Clinical and Laboratory Evaluation of a New Specific Point-of-Care Test for Intact Proinsulin

Abstract

Background:

Intact proinsulin is a biomarker for pancreatic ß-cell dysfunction. In large prospective studies in nondiabetic subjects, elevated intact proinsulin predicted development of type 2 diabetes and/or macrovascular events up to 7 years in advance. This study was performed to evaluate a new semiquantitative lateral flow-based point-of-care rapid test (POCT) for elevated intact proinsulin (cutoff: 15 pmol/L). The test requires 10 µL of capillary whole blood, with visual readout after 5 minutes. It is best applied at 2 hours after a glucose challenge or a meal.

Methods:

POCT results were obtained by health care professionals from 60 patients and healthy subject (33 female, 27 male, 28 type 2 diabetes, age: 53.6 ± 12.3 years). An additional venous blood sample was obtained from all participants for measurement of intact proinsulin by means of a quantitative ELISA reference method (TecoMedical, Sissach, Switzerland).

Results:

Elevated intact proinsulin levels (>15 pmol/L) were determined by the reference method in 26 participants, of whom 22 were also positive with the POCT (sensitivity: 85%). All 34 subjects with low intact proinsulin levels were tested negative by the POCT (specificity: 100%).

Conclusions:

The test successfully detected elevated postprandial intact proinsulin levels in 85% of the tested subjects and no false positive test result occurred. This POCT can therefore serve as a simple screening tool for identification of patients with prevalent ß-cell dysfunction, who are at high risk for development of type 2 diabetes and/or macrovascular events within the next 5-7 years.

Intact proinsulin is produced by pancreatic ß-cells, when the genetically driven ß-cell dysfunction, which is associated with development of type 2 diabetes, reaches a later stage (III) of deterioration.^{1, 2} In patients with type 2 diabetes, elevation of intact proinsulin has been described and confirmed to be a highly specific indicator of clinically relevant insulin resistance.^3,4 Intact proinsulin is able to lower glucose levels and may be prevalent in a nondiabetic subject with ß-cell dysfunction for several years before glucose metabolism deteriorates.⁵ In previous and recent prospective epidemiological studies in nondiabetic patients, elevated fasting and postmeal intact proinsulin levels have consequently been identified as an excellent predictor of consecutive type 2 diabetes development within 2-7 years.^6-9

However, binding of proinsulin to insulin receptors at the endothelial cells in diabetic and nondiabetic subjects is also promoting of macrovascular disease by activation of the MAP-Kinase pathway.¹⁰ This postreceptor signal-transduction pathway is known to result in endothelin-I secretion, release of plasminogen-activator-inhibitor I, vascular inflammation, and atherosclerosis.¹¹ In consequence, endogenously and exogenously derived elevated proinsulin concentrations are related to atherosclerosis and cardiovascular disease. Elevated intact and total proinsulin levels in the blood have therefore been identified as predictors of myocardial infarctions and stroke in people with or without diabetes in large-scale prospective clinical and epidemiological trials.^12-18

Intact proinsulin is in any case an interesting option for economic large-scale population screenings and diabetes awareness campaigns. However, and despite the excellent and annually growing evidence about its predictive diagnostic value, the biomarker has been more or less ignored by the scientific community, and has not yet been implemented into national or international diabetes diagnostic and care guidelines. Next to the lack of information about the biomarker itself, 1 reason for the low acceptance may be the unavailability of the biomarker in the test portfolio of routine laboratories in many countries. According to our knowledge, Germany is the only country where tests for intact proinsulin are currently offered by routine laboratories on a nationwide basis.

One solution to give interested health care providers the opportunity to access the biomarker and use it as a proof of ß-cell dysfunction in high-risk populations would be to provide a simple intact proinsulin-based screening tool, which does not require any specific laboratory equipment and special performance knowhow. A first test to serve this purpose has now been developed (PreDiabetes Care™, InsulinNG, Naples FL, also BelDia Pre®, PharmACT, Klein Machnow/Berlin, Germany). With a similar functionality and test procedure like a point-of-care pregnancy test, the test is supposed to be used for screening of high-risk subjects in daily practice. A positive test result should lead to further and in-depth diagnostic evaluation of the patient’s metabolic and vascular status. I may also serve to motivate patients to take appropriate measures (eg, lifestyle changes) to avoid or delay the development of type 2 diabetes and its associated macrovascular complications.

The purpose of this study was to investigate the analytical and laboratory performance of this first-generation point-of-care test (POCT) for assessment of elevated intact proinsulin levels indicating ß-cell dysfunction in comparison to a laboratory reference ELISA method.

Patients and Methods

The test performance and sample collection were conducted in accordance with local ethical and regulatory requirements in the context of an approved clinical study protocol. Patients gave written informed consent prior to test performance and venous blood draw. The intact proinsulin POCT was performed, and the EDTA whole blood sample was drawn, about 1 hour after the patient had his or her last major meal (breakfast or lunch).

The POCT is based on a lateral-flow technology. A scheme of this sandwich immunoassay is provided in Figure 1. The assay is using proprietary antibodies, and employs gold-labeled detection antibodies for final visualization of the antibody binding to the control and test line. It requires 10 µl of capillary whole blood, which can be obtained from a finger-prick and using lancinating devices similar to those used with patient self-test blood glucose monitoring systems. A ready to use disposable lancing device is provided in the POC test kit. The blood is transferred to the sample pad by means of a transfer pipette, which is also provided in the test kit. After complete sample transfer, 2 drops of a running buffer are applied to the sample pad and the test is allowed to incubate for 5 minutes at room temperature. After 5-6 minutes, the test is read out in a visual manner. A control line (C in Figure 1) indicates that the test has been properly performed and will provide a valid result. A second test line (T in Figure 1) is used to identify the proinsulin concentration of the test sample. If this second line is visible (weakly or more prominently), the test indicates an intact proinsulin concentration in the capillary blood sample of 15 pmol/L or higher and is indicative for stage III ß-cell dysfunction as defined previously.^1,2 A readout after more than 8 minutes can result in wrong positive results due to an aging process of the involved chemicals. It is recommended to make a photo for result documentation. For this evaluation study, the POCT was performed and analyzed by trained health care professionals.

Figure 1.

Schematic design of the point-of-care lateral-flow test for assessment of intact proinsulin.

A quantitative reference intact proinsulin result was determined by means of an ELISA reference method (TecoMedical, Sissach, Switzerland) as described previously¹⁹ using a 96-well microtiter plate and regular laboratory equipment. The assay was performed in accordance with the manufacturer’s protocol and using ISO 9001 compliant standard operating procedures.

Statistical Methods

Statistical analyses were performed by means of descriptive methods only. Sensitivity was calculated as the number of positive samples with values > 15 pmol/L in the ELISA test, which were also analyzed as positive with the POCT. Specificity was calculated based on the number of wrong positive POCT results in samples with intact proinsulin concentrations <15 pmol/L as determined by the ELISA reference method.

Results

In total, 60 subjects were enrolled into this evaluation protocol (27 male, 33 female, 28 type 2 diabetes, age: 53.6 ± 12.3 years, BMI: 30.4 ± 2.7 kg/m²). The patient characteristics of the subjects with and without diabetes are provided in Table 1. Patients without known diabetes were in the mean about 9 years younger. However, there was no significant difference with respect to the mean BMI values between the groups.

Table 1.

Patient Characteristics, POCT Results, and ELISA Test Results for the Entire Cohort and the Subgroups

	All	Known type 2 diabetes	Control
N	60	28	32
Gender
Male	27	12	15
Female	33	16	17
Age	53.6 ± 12.3	58.1 ± 8.7	49.5 ± 13.7*
BMI	30.4 ± 2.7	30.8 ± 2.7	30.1 ± 2.7
Obesity
BMI ≥ 30 kg/m2	31 (52%)	17 (61%)	14 (44%)
BMI < 30 kg/m2	29 (48%)	11 (39%)	18 (56%)
POCT
Positive	22	16	6
Negative	38	12	24
ELISA	12.4 ± 10.5	18.0 ± 9.6	7.6 ± 8.8**

^*P < .01 vs control. **P < .001 vs control.

All POC tests were performed in a valid way with visible control lines. Typical examples each for a positive and a negative POCT result is shown in Figure 2. The POCT result was positive in 22 subjects (36.7%). The reference ELISA revealed intact proinsulin concentrations > 15 pmol/L in 26 subjects (43.3%). The mean result for all samples was 12.4 ± 10.5 pmol/L. All samples tested with an intact proinsulin concentration < 15 pmol/L were also tested negative in the POCT. Only 4 samples tested > 15 pmol/L in the ELISA test were not positive with the POCT. In all 4 cases, the numerical intact proinsulin value was close to the cutoff value (15.8-17.3 pmol/L). These results are graphically displayed in Figure 3. They indicate a specificity of the POCT of 100% and a sensitivity of 87.5% in comparison to the ELISA test.

Figure 2.

Picture of a positive and a negative POCT result for intact proinsulin.

Figure 3.

Sensitivity and specificity of the POCT in comparison to the ELISA reference method.

It is of note that in 6 cases, the POCT results were positive in nondiabetic subjects. In all cases, the positive POCT result was confirmed by the ELISA test result, indicating stage III ß-cell dysfunction. These patients were overweight to obese (BMI range: 28.4-36.1 kg/m²), and their age range was from 49 to 71 years. In 4 of the 6 cases, a family history of type 2 diabetes was known for 1 of the parents and macrovascular disease was prevalent in the family history of the other 2 subjects. All 6 subjects with the positive POCT value agreed to enroll into further diagnostic and lifestyle intervention programs.

Discussion

According to the most recent published statistics, age-standardized diabetes prevalence in adults has increased, or at best remained unchanged since 1980, in every country on the globe. Together with population growth and ageing, this rise has led to a near quadrupling of the number of adults with diabetes worldwide. The burden of diabetes, both in terms of prevalence and number of adults affected, has increased faster in low-income and middle-income countries than in high-income countries.²⁰ These alerting numbers support the requirements for better ways to identify patients at risk for disease manifestation. Based on the underlying pathophysiological deteriorations, measurement of intact proinsulin provides a simple and efficient tool to identify patients, who are in the real process of disease development and not just having a certain risk based on family history, body weight, or waist-to-hip ratio.

In fact, there are only 3 reasons known why a human subject may have elevated intact proinsulin values in the plasma. First, the subject may suffer from a very rare and benign tumor of the pancreas, called proinsulinoma, which is a very rare disease and clinically associated with frequent episodes of hypoglycemia—and is easy to detect.^21,22 Second, the patient may suffer from type 1 diabetes and is in a situation close to clinical manifestation, where the pancreas secretes all possible means to lower glucose, including proinsulin.^23,24 Finally, and most likely, the subject is in the process of developing type 2 diabetes and only the glucose-lowering effect of intact proinsulin prevents disease detection as glucose remains normal due to a glucose lowering effect of the in-mature precursor molecule.

This glucose lowering effect has led to an attempt to develop proinsulin as an antidiabetic drug in the 1980s. The phase I results from this development demonstrated that proinsulin has about 10%-20% of the hypoglycemic effect of insulin. During the double-blind phase II studies, occurrence of macrovascular events in newly manifested diabetes patients exclusively in the proinsulin arm led finally to premature termination of the study and most likely also to the ultimate stop of the drug development.¹⁰ The glucose lowering effect of proinsulin and the fact that only a third of the obese population finally develop type 2 diabetes²⁵ explains the added predictive value of measuring intact proinsulin to measuring glucose or obesity alone for detection of prediabetes.

Several prospective population-based cohort studies in nondiabetic subjects have impressively confirmed the value of intact proinsulin measurement as an indicator for diabetes development.^6-9 In our opinion, it is of major practical clinical relevance that patients with ß-cell dysfunction and in the process of diabetes development are also developing macrovascular disease, while having still normal fasting and postchallenge blood glucose values. Elevated proinsulin has therefore been identified as a highly sensitive marker for prediction of macrovascular events as well.^11-18 This is the reason, why a positive proinsulin test in a nondiabetic subject should in any case also lead to further diagnosis of the vascular status.

Based on this background information, it is compelling to use intact proinsulin as an indicator of disease development and potentially redefine the stage of “prediabetes” even earlier and in addition to the glucose values. For this purpose, however, it is required to provide access to the determination of this biomarker to family doctors and general practitioners on a global basis.

These considerations lead to the development of the point-of-care rapid test for assessment of elevated intact proinsulin, which was evaluated in this study. The test achieved excellent results with respect to sensitivity (84.6%) and specificity (100%) in comparison to an ELISA reference method. It is therefore eligible for screening purposes and a positive result should have a variety of further consequences for the affected individual subject.

The following recommendations are based on our experience, while working with intact proinsulin as a diabetes biomarker for more than a decade.

1. If the screening test is positive, it is may be recommended to obtain an additional quantitative ELISA result as confirmation of the initial POCT findings, and also to get a baseline value to monitor the success of potentially following interventions.

2. The POCT-positive patient should be subjected to an oral glucose challenge (oral glucose tolerance test, OGTT). In many cases, this test will reveal an unknown diabetes or impaired glucose tolerance, and the patient should then be enrolled into an appropriate diabetes treatment program.

3. In case of a negative-OGTT, POCT-positive patient, the patient should be encouraged to perform lifestyle changes as an early intervention to delay disease manifestation. Diet and exercise have demonstrated to prevent disease onset and reduce proinsulin levels.^26,27

4. A proinsulin-positive/OGTT-negative subject should also be encouraged to regularly measure glucose (eg, on a weekly basis) before and after meals to capture the time point of disease onset.

5. A positive test should also lead to a thorough investigation of the vascular status of the patient with appropriate diagnostic procedures.

Any effective intervention should lead to relieve of stress to the pancreatic ß-cells and to a reduction of intact proinsulin levels, which would result in a negative POCT. If this is the case, it is recommended to repeat the screening test on an annual basis to catch the time of further disease progression.

In patients with overt diabetes, as detected through the POCT screening test with consecutive OGGT confirmation, ß-cell dysfunction is certainly one of the key disease driving factors. We, therefore, recommend in these patients to preferably use drugs that are known to protect ß-cells function under these circumstance (eg, insulin, pioglitazone, GLP-1 agonists, DPPIV inhibitors, SGLT2 inhibitors),^28-31 At least the used drugs should have a neutral effect on intact proinsulin levels (eg, metformin³²). Treatment with any of these drugs alone or in combination may have been the reason for the negative or low proinsulin test results in the POCT and ELISA test in our study in the patients with known type 2 diabetes. Use of drugs that actively target to lower glucose by enhancing ß-cell secretion and increasing circulatory proinsulin levels in an uncontrolled and unregulated way, such as sulfonylurea drugs or glinides should be avoided based on pathophysiological considerations.^28,31

In conclusion, the evaluated intact proinsulin POCT was shown to be an easy-to-use and fast test for an initial risk assessment and detection of ß-cell dysfunction and should lead to entering patients with elevated intact proinsulin levels into more sophisticated diabetes diagnostic and treatment programs.