MIGUEL BOYAYANFor certain types of diagnosis the always undesirable prick of a needle could be replaced by a small amount of saliva, in a less painful and invasive procedure than drawing blood. In studies and commercial trials in a number of countries, saliva is being used in various ways, such as identifying hormones, cancer and, more recently, to measure stress levels. In Brazil, the company Probiotic, established at the Ciaem Company Incubating Center at the Federal University of Uberlandia (UFU), in the state of Minas Gerais, is ready to start producing kits that use saliva as a source for diagnosis. The company, which is also part of the Local Production Arrangement for Biotechnology of the Triangulo Mineiro region, was founded by Professor Foued Salmen Espindola, from the UFU Institute of Genetics and Biochemistry and two biologists, Leonardo Gomes Peixoto and Rogerio de Freitas Lacerda, who completed their masters degrees in genetics and biochemistry at that institution.
The first proven products that they intend to market as soon as Anvisa, the National Sanitary Surveillance Agency releases the registration, are two kits that use saliva as a source for biomarkers of physical exercise and psychological stress, especially in situations linked to sport. Espindola says: “we use the total dosage of the saliva protein and the activity of the salivary alpha-amylase enzyme.” One of the aims of the development of these kits is to offer simplified technical innovation, to determine the anaerobic threshold, i.e., the relation between the consumption of oxygen and the ongoing increase of lactates in the blood during physical exercise tests such as those that rely on a stationary bicycle or a treadmill. The measurement of lactate thresholds is important in the physiology of exercise, sporting medicine, physical education and for athletes in many types of sport. “This threshold can be determined by an analysis of saliva. In this way we can offer a biochemical diagnosis of the metabolic adaptation and resistance to fatigue.”
Currently the concentration of blood lactates is used as an indication of this threshold and the test can be carried out in one of two ways; one kind yields an answer on the spot taking a reading of a strip impregnated with blood drawn from a fingertip and the other demands that blood drawn from the earlobe be taken to a laboratory for analysis with specific and expensive equipment. Both methods are invasive as they rely on the painful drawing of blood, an inconvenience that using saliva eliminates.
Countless studies published in recent scientific literature and carried out in the laboratories of UFU and by Probiotic indicate the measure of the activity of alpha-amylase as an interesting biomarker with great potential for evaluating physical and psychological stress. “It is even possible that the identification of stress in saliva could become an effective marker of stress , capable of replacing the analyses of levels of adrenaline and cortisol, which are also related to the physical and mental state. The alpha-amylases are produced by the pancreas and salivary glands and are responsible for digesting carbohydrates. In saliva, this enzyme is associated with other important mouth health functions and when extracted from blood it can show abnormal alterations associated with pancreatic pathologies.”
Cups and tubes
The new tests can be carried out using saliva collected after mouth cleansing and stimulated by briefly chewing on a piece of cotton wool, paraffin, a piece of plastic packaging film or even sugar free chewing gum . After chewing for two or three minutes, one spits out the saliva and saves it in a cup (a small plastic coffee cup is ideal) or in a small cellulose or polyester tube that will come with the collection kit. The saliva can then be transferred to a small plastic tube and sent to the laboratory on the same day it is collected. This can be done by mail and does not demand refrigeration. As in the case of drawing a blood sample for a lactate analysis, the saliva should be produced immediately before, during or about 15 minutes after engaging in physical exercise, as the total levels of protein and salivary amylase tend to quickly return to the levels found when at rest. Apart from producing the kit, Probiotic will also analyze the material and issue an evaluation report.
The use of saliva in diagnoses is being disseminated in the United States, where kits are offered to detect the levels of male and female hormone and also of cortisol. Many research groups around the world are betting on the study of saliva for other types of diagnosis. Espindola informs us that “in Japan, a group led by Professor Masaki Yamaguchi, of the Toyama School of Engineering, has developed and is testing a portable device that measures amylase on a paper strip (as in the traditional tests for glucose). It is indicated for use in psychological stress tests, principally in situations that require the test to be conducted on the scene, such as the diagnosis of a bus driver throughout the day”, says Espindola. “In Switzerland, Professor Urs Markus Nater of the Institute of Psychology at the University of Zurich is testing a series of psychological tests with amylase; the tests involve violent situations and as a biological marker in therapy sessions so that the psychologist can check whether the patient is stressed.”
Recent work on saliva proteomics, which aims at identifying all the proteins in this bodily fluid, should open the way to new types of examination. “More than 300 proteins in saliva have already been catalogued. Previously it was thought that the number was much lower. These discoveries will be very important in the development of diagnoses for human illnesses.” At the beginning of this year, researchers at the University of Texas in the United States announced a test with saliva, which identifies proteins linked to breast cancer. It might be a way of identifying the illness early. Espindola also speaks of the potential of saliva to diagnose mouth cancer and genetic polymorphisms, and to identify molecules and drugs that a person may have ingested, including doping in athletes and in forensic toxicology. With so many possibilities and biotechnological prospects involving the use of saliva as a means of diagnosis Probiotec will have a lot of work ahead. “Apart from the scientific and technological aspects with which we are fully conversant, we have been given aid by the National Council for Scientific and Technological Development (CNPq), through the Human Resources Training Program for Strategic Activities (RHAE) in 2005, which provided three grants for research; by Ciaem; by the Brazilian Aid Service for Small Companies (Sebrae), in the structuring of the business and in getting financing for it; and by Finep, the Financing Agency for Studies and Projects, which, through Fapemig, the Minas Gerais State Foundation for Research Aid, approved a project of the Program of Research Aid for Companies (Pappe), which has financed a substantial part of our company’s research,” says Espindola.
1. Saliva biomarkers for evaluating stress; Type Aid Program for Company R&D (Pappe); Coordinator Foued Salmen Espindola – UFU/Probiotec; Investment R$ 177,057.79 (Finep/Fapemig)
2. Development of diagnostic kits for saliva biomarkers; Type Rhae – Innovation; Coordinator Foued Salmen Espindola – UFU/Probiotec; Investment R$ 78,000.00 (MCT-CNPq)