ASSAY DEVELOPMENT

Figure 1. LabCD cap valve. The two inlet streams entering from the left and right are mixed and then directed out the top channel.

A prime target for the early detection of disordered glucose metabolism in DM is the measurement of accelerated protein metabolism that manifests as increased proteolytic activity, amino acid metabolism, and concomitant oxidative stress.³ The measurement of accelerated protein metabolism does not replace measurements of glycemia. Instead, it allows a more complete assessment of the altered metabolic state found in diabetes and prediabetes.

The human body misinterprets the energy-deficient state of impending or uncontrolled DM as starvation.² The metabolism seeks to restore homeostasis by finding other ways to maintain adenosine triphosphate (ATP) production. This restoration leads to a cascade of adverse biochemical reactions and oxidative stress.1,4 This state is atherogenic and, in the case of type 2 diabetes, can precede the diabetic state by 10 years or more before the compensatory measures are overwhelmed and the glucose levels rise above the accepted normal range.²

In this energy-deficient state, intracellular proteins are selectively sacrificed to restore ATP production by oxidating amino acids in the Krebs cycle.^4–7 Alanine and glutamine are released into the blood to become substrates for gluconeogenesis in the liver and kidney, respectively, and the oxidation of other amino acids is increased.1,8 Net gain or loss of proteins is determined by balancing two opposite processes, protein synthesis and protein degradation. However, the techniques used by biochemists to measure this parameter are complex and time-consuming, which has limited their widespread use in the clinical setting.^9–11

Proteolysis of functional skeletal muscle protein releases amino acids, resulting in the formation of glutamate and glutamine.⁵ Glutamine and aspartate regulate proteolysis.^1,12–14 In the diabetic state, muscle glutamine synthetase (GS) increases by up to 200%.¹⁵ Insulin reverses such increased GS levels and the concomitant increased GS activity (GSA).^15,16

At the same time, glutamine production increases in diabetes by the activation of GS. Higher GS hepatic mRNA occurs in diabetics even with lower total cell RNA, which means the diabetic state selectively focuses on increased GS production.¹⁵ Correcting the diabetic state by treatment with insulin restores GS and GS mRNA to normal levels. The activation of GS in diabetes is independent of adrenal corticosteroid action. Active GS binds to promoter regions of DNA to activate transcription of a variety of genes, including those for nitrogenase.^12,15

Proteolysis of skeletal muscle releases an abundance of the sulphur-containing amino acids methionine and cysteine, most of which are metabolized to glutamate. This glutamate production requires increased pathways leading to the removal of the remaining sulphur load, such that methionine is converted to homocysteine and thiolactone.^7,8,14–16

With consequent production of glutamate and ammonia, the proteolytic state requires activation of the central enzyme of protein metabolism, GS.⁴ This enzyme is complex, with nine allosteric feedback inhibitors. Several enzyme systems regulate GS, and each of them is in turn regulated. Glutamine passes freely across skeletal muscle and liver plasma membranes.^4,7 For such reasons, glutamine is the principal soluble, nontoxic nitrogen carrier that is used for amino acid precursors of the Krebs cycle and is a central point in nitrogen (amino acid) metabolism.^4,7

Glutamate is neurotoxic and does not pass across plasma membranes freely. Metabolizing glutamate to glutamine prevents significant amounts of glutamate from entering the blood.¹⁵ In this way, proteolysis causes the formation of glutamate that is transformed by GS into glutamine. This final product of activated GS passes freely across cell membranes in order to provide precursors for the Krebs cycle when amino acids are needed for energy (ATP) production.⁴ This occurs when glucose, the preferred fuel, is unavailable at the intracellular level due to prolonged fasting or DM.

Significant glutamine production is also required to remove the resulting ammonia load that accompanies heavy metabolism of amino acids for energy production.^7,8,15 The hydrolysis of glutamine and thiocyanate during the proteolytic state causes quantitative changes in g-glutamyl-hydroxamate and hydroxyamine-N-thiocarboxylate in extracellular fluids. These products correlate with increased activity of GS, protein turnover, and high-level oxidative stress. Accordingly, these compounds can be monitored in extracellular fluids such as saliva and can serve as markers for GS activity and the degree of metabolic disturbance in diabetes and prediabetes.17–19

Measuring GSA Levels

New DiaTech Diagnostics Technology Corp. (Victoria, BC, Canada) has developed a noninvasive alternative to already existing laboratory-based methods to assess the activation of glutamine synthetase. This test can be used in laboratories, medical offices, or at home, and is based on the fundamental biochemical pathways described above.^8,15,16,20 The test is a sensitive reflection of GSA and measures the degree to which metabolism has shifted away from glucose with increasing severity of DM.

This noninvasive test uses human saliva samples on colorimetric test strips that are compared with a graduated color scale. An immediate change in color occurs when the test strip comes in contact with saliva. The colorimetric test strip is a nontoxic, absorbent strip that is dipped in an expectorated saliva sample for a contact period of 1–2 seconds. The color on the test strip is compared with a standard color chart 40–60 seconds after contact with saliva. The test results are classified into two categories. A yellow to light-orange color indicates normal metabolism. Dark orange, shades of red from lighter to darker, and brown to black colors indicate increasing degrees of metabolic disturbance. The color depends on the degree of GSA and reflects the amount of proteolysis and oxidative stress of amino acid metabolism. The degree of color change provides a rapid measure of the compensatory metabolic changes accompanying early disturbance in glucose metabolism.

Materials and Methods

Table I. (click to enlarge) Characteristics of subjects in clinical studies using the saliva GSA test.

Data collected from the studies included the following: age, sex, weight, height, presence of dentures, degree of success in controlling DM, blood pressure, smoking habits, family history of diabetes or atherosclerosis, clinically verified comorbid disease states, and the names and doses of medications taken for treating diabetes. In the studies, subjects underwent standard clinical tests for glucose and lipid metabolism. The degree of altered metabolism and the assessment of the subjects’ responses to treatment for carbohydrate and lipid disorders were determined by the standard clinical criteria.^1,21

While certain subjects were assigned to the normal study group if they did not meet the standard diagnostic criteria for DM, they were still chosen due to having a strong metabolic syndrome with borderline glucose metabolism. Their inclusion explains the high normal mean glycemic values for the nondiabetic group. Prestudy treatments were continued for all subjects, including those with DM, in order to conduct the studies according to standards acceptable to research ethics review boards.

Blood, saliva, and urine samples were simultaneously collected. The first fasting saliva samples of 500–1000 ml were collected and stored at –27°C until the analyses were performed. After patients with diabetes were given insulin or other medications and a light breakfast, the second blood and saliva samples were collected two hours later. Similarly, two hours after the nondiabetics were given 75 g of glucose, a second sample was collected.

The saliva samples were tested using the saliva GSA test. Data were collected to determine whether the saliva test could detect prediabetic metabolic syndrome and DM. The saliva GSA test and other standard diagnostic tests were performed on the samples from 701 apparently healthy subjects. Within this group, 392 subjects had type 1 and type 2 DM, 408 subjects had metabolic syndrome, and 150 were heavy smokers. All of these subjects had previously been found by their clinicians to be free from DM.

In addition, 2272 saliva and blood samples were collected from patients with type 2 diabetes over a period of three years. These samples were examined to evaluate the potential usefulness of the saliva GSA test to assess and monitor metabolic disturbances in type 2 DM. These data were gathered to investigate whether the saliva test could detect improvements in controlling DM before glucose levels normalized with improved treatment and physical activity. The samples were collected before breakfast and medications were taken, after medications were taken, following 1–1.5 hours of mild to moderate exercise, and after an increase in the diabetes medication dosage.

The results of the saliva GSA tests were compared with the standard diagnostic tests for DM using the morning and evening samples (before and after medication and exercise), and before and after an increase of the diabetes medication.

Statistical analyses used Mann-Whitney nonparametric tests because of the nonnormal distributions of blood test values. The results were considered statistically significant by reaching a p value of 0.05 or less. The blood glucose values were compared by two samples of t-tests, and the saliva frequency was compared by Pearson Chi-square tests.

Results

Table II. (click to enlarge) Saliva GSA test performance.

The data from the studies listed in Table II demonstrated strong correlations between the saliva GSA test and type 1 and type 2 DM. The test also has a strong correlation with prediabetic metabolic syndrome that manifests primarily as essential hypertension or borderline results on oral glucose tolerance tests (OGTT). The saliva GSA test has high sensitivity and specificity, and high positive and negative predictive values for type 1 and type 2 DM and prediabetic metabolic syndrome.

The data from study group 7 showed that 93% of the nondiabetic subjects with lung cancer had normal saliva GSA test results, and 95% of nondiabetic subjects with stomach cancer had normal saliva GSA test results. Patients with such malignancies were tested to determine whether the stressful cancer metabolic state would cause abnormal saliva GSA test results, which they did not.

The studies with the saliva GSA test found 80 individuals with abnormal results among 701 apparently normal nondiabetic subjects, 408 subjects with metabolic syndrome, and 150 heavy smokers. A careful assessment of these individuals found that 21 subjects met the diagnostic criteria for DM, 37 subjects had essential hypertension, and 27 subjects had abnormal OGTT and abnormal levels of immunoreactive insulin. Five subjects also had essential hypertension, abnormal OGTT, and immunoreactive insulin levels. The saliva GSA test allowed 99% of these previously undiagnosed subjects to be accurately reclassified as having DM or prediabetic metabolic syndrome.

Hypertension was found to be a component of metabolic syndrome, a condition with a high risk for future development of DM. One hundred fifty subjects were heavy smokers, and those subjects who had suboptimal blood test parameters also had significantly positive results using the saliva GSA test. Practically all of the patients with positive saliva GSA test results had significantly elevated risk for atherosclerosis, and most had either diabetes or prediabetic metabolic syndrome, particularly suboptimal OGTT or essential hypertension. These data demonstrate a strong correlation between the saliva test and prediabetic metabolic syndrome in which glucose levels are still near the normal range.

Figure 1. (click to enlarge) Parallel samples of fasting blood glucose (mmol/L) and fasting saliva prior to intake of morning medication (4 diabeta and 2 glucophage per day).

Figure 2. (click to enlarge) Parallel samples of saliva and blood from same subject after medication (4 glyburide and 2 metformin) and evening exercise.

Figure 3. (click to enlarge) Parallel samples of fasting blood glucose (mmol/L) and fasting saliva from same subject after increase in medication (4 glyburide, 4 metformin, and 2 enalapril) prior to intake of morning medication.

Conclusion

Figure 4. (click to enlarge) Parallel samples of saliva and blood from same subject after medication (4 glyburide, 4 metformin, and 2 enalapril) and evening exercise.

Studies also demonstrated that the saliva test correlates with prediabetic metabolic syndrome. Subjects with metabolic syndrome and essential hypertension have abnormal saliva test results at an early stage in the progression toward diabetes, which precedes the development of abnormal glycemic levels.

The saliva test does not replace glucose measurements or other measurements of carbohydrate metabolism. The saliva GSA test provides additional information about the degree of protein turnover, proteolytic activity, amino acid metabolism, and oxidative stress that are characteristic of the disturbed metabolism of DM and prediabetes. Detecting the initial shift in metabolism away from glucose toward amino acid metabolism by measuring the degree of GSA can alert the need for additional interventions to reduce the negative consequences of arterial vascular disease in DM. While measuring, monitoring, and controlling glucose and lipid levels have provided clinicians with important tools to mitigate risk in diabetic patients, there is still room for improvement.^1,22,23

Studies of the saliva GSA test have demonstrated its value as a noninvasive, rapid test for detecting, assessing, and monitoring disordered protein metabolism that accompanies early changes in DM. Disturbed protein metabolism is characterized by accelerated proteolysis, increased amino acid metabolism, and activation of glutamine synthetase. Being able to measure such changes in protein metabolism could prove valuable for patients living with obesity, prediabetes, and DM.

Sergei Khartchenko, MD, PhD, is the president and chief executive officer of New DiaTech Diagnostics Technology Corp. (Victoria, BC, Canada). He can be reached at newdiatech@shaw.ca.	Nigel Flook, PhD, is president of NWF Consulting Inc. (Edmonton, AB, Canada). He can be reached at nflook@shaw.ca.