Clinical Relevance of C-peptide Indicators of β-cell function than peripheral insulin concentration. Primary indicator for evaluation of fasting hypoglycemia.

 The relationship of parathyroid hormone assay to

serum or ionized calcium levels are the best discriminators

of parathyroid activity.

PARATHYROID HORMONE (INTACT) ELISA

(Courtesy: MD Biosciences)

Intended Use

The intact-PTH ELISA is intended for the quantitative

determination of intact-PTH (parathyroid hormone) in

human serum. This assay is intended for in vitro diagnostic

use.

Summary and Explanation

PTH (parathyroid hormone, parathormone, parathyrin)

is biosynthesized in the parathyroid gland as a

preproparathyroid hormone, a larger molecular precursor

consisting of 115 amino acids. Following sequential

intracellular cleavage of a 25-amino acid sequence,

preproparathyroid hormone is converted to an intermediate, a 90-amino acid polypetide, proparathyroid

hormone. With additional proteolytic modification,

proparathyroid hormone is then converted to parathyroid

hormone, an 84 amino acid polypeptide. In healthy

individuals, regulation of parathyroid hormone secretion

normally occurs via a negative feedback action of

serum calcium on the parathyroid glands. Intact PTH

is biologically active and clears very rapidly from the

circulation with a half-life of less than 4 minutes. PTH

undergoes proteolysis in the parathyroid glands, but

mostly peripherally, particularly in the liver but also in the

kidneys and bone, to give N-terminal fragments and longer

lived C-terminal and midregion fragments. In subjects with

renal insufficiency, C-terminal and midregion PTH assays

typically give elevated PTH results, as reflected by impaired

renal clearance.

Clinical Significance

Intact PTH assays are important for the differentiation

of primary hyperparathyroidism from other (nonparathyroid-mediated) forms of hypercalcemia, such

as malignancy, sarcoidosis and thyrotoxicosis. The

measurement of parathyroid hormone is the most specific

way of making the diagnosis of primary hyperparathyroidism. In the presence of hypercalcemia, an elevated

level of parathyroid hormone virtually establishes the

diagnosis. In over 90% of patients with primary hyperparathyroidism, the parathyroid hormone will be elevated.

The most common other cause of hypercalcemia,

namely hypercalcemia of malignancy, is associated with

suppressed levels of parathyroid hormone or PTH levels

within the normal range. When intact PTH level is plotted

against serum calcium, the intact PTH concentration for

patients with hypercalcemia of malignancy is almost

always found to be inappropriately low when interpreted

in view of the elevated serum calcium. Unlike C-terminal

and midregion PTH, which typically are grossly elevated

in subjects with renal insufficiency, intact PTH assays are

less influenced by the declining renal function. PTH values

are typically undetectable in hypocalcemia due to total

hypoparathyroidism, but are found within the normal

range in hypocalcemia due to partial loss or inhibition of

parathyroid function.

Principle of the Test

The intact PTH immunoassay is a two-site ELISA [enzymelinked immunosorbent assay] for the measurement of

the biologically intact 84 amino acid chain of PTH. Two

different goat polyclonal antibodies to human PTH have

been purified by affinity chromatography to be specific for

well-defined regions on the PTH molecule. One antibody

is prepared to bind only the mid-region and C-terminal

PTH 39–84 and this antibody is biotinylated. The other

antibody is prepared to bind only the N-terminal PTH 1-34

and this antibody is labeled with horseradish peroxidase

[HRP] for detection.

Streptavidin Well - Biotinylated Anti-PTH

(39-84) -Intact PTH —HRP conjugated

Anti-PTH (1-34)

Although mid-region and C-terminal fragments are

bound by the biotinylated anti-PTH (39-84), only the

intact PTH 1-84 forms the sandwich complex necessary

for detection. The capacity of the biotinylated antibody

and the Streptavidin coated microwell both have been

adjusted to exhibit negligible interference by inactive

fragments, even at very elevated levels.

In this assay, calibrators, controls, or patient samples are

simultaneously incubated the enzyme labeled antibody

and a biotin coupled antibody in a streptavidin-coated

microplate well. At the end of the assay incubation, the

microwell is washed to remove unbound components and

the enzyme bound to the solid phase is incubated with the

substrate tetramethylbenzidine (TMB). An acidic stopping

solution is then added to stop the reaction and converts

the color to yellow. The intensity of the yellow color is

directly proportional to the concentration of intact PTH in

The Endocrine System 757

FIG. 24.8: Proinsulin and cleaved products

the sample. A dose response curve of absorbance unit vs.

concentration is generated using results obtained from the

calibrators. Concentrations of intact-PTH present in the

controls and patient samples are determined directly from

this curve.

Test Significance

Investigation is of importance in distinguishing nonparathyroid from parathyroid causes of hyper-calcemia. A

decrease in the level of ionized calcium is the primary

stimulus for PTH secretions, while a rise in calcium

inhibits secretions. This relationship is lost in hyperparathyroidism, and PTH will be inappropriately high

in relation to calcium. The C assays tend to have higher

values and are more widely accepted as better indication

of hyperparathyroidism. Creatinine is usually determined

with all PTH assays to determine kidney function.

Clinical Relevance

1. Increased PTH values are seen in:

a. Chronic renal failure. This is a cause of secondary

hyperparathyroidism

b. Pseudohyperparathyroidism. There is a primary

defect in renal tubular responsiveness to PTH

(slight increase)

c. Vitamin D deficiency (moderate)

d. Malabsorption (moderate)

e. Rickets (moderate)

f. Osteomalacia (moderate).

2. Decreased PTH values occur in nonparathyroid

hypercalcemia, as in:

a. Use of thiazide diuretics

b. Milk alkali syndrome

c. Vitamin A and D intoxication

d. Hematologic malignancies (some of them)

e. Sarcoidosis

f. Graves’ disease

g. Permanent postoperative hypoparathyroidism.

3. Increased PTH-N values occur in:

a. Pseudohypoparathyroidism

b. Secondary hyperparathyroidism

c. Primary hyperparathyroidism.

4. Decreased PTH-N values are seen in:

a. Hypoparathyroidism

b. Neoplasms

c. Nonparathyroid hypercalcemia.

5. Increased PTH-C values are seen in:

a. Pseudohypoparathyroidism

b. Secondary hyperparathyroidism

c. Primary hyperparathyroidism

d. Neoplasms.

6. Decreased PTH-C values occur in:

a. Hypoparathyroidism

b. Nonparathyroid hypercalcemia.

Interfering Factors

A. Fasting sample should be obtained:

1. Elevated blood lipids interfere with results

2. Milk ingestion will lower PTH levels.

PANCREAS

The pancreas is a mixed exocrine and endocrine gland.

The exocrine portion makes many of the digestive enzymes

necessary for gastrointestinal function. The endocrine

portion is comprised of discrete islands of cells called the

islets of Langerhans. Cells within the islets produce two

hormones that regulate the concentration of glucose in

the blood.

Insulin

It is a small protein (MW 6000 Daltons) and is composed of

two chains (A and B) held by disulfide bonds. It is secreted

only by the β-cells of the pancreas. The α-cells of the

pancreas secretes glycogen.

Insulin is secreted in a precursor form, Proinsulin. This

is cleaved to release Insulin and C-peptide. C-peptide is the

connecting peptide between the A and B chains of insulin.

All the three—proinsulin, insulin and C-peptide are found

in the blood. Estimation of these is important in different

conditions (Fig. 24.8).

758 Concise Book of Medical Laboratory Technology: Methods and Interpretations Functions of Insulin

¾ Reduces blood sugar level by stimulating uptake of

sugar by various tissues

¾ Stimulates liver to store glucose in the form of glycogen.

¾ Promotes synthesis of fatty acids in liver

¾ Stimulates uptake of amino acids, contributing to

overall anabolic effect

¾ Increase the permeability of many cells to potassium,

magnesium and calcium ions.

Clinical Relevance of C-peptide

Indicators of β-cell function than peripheral insulin

concentration.

Primary indicator for evaluation of fasting hypoglycemia.

Condition Insulin C-peptide

Insulinoma Increased Increased

Insulin producing Normal Increased

β-cell tumors

Hypoglycemia

(injected/exogenous)