in some assays.…”

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confidence: 98%

A Rapid and Sensitive Radioimmunoassay for the Measurement of Proinsulin in Human Serum

1992

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RIA methodology is used widely to measure proinsulin in human serum. However, some RIAs lack the sensitivity necessary to quantify proinsulin in unextracted serum and require long incubation periods. We developed an RIA with a sensitivity of 3.5 pM that permits the routine measurement of proinsulin in less than 48 h. This was accomplished by using a nonequilibrium binding reaction at room temperature and PEG-assisted second antibody precipitation as the method for separating bound and free proinsulin. We obtained a specific antiproinsulin antibody by absorbing the initial goat antiserum with human C-peptide-agarose. Proinsulin produced 50% displacement of tracer at 25.6 pM, whereas both human insulin and C-peptide failed to displace tracer at concentrations as high as 1 microM. We evaluated several cleaved derivatives of proinsulin for cross-reactivity with the antibody. B-chain-C-peptide cleaved derivatives (less than or equal to 50% cross-reactivity) were more potent than A-chain-C-peptide cleaved derivatives (less than 5% cross-reactivity). However, all derivatives cleaved in the region from 56-60 failed to cross-react with the antiserum. These data indicate that a major antigenic determinant is present on the C-peptide region of proinsulin adjacent to the A-chain-C-peptide junction. After administration of an oral glycemic challenge, the mean fasting serum concentration of proinsulin in normal adults rose from 4.1 +/- 0.28 to 23.6 +/- 3.8 pM. We found a significant difference in the proinsulin concentrations in 6 adults before and after a glycemic challenge when two different antibodies were used in the RIA.(ABSTRACT TRUNCATED AT 250 WORDS)

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Proinsulin in Human Serum: Problems in Measurement and Interpretation

Cited by 27 publications

References 10 publications

Serum proinsulin levels at fasting and after oral glucose load in patients with Type 2 (non-insulin-dependent) diabetes mellitus

Serum proinsulin levels at fasting and after oral glucose load in patients with Type 2 (non-insulin-dependent) diabetes mellitus

Characterization and Use in Immunoradiometric Assay of Monoclonal Antibodies Directed Against Human Proinsulin

A Rapid and Sensitive Radioimmunoassay for the Measurement of Proinsulin in Human Serum

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