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Link:  Pharm/Biotech Resources


Title:  Method of determining biological/molecular age

United States Patent:  6,933,120

Issued:  August 23, 2005

Inventors:  Seidman; Michael D. (5310 Putman, West Bloomfield, MI 48323)

Appl. No.:  271469

Filed:  October 15, 2002

Abstract

Methods of obtaining a measurement indicative of oxidative stress and the molecular age of an individual include the step of detecting a mitochondrial DNA deletion and correlating the quantity of the deletion with a measurement of a parameter related to oxygen metabolism.

SUMMARY OF THE INVENTION

A method is provided for obtaining a measurement indicative of oxidative stress in an individual subject. Such a method includes the steps of obtaining a specimen from the individual and performing an assay on the specimen in order to detect a quantity of a mitochondrial DNA deletion. The quantity of the mitochondrial DNA deletion detected is compared to a reference and a first value is generated by this comparison. Additionally, a parameter related to the individual's oxygen metabolism is assessed and a second value is obtained as a result of this assessment. Then, the first and second values are correlated in order to obtain a measurement indicative of oxidative stress.

In a further method for obtaining a measurement indicative of oxidative stress in an individual subject, a specimen from an individual subject is obtained and tested in order to detect a quantity of a mitochondrial DNA deletion. The quantity of the mitochondrial DNA deletion detected is compared to a reference and a first value is generated by this comparison. Additionally, an antioxidant indicator is assayed and a second value is obtained thereby. The first and second values are correlated in order to obtain a measurement indicative of oxidative stress.

DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT

Accordingly, an inventive method of obtaining a measurement indicative of oxidative stress in an individual subject is presented herein, the method including the step of obtaining a specimen from the individual subject. Mitochondrial DNA (mtDNA) is then isolated from the specimen and the mtDNA is subjected to an assay in order to detect a quantity of a deletion present therein. In a further step of an inventive method, the quantity of the deletion is compared to a reference to obtain a first value indicative of the level of an mtDNA deletion present in the specimen. In addition, an indicator related to the individual's oxygen metabolism is assessed to obtain a second value. Then, the first value and the second value are correlated over value dependant time scales to obtain a measurement indicative of oxidative stress. This information is communicated to the individual subject along with suggestions regarding treatment options and lifestyle changes.

An individual's level of oxidative stress is measured by assessing various physiological parameters related to oxygen metabolism. These data are immediate measures of metabolism. For example, such parameters include the amount of oxygen consumed, CO2 produced, the ventilatory rate, a respiratory volume such as an inhalation volume or tidal volume, arterial pO2, respiratory quotient and the like. Oxygen metabolism may also be estimated by measuring metabolic rate of an individual such as basal metabolic rate or resting metabolic rate. Physiological measurements relating to oxygen metabolism include the amount of reactive oxygen species, such as superoxide, which cells or tissues are exposed to. The level of reactive oxygen species is related to the amount of oxygen which a tissue or cell is exposed to and to the amount of oxygen metabolism occurring in the cell.

Further physiological parameters related to oxygen metabolism relating to oxidative stress include levels of antioxidants present in a sample obtained from an individual. An antioxidant is a substance present that inhibits oxidation of another substance. Preferably, a measured antioxidant includes those that inhibit oxidation of a substance which is a natural component of an organism, such as a lipid, a protein, a nucleic acid, or a carbohydrate, that is subject to oxidation. Exemplary antioxidants of this type include enzyme antioxidants such as superoxide dismutase, glutathione peroxidase, and catalase. Other antioxidants found naturally in an organism include tocopherols such as vitamin E, carotenoids such as beta carotene, flavonoids, glutathione, an ubiquinone such as coenzyme Q-10, and ascorbate. In another embodiment, a measured antioxidant is an antioxidant not usually found in the subject organism, such as a synthetic drug or naturally-occurring pharmaceutical substance derived from a second organism. Illustrative examples include 3,4-dihydroxybenzohydroxamic acid, 3,4,5-trihydroxybenzohydroxamidoxime and others known the art such as are found in Merck Index: An Encyclopedia of Chemicals, Drugs, & Biologicals, O'Neil et al. (Eds.), 13th edition, 2001.

An antioxidant is assayed in any cell, tissue or bodily fluid. For instance, antioxidant levels are measured in blood or fractions thereof such as plasma or serum, or other body fluids such as saliva, mucus, tears or urine. Antioxidant levels may also be measured in a cell sample from an individual including cell sources such as epidermal cells; mucosal cells; epithelial cells; hair roots; spermatozoa; and leukocytes, in particular, lymphocytes and platelets.

In a method according to the present invention, a measurement indicative of oxidative stress in an individual subject which relates to an individual's molecular age is obtained by assaying an antioxidant in a specimen obtained from the individual. An assay of an antioxidant includes assay of levels and/or the oxidation state of the particular antioxidant. For example, measurement of the ratio of reduced glutathione to oxidized glutathione is performed. Antioxidant levels are measured by techniques known in the art including any of various methods such as spectrophotometric assays, antibody-based assays such as ELISA, and activity based assay among others. For example, superoxide dismutase activity is measured by an indirect assay using a nitroblue tetrazolium. Commercial kits are available for assay of antioxidants, such as the glutathione peroxidase ELISA kit from CalBiochem. Other common antioxidants such as vitamin E and vitamin C are measured for instance, by high performance liquid chromatography or other suitable methods known in the art.

In a further step to obtain a measurement indicative of oxidative stress, an assay for mitochondrial deletions in a specimen obtained from the individual is performed. These data are representative of a time period beyond the moment of measurement. Assay of mitochondrial DNA deletions includes a step of detection of mitochondrial DNA deletions. There are a large number of mitochondrial DNA deletions that are known to occur and human deletions are well-studied. Information on the mitochondrial genome, including DNA sequence and base pair reference numbers is available, for example see reference 51. In particular, the deletion known as 4977 which occurs at nucleotide 8469 to 13447 in human mt DNA is commonly observed, for example, see references 21 and 45-48. DNA in the region of the 4977 deletion encodes NADH dehydrogenase subunit 5 and mitochondrial ATP synthase subunit 8. Further commonly observed human mitochondrial DNA deletions include a 6063 deletion occurring at nucleotide 7841 to 13905 (refs. 47, 49-50), a 7436 base pair deletion occurring at 8648 to 16085 (refs. 28-29), and other common DNA deletions such as is detailed in references 18-44 below and other references cited herein. RNA molecules transcribed and proteins encoded by the regions encompassed by the various deletions are known in the art.

Deletions are detected by, for example, molecular assays such as polymerase chain reaction (PCR), primer extension, restriction fragment length polymorphism, in situ hybridization, reverse transcription-PCR, differential display of RNA, and antibody based protein detection.

A particularly preferred method of deletion detection includes a polymerase chain reaction. PCR methods are known in the art, as are guidelines for choosing specific primers to detect specific deletions. Details of reaction protocols and parameters considered in choosing appropriate primers are found in standard references such as Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, 3rd Edition, 2001; Dieffenbach and Dveksler, PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratory, 1995; PCR Protocols: Current Methods and Applications (Methods in Molecular Biology, 15) by Bruce A. White (Ed.), 1993; and in the examples below.

In a further embodiment, an assay for mtDNA deletions includes an assay for protein products of mtDNA. In this assay, the level of a protein product encoded by a deleted region of mtDNA is compared with the level of another protein encoded by intact nuclear DNA or mtDNA. For example, mammalian cytochrome oxidase consists of thirteen subunits of which subunits I, II and III are encoded by mitochondrial DNA and the remaining subunits are encoded by nuclear DNA. An assay for mitochondrial DNA deletions may include assay of presence or absence of mitochondrial encoded cytochrome oxidase subunits I, II or III compared to a nuclear encoded subunit. In another example, an assay for the 4977 mtDNA deletion may include an assay of NADH dehydrogenase subunit proteins or ATP synthase subunit proteins. In this assay, levels of mtDNA encoded NADH dehydrogenase subunit 5 or mitochondrial ATP synthase subunit 8 are assayed.

A detected mitochondrial DNA deletion is preferably compared to a reference in order to provide a quantitative assessment of a mitochondrial deletion. Where the mtDNA deletion is detected by assessing a nucleic acid, a reference is preferably another nucleic acid in the specimen, such as non-deleted mitochondrial DNA, genomic DNA, mitochondrial RNA including messenger RNA, ribosomal RNA and transfer RNA, and RNA encoded by nuclear DNA, including messenger RNA, ribosomal RNA and transfer RNA. Quantitative PCR methods and standards are known in the art and are detailed in the examples below and in references such as Quantitative PCR Protocols (Methods in Molecular Medicine, Vol 26), Kochanowski and Reischl (Eds.), Humana Press; 1999. Where proteins encoded by mtDNA deletions are assayed, an appropriate protein reference, such as a nuclear encoded protein or a mitochondrial protein from a distinct region of the mitochondrial genome is assayed.

Since mitochondria exist in most cells of the body, any of various cells or tissues is used as a specimen for assay of a mitochondrial deletion. Previously, measurements of mtDNA deletions required invasive sampling or biopsy of organs such as liver and post-mitotic tissues such as brain. An inventive method allows non-invasive sampling for mtDNA deletion analysis and provides practical methods for achieving the obtention of a measure of oxidative stress. In particular, samples from an individual for use in a method according to the present invention are obtained from easily accessed material such as epidermal cells; mucosal cells, such as from the buccal or nasal cavity; epithelial cells; hair roots; spermatozoa; and blood cells such as leukocytes, in particular, lymphocytes and platelets. In addition, various bodily fluids and secretions are assayed for mitochondrial deletions in cells contained therein. For instance blood, urine, saliva, mucus and tears are sampled. In addition, different tissues are compared with each other, for instance a tissue containing cells which have relatively low turnover rates such as muscle cells might be compared with cells that are continually renewed, such as epidermal cells, in order to obtain temporal information about when deletions have occurred. In another embodiment a measurement in a sample from one cell type is compared with previous measurements from that cell type in order to obtain information on ongoing oxidative stress in the individual. Measurements of the quantity of mitochondrial DNA deletions are taken at intervals, the interval length dependent on the typical lifetime of the cell to be assayed. For instance, specimens of scalp hair including roots are taken over a period of time ranging from days to years.

Once a sample is obtained, assay for mtDNA deletions is performed on whole lysed cell preparations, isolated total nucleic acid preparations, isolated mitochondria or isolated mitochondrial nucleic acids. For assay of mitochondrial DNA deletions include preparation of total DNA from cell samples and purified mitochondrial DNA. Techniques for isolation and purification of mitochondria and various nucleic acids are known in the art. For example, various protocols are known for purification of mitochondria such as those found in Current Protocols in Cell Biology, Bonifacino, Dasso, Lippincott-Schwartz, Harford, and Yamada (Eds.), John Wiley & Sons, Inc. Further, kits for isolation of mitochondria are commercially available, for example from Sigma-Aldrich.

In a preferred method, mtDNA deletions are detected in blood cells, such as leukocytes. These cells are easily sampled, for example by a blood draw via a needle inserted into a blood vessel. Blood samples ranging in volume from 0.1 milliliter to 10 milliliters are typically used in detecting deletions, although more or less may be used. Particular populations of blood cells are optionally purified for analysis. For example, platelets are a preferred cell type for some applications since they contain mitochondria but no nucleus, facilitating assay of mtDNA. Protocols for isolation of specific populations of blood cells, including platelets, are known in the art, as are methods for purification of mitochondria, nucleic acids and proteins.

It will be appreciated that a method according to the present invention is applicable to various species, including humans, horses, cows, pigs, sheep, goats, rats, mice and avian species such as chickens. In species having organelle-containing erythrocytes, these cells are also appropriate for DNA analysis.

In a further step according to an inventive method, the measure of an oxygen related parameter and the measure of the quantity of an mtDNA deletion are related to obtain a measurement of oxidative stress in the individual. The relation of these measurements yields a significantly more informative measure of oxidative stress in an individual than either measure alone. For example, comparison of a measure of a parameter related to oxygen metabolism and the measure of the quantity of an mtDNA deletion present in an individual's blood cell sample shows a high level of the antioxidant vitamin E and a high level of an mtDNA deletion relative to levels of each usual for an individual of like age. Consideration of either measure alone yields some information regarding the health of the individual. For example, considered separately, the high level of mtDNA deletion signifies a high level of oxidative stress, indicating a need for medical intervention. A high vitamin E level represents a healthy state. Combined, these measurements not only inform the individual of various values representing aspects of oxidative metabolism, but, additionally, the combination of these measurements provides a measure that achieves a higher level of information, since the measurements represent a physiological marker of past metabolic events, a measure of actual damage done and a contemporary state of the patient's physiology in a single test. Thus, the patient and physician can effectively contemplate treatment options necessitated by the patient's molecular history and immediate molecular situation on the basis of one exam, rather than the usual basis of a series of lab test records compiled over a period of time in order to establish a history.

EXAMPLES

Example 1

Protocol

Detection and Quantification of MtDNA Deletion (MtDNA4977)

A detailed protocol is found in reference 35, N-W Soong and N. Arnheim, Meth Enzymol., 421-431, 1996.

Primers (designed in our laboratory):

Mt1C: (SEQ ID NO:1)
AGG CGC TAT CAC CAC TCT TGT TCG (13,176-13198) 
Mt2: (SEQ ID NO:2)
AAC CTG TGA GGA AAG GTA TTC CTG C (13,501-13,477)
Mt1A: (SEQ ID NO:3)
GAA TTC CCC TAA AAA TCT TTG AAA T (8224-8247)

Primers are end-labeled with (γ-32P) ATP using T4 Polynucleotide Kinase. Unincorporated nucleotides are removed by spinning through P4 columns. These primer lots are prepared to give approximately 10 concentration for PCR (5 micromolar) and are diluted directly into the PCR mix.

Example 2

PCR Analysis

PCR is carried out in 50 microliter volumes in 1 PCR buffer, containing 1.5 mM MgCl2.

32P end-labeled primer concentration is 0.5 micromolar.

Deoxy-nucleoside triphosphate (dNTPs) 200 micromolar.

2.5 Units of Taq polymerase.

100-1000 ng of genomic DNA.

Primers for Total MtDNA: Mt1C and Mt2, fragment size 324 bp.

Primers for Deletion: Mt1A and Mt2, fragment size 303 bp.

Cycle Parameters:

Initial denaturation at 94 C. for 3 min.    
Denaturation at 94 C. for 30 sec.
Annealing at 54 C. for 30 sec   30 cycles
Extension at 72 C. for 1 min.
Followed by 7 min extension at 72 C.

PCR conditions are identical for total and deletion-specific reactions except that deletion-specific reactions are run for 30 cycles and control PCR is carried out for 15 cycles.

Example 3

Polyacrylamide Gel Electrophoresis

After PCR, 10% (5 microliters) of each reaction is electrophoresed through 8% polyacrylamide gel. The gel is dried and counts from each specific band are quantitated with a Phosphorlmager (Biorad) after 15-24 hr exposure.

Example 4

Preparation of External Standards

For the construction of standard curves for deletion and control PCR, the respective PCR products are purified as a source of templates for the amplification reactions. Genomic DNA from aged heart tissues is used as a template for these preparative PCRs. The product bands are excised, electroeluted and concentrated by centrifuging through Centricon-10. These are aliquoted and stored at -;20 C. In order to develop "normal ranges" an acceptable standard is created by studying as few as 10 people and as many as 10,000 people in each decade ranging from 0-10, 11-20, 21-30, 31-40, 41-50, 51-60, 61-70, 71-80, 81-90, 91-100, 101-110, 111-120 years etc. Individual patients are then compared to a "normal" range thus providing a range for an acceptable amount of MtDNA deletion.

Serial dilutions of external standards were made and the range of dilutions over which the amplifications were exponential was determined. The plot of log counts versus log dilutions provides a good linear fit with a slope close to 1.

Example 5

Quantitation of Samples

Preliminary deletion and control PCR with unlabeled primers are performed on dilutions of DNA samples. The products signals are visually compared in ethidium bromide stained gels along with those of generated by amplification of the most concentrated standard dilution in the exponential range. The samples can then be diluted not to exceed the exponential range of the standard.

The PCR is then repeated with 32P labeled primers. Both control and deletion standards are amplified in parallel with the samples. The products are quantified and the signal generated by each sample is then extrapolated from the appropriate standard curve to obtain the equivalent dilution of the standard stock that would have given the same signal. The percentage of the ratio of the deletion dilution to that of control dilution would then give the % ratio of MtDNA del to total MtDNA.

Example 6

Mitochondrial DNA Deletion Analysis by Serial Dilution

A detailed protocol is found in reference 27, N. S. Hamblet and F. J. Castora, Biochem Biophys Res Commun., 207:839-847, 1995.

Primers: Same as above.

PCR Reaction and Cycle Parameters: Same as above.

Protocol:

Total DNA was diluted in two ranges: one for deleted MtDNA amplification (250,000-976 pg) and one for wild type MtDNA amplification (500-1.95 pg). Samples were linearized with Bam H1 before amplification. PCR products were electrophoresed on an 8% polyacrylamide gel and visualized by ethidium bromide staining. Photographs were taken and negatives were scanned using a laser densitometer. The ratio of deleted to wild type MtDNA was determined by densitometric measurement of the intensity of each band and subsequent plotting of the optical density (OD) versus the log of weight of DNA in the reaction mix. The OD was adjusted so that the area of each DNA band was normalized by the size of the DNA fragment. The plots of deleted and wild type PCR products were examined to determine the logarithmic values on the x axis at which ODs of the deleted and undeleted PCR products were equivalent. The selected OD should be within the linear range of the density curve and has low standard deviation.
 

Claim 1 of 8 Claims

1. A method of obtaining a measurement indicative of oxidative stress in an individual subject, the method comprising the steps of:

a) obtaining a specimen from an individual, wherein the obtaining a specimen does not include obtaining a specimen by organ biopsy;

b) performing an assay on the specimen to detect a quantity of a mitochondrial DNA deletion;

c) comparing the quantity of the deletion to a reference to obtain a first value;

d) making a quantitative measurement of a parameter related to oxygen metabolism in the individual to obtain a second value wherein the parameter related to oxygen metabolism is selected from the group consisting of: breathing rate, respiratory quotient, heart rate, basal metabolic rate, caloric intake, and calculating caloric expenditure; and

e) relating the first and second values to obtain a measurement indicative of oxidative stress.

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