Diabetes Mellitus
and the Utility of Insulin, C-Peptide and Urinary Albumin Assays

Glucose plays multiple essential roles in life processes. It is a major metabolic precursor for structural components of DNA and RNA, and serves as the primary energy source for virtually every living organism. In humans, glucoregulatory mechanisms normally maintain plasma glucose levels within a relatively narrow concentration range (70 to 150 mg/dL), despite wide variations in glucose influx and efflux associated with meals and exercise. Inadequate glucose levels (hypoglycemia) can cause profound brain dysfunction and death. Chronic, abnormally elevated levels (hyperglycemia) can lead to a number of microvascular diseases that can severely damage eyes (retinopathy), kidneys (nephropathy), nerves, and blood vessels.

Defects in insulin secretion and/or insulin action are the most common failures in the regulation of plasma glucose levels. Insulin deficiency causes hyperglycemia, a condition referred to as diabetes mellitus. The word diabetes means an excessive discharge of urine, and the term mellitus means honey. Diabetes mellitus refers to metabolic disorders that are characterized by abnormally elevated levels of sugar in the blood (hyperglycemia) and urine. Hypoglycemia is most often iatrogenic and is usually associated with side effects of diabetes treatment regimens.

Diabetes is a major cause of morbidity and the fourth leading cause of death in the US. In 1992, diabetics constituted 4.5 percent of the US population (approximately 11 million people); the disease thus represents a major public health issue.^1,2

Long-term complications of diabetes mellitus include retinopathy, the leading cause of adult blindness; and nephropathy, the leading cause of end-stage renal failure. Diabetes is also a major cause of gangrene, myocardial infarction and stroke, which result from the associated increased incidences of atherosclerotic, cardiovascular, peripheral vascular and cerebrovascular diseases.^3-6

The cost of diabetes-associated expenditures to individuals and society is enormous. In 1992, these expenditures accounted for $105 billion of the total US healthcare cost, representing approximately one in seven healthcare dollars spent.⁷

Classification of diabetes mellitus
The revised 1997 American Diabetes Association classification scheme⁸ identifies two major classes of diabetes mellitus: type 1 and type 2. These designations replace the historic nomenclature--insulin-dependent diabetes mellitus (IDDM) and non-insulin-dependent diabetes mellitus (NIDDM), respectively.

Type 1 represents approximately 5 to 10 percent of all diabetes cases. Patients present with abrupt onset of symptoms that include polyuria, polydipsia, and rapid weight loss, with peak incidence in early childhood and adolescence. Destruction of pancreatic islet cells, usually by an autoimmune process, leads to absolute insulin deficiency. These patients require insulin administration to sustain life and prevent ketosis.

Type 2 occurs mostly in patients older than 40 years of age. These patients have minimal symptoms, are not prone to ketosis, and represent approximately 90 percent of all diabetics. The abnormality is associated with impaired physiological effectiveness of insulin: levels may be normal, decreased or increased. Control of hyperglycemia may be achieved by weight loss, and may require treatment regimens involving specialized diets, ingestion of oral hypoglycemic agents or administration of insulin.

Diagnosis of diabetes mellitus
The diagnosis of diabetes mellitus depends solely on the demonstration of hyperglycemia; glucose measurements therefore serve as the front-line laboratory test. According to the 1997 revised diagnostic criteria,⁸ one of the following three conditions must be identified, and then confirmed on a subsequent day:

The armamentarium of diabetes-related laboratory tests is much broader, however. It includes tests for insulin, C-peptide, urinary albumin (microalbuminuria), glycated proteins, associated autoantibodies and genetic markers. These important laboratory tools are used for screening at-risk individuals, for characterizing disease status, or for long-term management of diabetic patients.

The focus here is on the associated physiology and clinical utility of three analytes that are generally measured by immunoassay: insulin, C-peptide and urinary albumin.

Insulin and C-peptide
Elevated blood glucose levels stimulate pancreatic beta cells to produce and release insulin into the circulation. Insulin is a polypeptide hormone that exerts an immediate hypoglycemic effect by promoting cellular uptake of glucose in muscle and adipose tissue, and by suppressing endogenous glucose production. Its function is to shift excess extracellular glucose to intracellular storage sites, mostly in the form of glycogen. Counterregulatory hormones--glucagon, epinephrin, growth hormone and cortisol--have an opposite effect. They increase plasma glucose levels by stimulating the breakdown of glycogen to glucose (glycogenolysis) and the synthesis of glucose (gluconeogenesis).

The immediate precursor of insulin is proinsulin (MW 9 kDa), a single-chain polypeptide consisting of 86 amino acids with three disulfide bridges. Proinsulin is the major storage form of insulin in the Golgi complex of the pancreatic beta cells. Proteolytic cleavage produces insulin (MW 6 kDa), which consists of 51 amino acids in two chains joined by two disulfide bridges; and the connecting peptide (C-peptide; MW 3 kDa), a single polypeptide chain containing 31 amino acids. (See Figure 1.) Equimolar amounts of insulin and C-peptide are then secreted into the circulation. Circulating C-peptide concentrations are approximately 5- to 10-fold higher than those of insulin, however, as a result of the much longer half-life of C-peptide (approximately 35 minutes, compared to 5 to 10 minutes for insulin).

Human Proinsulin
Figure 1. Proinsulin undergoes enzymatic cleavage to yield insulin and C-peptide.

For an enlarged view, click on the image.

The primary clinical application of insulin and C-peptide measurements is in the differential diagnosis of fasting hypoglycemia. Elevated levels suggest an insulinoma. However, elevated insulin results may be due to factitious hypoglycemia (possibly surreptitious insulin administration), which must be ruled out before results are accepted as evidence for an insulinoma. C-peptide measurements are used to resolve the uncertainty. Levels are low in cases of exogenous insulin administration--commercial insulin preparations do not contain C-peptide--but are elevated in hyperinsulinemia resulting from pancreatic secretory activity.

Insulin and C-peptide measurements may be relied on to distinguish between type 1 and type 2 diabetes by assessing pancreatic reserve.⁹ Furthermore, insulin determinations have been used for distinguishing between insulin-requiring type 2 diabetics and type 2 diabetics whose disease can be controlled with diet and exercise alone. Subjects with peak plasma insulin levels greater than 60 µIU/mL during a glucose tolerance test have been reported to be unlikely to develop microvascular complications and likely to be controlled by diet alone. In contrast, peak insulin levels below 40 µIU/mL reportedly identify patients who require insulin treatment and have a higher probability of developing microvascular disease.¹⁰ C-peptide determinations have been used in a similar manner. For example, glucagon-stimulated C-peptide levels greater than 1.8 ng/mL have been reported to identify type 2 diabetics who could be managed without insulin treatment. In contrast, levels less than 0.5 ng/mL reportedly identify type 1 patients who require insulin treatment.¹¹ C-peptide measurements, both basal and glucagon- or glucose-stimulated, have also been used to reevaluate the necessity for continuing insulin treatment regimens in patients who may be better managed with diet alone. Furthermore, the demonstration of glucagon- or glucose-stimulated residual beta-cell function in type 1 diabetics, as evidenced by a significant rise in C-peptide values, is considered a better prognostic indicator than no C-peptide response at all.

Insulin-treated patients present special problems, as insulin assays do not distinguish between exogenous and endogenous insulin. Furthermore, such patients are likely to have developed anti-insulin antibodies, which interfere with insulin immunoassays. Results may be either falsely elevated or falsely decreased, depending on the characteristics of the insulin assay. In contrast, C-peptide assays provide a reliable indication of beta-cell function despite the presence of exogenous insulin and insulin antibodies.

Urinary albumin (microalbuminuria)
Diabetes is the most common cause of renal failure in the US. Both type 1 and type 2 diabetics are at high risk for developing diabetic nephropathy with the associated threats to quality of life and survival. Overt nephropathy is likely to progress within 5 years to end-stage renal failure requiring dialysis or renal transplantation.¹²

Historically, diabetic nephropathy was recognized only when routine urinalysis revealed protein levels exceeding the detection limits of these assays, i.e., 200 to 300 mg/L. However, approximately 17 years ago, immunoassays for measuring urinary albumin became available which allowed for far more sensitive determinations. These opened up a new window onto the diabetic kidney, exposing diabetic nephropathy as a continuum that starts long before urinary protein levels exceed 200 to 300 mg/L.¹³ The term microalbuminuria was coined to refer to the early phase of diabetic nephropathy which is characterized by abnormally elevated albumin excretion rates; albumin levels nevertheless remain below the 200 mg/L threshold. Microalbuminuria has been defined¹⁴ in terms of abnormally elevated excretion rates, albumin/creatinine ratios, or albumin concentrations as follows:

Many studies have confirmed that microalbuminuria is an early predictor of the renal and cardiovascular complications of diabetes.¹⁵ It identifies at-risk patients before major organ damage has taken place, allowing for early intervention with preventative and cost-saving measures. For example, intensive blood glucose control has been reported to reduce the incidence of microalbuminuria and progression to overt renal disease.¹⁶

Blood pressure is an important, modifiable risk factor for the progression of renal disease. Antihypertensive treatment regimens postpone end-stage renal failure in patients with overt diabetic nephropathy. Furthermore, initiation of early antihypertensive treatment lowers urinary albumin excretion rates and appears to postpone or prevent progression to nephropathy. Although final conclusions await the completion of long-range studies, indications are that treatment strategies guided by urinary albumin determinations can improve patient outcome, save lives, and allow significant reduction in associated healthcare costs.¹⁷ Not surprisingly, recommendations for routinely assessing microalbuminuria in diabetic patients at least once a year, if not more often, have gained wide acceptance.^13,14

Summary
Diabetes mellitus comprises a group of common but serious and costly disorders characterized by chronic hyperglycemia due to defective insulin secretion and/or action. The two major classifications of the disease are type 1, which involves pancreatic beta-cell destruction, usually by an autoimmune process; and type 2, the more common, which is characterized by the impaired physiological effectiveness of insulin.

Diabetes mellitus is diagnosed by the demonstration of hyperglycemia through the use of random or fasting plasma glucose determinations, or by an oral glucose tolerance test. Once diabetes is diagnosed, assays for insulin and C-peptide can be used to differentiate type 1 from type 2 diabetes and, among type 2 diabetics, to distinguish those who require insulin treatment from borderline cases who can be managed with changes in diet and exercise alone. Insulin determinations are also useful for identifying patients at risk of developing the complications of diabetes, such as microvascular disease.

Measurement of C-peptide, a by-product of insulin production, provides an index of endogenous insulin production and pancreatic beta-cell function. C-peptide levels, unaffected by exogenous insulin or insulin autoantibodies, can also be used to distinguish between insulinoma and contrived hypoglycemia, such as might be caused by surreptitious insulin injection.

Detection of microalbuminuria can identify diabetic patients at risk for renal and cardiovascular complications before the occurrence of major organ damage. Lowering albumin excretion rates with antihypertensive therapy or other measures can either postpone or prevent progression to clinical nephropathy, thereby saving lives, improving patient outcomes, and reducing costs.

Blood tests for insulin and C-peptide as well as urinary albumin tests are commonly performed by immunoassays that provide valuable information to assist the physician in the identification, characterization and management of the diabetic patient.

DPC offers assays for the measurement of insulin, C-peptide and urinary albumin on the automated IMMULITE^® platform. Assay characteristics are summarized below.

References

1. Prevalence and incidence of diabetes mellitus: United States, 1980-1987. MMWR Mortality and Morbidity Weekly Report 1990;39:809-12.

2. Harris MI. Impaired glucose tolerance in the U.S. population. Diabetes Care 1989;12:464-74.

3. Nathan DM. Long-term complications of diabetes mellitus. N Engl J Med 1993:328:1676-85.

4. Davis MD. Diabetic retinopathy: a clinical overview. Diabetes Metab Rev 1988;4:291-322.

5. Geiss LS, Herman WH, Goldschmid MG, DeStefano F, Eberhardt MS, Ford ES, et al. Surveillance of diabetes mellitus: United States, 1980-1989. MMWR Mortality and Morbidity Weekly Report 1993;42:1-20.

6. National Diabetes Advisory Board. Diabetes in the 1980¹s: challenges for the future. US Department of Health and Human Services, Public Health Service. NIH Publication No. 82-2143. Washington, DC: Government Printing Office, 1982.

7. Rubin RJ, et al. Health care expenditures for people with diabetes mellitus, 1992. J Clin Endocrinol Metab 1994;78:809 A-F.

8. American Diabetes Association. Position statement: Report of the expert committee on the diagnosis and classification of diabetes mellitus. Diabetes Care 1997;20:1183-201.

9. Katzeff HL, Savage PJ, Barclay-White B, Nagulesparan M, Bennett PH. C-peptide measurement in the differentiation of type 1 (insulin-dependent) and type 2 (non-insulin-dependent) diabetes mellitus. Diabetologia 1985;28:264-8.

10. Turkington RW, Weindling HD. Insulin secretion in the diagnosis of adult-onset diabetes mellitus. JAMA 1978;240: 833-6.

11. Hoekstra JBL, van Rijn HJM, Thijssen JHH, Erkelens DW. C-peptide reactivity as a measure of insulin dependency in obese diabetic patients treated with insulin. Diabetes Care 1982;5:585-91.

12. Hans-Henrik P, et al. Diabetic nephropathy. In: Brenner MB, ed. The kidney. 5th ed. Philadelphia: WB Saunders, 1996:1864.

13. Watts NB. Albuminuria and diabetic nephropathy: an evolving story. Clin Chem 1991;37:2027-8.

14. Mogensen CE, et al. Prevention of diabetic renal disease with special reference to microalbuminuria. Lancet 1995;346:1080-84.

15. Messent JWC, Elliott TG, Hill RD, Jarrett RJ, Keen H, Viberti G. Prognostic significance of microalbuminuria in insulin-dependent diabetes mellitus: a twenty-three year follow-up study. Kidney Int 1992;41:836-9.

16. The Diabetes Control and Complications Trial Research Group. The effect of intensive treatment of diabetes on the development and progression of long-term complications in insulin-dependent diabetes mellitus. N Eng J Med 1993;329: 977-86.

17. Borch-Johnsen K, et al. Is screening and intervention for microalbuminuria worthwhile in patients with insulin dependent diabetes? BMJ 1993;306:1722-5.

Additional Reference

Sacks DB. Carbohydrates. In: Tietz textbook of clinical chemistry. 3rd ed. Burtis CA, Ashwood ER, editors. Philadelphia: W.B. Saunders, 1999: 750-808.