Insulin

• U type, U-E type: Highly sensitive. Suitable for the measurement of low concentrations of insulin (e.g., fasting insulin level).
• S type: Enables insulin-specific measurement by suppressing cross-reactivity with proinsulin.
• H type: Suitable for the measurement of high concentrations of insulin. Use this product for the measurement of highly concentrated samples.
• RTU type: Ready-to-use kits available in 6 concentrations of standards. Diluted antibody and POD-avidin solutions are included.
• T type: A normal type, standard insulin measurement kit.

Specificity for insulin

Ordinary insulin assay kits: All ELISA kits except for S type.

Ordinary assay kits show the affinity to both insulin and proinsulin. Normally, in circulating blood, both proinsulin and processed insulin are secreted with minor ratio of proinsulin to insulin, and secretion of proinsulin is in parallel with insulin. So, in usual studies, it is enough to measure both of them by using ordinary assay kit.

Insulin-specific assay kit: S type (at present for rat insulin)

This type of kit shows a very low cross-reactivity to proinsulin (see data in specificity section), and practically can measure only insulin in the presence of proinsulin. This type of kit is useful when levels of proinsulin change owing physiological conditions. From the difference of assay values between ordinary and specific assay kits we can estimate proinsulin secretion.

From assay sensitivity

Kits of ordinary sensitivity: TMB

These types of kits are for measurement of insulin in normal state animals. In any species, a good standard curve is obtained in a range of 0.156 to 10ng/ml.

Ultra-sensitive assay kits: U type, U-E type

For rat and mouse, we are providing ultra-sensitive assay kits, U type. This type of kit is improved to be able to measure insulin levels in fasted animals with an excellent precision. The assay ranges are 25-1,500 pg/ml for rat, and 39-2,500 pg/ml for mouse. Because rat U type needs an overnight incubation, we improved the incubation period, and prepared U-E type the assay range of which is 39-2,500 pg/ml. The highly specific assay kit of S type also attained higher sensitivity than ordinary TMB the assay range of which is 100 to 10,000 pg/m.

Kit for insulin-related substance: rat and mouse C-peptide ELISA kits

We provide assay kits for C-peptide that has closely related to insulin. Amino acid sequences of C-peptides are to some extent different between rat and mouse. C-peptide is secreted with insulin after processing of proinsulin to insulin and C-peptide. C-peptide has a longer biological half-life than insulin. So, C-peptide is measured in observation of biosynthesis and secretory functions for insulin. C-peptide is also found in urine in considerably high concentration, and the level is parallel to that in plasma, so, can be applied to urinary samples.
Measurement of C-peptide is also useful in estimation of insulin secretion to culture medium of e.g. isolated islets more than measurement of insulin itself, because very often insulin is added to a culture medium to make the measurement of insulin secreted to the medium by cultured cells. As C-peptide is secreted into the medium in equi-molar base, we can estimate insulin secretion by measuring C-peptide. Our C-peptide kit recognizes both C-peptide 1 and 2, the total amount of C-peptide is measured by the kit.

What is proinsulin?

The amino acid sequence of proinsulin produced in B-cells of pancreatic islet of Langerhans is shown in the following figure (in the case of rat).

This proinsulin is processed by an enzyme at the position where two basic amino acids (K, R) moieties are present side by side shown by black arrows, yielding insulin and C-peptide (connecting peptide). Most proinsulin was processed while it passes Golgi apparatus to secretory granules, however, some remains without processing and present in secretory granules, and secreted into the blood circulation with insulin. The biopotency of proinsulin is estimated to be about 5-10% of insulin.
When we measure insulin or C-peptide in the blood, in general, we also measure the amount proinsulin because the structure of proinsulin includes those of insulin and C-peptide, if we ignore the three-dimensional structure. So, when we measures insulin by immunological method, we call the assay value IRI ( immuno-ractive insulin ) , discriminating from the real insulin level. IRI includes both insulin and proinsulin levels which reacted with the antibody.

Amount of proinsulin in IRI

Let's try to estimate the ratio of proinsulin to insulin from data for human blood samples. The molecular weight of proinsulin is about 9,400, and that of insulin is about 5,800. So, the ratio of molecular weight is 1: 0.62. We will calculate the amount of insulin by weight as 24U/mg according to international standard preparation.

Normal levels (fasting)

Proinsulin, pmol/l	Reporter
3.3 ~ 10.1 (mean:4.47)	Hampton, 1988
1.5 ~ 27 (mean:14.25)	Naylor, 1987
5.8 ±3.3	Yoshioka, 1988

If we takes the mean, it ranges 4.5 ~ 14.28 pmol/ml
1 pmol/l of proinsulin is 9400pg/ml = 9.4pg/ml
so 4.5 ~ 14.28 pmol/l = 43 ~ 134 pg/ml
Insulin equivalent from the difference in molecular weight, 27 ~ 83 pg/ml
As normal fasting level of insulin in human subject is about 10mU/ml
= 10/24 ng/ml=420pg/ml. This means 10~30% of IRI may be proinsulin.

Does ratio of proinsulin to insulin change?
According to clinical reports, the blood proinsulin ratio increases in various cases.

NIDDM(Type II diabetes mellitus)
In both fasting state and glucose administration, blood levels of proinsulin and the ratio to insulin were reported to be higher in NIDDM patients than in normal subjects. This tendency is more obvious in those patients who show high fasting blood sugar and obesity. Continuous insulin secretion caused by higher blood sugar may results in the secretion of immature secretory granules.

IDDM(Type I diabetes mellitus)
In the case of untreated subjects, blood insulin levels are low because of impaired biosynthesis of insulin. But in many cases, insulin-treated patients have produced insulin antibody to exogenous insulin, and show higher blood proinsulin levels. This may be due to the binding of proinsulin to insulin antibody which causes the retardation of metabolism of proinsulin. Even some untreated patients show higher proinsulin levels because the presence of antibody.

Obesity
High proinsulin levels are observed in obesity.
As in NIDDM, acceleration of biosynthesis-secretion process by the increase of insulin secretion may cause the release of immature secretory granules.

Insulinoma
In insulinoma patients, hyperproinsulinemia is more obvious than the increase in insulin and C-peptide levels, which is helpful in diagnosis of insulinoma.

Familial hyperproinsulinemia
In hereditary occurring hyperproinsulinemia observed in some families, IRI in secretory granules consisted mostly of proinsulin. This is caused by some abnormality of processing enzyme from proinsulin to insulin, or by possible point mutation in proinsulin gene which produces stable proinsulin against processing enzyme.

Hyperthyroidism
Increase in gluconeogenesis caused by thyroid hormones results in hyperglycemia, and promote insulin biosynthesis and release, which also increases proinsulin secretion.
As stated above, in most cases, the ratio of proinsulin to insulin may reflect the situation of insulin biosynthetic process.

Highly specific insulin ELISA kit, S-type
We are providing ordinary assay kits(T, U, UE) and high specific assay kits for insulin(S). S-type has a very low cross-reactivity to proinsulin. We tried a recovery test by adding proinsulin to assay samples. The assay results are shown below. It proved that proinsulin added to the samples only slightly increased the insulin assay value. This means we can measure insulin precisely in the presence of proinsulin.

Recovery test of proinsulin to rat serum sample n=2

Rat proinsulin added ng/ml	Assay value as insulin ng/ml	Influence Ðassay value/added (%)
0	1.00	-
1.0	1.01	1.0
2.0	1.03	1.5
4.0	1.07	1.8
8.0	1.19	2.4

Recovery test of proinsulin to mouse serum sample n=2

Mouse proinsulin added ng/ml	Assay value as insulin ng/ml	Influence Ðassay value/added (%)
0	106.0	-
100	106.9	0.9
500	112.5	1.3
5000	286.0	3.6

Comparison of assay values obtained by ordinary kit (T) and specific kit (S) with rat and mouse serum samples.

As shown in the figures, very high correlation coefficients were obtained with both rat(R=0.9946) and mouse(R=0.9868) samples. The slopes of the regression lines were 1.209 in rat and 1.198 in mouse. This means that type S kits gave about 20% lower assay values than ordinary T-type kits with both rat and mouse samples. This must be caused by improvement of specificity in S-type. Though we should be careful in thinking that the difference is directly related proinsulin, difference of 20% is a reasonable value of proinsulin ratio to IRI in human subjects.
We recommend our customers to use our highly specific insulin kit S-type in measurements of rat and mouse insulin.