News Summary
According to researchers at Stockholm's Karolinska Institute, c-peptide, the connecting piece of the basic insulin structure, may be useful in preventing the onset of the complications common to diabetes sufferers. They report that, when injected with usual insulin doses, c-peptide limits the likelihood of diabetes-related complications, such as vision problems, loss of nerve sensitivity and kidney problems. While the researchers admit that very little is known about the potential function or absolute benefit of c-peptide, their early studies have shown encouraging results. The researchers also state that research groups in the United States and Germany have observed similar benefits.

IDC Commentary
The reports of the benefits of c-peptide have been reported in small numbers for several years. To date, no large research trial or scientific report has confirmed the news reports outlined above.

While c-peptide may be among a number of potentially helpful treatments to reduce the risk of diabetes complications, the benefits are still not proven. The small research studies outlined in this news report have not yet been critically reviewed by other scientists. Furthermore, the researchers do not comment on the occurrence of complications in patients with type 2 diabetes, many of whom have normal or even elevated levels of c-peptide in their blood.

The most important treatments to prevent or reduce the complications of diabetes include better blood glucose control, control of high blood pressure, regular eye and foot examinations, and control of cholesterol levels. While newer treatments will undoubtedly add to our list of helpful therapies, at present, c-peptide has not been proven to prevent the complications of diabetes.

If you have questions about this or other diabetes treatments, talk to your health care provider.

A Surprise Discovery That May Help Diabetics

By WILLIAM ALLEN
St. Louis Post-Dispatch

A chemical once thought to be a minor supporting actor on the stage of human physiology may prevent or even reverse some of the most devastating effects of diabetes, Washington University researchers have found.

The chemical, called C-peptide, is normally produced by the body but is scarce or absent in people with diabetes. In a major scientific surprise, the researchers found that C-peptide repaired damaged blood vessels and nerves in diabetic rats.

The discovery raises hopes that the 16 million Americans with diabetes might avoid the heart disease and other debilitating and often fatal complications.

"It could have a real impact on their lifestyle," said Dr. Joseph Williamson, a researcher with the university's School of Medicine and one of the authors of the study, which was reported in the weekly journal Science.

Two former skeptics of the role of C-peptide praised the work. It raises "tantalizing possibilities," Drs. Donald Steiner and Arthur Rubenstein of the University of Chicago wrote in a separate report in Science. Said Dr. Gerald Bernstein, president-elect of the American Diabetes Association: "It's fascinating, and it opens up a big, big door."

In diabetes, the body does not produce or respond properly to insulin. The resulting high blood sugar can severely damage the heart, blood vessels, kidneys, eyes and nerves.

Diabetes is the fourth-leading cause of death by disease in the United States and the leading cause of blindness in people ages 20 to 74. More than 178,000 people die each year from the disease and its complications.

Nerve damage related to the disease results in foot or leg amputations in 54,000 people a year. People with diabetes are up to four times more likely to have a heart attack or stroke than someone without the disease.

The discovery at Washington University applies to both kinds of diabetes ╢ one that requires daily injections of insulin and the other, which is controlled with or without a pill.

The discovery comes out of a field of science where researchers focus on the interactions of chemicals that run the basic machinery of cells in the body. Among these chemicals are ones known as proteins.

C-peptide is a small protein. Scientists have long known that it helped assemble insulin, a much larger protein.

That assembly goes on in the pancreas, which secretes insulin into the bloodstream when needed. Insulin helps carry energy, in the form of sugar, from the bloodstream into cells. Once inside, the sugar helps power the cells.

Until now, C-peptide was thought to be a useless byproduct of the process, "shavings from the carpenter's bench," as scientists put it.

But the new research shows that C-peptide enters the bloodstream and teams up with insulin to regulate indispensable processes in cells. When diabetes disrupts these processes, nerve cells and cells that line the blood vessels are damaged, setting the stage for potentially fatal complications.

"Perhaps the most surprising finding was that this small protein turns out to have significant biological effects that are very beneficial in terms of preventing or reducing the effects of nerve damage and cardiovascular disease," Williamson said.

Another key discovery was that C-peptide apparently affects the cell without fitting into a structure called a receptor, a step long thought to be necessary for all proteins. This new insight may cause scientists to entirely re-think ideas about how proteins of all kinds affect cell function.

The study was led by the St. Louis researchers but also included researchers from Eli Lilly and Co. It was paid for by the pharmaceutical company, the National Institutes of Health and the Kilo Diabetes and Vascular Research Foundation.

(Distributed by Scripps Howard News Service.)

July 28, 1997

C-Peptide (ICMA)

FEATURES

• Highly sensitive two-site immunochemilumino-metric assay (ICMA) specific for human C-peptide.
• A useful laboratory test for monitoring beta-cell function.
• Values correlate closely with endogenous insulin secretion during glucose tolerance testing.
• Laboratory aid for the detection of surreptitious insulin injection.

INTRODUCTION

Insulin is formed by enzymatic cleavage of a single-chain high molecular weight precursor, proinsulin, within the pancreatic beta-cell. The residual connecting peptide, C-peptide, is stored with insulin in secretory granules, where it remains until stimulation of insulin secretion. C-peptide and insulin are released into the circulation in equimolar quantities, along with small amounts of proinsulin and heterogeneous cleavage fragments. The peripheral levels of C-peptide provide a valid index of beta-cell secretory activity. In cases where insulin levels are not helpful due to interference by exogenous insulin or insulin antibodies, the determination of C-peptide provides an accurate evaluation of beta-cell function.

CLINICAL SIGNIFICANCE

C-peptide determinations are useful in the evaluation of beta-cell secretory activity in a number of clinical situations. Because of the lack of interference by insulin and insulin antibodies, the most obvious application of C-peptide measurement is in cases where the assay of insulin would normally be used. In diabetic patients, the long-term administration of insulin often leads to the formation of insulin antibodies. C-peptide levels have been used to evaluate residual beta-cell function in the diagnosis and long-term management of diabetic patients.² A number of patients recover from transient forms of diabetes mellitus,³ and C-peptide levels have been applied in evaluating the insulin requirements of such patients as well.

C-peptide measurements have also been utilized with simultaneous glucose measurements in the diagnosis of insulinomas in both normal and diabetic individuals after prolonged fasting.⁴ After total pancreatectomy, C-peptide measurements have been used for assessing the presence of residual tissue, tumor recurrence or functional metastases. C-peptide determinations have also been employed in the diagnosis of surreptitious injection of insulin.⁵

METHODOLOGY

Numerous reports concerning C-peptide immuno-reactivity have appeared in the literature, and the serum C-peptide levels determined by different investigators have varied considerably. The basis for these discrepancies has not been completely resolved, but lies in both the assay methodology and in the molecular nature of C-peptide itself. Assay procedures for C-peptide have employed a number of different preparations for the production of antisera, as standards and as analogues for radioiodination. The use of combinations of these reagents in various laboratories has given rise to a number of assays with different immunoreactive characteristics. Variable results may be obtained with different antisera, even when the standards and radioiodinated tracers are equivalent.⁷

Endocrine Sciences has developed a specific two-site immunochemiluminescent assay (ICMA) which yields results highly correlated with our well-characterized RIA method. In this new method, patient samples are sandwiched with a solid-phase monoclonal antibody and a second monoclonal antibody conjugated to a chemiluminescent label. The C-peptide concentration is quantitated by chemiluminescent signal and compared to synthetic 31-amino acid C-peptide standard. Sensitivity of this assay is 0.025 ng/ml. The intra-assay and interassay variations are 5.8% and 7.7%, respectively.

This assay exhibits no cross-reactivity with insulin or insulin antibodies. Cross-reactivity with proinsulin is 60% on molar basis. However, proinsulin concentration represents less than 5% of C-peptide concentration in samples obtained from fasting or post glucose stimulation. Thus, the cross-reactivity with proinsulin does not significantly affect the C-peptide results. For patients suspected to be hyperproinsulinemic, the specific measurement of proinsulin (available at Endocrine Sciences) should be performed.

EXPECTED VALUES

Using Endocrine Sciences’ assay, values are reported for normoglycemic adults during fasting conditions, after 75 gm glucose load (Glucola) and after mixed meal (Sustacal). Following an overnight fast, the mean C-peptide level was 1.1 + 0.3 ng/ml (M + S.D.) with a range of 0.4 - 2.1 ng/ml. Two hours post Sustacal, values increased significantly to 1.8 + 0.9 ng/ml (M + S.D.) with a range of 1.2 - 3.4 ng/ml. Values two hours post glucose were 3.0 + 1.0 (M + S.D.) with a range of 2.0 - 4.5 ng/ml. These results are shown in Figure 1.

NORMAL C-PEPTIDE LEVELS

NORMAL VALUES

SPECIMEN REQUIREMENTS

• Adult: 1.0 ml EDTA plasma. Separate within one hour. Store and ship frozen in plastic vial.
• Pediatric: 0.5 ml EDTA plasma. Collect as indicated above.
• Minimum Pediatric: 0.2 ml EDTA plasma. Collect as indicated above. Please note: This volume does not permit repeat analysis.

SAMPLE COLLECTION

Samples for C-peptide determination must be collected and stored carefully. We recommend that all samples for C-peptide, glucagon, insulin and proinsulin be collected in EDTA (lavender top) venipuncture tubes. Samples should be collected, centrifuged and the plasma frozen without delay. Hemolysis significantly reduces measurable C-peptide and should be avoided.

STABILITY

Long-term sample stability is measurably improved in EDTA plasma. Samples should not be subjected to repeated freezing and thawing. For prolonged storage, samples should be kept at -70╦C. Stability studies presented in Figure 2 were performed under carefully controlled conditions on samples from normal subjects. Stability of samples from patients with various disease states may be considerably reduced due to proteolytic degradation of C-peptide.

STABILITY OF C-PEPTIDE

Effect of Anticoagulant and Storage Conditions

* RT = room temperature

REFERENCES

1. Horowitz DL, Starr JI, Muke ME, Blachard WG and Rubenstein AH. Proinsulin, insulin and C-peptide concentrations in human portal and peripheral blood. J Clin Invest. 55:1278-1283, 1975.
2. Yue DK, Baxter RC and Tuttle JR. C-peptide secretion and insulin antibodies as determinants of stability in diabetes mellitus. Metab. 27:35-44, 1978.
3. Block, MB, Rosenfield RL, Mako ME et al. Sequential changes in beta-cell function in insulin treated diabetic patients assessed by C-peptide immunoreactivity. N Engl J Med. 288:1144-1148, 1973.
4. Ohneda A, Sukal R, and Goto Y. C-peptide in patients with insulinoma in proinsulin, insulin and C-peptide, S Baba, T Kaneko and N Yahaihara, eds. Excerpta Medical. Amsterdam/Oxford. pp 385-390, 1979.
5. Service FJ. Factitial hypoglycemia. The Endo. 2:173-176, 1992.
6. Kuzuya H, Blix DM, Horwitz DM, Rubenstein AM, Steiner DR, Binder C and Faber OK. Heterogeneity of circulating C-peptide. JCEM. 44:952-962, 1977.

TEST CODE: 104 Rev. 7/96

Copyright c 1997 Endocrine Sciences Inc.

Prevention of Vascular and Neural Dysfunction in Diabetic Rats by C-Peptide
Y. Ido, A. Vindigni, K. Chang, L. Stramm, R. Chance, W. F. Heath, R. D. DiMarchi, E. Di Cera, J. R. Williamson^*

C-peptide, a cleavage product from the processing of proinsulin to insulin, has been considered to possess little if any biological activity other than its participation in insulin synthesis. Injection of human C-peptide prevented or attenuated vascular and neural (electrophysiological) dysfunction and impaired Na⁺- and K⁺-dependent adenosine triphosphate activity in tissues of diabetic rats. Nonpolar amino acids in the midportion of the peptide were required for these biological effects. Synthetic reverse sequence (retro) and all-D-amino acid (enantio) C-peptides were equipotent to native C-peptide, which indicates that the effects of C-peptide on diabetic vascular and neural dysfunction were mediated by nonchiral interactions instead of stereospecific receptors or binding sites.

Y. Ido, K. Chang, J. R. Williamson, Department of Pathology, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110, USA.
A. Vindigni and E. Di Cera, Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110, USA.
L. Stramm and R. Chance, Endocrine Division, Lilly Research Labs, Lilly Corporate Center, Indianapolis, IN 46285, USA.
W. F. Heath and R. D. DiMarchi, Research Technologies and Protein, Lilly Research Labs, Lilly Corporate Center, Indianapolis, IN 46285, USA.
^* To whom correspondence should be addressed.

SCIENCE Volume 277, Number 5325, Issue of 25 July 1997, pp. 563-566