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Title:  13C glucose breath test for the diagnosis of diabetic indications and monitoring glycemic control

United States Patent:  6,878,550

Issued:  April 12, 2005

Inventors:  Yatscoff; Randall W. (Edmonton, CA); Foster; Robert T. (Edmonton, CA); Aspeslet; Launa J. (Edmonton, CA); Lewanczuk; Richard (Edmonton, CA)

Assignee:  Isotechnika Inc. (Scottsdale, AZ)

Appl. No.:  439290

Filed:  May 16, 2003

Abstract

Use of 13 C glucose in an analytical assay to monitor glucose metabolism by measurement of labeled exhaled CO2 is provided. A breath test and kit for performing the breath test are described for the diagnosis of diabetic indications and monitoring of glycemic control. The breath test utilizes the measurement of expired 13 C-labeled CO2 following the ingestion of a 13 C-enriched glucose source.

Description of the Invention

FIELD OF INVENTION

Use of 13 C glucose in an analytical assay to monitor glucose metabolism by measurement of labeled exhaled CO2 is provided. A breath test and kit for performing the breath test are described for the diagnosis of diabetic indications and monitoring of glycemic control. The breath test utilizes the measurement of expired 13 C-labeled CO2 following the ingestion of a 13 C-enriched glucose source.

BACKGROUND OF THE INVENTION

Glucose tolerance is defined as the ability to properly utilize glucose. Diabetes is not a single disease, but an array of diseases that exhibit the common symptom of glucose intolerance, an impairment in glucose utilization.

The prevalence of diabetes in the general population is approximately 6-7%. Only about half of diabetics are actually diagnosed. Studies have shown that rates for persons with glucose intolerance are equal by sex and greater for blacks than for whites.

In general, the following types of diabetes have been recognized: type I diabetes mellitus, type II diabetes mellitus, secondary diabetes mellitus, impaired glucose tolerance and gestational glucose mellitus. The general characteristics of the symptoms of diabetes include the following:

Polyuria (high urine blood volume)

Hyperglycemia (high blood glucose levels)

Glucosuria (loss of glucose in urine)

Polydipsia (excessive thirst)

Polyphagia (excessive hunger)

Sudden weight loss

It has been observed that complications resulting from diabetes mellitus are the third leading cause of death in most developed countries. Diabetes is a risk factor for a variety of conditions including coronary heart disease, cerebrovascular stroke, neuropathy (nerve damage), nephropathy (kidney damage), retinopathy (eye damage), hyperlipidemia (excessive blood lipids), angiopathy (damage to blood vessels) and infection.

A number of different methods exist for determining a condition of intolerance for glucose. These include postprandial blood glucose, oral glucose tolerance test (OGTT), O'Sullivan glucose tolerance test (gestational test), hemoglobin Alc (Hb A1, Hb A1c), islet cell antibodies, glutamic acid decarboxylase (GAD) antibodies and insulin antibodies. Diabetes, however, is most readily detected when the carbohydrate metabolic capacity is tested. This is done by stressing the system with a defined glucose load as in the oral glucose tolerance test (OGTT).

The OGTT has been criticized, however, because many of the variables affecting test results are difficult to control. For instance: patients must be on a standardized carbohydrate diet at least three days before the test; the test requires an 8 to 16 hour fast; the test should only be performed on ambulatory patients; stress should be avoided; exercise should be avoided; various hormone imbalances can affect validity such as with: thyroxine, growth hormone, cortisol and catecholamines; various drugs and medications can affect validity such as: oral contraceptives, salicylates, nicotinic acid, diuretics and hypoglycemics; and evaluation should normally be corrected for age. The greatest disadvantage of the OGTT is that it is poorly reproducible and this limits its diagnostic usefulness.

Type 2 diabetes is a common condition, associated with significant morbidity and mortality. It is generally acknowledged that overt type 2 diabetes is preceded by a period of glucose intolerance which itself is preceded by a significant period of insulin resistance (1-5). It is now further recognized that typical diabetic complications can begin to develop during this "pre-diabetic" phase (3,6). The identification of persons at risk of developing overt type 2 diabetes has therefore taken on even greater importance. It has been suggested that if such persons could be easily identified, a lifestyle modification strategy could be implemented which might prevent their progression to type 2 diabetes with its attendant morbidities.

Because of the public health importance of type 2 diabetes, regular screening for this condition is now advocated (5,7,8). However, such screening programs, whether by fasting plasma glucose or by the 75-g OGTT, only identify diabetic or glucose-intolerant patients. The homeostasis model assessment (HOMA) index has been advocated as a method of detecting persons with insulin resistance and therefore presumably at risk of progressing to overt type 2 diabetes (9-11). However, the HOMA index requires a serum insulin measurement and, some argue, the use of a computer program. Thus, this index is not as simple or accessible as a fasting blood glucose level. Similarly, the gold standard euglycemic, hyperinsulinemic clamp is clearly not appropriate for mass screening campaigns.

The current methods of diagnosing diabetes involve either invasive testing (i.e., repeated blood collections), or use blood-borne markers (i.e., glycosylated proteins, or antibodies) which offer an indirect assessment of glucose regulation. Accordingly, it is an object of the present invention to avoid the need for invasive testing or the use of blood-borne markers in determinations of glucose regulation.

SUMMARY OF THE INVENTION

The above and other objects of the invention are attained by a 13 C breath test and a kit for determining glucose regulation in a patient in need thereof.

Based on our experience in the use of 13 C breath tests, we propose a simple, sensitive test of insulin resistance. In normal individuals, in the presence of insulin, glucose is taken up by cells where it undergoes glycolysis and then enters the citric acid cycle or is shunted to fat synthesis. In either case, CO2 is produced as a metabolic by-product. This CO2 then re-enters the circulation and is eliminated in the lungs. We found that if glucose was labeled with 13 C, the resultant CO2 could be detected in the expired air. In type 2 diabetes and other states of insulin resistance, glucose uptake is impaired and the generation of 13 CO2 is likewise blunted. Accordingly, we have developed a 13 C-glucose breath test for the diagnosis of type 2 diabetes and insulin resistance. In particular, the test provides a means to detect insulin resistance when blood glucose levels are still in the normal range and before .beta.-cell destruction leading to diabetes has occurred. Early detection of insulin resistance will allow intervention in time to prevent the development of type 2 diabetes. In addition, the test allows the success of intervention therapies, including diet and exercise to be monitored.

An analytical assay is described that is based on the use of non-radioactive 13 C. Labeled expired 13 CO2 is measured in the present assay. Isotope ratio mass spectroscopy (IRMS) is used as a detection method for 13 C, a non-radioactive isotope that occurs naturally in food and animal tissues. Non-dispersive infrared spectroscopy (NDIRS) analysis and analysis methods known in the art may be employed. The test protocol is as follows: after an overnight fast, the oral dose of 13 C uniformly labeled glucose (containing about 25 mg of 13 C glucose in combination with about 15 g of unlabeled glucose in 100 ml of tap water) is administered. Breath samples will be collected before the dose and then 11/2 hours after 13 C glucose ingestion. Levels of 13 CO2 in expired air will be measured by an IRMS method.

Advantages of this test are the following:

it is practical, sensitive and specific;

the validity of the test is not influenced by stress, exercise, hormone imbalances, or some drugs and medications;

it is a non-invasive method;

it is simple to perform and can be readily used in physicians' offices or medical laboratories;

it is safe since 13 C is a naturally occurring isotope found in all carbon-containing substances;

it involves no radioactivity, and may be used in children and women.

The 13 C glucose test is safe, reliable, and specific in diagnosis of diabetes and measurement of the severity of insulin resistance in patients. The invention is also preferred to diagnose gestational diabetes and to monitor glycemic control in diabetes patients. A preferred embodiment of the invention is a kit containing the necessary material for performing the described method. This kit may contain, but is not limited to, a source of 13 C enriched glucose (preferably uniformly labeled D-glucose); a source of unenriched glucose; and a breath collection device. The kit may also contain a set of patient instructions for its use. In another embodiment, the kit may additionally contain a blood collection device, such, as a lancet or hypodermic needle and vacutainer for the additional determination of blood glucose levels.

Accordingly, in one aspect the invention provides diagnostic kits for the determination of glycemic control in a subject comprising: a predetermined quantity of 13 C-enriched glucose; and a breath collection container. A plurality of breath containers and/or instructions for use may be included. The kits may be used for the diagnosis of diabetes, insulin resistance, gestational diabetes, and the like or to determine the adequacy of antihyperglycemic therapy.

In a further aspect, the invention provides a use of 13 C-enriched glucose for the determination of glycemic control in a subject.

In another aspect, the invention provides 13 C-enriched glucose for use in the manufacture of diagnostic kits for the determination of glycemic control in a subject. The kits may be used for the diagnosis of diabetes, insulin resistance, gestational diabetes, and the like or to determine the adequacy of antihyperglycemic therapy.

In yet a further aspect, the invention provides diagnostic kits for the determination of glycemic control in normal, diabetic and insulin resistant subjects by comparing blood glucose levels with breath levels of 13 C-enriched CO2

In a still further aspect, the invention provides method of diagnosing a condition in a subject, said condition selected from the group consisting of diabetes, insulin resistance impaired glucose tolerance, impaired fasting glucose and gestational diabetes, said method comprising collecting a first breath sample from said subject in a first breath collection container; administering 13 C-enriched glucose to said subject; collecting a second breath sample from said subject in a second breath container at a time point after administration of said 13 C-enriched glucose; measuring the 13 CO2 in each of said first and second breath samples; and comparing the amount of 13 CO2 in said second breath sample with the amount of 13 CO2 in said first breath sample to obtain a delta value, wherein the presence of less 13 CO2 in said second breath sample compared to normal control values indicates the presence of said condition. Using an ROC curve, a delta cutoff is chosen wherein the sensitivity and specificity are such as to maximize diagnostic accuracy. In particular, when the condition is insulin resistance, a range of deltas from 8 to 10 is preferred. A delta of 9 is most preferred.

In yet an additional aspect, the invention provides method of predicting a subject's risk of developing diabetes, said method comprising collecting a first breath sample from said subject in a first breath collection container; administering 13 C-enriched glucose to said subject; collecting a second breath sample from said subject in a second breath container at a time point after administration of said 13 C-enriched glucose; measuring the 13 CO2 in each of said first and second breath samples; and comparing the amount of 13 CO2 in said second breath sample with the amount of 13 CO2 in said first breath sample, wherein the presence of less 13 CO2 in said second breath sample compared to normal control values indicates risk of developing diabetes. The comparison may be made by choosing a cutoff of ROC values wherein the sensitivity and specificity are such as to maximize diagnostic accuracy. In particular, a range of ROC's from 8 to 10 is preferred. An ROC of 9 is most preferred.

The 13 C-glucose breath test is superior to currently used laboratory criteria in the diagnosis of type 2 diabetes. Its predictive value for clinical status, as well as its correlation with the HOMA index, make it a simple but useful test for detecting early evidence of insulin resistance and hence, risk for type 2 diabetes.

DETAILED DESCRIPTION OF THE INVENTION

The introduction of a 13 C breath test offers a novel, non-invasive, direct means to monitor glucose metabolism by measurement of exhaled CO2 using highly enriched, uniformly labeled 13 C-glucose. Glucose metabolism will generate labeled CO2, which is then exhaled and collected in tubes. Enrichment of labeled CO2, over a determined time course, can be used as a quantitative index of glucose metabolism. Comparison is made against age-specific reference intervals.

The present invention has a number of advantages, including lower dose of glucose needed (overcomes inconsistencies due to malabsorptive disorders or previous gastric or intestinal surgery), reduction in testing time (from the current 2 hours required for the OGTT) and fewer interpretational ambiguities (greater sensitivity and specificity).

The 13 C glucose breath test is based on the metabolism of glucose. Following a baseline breath sample, a 13 C glucose solution containing about 25 mg of 13 C glucose in combination with about 15 g of unlabeled glucose in 100 ml of tap water is administered. Breath samples will be obtained before the dose and then 12 hours after 13 C glucose ingestion. Measurement of the expired air will be detected by an isotope ratio mass spectroscopy assay method. Elevated or excessive breath of 13 CO2 concentrations will be seen in individuals who have normal glucose metabolism.

The 13 C-glucose breath test provides a more sensitive and diagnostically accurate indicator of the presence of type 2 diabetes than do currently used common methodologies. However, a problem arises in that the definition of diabetes is made on the basis of fasting plasma glucose or glucose-tolerance test values. Thus, these tests are the de facto "gold standards" and theoretically should be the most accurate. In the well-characterized group of diabetic patients studied in this investigation, the pitfalls of a single fasting blood glucose value or a glucose tolerance test are evident. Indeed, numerous reports of the poor overall diagnostic accuracy of the glucose tolerance test or fasting plasma glucose as a diagnostic tool for diabetes exist (13-17). Moreover, the requirement for confirmation of an abnormal fasting plasma glucose reduces sensitivity of this test albeit at a gain in specificity. It could be argued, however, that for screening purposes, sensitivity is perhaps preferable to specificity. However, because of the theoretical advantage of diagnosing subjects at risk of diabetes prior to the actual onset of the disease, various indices of insulin resistance or glucose intolerance have been devised (for a review see 18). The hypothesis associated with these latter measurements is that insulin resistance and abnormalities in glucose homeostasis occur well before the onset of overt type 2 diabetes. If patients demonstrating such abnormalities can be detected through screening programs, it has been suggested that the development of overt diabetes may be prevented or delayed (4,5,19). The importance of such an approach is further underscored by the finding that at the time of type 2 diabetes onset, a significant number of patients already have diabetic complications (3,6).

In order to address the need for a relatively simple index of insulin resistance, the HOMA index was developed. This index has been shown to correlate with results from the gold-standard hyperinsulinemic, euglycemic clamp (9,11,20,21). Although the HOMA index was significantly higher in the diabetics in this study, it was diagnostically inferior in all aspects to the 13 C-glucose breath test. Indeed, when both the HOMA index and the 13 C-glucose breath test results were entered into a logistic regression which included fasting blood sugar, age, sex and BMI as variables, only the 13 C-glucose breath test gave a statistically significant partial correlation coefficient. Similarly, when each of the two variables of interest was individually included in a similar logistic regression, which also included the 2 hour OGTT value as a further variable, the 13 C-glucose breath test retained a statistically significant predictive value whereas the HOMA index did not. Indeed, in all possible iterations of the logistic regression, the 13 C-glucose breath test was always the strongest predictor of diabetic status. Although it may be argued that a HOMA is an easier test, requiring only a single blood sample, there are disadvantages to this test as well. First of all, a serum insulin measurement must be carried out in a reasonably advanced medical laboratory by trained technicians. This adds time and cost to the screen. The 13 C-glucose breath test, however, can be analyzed using a point of care instrument that requires very little training to use. Thus, screening can be carried out in the field with results available almost as soon as the last breath sample is complete. The HOMA index requires blood samples with the attendant infectious precautions. The 13 C-glucose breath test is carried out on breath and therefore only general infectious precautions are necessary. Similarly, phlebotomy requires trained medical personnel whereas the 13 C-glucose breath test does not necessarily require any supervision--a package insert can provide all the necessary instructions. Thus, the 13 C-glucose breath test can also be made available to remote locations via post. Finally, although the HOMA provides added diagnostic accuracy to the diagnosis of diabetes when compared to a fasting blood sugar, as can be seen from Table 1, the traditional OGTT is superior to both. Compared to the OGTT, however, the 13 C-glucose breath test has even greater accuracy and has the advantage of requiring a lower glucose load and a shorter time requirement along with all the other advantages listed above. One final consideration is the possibility of false negative results with the breath test in subjects with delayed gastric emptying. Given the relatively low volume and lower osmolarity of the breath test compared with the OGTT, problems with gastric emptying are likely to be less than those associated with the OGTT. Indeed, based on 1, 1.5 and 2 hour breath test values in this study, no subjects showed evidence of delayed gastric emptying. As this test is most likely to find use early in the course of insulin resistance/type 2 diabetes, it is unlikely that diabetic gastroparesis will be a significant confounder. Thus, the 13 C-glucose breath test offers a simple, sensitive and accurate method for the diagnosis of type 2 diabetes.

In terms of insulin resistance, studies are underway to validate the 13 C-glucose breath test against the hyperinsulinemic, euglycemic clamp. However, even with the current results, there is evidence that the 13 C-glucose breath test is an indicator of insulin resistance. First, the 13 C-glucose breath test results do correlate with the HOMA. Secondly, there is a strong correlation between the breath test and body mass index whereas the correlation between the HOMA index is less strong. Third, the superior diagnostic parameters of the breath test and the fact that a type 1 diabetic had a breath result of <1.2 show a correlation between insulin resistance and the 13 C-glucose breath test result. Finally, the underlying principal of the 13 C-glucose breath test is based on resistance to glucose uptake by target tissues. Thus, the 13 C-glucose breath test also offers a simple, sensitive, specific test for the diagnosis of insulin resistance.

One final advantage of the 13 C-glucose breath test is its application for following insulin resistance. This test has the potential to allow the effectiveness of various interventions in type 2 diabetes to be monitored. Whether these interventions be lifestyle or pharmacological, the 13 C-glucose breath test offers a sensitive, dynamic method to assess effectiveness of type 2 diabetes treatments.

Thus, the 13 C-glucose breath test may be used not only to diagnose diabetes, but also to determine insulin sensitivity and insulin resistance. The test may reliably be used to diagnose other difficult to detect pre-diabetic conditions. Thus, it is a useful tool to determine whether a patient is at risk of developing diabetes.

It is important that any diagnostic test procedure have diagnostic accuracy, i.e., that it accurately predicts positive and negative values. The receiver operated characteristics (ROC) value describes the balance between the sensitivity (i.e., the number of hits detected) and the specificity (i.e., the accuracy) of a test. These two variables may also be considered positive predictive value and negative predictive value, and are correlated with diagnostic accuracy. The ROC curve shows the relationship of the probability of a positive test, given no disease, to the probability of a positive test, given disease. An ROC cutoff value is chosen to maximize diagnostic accuracy of the test in question.

Claim 1 of 29 Claims

What is claimed is:

1. A diagnostic kit for the determination of glycemic control in a subject comprising:

(a) a predetermined quantity of uniformly labeled 13 C-enriched glucose; and

(b) a plurality of breath collection containers.


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