| PATENT NUMBER | This data is not available for free |
| PATENT GRANT DATE | August 29, 2000 |
| PATENT TITLE |
Breath test for assessing hepatic function |
| PATENT ABSTRACT |
Provided herein is a novel breath test for assessing hepatic function. The test involves administration of a labeled methionine or methionine metabolite to a subject and measurement of the expired label. |
| PATENT INVENTORS | This data is not available for free |
| PATENT ASSIGNEE | This data is not available for free |
| PATENT FILE DATE | January 28, 1999 |
| PATENT REFERENCES CITED |
E Hayama., Eur, J. Drug Matab. Pharmocokinet 16:287-297. Dorothy S. Luciano, Ph.D., Human Function and Structure, 1978. Albert L. Lehninger, The John Hopkins University School of Medicine, Biochemisty . . . p. 189. Albert L. Lehninger, The John Hopkins University School of Medicine, Biochemistry . . .pp. 381-382, 502. J. G. Salway, Senior Lecturer in Medical Biochemistry, School of Biological Sciences, University of Surrey, Guildford . . . Metabolism at a Glance, 1994. |
| PATENT PARENT CASE TEXT | This data is not available for free |
| PATENT CLAIMS |
What is claimed is: 1. A method of assessing hepatic mitochondrial function in a subject comprising the steps of: a) orally administering to said subject an effective amount of carbon-labeled methionine to said subject; b) collecting expired breath from said subject; and c) measuring the amount of labeled carbon in said expired breath to assess hepatic mitochondrial function in said subject. 2. The method of claim 1, wherein said carbon-labeled methionine is selected from the group consisting of .sup.14 C methionine, .sup.13 C methionine, and mixtures thereof. 3. The method of claim 1, wherein said carbon-labeled methionine is .sup.13 C methionine. 4. The method of claim 1, wherein said carbon-labeled methionine is labeled at the 1-position of methionine. 5. The method of claim 1, wherein said carbon-labeled methionine comprises a plurality of labeled carbons. 6. The method of claim 1, wherein said labeled carbon in said expired breath is labeled carbon dioxide. 7. The method of claim 6, wherein said labeled carbon dioxide is .sup.13 C carbon dioxide. 8. The method of claim 1, wherein said administering step comprises administering carbon-labeled methionine in a pharmaceutically acceptable carrier. 9. The method of claim 1, further comprising comparing said amount of expired labeled carbon with a standard, whereby said comparing yields a measure of hepatic mitochondrial function. 10. The method of claim 9, wherein said standard comprises the mean value of expired label in a control population without hepatic disease or hepatic dysfunction. 11. The method of claim 10, wherein said assessing mitochondrial function is used to diagnose hepatic disease or hepatic dysfunction. 12. The method of claim 11, wherein said hepatic disease or dysfunction is selected from the group consisting of chronic liver disease, fulminant hepatic failure, viral-induced liver disease, metabolic liver disease, and hepatic dysfunction associated with sepsis or liver trauma. 13. The method of claim 1, wherein said assessing mitochondrial function is used to diagnose hepatic disease or hepatic dysfunction. 14. The method of claim 13, wherein said hepatic disease or dysfunction is selected from the group consisting of chronic liver disease, fulminant hepatic failure, viral-induced liver disease, metabolic liver disease, and hepatic dysfunction associated with sepsis or liver trauma. 15. The method of claim 1, wherein said measuring comprises isotopic measurement of labeled carbon. 16. The method of claim 15, wherein said measurement is selected from the group consisting of mass spectrometric measurement, laser measurement, infrared detection, nuclear magnetic resonance and liquid scintillation counting of labeled carbon. |
| PATENT DESCRIPTION |
BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for monitoring hepatic disease or dysfunction. More specifically, the invention relates to administering labeled methionine or methionine metabolites to an individual and assessing expired labeled carbon dioxide. 2. Description of the Prior Art Standard serologic and biochemical serum liver tests have been used to determine the presence of liver disease. However, these tests do not provide an accurate of hepatic functional capacity nor do they detect changes in hepatic disease severity (Gitnick, G. Surg. Clin. N. Am. 61:197-207 [1981]). Increasing prothrombin time and decreasing serum albumin concentrations have been used as prognostic indicators of progressive liver disease (Rydning, A., et al. Scand. J. Gastroenterol. 25:119-126 [1990]). Significant changes in prothrombin time and albumin may occur in patients for reasons other than liver dysfunction and, at times, only after severe liver decompensation. Further, radiological testing and histological examination of liver biopsies are poor indicators of decreasing hepatic function. The Child-Pugh (CP) classification is used to determine the degree of liver disease severity. The CP classification reflects the sum of scores derived from clinical and laboratory parameters. Disadvantages of the CP classification include subjective measures (degree of ascites and encephalopathy) and dependence on serum tests (bilirubin, albumin, and prothrombin time) that may be influenced by extrahepatic factors. As a result, the CP classification is a poor measure of patient status and is insensitive to small changes in the patient's condition. During the last twenty years, much work has been devoted to devising quantitative liver tests. Liver function can be subdivided into three compartments: 1) cytosolic, 2) microsomal, and 3) mitochondrial. Each compartment's function can be evaluated with both quantitative serum and breath tests. Blood and breath tests have been used for assessing mitochondrial-compartment hepatic function. A typical blood test is the measurement of the arterial ketone body ratio (AKBR). The hepatic mitochondrial redux potential ratio (ratio of NAD/NADH) correlates with the ketone body ratio (acetoacetate/.beta.-hydroxy butyrate) in liver disease. Serial changes in the AKBR can predict hepatic dysfunction, postoperative graft viability, and acute rejection (Asonuma K., S., et al. Transplantation. 51:164-171 [1991], Mori K, K., et al. Ann. Surg. 211:438-446 [1990]) post liver transplantation. However, recent experiments by Matsushita et al. determined that extrahepatic metabolism of ketone bodies diminishes the value of the AKBR (Matsushita K. et al. Hepatology. 20:331-335 [1994]). Additional disadvantages of the AKBR are its labor-intensiveness and the requirement for arterial blood. Breath tests can also be used to access the mitochondrial compartment hepatic function. The first substrate used as a breath test to measure mitochondrial function was the keto-analog of leucine ketoisocaproic acid (KICA) (Michaletz P. A., et al. Hepatology. 10:829-832 [1989]). Decarboxylation of KICA occurs mainly in hepatic mitochondria since anhepatic animals have a 75% reduction in .sup.14 CO.sub.2 production. Alcohol, which is known to alter the NAD/NADH ratio, deceases KICA decarboxylation. Further experiments with sodium salicylate, an uncoupler of mitochondrial respiration, showed an increase in KICA decarboxylation. The .sup.13 C and .sup.14 C-KICA breath tests have been used to access mitochondrial function in controls and in patients with alcoholic and non-alcoholic liver disease (Lauterburg B H, et al. Hepatology 17:418-422 [1993]). The KICA breath test showed impaired mitochondrial function in the alcoholic patients compared to controls and non-alcoholic patients. Patients with alcoholic disease had normal aminopyrine breath test and galactose elimination capacity (both measurements of cytosolic function) despite decreased mitochondrial function. These results suggest that KICA decarboxylation reflects hepatic mitochondrial function in patients with chronic alcoholic liver disease. The .sup.13 C-KICA breath test has also been used to differentiate between alcoholic and nonalcoholic liver-diseased patients (Witschi, A., et al. Alcohol Clin. Exp. Res. 18:951-955 [1994], Mion F, et al. Metabolism. 44:699-700 [1995]). Lauterburg and co-workers have shown that the .sup.13 C-KICA test can detect mitochondrial changes with the ingestion of the equivalent of two alcoholic drinks or with therapeutic doses of acetylsalicylic acid (ASA, aspirin) (Lauterburg B H, et al. J. Lab. Clin. Med. 125:378-383 [1995]). However, the KICA breath test is not widely used. Disadvantages of the KICA breath test are the high cost of the stable isotope and its instability in solution. These and other disadvantages of the prior art are overcome by the present invention. As shown herein, we provide a novel breath test for assessing hepatic disease or dysfunction. SUMMARY OF THE INVENTION The present invention overcomes the limitations of the prior art and provides a method and kit for the assessment of mitochondrial-compartment hepatic function. Provided herein is a method of assessing hepatic mitochondrial function in a subject comprising the steps of: a) administering to said subject an effective amount of carbon-labeled methionine or carbon-labeled methionine metabolite to said subject; b) collecting expired breath from said subject; and c) measuring the amount of label in said expired breath to assess hepatic mitochondrial function in said subject. The label is a carbon label. Preferably the labeled compound administered is .sup.13 C methionine or .sup.13 C methionine metabolite, or mixtures thereof. The labeled methionine metabolite is selected from the group consisting of carbon-labeled S-adenosylmethionine, S-adenosylhomocysteine, homocysteine, cystathionine, homoserine and .alpha.-ketobutyrate. The carbon-labeled compound may comprise a plurality of labeled carbons. The method further comprises comparing said amount of expired labeled carbon with a standard, whereby said comparing yields a measure of hepatic mitochondrial function. The standard comprises the mean value of expired label in a control population without hepatic disease or hepatic dysfunction. The hepatic disease or dysfunction that may be assessed with this method or kit includes liver disease or dysfunction associated with an impairment in the mitochondrial compartment of hepatic tissues. The disease or dysfunction is selected from the group consisting of chronic liver disease, fulminant hepatic failure, viral-induced liver disease, metabolic liver disease, and hepatic dysfunction associated with sepsis or liver trauma. The label may be measured by techniques commonly used for measuring the presence of labeled species. Isotopic measurement of label is selected from the group consisting of mass spectrometric measurement, laser measurement, infrared detection, nuclear magnetic resonance and liquid scintillation counting of labeled carbon. The present invention also provides a kit for assessing hepatic mitochondrial function comprising carbon-labeled methionine or carbon-labeled methionine metabolite in a pharmaceutically acceptable carrier, and a means for collecting expired breath. The advantages of the present invention may be gleaned from the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1: Metabolism of methionine. FIG. 2: MBT Test 1 vs Test 2 data for controls and liver patients. FIG. 3: MBT vs CP score. DETAILED DESCRIPTION OF THE INVENTION Methionine is an essential amino acid which has important roles in various metabolic processes, including protein synthesis (Lehninger, A. L. "Biochemistry." 1977 Worth Publishers, Inc. New York., Storch, K. J., et al. Am.J. Physiol. 255:E322-E331 [1988]). .sup.13 C-Methionine is metabolized via a transsulfuration pathway to cystathionine which is subsequently metabolized to alpha-ketobutyrate. Metabolism of alpha-ketobutyrate occurs solely in the hepatic mitochondria with release of .sup.13 CO.sub.2 and reduction of NAD to NADH. These reactions are illustrated in FIG. 1. Methionine metabolism is impaired in liver disease. Fasting levels of plasma methionine are elevated and the intravenous plasma clearance of methionine is reduced in liver disease when compared to healthy controls (Kinsell, L. W., et al. Science. 106:589-590 [1947]; Clowes, G. H. A. J., et al. Surgery. 96:675-684 [1984]; Horowitz J H, et al. Gastroenterology. 81:668-675 [1981]; Marchesini G, et al. Hepatology. 16:149-155 [1992]). Urinary sulfate excretion was also significantly decreased in cirrhotic patients. This suggests that a block in the transsulfuration pathway occurs with liver disease. Methionine plasma clearance was reduced in cirrhotic patients compared with controls. Methionine clearance correlated with the GEC (r=0.82) and with the CP score (r=-0.80). The reduced metabolism of methionine and the decreased formation of methionine end products indicate that mitochondrial function is impaired in cirrhotics. The present invention features a method of determining hepatic function in a patient using a breath test. This breath test is sufficiently sensitive to allow detection of not just chronic hepatic conditions where the liver is already irreparably damaged but it also is able to uncover these conditions at an early stage because of its dynamic nature. The method of the invention commences with a step of administering, preferably orally, a dose of a carbon-labeled methionine to a subject. In an additional embodiment, a carbon-labeled methionine metabolite may be administered to the subject. Useful methionine metabolites include compounds which are transported across the mitochondrial membrane into the mitochondria. Useful metabolites include, but are not limited to metabolites selected from the group consisting of S-adenosylmethionine, S-adenosylhomocysteine, homocysteine, cystathionine, homoserine and .alpha.-ketobutyrate, and mixtures thereof. As used herein, the term "compound" refers to labeled methionine or methionine metabolite. Oral administration ensures that the liver, rather than some other organ, gets the first chance to metabolize the labeled compound. The compound is oxidized in the patient, the expired breath from the patient is collected, and the amount of isotope in the expired breath is analyzed. The amount of expired isotope determined is compared with the standard and this comparison yields a measure of hepatic function. If the compound is administered orally, it preferably is in a pharmaceutically acceptable carrier such as water or a sugar solution but may be delivered chemically bound as a peptide or similar entity and released upon digestion. Alternatively, it may be administered intravenously. The preferred labeled isotope is a carbon isotope which yields expired carbon dioxide. The preferred carbon isotopes are .sup.13 C and .sup.14 C. .sup.13 C is more preferred because it is a stable rather than a radioactive isotope. Methionine or methionine metabolite having an isotope label at the 1-carbon position are preferred. This is because the 1-carbon is excised and exhaled as carbon dioxide at an early step in the oxidative process. However, in an alternative embodiment of the present invention, the compound may have a plurality of carbons labeled. If a .sup.13 C isotope is used, the preferred method of measurement is with a mass spectrometer, but other detection methods can be used, as would be known to one skilled in the art. These include, but are not limited to laser measurement, infrared detection, nuclear magnetic resonance and liquid scintillation counting of labeled carbon. The method of the invention can be used to detect hepatic dysfunction and disease by comparing a standard in the form of the mean value of expired isotope in a normal population with value determined from the test subject. This test can be used in identifying the presence of liver dysfunction caused by chronic liver diseases, fulminant hepatic failure, metabolic liver diseases, and liver dysfunction seen in septic or injured patients. For a further and more detailed description of these states, see Hepatology Textbook of Liver Disease, Zakim and Bayer, (W. B. Saunders 1990), incorporated by reference herein. The present invention features a breath test for determining problems in hepatic function. This test is a dynamic rather than a static test and shows hepatic function rather than merely liver cell degradation. The test is relatively inexpensive to carry out and yields rapid results. While radioactive isotopes can be used in the test, it preferably is carried out with stable isotopes. In the preferred embodiment, the individual is first required to fast overnight. This minimizes metabolic effects of meal absorption and the contribution of endogenous label appearing in the breath from natural levels of the endogenous isotope in the diet. However, the test may be conducted without requiring overnight fasting. Preferably at least two baseline breath samples are collected and the mean isotope value in these samples is used as a background. In an alternative embodiment, only one background measurement is made or, alternatively, no background measurement is made. If no background measurement is made, the amount of label in the expired breath is taken as the measurement for comparison with a control value. If a background measurement is made, this background is subtracted from the labeled carbon levels determined following isotopic administration in order to obtain the change in labeled species level. If the compound is administered orally, it is preferable to wait a sufficient amount of time after administration before collecting the breath sample(s) to allow metabolism of the labeled compound to release the labeled species. A sufficient amount of time is about 5 minutes to about 120 minutes, preferably about 10 minutes to about 90 minutes, and most preferably about 30 minutes to about 60 minutes. If the compound is administered intravenously, the sufficient amount of time is about 5 minutes to about 120 minutes, preferably about 10 minutes to about 90 minutes, and most preferably about 30 minutes to about 60 minutes. If the compound is an amino acid or methionine metabolite with a chiral center, it is preferably the L-stereoisomer, but may also be the D-stereoisomer or a racemic mixture thereof. All breath samples, both those collected prior to administration of the isotope and those after administration, may be collected with a commercially available breath sampler. These include, but are not limited to a Quintron AlveoSampler (Milwaukee, Wis.). These samplers have a mouthpiece and a collection bag with a one-way valve there between. The breath samples are trapped in a collection bag or other suitable breath collection device and the contents are injected into an evacuated tube such as, but not limited to an Exetainer tube (Labco Ltd, U.K.). |
| PATENT EXAMPLES | available on request |
| PATENT PHOTOCOPY | available on request |
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