Use of .sup.13C glucose in an analytical assay to monitor glucose metabolism by measurement of labeled exhaled CO.sub.2 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 .sup.13C-labeled CO.sub.2 following the ingestion of a .sup.13C-enriched glucose source.

SUMMARY OF THE INVENTION

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

Based on our experience in the use of .sup.13C 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, CO.sub.2 is produced as a metabolic by-product. This CO.sub.2 then re-enters the circulation and is eliminated in the lungs. We found that if glucose was labeled with .sup.13C, the resultant CO.sub.2 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 .sup.13CO2 is likewise blunted. Accordingly, we have developed a .sup.13C-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 .sup.13C. Labeled expired .sup.13CO.sub.2 is measured in the present assay. Isotope ratio mass spectroscopy (IRMS) is used as a detection method for .sup.13C, 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 .sup.13C uniformly labeled glucose (containing about 25 mg of .sup.13C 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 .sup.13C glucose ingestion. Levels of .sup.13CO.sub.2 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 .sup.13C is a naturally occurring isotope found in all carbon-containing substances; it involves no radioactivity, and may be used in children and women.

The .sup.13C 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 .sup.13C 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 .sup.13C-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 .sup.13C-enriched glucose for the determination of glycemic control in a subject.

In another aspect, the invention provides .sup.13C-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 .sup.13C-enriched CO.sub.2

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 .sup.13C-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 .sup.13C-enriched glucose; measuring the .sup.13CO.sub.2 in each of said first and second breath samples; and comparing the amount of .sup.13CO.sub.2 in said second breath sample with the amount of .sup.13CO.sub.2in said first breath sample to obtain a delta value, wherein the presence of less .sup.13CO.sub.2 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 .sup.13C-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 .sup.13C-enriched glucose; measuring the .sup.13CO.sub.2 in each of said first and second breath samples; and comparing the amount of .sup.13CO.sub.2 in said second breath sample with the amount of .sup.13CO.sub.2 in said first breath sample, wherein the presence of less .sup.13CO.sub.2 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 .sup.13C-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 .sup.13C breath test offers a novel, non-invasive, direct means to monitor glucose metabolism by measurement of exhaled CO.sub.2 using highly enriched, uniformly labeled .sup.13C-glucose. Glucose metabolism will generate labeled CO.sub.2, which is then exhaled and collected in tubes. Enrichment of labeled CO.sub.2, 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 .sup.13C glucose breath test is based on the metabolism of glucose. Following a baseline breath sample, a .sup.13C glucose solution containing about 25 mg of .sup.13C 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 .sup.13C glucose ingestion. Measurement of the expired air will be detected by an isotope ratio mass spectroscopy assay method. Elevated or excessive breath of .sup.13CO.sub.2 concentrations will be seen in individuals who have normal glucose metabolism.

The .sup.13C-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 defacto "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 .sup.13C-glucose breath test. Indeed, when both the HOMA index and the .sup.13C-glucose breath test results were entered into a logistic regression which included fasting blood sugar, age, sex and BMI as variables, only the .sup.13C-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 .sup.13C-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 .sup.13C-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 .sup.13C-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 .sup.13C-glucose breath test is carried out on breath and therefore only general infectious precautions are necessary. Similarly, phlebotomy requires trained medical personnel whereas the .sup.13C-glucose breath test does not necessarily require any supervision--a package insert can provide all the necessary instructions. Thus, the .sup.13C-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 .sup.13C-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 .sup.13C-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 .sup.13C-glucose breath test against the hyperinsulinemic, euglycemic clamp. However, even with the current results, there is evidence that the .sup.13C-glucose breath test is an indicator of insulin resistance. First, the .sup.13C-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 .sup.13C-glucose breath test result. Finally, the underlying principal of the .sup.13C-glucose breath test is based on resistance to glucose uptake by target tissues. Thus, the .sup.13C-glucose breath test also offers a simple, sensitive, specific test for the diagnosis of insulin resistance.

One final advantage of the .sup.13C-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 .sup.13C-glucose breath test offers a sensitive, dynamic method to assess effectiveness of type 2 diabetes treatments.

Thus, the .sup.13C-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 5 Claims

1. A method of diagnosing insulin resistance in a subject, said method comprising: a) collecting a first breath sample from said subject in a first breath collection container; b) administering .sup.13C-enriched glucose to said subject; c) collecting a second breath sample from said subject in a second breath container at a time point after administration of said .sup.13C-enriched glucose; d) measuring the .sup.13CO.sub.2 in each of said first and second breath samples; and e) comparing the amount of .sup.13CO.sub.2in said second breath sample with the amount of .sup.13CO.sub.2in said first breath sample, wherein the presence of less .sup.13CO.sub.2in said second breath sample compared to normal control comparison values indicates insulin resistance in the subject wherein said comparison is made by choosing a cutoff of receiver operated characteristics (ROC) values wherein the sensitivity and specificity are such as to maximize diagnostic accuracy.

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