| PATENT NUMBER | This data is not available for free |
| PATENT GRANT DATE | April 1, 2003 |
| PATENT TITLE |
Breath test for the detection of various diseases |
| PATENT ABSTRACT | The alkane profile (FIGS. 12-14) comprising the alveolar gradients of n-alkane in breath having 4 carbons to 20 carbons, and the methyl alkane profile (FIGS. 30-34) are determined for the diagnosis of disease in mammals, including humans |
| PATENT INVENTORS | This data is not available for free |
| PATENT FILE DATE | September 18, 2001 |
| PATENT CT FILE DATE | January 12, 2000 |
| PATENT CT NUMBER | This data is not available for free |
| PATENT CT PUB NUMBER | This data is not available for free |
| PATENT CT PUB DATE | July 20, 2000 |
| PATENT REFERENCES CITED |
Phillips M. and Greenberg J., "Detection of Endogenous Ethanol and Other Compounds in the Breath by Gas Chromatography With On-Column Concentration of Sample" Analytical Biochemistry, 1987; 163:165-169. Phillips, M., "Method For the Collection and Assay of Volatile Organic Compounds in the Breath" Analytical Biochemistry, 1997; 247:272-278. Phillips M., " Breath Tests in Medicine" Scientific American 1992; 267(1): 74-79. Phillips, M., Sabas M. & Greenberg J., "Alveolar Gradient of Pentanein Normal Human Breath" Free Radical Res. Commun. 1994; 20:333-337. |
| PATENT PARENT CASE TEXT | This data is not available for free |
| PATENT CLAIMS |
I claim: 1. A process for determining the presence or absence of aging in a mammal which comprises: collecting a representative sample of alveolar breath from the mammal; collecting a representative sample of ambient air; analyzing the samples of breath and air to determine content of n-alkanes having 2 to 20 carbon atoms inclusive; calculating the alveolar gradients of the n-alkanes having 2 to 20 carbon atoms, inclusive, in the breath sample in order to determine the alkane profile; and comparing the alkane profile to baseline alkane profiles calculated for mammals; the finding of differences in the alkane profile from the baseline alkane profile being indicative of the presence of aging. 2. The process of claim 1 wherein the mammal is a human. 3. The process of claim 1 further comprising: analyzing the samples of breath and air to determine the methylation site, if any of n-alkanes having 3 to 20 carbon atoms, inclusive; wherein determining the alkane profile further comprises calculating the alveolar gradients of methylated alkanes having 3 to 20 carbon atoms, inclusive, in the breath sample in order to determine a second component in the alveolar profile; and comparing the alkane profile to baseline alkane profiles calculated for mammals; the finding of differences in the alkane profile from the baseline alkane profile being indicative of the presence of aging. 4. The process of claim 3 wherein the mammal is human. -------------------------------------------------------------------------------- |
| PATENT DESCRIPTION |
BACKGROUND OF THE INVENTION 1. Field of the Invention The invention relates to the diagnosis of disease in mammals and more particularly to a method employing breath testing for the detection of particular diseases in humans. 2. Brief Description of Related Art Volatile Organic Compounds in Human Breath Alveolar breath is a distinctive gas whose chemical composition differs markedly from inspired air. Volatile organic compounds (VOCs) are either subtracted from inspired air (by degradation and/or excretion in the body) or added to alveolar breath as products of metabolism. Some features of this transformation have been well understood for many years: e.g. oxygen is subtracted and carbon dioxide is added by the oxidative metabolism of glucose (Phillips M., Breath tests in medicine, Scientific American 1992:267(1):74-79). Pauling et al, in 1971, employed cold trapping to concentrate the VOCs in breath and found that normal human breath contained several hundred different VOCs in low concentrations (Pauling L. Robinson A B, Teranishi R and Cary P: Quantitative analysis of urine vapor and breath by gas-liquid partition chromatography, Proc Nat Acad Sci USA 1971:68:2374-6). This observation has been subsequently confirmed in many different laboratories, employing progressively more sophisticated and sensitive assays. More than a thousand different VOCs have been observed in low concentrations in normal human breath (Phillips M: Method for the collection and assay of volatile organic compounds in breath, Analytical Biochemistry 1997;247:272-278). Reactive oxygen species (ROS) are toxic byproducts of energy production in the mitochondria. "Oxidative stress" is the constant barrage of oxidative damage which ROS inflict upon DNA, proteins, lipids and other biologically important molecules Fridovich I. The biology of oxygen radicals. Science 201:875-880;1978; Pryor W A: Measurement of oxidative stress status in humans. Cancer Epidemiol Biomarkers Prev 2 (3):289-292; 1993 (FIG. 1). Oxidative stress has been implicated as a pathologic mechanism in aging and several diseases. Ashok B T; Ali R: The aging paradox: free radical theory of aging Exp Gerontol 1999 34(3):293-303; Saretzki G and von Zglinicki T: (Replicative senescence as a model of aging: the role of oxidative stress and telomere shortening--an overview) Z Gerontol Geriatr 1999;32(2):69-75; Halliwell B, Gutteridge J M C, Cross C E: Free radicals, antioxidants, and human disease: Where are we now? J Lab Clin Med 119: 598-620;1992. Consequently, oxygen is now recognized as both beneficial and harmful: it is essential to sustain mammalian life because it is the final acceptor of electrons in oxidative metabolism, but in this process it also causes oxidative stress and tissue damage. Breath Alkanes as Markers of Disease Analysis of VOCs in inspired air and alveolar breath is a useful research tool with potential applications in clinical medicine. Breath analysis opens a non-invasive window on normal metabolic pathways, and also illustrates how these pathways are altered in disease. Alkanes in breath are markers of oxygen free radical (OFR) activity in vivo. OFR's degrade biological membranes by lipid peroxidation, converting polyunsaturated fatty acids (PUFAs) to alkanes which are excreted through the lungs as volatile organic compounds (VOCs); (Kneepkens C M F, Ferreira C. Lepage G and Roy C C: The hydrocarbon breath test in the study of lipid peroxidation; principles and practice, Clin Invest Med 1992; 15(2):163-186; Kneepkens C M F, Lepage G and Roy C C: The potential of the hydrocarbon breath test as a measure of lipid peroxidation, Free Radic Biol Med 1994;17:127-60) (FIG. 5). Increased pentane in the breath has been reported as a marker of oxidative stress in several diseases including breast cancer (Hietanen E, Bartsch H, Beireziat J-C, Camus A-M, McClinton S. Eremin O, Davidson L and Boyle P: Diet and oxidative stress in breast, colon and prostate cancer patients: a case control study, European Journal of Clinical Nutrition 1994;48:575-586), heart transplant rejection (Sobotka P A, Gupta D K, Lansky D M, Costanzo M R and Zarling E J: Breath pentane is a marker of acute cardiac allograft rejection. J. Heart Lung Transplant 1994; 13:224-9), acute myocardial infarction (Weitz Z W, Birnbaum A J, Sobotka P A, Zarling E J and Skosey J L: High breath pentane concentrations during acute myocardial infarction. Lancet 1991;337:933-35), schizophrenia (Kovaleva E. S, Orlov O. N, Tsutsul'kovskaia Mia, Vladimirova T. V, Beliaev B. S: Lipid peroxidation processes in patients with schizophrenia. Zh Nevropatol Psikiatr 1989:89(5): 108-10), rheumatoid arthritis (Humad S. Zarling E. Clapper M and Skosey J L: Breath pentane excretion as a marker of disease activity in rheumatoid arthritis, Free Rad Res Comms 198;5(2):101-106) and bronchial asthma (Olopade C O, Zakkar M, Swedler W I and Rubinstein I: Exhaled pentane levels in acute asthma, Chest 1997;111(4):862-5). Analysis of breath alkanes could potentially provide a new and non-invasive method for early detection of some of these disorders (Phillips M: Breath tests in medicine, Scientific American 1992;267(1):74-79). Alkanes are degraded to other VOCs such as alkyl alcohols and possibly to methyl alkanes (Phillips M: Method for the collection and assay of volatile organic compounds in breath, Analytical Biochemistry 1997; 247:272-78) but there is little information about the excretion of these compounds in the breath, where they might also provide clinically useful markers of disease. Breath testing for VOC markers of oxidative stress is a comparatively new field of research, and published information is scanty in a number of areas: First, studies of breath alkanes have focused near-exclusively on ethane and pentane which are degradation products of n-3 and n-6 PUFAs respectively. Hexane and octane have also been observed in the breath of animals, but there is little information about longer chain VOCs in normal human breath. Second, most studies have taken little or no account of the presence of alkanes in the inspired ambient air, where they appear to be near-universal contaminants. Cailleux and Allain questioned whether pentane was a normal constituent of human breath, because the concentrations in breath and inspired air, were frequently so similar. (Cailleux A & Allain P: Free Radicals Res Commun 1993; 18:323-327). This problem may be resolved by determination of the alveolar gradient of a VOC, the difference between its concentration in the breath and in the ambient air. (Phillips M. Sabas M & Greenberg J: Free Radical Res Commun. 1994; 20:333-337). Breath Alkanes As Markers of Breast Cancer Breast cancer is a common disease which now affects approximately one in every ten women in the United States. Early detection by periodic screening mammography can reduce mortality by 20-30%. However, mammography is expensive, frequently requires painful breast compression, entails exposure to radiation, and generates false-positive results in one third of all women screened over a 10 year period (Elmore J G, Barton M B, Moceri V M, Polk S, Arena P J and Fletcher S W: Ten-year risk of false positive screening mammograms and clinical breast examinations). There is a clinical need for a screening test for breast cancer which is at least as sensitive and specific as mammography, but is simpler, safer, less painful and less expensive. The cytochrome P450 (CYP) system comprises a group of mixed function oxidase enzymes which metabolize drugs and other xenobiotics. This system also metabolizes alkanes to alcohols e.g. n-hexane to 2- and 3-hexanol (Crosbie S J, Blain P G and Williams F M: Metabolism of n-hexane by rat liver and extrahepatic tissues and the effect of cytochrome P40 inducers. Hum Exp Toxicol 1997; 16(3):131-137). Rats treated with a potent cytochrome P-450 inhibitor exhibited a ten-fold increase in hexane and other breath VOCs with no increase in hepatic lipid peroxidation, demonstrating the significance of this pathway for VOC clearance (Mathews J M, Raymer J H, Etheridge A S, Velez Gr and Bucher J R: Do endogenous volatile organic chemicals in breath reflect and maintain CYP2E1 levels in vivo? Toxicol Appl Pharmacol 1997; 146(2):255-60). Studies in normal animals initially have demonstrated that the liver is a major site of clearance of alkanes from the body by cytochrome P450 metabolism (Burk-R J; Ludden-T M; Lane-J M: Pentane clearance from inspired air by the rat: dependence on the liver. Gastroenterology. 1983 84(1): 138-42: Daugherty-M S; Ludden-T M; Burk-R F: Metabolism of ethane and pentane to carbon dioxide by the rat, Drug-Metab-Dispos. 1988; 16(5):666-71). However, several recent reports have demonstrated that cytochrome P450 metabolism is not confined to the liver. Metabolism of alkanes to alcohols has also been observed in lung, brain and skeletal muscle microsomes expressing cytochrome P450 2E1 or 2B6 (Crosbie S J, Blain P G and Williams F M: Metabolism of n-hexane by rat liver and extrahepatic tissues and the effect of cytochrome P-450 inducers. Hum Exp Toxicol 1997; 16(3):131-137). The cytochrome P450 system is also present in human breast tissue. Murray et al reported that cytochrome P450 CYP1 B1 was expressed in cancers of breast as well as other tissues (Murray G I, Taylor M C, McFadyen M C, McKay J A, Greenlee W F, Burke M D and Melvin W T: Tumor-specific expression of cytochrome P450 CYP1B1. Cancer Res 1997;57(14):3026-31). Huang et al detected activity of the xenobiotic-metabolizing CYP1, CYP2 and CYP3 subfamilies of cytochrome P450 in human breast tissue (Huang Z, Fasco M J, Figge H L, Keyomarsi K and Kaminsky L S: Expression of cytochromes P450 in human breast tissue and tumors. Drug Metab Dispos 1996;24(8):599-905). They observed: " . . . When normal and tumor tissues were from the same individuals, higher amplification occurred in normal tissues . . . The machinery of possible in situ bioactivation of xenobiotics and modification of therapeutic drugs is thus present in human breast tissue". Taken together, these studies demonstrate: 1. Alkanes are metabolized in vivo by cytochrome P450 enzymes 2. Cytochrome P450 enzymes are present in normal and neoplastic human breast tissues 3. Breast cancer induces increased cytochrome P450 activity in normal breast tissue 4. Breast cancer may therefore induce increased metabolism of alkanes. Hietanen et al studied 20 women with histologically proven breast cancer and a group of age and sex-matched controls (Hietanen E, Bartsch H, Beireziat J-C, Camus A-M, McClinton S. Eremin O, Davidson L and Boyle P: Diet and oxidative stress in breast, colon and prostate cancer patients: a case control study, European Journal of Clinical Nutrition 1994;48:575-586). Mean breath pentane concentration in the cancer patients (2.6 ppb, SD=2.8) was significantly higher than in the controls (0.6 ppb, SD=1.1, p<0.01). They did not report concentrations of pentane in ambient air, nor the alveolar gradients of pentane. Breath Alkanes as Markers of Ischemic Heart Disease More than 3 million patients are hospitalized every year in the United States for chest pain. The cost is over $3 billion just for those found to be free of acute disease. Many patients with acute chest pain but without myocardial infarction are admitted to specialized services to determine the cause of their pain (Hoekstra J W and Gibler W B; Chest pain evaluation units: an idea whose time has come, JAMA 1997;278(20):1701-2). The main objective is to detect unstable angina, which is potentially life threatening. Evaluation of these patients is frequently extensive and expensive, entailing a comprehensive battery of tests such as echocardiography, exercise electrocardiography (ECG), myocardial scintigraphy and Holter monitoring. Employing such a battery of tests, Fruergaard et al evaluated 204 patients with acute chest pain but without myocardial infarction. They found the commonest etiology was gastro-esophageal disease, followed by ischemic heart disease and chest wall syndrome. The high risk subset comprised less than a third of all diagnoses (Fruergaard P, Laundbjerg J, Hesse B et al: The diagnoses of patients admitted with acute chest pain but without acute myocardial infarction. Eur Heart J 1996; 17(7):1028-34). McCullough et al determined that the practice of hospital admission for patients with chest pain and essentially normal ECGs was not cost favorable, at $1.7 million dollars per life saved (McCullough P A, Ayad O, O'Neill W W and Goldstein J A: Costs and outcomes of patients admitted with chest pain and essentially normal electrocardiograms. Clin Cardiol 1988;21(1):22-6). Despite these and other well-documented studies, patients with acute chest pain but without myocardial infarction are commonly hospitalized because physicians are generally reluctant to discharge a patient if there is a risk of unstable angina and sudden death. Hence there is a clinical need and an economic need for a diagnostic test which differentiates between the high-risk patient with cardiac chest pain who could benefit from hospitalization, and the low-risk patient with non-cardiac chest pain who could be safely discharged home and evaluated as an out-patient. Such a test could potentially reduce mortality and morbidity from unrecognized heart disease, while at the same time reducing costs to the health care system by reducing the number of unnecessary hospitalizations. There is now new evidence that a non-invasive breath test could provide such a test. There is an increasing body of evidence that myocardial oxygen free radical activity is increased in ischemic heart disease. Oxidative stress also increases during surgical reperfusion of the heart, or after thrombolysis, and it is related to transient left ventricular dysfunction, or stunning (Ferrari R; Agnoletti L; Comini L; Gaia G; Bachetti T; Cargnoni A; Ceconi C; Curello S; Visioli O; Oxidative stress during myocardial ischaemia and heart failure, Eur Heart J 1998;19 Suppl B:B2-11). The two major hypotheses which explain the mechanism of stunning are that it either results from a burst of oxygen free radical activity or from a loss of sensitivity of contractile filaments to calcium. These hypotheses are not mutually exclusive, and are likely to represent different facets of the same pathophysiological cascade. Myocardial stunning occurs clinically in various situations in which the heart is exposed to transient ischemia, such as unstable angina, acute myocardial infarction with early reperfusion, exercised-induced ischemia, cardiac surgery and cardiac transplantation (Bolli R: Basic and clinical aspects of myocardial stunning, Prog Cardiovasc Dis 1998;40(6): 477-516: Miura H; Morgan D A; Gutterman D D; Oxygen-derived free radicals contribute to neural stunning in the canine heart, Am J Physiol 1997;273(3 Pt 2): H1569-75). In 1991, Weitz et al reported that breath pentane was significantly increased in 10 patients with acute myocardial infarction compared to 10 healthy controls (Weitz Z W, Birnbaum A J, Sobotka P A, Zarling E J and Skosey J L: High breath pentane concentrations during acute myocardial infarction. Lancet 1991;337:933-35). However, these results were called into question by a subsequent study from the same institution which found no significant differences in breath pentane between 15 patients with acute myocardial infarction, 15 with stable angina and 15 normal controls (Mendis S. Sobotka P A and Euler D E: Expired hydrocarbons in patients with acute myocardial infarction, Free Radic Res 1995;23(2):117-22). They did observe a significant increase in breath pentane following balloon deflation in five patients with unstable angina undergoing coronary angioplasty (Mendis S, Sobotka P A, Leja F L and Euler D E: Breath pentane and plasma lipid peroxides in ischemic heart disease, Free Radic Biol Med 1995;19(5):679-84). However, Kohlmuller and Kochen demonstrated a fundamental flaw in the breath pentane assays: the column employed in the gas chromatograph (GC) did not separate pentane from isoprene, the most abundant compound in breath. What the investigators had reported as breath pentane was probably a mixture of pentane and isoprene (Kohlmuller D; Kochen W: Is n-pentane really an index of lipid peroxidation in humans and animals? A methodological reevaluation. Anal Biochem 1993 May 1;210(2):268-76). The GC columns employed in this research separate pentane and isoprene from one another (Phillips M, Sabas M and Greenberg J. Alveolar gradient of pentane in normal human breath. Free Radical Research Communications 1994;20(5):333-337). Breath Alkanes as Markers of Heart Transplant Rejection In December 1967, Christiaan Barnard, a South African surgeon, performed the first human heart transplant. Three days later, a surgical team in Brooklyn performed the first heart transplant in the United States. Since then, more than 36,000 heart transplants have been performed at over 271 centers throughout the world, including approximately 165 centers in the United States. There are nearly 20,000 people alive today in the United States who are the recipients of transplanted hearts. Refinements in patient selection, improved surgical techniques, newer antimicrobial agents, better myocardial protection, and the application of right ventricular endomyocardial biopsy to identify allograft rejection have resulted in better overall survival rates. Nevertheless, the most significant change in the management of transplant recipients came with the introduction and widespread commercial availability of cyclosporine in the early 1980s. Today, overall one year survival exceeds 80% and reported five and ten year survival approaches 65-70%. With the introduction of cyclosporine in the early 1980s, the incidence of life threatening acute allograft rejection decreased considerably. Unfortunately, patients receiving cyclosporine based triple drug immunosuppression regimens seldom have physical complaints suggestive of allograft rejection until very late in the rejection process. Even prior to the introduction of cyclosporine, however, signs and symptoms of allograft rejection were quite non-specific; generally ranging from subtle electrocardiographic changes to malaise, fatigue, dyspnea, edema, and anorexia (Winters G L, Loh E, Schoen F J: Natural history of focal moderate cardiac allograft rejection, Circulation 1995;91:1975. Billingham M E, Cary N R B, Hammond E H et al: A working formulation for the standardization of nomenclature in the diagnosis of heart and lung rejection study group. Heart Transplant 1990;9:587). Non-invasive techniques to diagnose rejection, such as electrocardiographic changes or echocardiographic indices suggestive of diastolic dysfunction, are relatively insensitive and have not routinely been used in clinical practice. Likewise, thallium and magnetic resonance imaging have not proven useful. Hence, right ventricular endomyocardial biopsy has remained the standard against which all other techniques are compared. The primary purposes of the right ventricular endomyocardial biopsy in the heart transplant recipient are to identify allograft rejection, assess the efficacy of treatment, and to rule out infectious etiologies. Biopsies are performed weekly for the first six post-operative weeks, biweekly until the third post-operative month, and monthly until month six. Subsequent intervals are generally determined on an individual basis. Unfortunately, right ventricular endomyocardial biopsy is associated, albeit infrequently, with complications including hematoma, infection, arrhythmia, ventricular perforation, and the development of coronary artery to right ventricle fistulas. There is a clinical need for an alternative method of detecting heart transplant rejection with a safe and non-invasive diagnostic test. There is a well-documented biochemical basis for breath testing provides for the early detection of transplant rejection. Tissue damage arising from inflammation is accompanied by an accumulation of intracellular oxygen free radicals (OFR'S) which cause lipid peroxidation of lipid membranes (Kneepkens C M F, Ferreira C, Lepage G and Roy C C: The hydrocarbon breath test in the study of lipid peroxidation: principles and practice. Clin Invest Med 1992; 15(2):163-186. Kneepkens C M F, Lepage G, Roy C C. The potential of the hydrocarbon breath test as a measure of lipid peroxidation. Free Radic Biol Med 1994;17:127-60). This process is accompanied by the evolution of alkanes which are excreted in the breath. One of these alkanes, pentane, is the best documented marker of OFR activity. Sobotka et al studied 37 outpatients with stable cardiac allograft function.(Sobotka P A, Gupta D K, Lansky D M, Costanzo M R and Zarling E J: Breath pentane is a marker of acute cardiac allograft rejection. J Heart Lung Transplant 1994; 13:224-9). Breath pentane was measured by gas chromatography and the results were compared with routine surveillance endomyocardial biopsy. Histopathologic findings consistent with rejection were present on endomyocardial biopsy in 52% of the subjects. Average pentane excretion for subjects with mild rejection (4.2 nmol/l, SD=2.8) or moderate rejection (5.4 nmol/l, SD=2.6) exceeded that seen in subjects who did not have rejection (1.7 nmol/l, SD=0.9) (p<0.02). A pentane cutoff value of 2.43 nmol/l, chosen to give the highest negative predictive value, had a sensitivity of 0.80. The authors concluded that breath pentane excretion was a sensitive noninvasive screening test for the detection of cardiac allograft rejection. These encouraging results have attracted criticism: Holt et al noted that the details of their analytic technique were sketchy; they may not have really been observing isoprene because most chromatographic columns do not separate pentane from isoprene, the most abundant compound in human breath. (Holt D W, Johnston A and Ramsey J D: Breath pentane and heart rejection. J Heart Lung Transplant 1994; 13:1147-8. Kohlmuller D, Kochen W: Is n-pentane really an index of lipid peroxidation in humans and animals? A methodological reevaluation. Anal Biochem 1993;210:266-76). Breath Alkanes as Markers of End-stage Renal Disease End-stage renal disease (ESRD) is a fatal condition unless it is treated with either kidney transplantation or dialysis of blood or peritoneal fluid. However, dialysis is not a cure for ESRD. The yearly gross mortality rate of patients in the ESRD program in the United States has increased from 20% in 1982 to approximately 24% in 1991, which has persisted (1). This high mortality may be due in part to case-mix factors e.g. the acceptance of older patients with severe concomitant disease to the ESRD program. Despite this, there is no clear understanding of why the mortality is so high, and what potentially reversible factors may be contributing. Clinicians who come into contact with patients with chronic renal failure are familiar with the classic odor of uremic breath. It has been variously described as "fishy", "ammoniacal" and "fetid" (2). Schreiner and Maher in their review of uremia described it as ammoniacal, comparing it to the smell of stale urine (3). Since the introduction of early dialysis in chronic renal failure, the debilitated patient with a foul mouth due to ulceration and bacterial overgrowth is seldom seen. However, a consistent fishlike odor is still noticeable in ESRD patients, suggesting that it is systemic in origin, rather than from bacteria in the mouth. As early as 1925, the uremic breath odor was described as arising from trimethylamine, in Osler's textbook (4). In 1963, Simenhoff et al reported increased levels of dimethylamine in the blood, cerebrospinal fluid and brain of patients with severe uremia (5). He extended this research with analysis of breath of uremic patients, employing GC (2). He found increased concentrations of secondary and tertiary amines, dimethylamine and trimethylamine. Both were significantly reduced by hemodialysis as well as by treatment with nonabsorbable antibiotics. He concluded that these VOCs were responsible in part for the classic fishy odor in uremic breath, and arose from bacterial overgrowth in the intestine. The use of no-load breath tests for the study of altered breath VOCs in renal failure has attracted little subsequent attention during the past 20 years, though a number of studies employing pre-load breath tests have been reported. Scherrer et al used the breath aminopyrine test to demonstrate accelerated hepatic microsomal metabolism in patients with chronic renal failure resulting from analgesic abuse (6). Heinrich et al performed a similar study with radiolabelled aminophenazone in patients with ESRD, and observed a depression of cytochrome P450 mixed function oxidases which was significantly reversed by dialysis (7). Maher et al investigated the potential role of free radical-mediated pulmonary injury during hemodialysis. They studied breath hydrogen peroxide levels during the first two hours of hemodialysis, but found no significant changes (8). Epstein et al studied excretion of radiolabelled CO.sub.2 in breath to demonstrate altered decarboxylation of alpha-ketoisovaleric acid in patients with chronic renal failure (9). In an animal model of trichlorethylene-induced nephrotoxicity, Cojocel et al found increased ethane in breath, demonstrating the role of oxygen free radical-induced lipid peroxidation (10). In addition, breath tests employing radiolabelled urea have been employed to demonstrate an increased rate of infection with H. pylori in ESRD patients. The Breath Methylated Alkane Contour Phillips et al also previously observed that methylated alkanes are common components of the breath in normal humans as well as in those suffering from lung cancer. Phillips M, Herrera J, Krishnan S, Zain M, Greenberg J and Cataneo R N: Variation in volatile organic compounds in the breath of normal humans. Journal of Chromatography B 629 (1-2):75-88; 1999; Phillips M, Gleeson K, Hughes J M B, Greenberg J, Cataneo R N, Baker L and McVay W P: Volatile organic compounds in breath as markers of lung cancer: a cross-sectional study. Lancet 353:1930-33; 1999. These VOCs appeared to provide additional markers of oxidative stress. SUMMARY OF THE INVENTION Improved analytical technology was employed to determine the most abundant volatile organic compounds (VOCs) in the breath of 50 normal humans. Kinetic analysis was employed to demonstrate that the alveolar gradient of a VOC (abundance in breath minus abundance in room air) varies with the difference between the rate at which a VOC is synthesized in the body and the rate at which it is cleared from the body by metabolism and excretion. A new marker of oxygen free radical (OFR) activity in the body was developed: the breath alkane profile. This comprised the alveolar gradients of a wide spectrum of VOCs ranging from C2 to C20 alkanes plotted as a function of carbon chain length. Similar profiles were developed for two alkane metabolites in breath: alkyl alcohols and 2-methyl alkanes. These profiles provide a new and non-invasive probe of human metabolism by demonstrating the relative predominance of synthesis versus clearance of a VOC in vivo. These breath profiles were evaluated in clinical studies of breast cancer, cardiac chest pain, renal disease, and aging. The breath profiles of controls and patients with disease were compared by logistic regression analysis. The breath alkane profile was determined in 35 women undergoing screening mammography. 10 had biopsy-proven breast cancer. The breath alkane profiles identified the women with breast cancer with 100% sensitivity and specificity. The breath alkane profile was determined in 8 patients with unstable angina pectoris and in 50 normal controls with no known history of heart disease. The breath alkane profiles identified the patients with unstable angina pectoris with 100% sensitivity and specificity. The changes in the breath alkane profile were exaggerated during subsequent coronary angioplastry. The breath alkane profile was determined in 19 patients with acute onset chest pain in a hospital emergency department. Ten had unstable angina pectoris and nine had an acute myocardial infarction. Compared to 50 normal controls with no known history of heart disease, the breath alkane profiles identified the patients with cardiac chest pain, and distinguished unstable angina pectoris from acute myocardial infarction with 100% sensitivity and specificity. The breath alkane profile and breath alkyl alcohol profile were determined in 213 studies of heart transplant recipients. Two pathologists reviewed the endomyocardial biopsies independently, and agreed that no treatment was required in 182, but treatment was required in 13. The combination of the breath alkane profile and the breath alkyl alcohol profile identified heart transplant rejection requiring treatment with 84.6% sensitivity and 80.2% specificity. The advanced new breath test appears to provide a highly, sensitive and specific test for breast cancer and cardiac chest pain. The profiles were different from one another in all conditions. The breath alkane profile was displaced downward in the patients with breast cancer, and upward in the patients with ischemic heart disease. Both the breath alkane profile and the alkyl alcohol profile were displaced upward in heart transplant rejection. These results of the breath tests are consistent with the documented pathophysiology of OFR'S in these disorders. In a further aspect of the present invention, a new marker of oxygen free radical (OFR) activity in the body was developed: the breath alkane profile. This comprised the alveolar gradients of a wide spectrum of VOCs ranging from C2 to C20 alkanes plotted as a function of carbon chain length. Similar profiles were developed for two alkane metabolites in breath: alkyl alcohols and 2-methyl alkanes. These profiles provide a new and non-invasive probe of human metabolism by demonstrating the relative predominance of synthesis versus clearance of a VOC in vivo. In the present inventive method, methylated alkanes were combined with the breath alkane profile in order to construct the breath methylated alkane contour (BMAC), a new three-dimensional marker of oxidative stress. This technique has been refined herein by determining the alveolar gradient of methylated alkanes and incorporating this data into a three dimensional plot. That is, where alveolar gradient versus the carbon chain length of n-alkanes was previously plotted, a third dimension has been added to the plot, which is the location of methylation along the carbon chain of the n-alkane. The information obtained from identifying the methylation site, in addition to the alveolar gradient and the carbon chain length of the nalkane, has produced a new and uniquely sensitive marker of oxidative stress in humans. In the data presented herein, collected in tests upon normal human beings and in those suffering form heart transplant rejection, it is shown that 1. Oxidative stress was greater in heart transplant recipients than in age-matched normal controls; 2. Oxidative stress increased with the severity of heart transplant rejection; and 3. The breath test was sensitive and specific for clinically significant rejection. |
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