WO2003044220A1 - Total cysteine assay - Google Patents

Total cysteine assay Download PDF

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Publication number
WO2003044220A1
WO2003044220A1 PCT/US2002/037420 US0237420W WO03044220A1 WO 2003044220 A1 WO2003044220 A1 WO 2003044220A1 US 0237420 W US0237420 W US 0237420W WO 03044220 A1 WO03044220 A1 WO 03044220A1
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Prior art keywords
homocysteine
cysteine
desulfurase
concentration
total
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PCT/US2002/037420
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French (fr)
Inventor
Qinghong Han
Mingxu Xu
Yuying Tan
Li Tang
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Anticancer, Inc.
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Priority to JP2003545841A priority Critical patent/JP4319545B2/en
Priority to CA2466503A priority patent/CA2466503C/en
Priority to AU2002346474A priority patent/AU2002346474B2/en
Priority to DE60212998T priority patent/DE60212998T2/en
Priority to EP02784538A priority patent/EP1456399B1/en
Publication of WO2003044220A1 publication Critical patent/WO2003044220A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/527—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving lyase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6806—Determination of free amino acids
    • G01N33/6812—Assays for specific amino acids
    • G01N33/6815—Assays for specific amino acids containing sulfur, e.g. cysteine, cystine, methionine, homocysteine
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/975—Kit
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00—Chemistry: analytical and immunological testing
    • Y10T436/18—Sulfur containing
    • Y10T436/182—Organic or sulfhydryl containing [e.g., mercaptan, hydrogen, sulfide, etc.]

Definitions

  • the invention relates to an enzymatic assay for total cysteine in blood or plasma.
  • Pathol (2001) 116:56-60 It appears that the levels of cysteine and homocysteine are interrelated since cysteine is, along with albumin, a covalent carrier of the homocysteine in circulation and cysteine is also a competitor of homocysteine for cellular uptake as well as a homocysteine metabolite via the transsulfuration pathway. Hortin, et al, J. Clin. Chem. (2001) 47:1121-1124.
  • total cysteine levels are correlated with age, total cholesterol concentration, diastolic blood pressure and coffee consumption, but unlike homocysteine levels, not smoking status, folate and vitamin intake, heart rate and physical activity.
  • SAHH S-adenosyl homocysteine hydrolase
  • adenosine is added to the sample containing a non-specific desulfurase, where the SAHH is present in sufficient quantity to catalyze the conversion of all of the homocysteine in the sample to SAH, thus protecting it from the action of the desulfurase.
  • SAHH S-adenosyl homocysteine hydrolase
  • the present invention provides an alternative enzymatic based assay for total cysteine.
  • the availability of both assays permits not only each total homocysteine and total cysteine to be determined, but also the tCys/tHcy ratio.
  • the invention is directed to determination of total cysteine in biological fluids, preferably plasma, by concomitant or otherwise controlled determination of both total homocysteine and the sum of the concentrations of homocysteine and cysteine.
  • an enzyme which is reactive with both homocysteine and cysteine to produce ammonia, hydrogen sulfide, and the respective ⁇ -keto-carboxylic acids ( ⁇ -ketoglutarate or pyruvate) in one reaction vessel and an enzyme which- carries out this desulfurase (lyase) reaction only with homocysteine in another reaction vessel the total cysteine level can be determined by the difference in concentrations.
  • the results are linear in the range of 2 ⁇ M- 1,000 ⁇ M in plasma.
  • the invention is directed to a method to determine the total cysteine concentration (tCys) in a biological fluid
  • a method to determine the total cysteine concentration (tCys) in a biological fluid comprises a) treating a first sample of said fluid with a desulfurase which utilizes both homocysteine and cysteine as substrates and measuring the level of at least one product of said desulfurase to determine the sum of the concentrations of cysteine (tCys) and of homocysteine (tHcy); b) treating a second sample of said biological fluid with a desulfurase that uses homocysteine specifically as a substrate and measuring the level of at least one product to determine the total concentration of homocysteine (tHcy) and c) subtracting the tHcy obtained in step b) from the tCys plus tHcy obtained in step a) to obtain total cysteine concentration.
  • the invention is directed to a kit for determining total cysteine concentration which comprises, in suitable containers, a desulfurase which utilizes both cysteine and homocysteine as a substrate, a desulfurase which utilizes homocysteine specifically as a substrate, and optionally, a reducing agent effective to reduce disulfide bonds, and optionally reagents for detection of at least one product, along with instructions for conducting the assay.
  • Figure 1 is a graph showing a typical standard curve of the assay of the invention.
  • the invention takes advantage of the specificity characteristics of desulfurase enzymes such that the levels of both homocysteine and cysteine can be determined in a biological fluid.
  • Suitable biological fluids include, for example, urine, cerebrospinal fluid, blood and plasma or serum. Much of the literature concerning the significance of these levels relates to plasma or serum. It is usually necessary to treat the sample with a reducing agent that is effective to reduce disulfide bonds (such as dithioerythritol) before the assay is conducted in order to liberate cysteine and homocysteine in their sulfhydryl form so as to be subject to the action of a desulfurase.
  • a reducing agent that is effective to reduce disulfide bonds (such as dithioerythritol) before the assay is conducted in order to liberate cysteine and homocysteine in their sulfhydryl form so as to be subject to the action of a desulfurase.
  • the desulfurases useful in the invention convert homocysteine to ⁇ -ketoglutarate, ammonia and hydrogen sulfide and may convert cysteine to pyruvate, ammonia and hydrogen sulfide.
  • the assays may involve measurement of any of these products. However, measurement of ammonia or hydrogen sulfide may be more convenient since the same methodology can be employed with respect to the products of both amino acids.
  • the ' measurement of hydrogen sulfide is particularly preferred, as methods for this measurement are relatively easy. However, means are well known in the art for measurement of ⁇ -ketoglutarate, pyruvate, and ammonia and measurement of these products' concentrations could be utilized as well.
  • One method to conveniently measure the levels of hydrogen sulfide produced is described in the above-referenced U.S. 6,468,762. Briefly, this method involves a first chromogenic reagent which contains an oxidizing agent such as potassium ferricyanide and a second chromogenic reagent which comprises an N,N-dialkyl phenylenediamine. These reagents react with the resulting hydrogen sulfide to form a colored complex which can be measured either spectraphotometrically by absorbance, or, with more sensitivity, by utilizing the fluorescence of the complex.
  • a preferred dialkylphenylenediamine is N,N-dipropyl phenylenediamine (DPPDA) or N,N-dibutyl phenylenediamine (DBPDA).
  • one of the desulfurases will utilize both cysteine and homocysteine as a substrate so that when a biological fluid is treated with this desulfurase, after a suitable reaction time, such as 10 minutes or 20 minutes, the desulfurase will have converted both the homocysteine and the cysteine in the biological sample to the above described products.
  • Such enzymes can suitably be prepared from microorganisms.
  • One example is the recombinantly prepared desulfurase from Pseudomonas putida. This enzyme is described by Tan, et al, Protein Purification & Expression (1997) 9:233-245.
  • a desulfurase specific for homocysteine referred to herein as a "homocysteinase” is defined as an enzyme having desulfurase activity with respect to homocysteine as a substrate in preference to cysteine as a substrate such that the amount of hydrogen sulfide liberated from treatment of a sample of blood, urine, tissue fluid, serum, or plasma of a subject with said enzyme is substantially generated from the homocysteine and not from the cysteine in said sample, even when the concentration of homocysteine is ten fold less than the concentration of cysteine in said sample.
  • the homocysteinase has the property that at least about 90% of the hydrogen sulfide produced by action of said homocysteinase upon contacting a biological fluid is contributed by homocysteine, when the concentrations of homocysteine and cysteine in said fluid are, respectively, about 5-15 ⁇ M and about 100-300 ⁇ M, respectively or wherein at least about 90%) of the hydrogen sulfide produced by action of said homocysteinase upon contacting a biological fluid is contributed by homocysteine when the fluid contains 5 ⁇ M homocysteine 1 ,000 ⁇ M cysteine.
  • homocysteinase enzymes are described in the above-referenced patent; and by Han, et al, Protein Expression & Purification (1998) 14:267-274.
  • the biological fluid is divided into two samples preferably treated identically.
  • the desulfurase is added to a sample in an amount needed to achieve a final concentration of 0.01-1,000 ⁇ g/ml; and reagents are added in amounts needed to achieve final concentrations of 1-100 mM of buffer, more preferably 5-50 mM; 0.01-100 mM reducing agent for disulfide bonds, even more preferably 0.1-10 mM.
  • the nature of the remaining reagents depends on the detection system chosen for the assay.
  • the level of products is determined by measuring the concentration or amount of hydrogen sulfide produced.
  • Particularly preferred is the formation of a chromophore which also has the property of being fluorescent.
  • the complex which is able to absorb light and to fluoresce, is obtained by reaction with N,N-dialkyl phenylenediamine, preferably N,N-dipropyl phenylenediamine (DPPDA) or N,N-dibutyl phenylene diamine (DBPDA).
  • DPPDA N,N-dipropyl phenylenediamine
  • DBPDA N,N-dibutyl phenylene diamine
  • the complex is then oxidized with a suitable oxidizing agent, such as ferric ion in the form of, for example, ferricyanide.
  • the level of the colored reagent can be measured by absorbance, typically at approximately 670-680 nm
  • the fluorescence of this complex could be measured by using an excitation wavelength in the range of approximately 665 nm or 640 nm and the measuring corresponding emission at lower wavelengths.
  • the use of fluorescence detection as opposed to absorbance is illustrated below and is shown to enhance the sensitivity of the assay.
  • the results in the sample containing the non-specific desulfurase provide concentration levels of tHcy plus tCys.
  • the results in the sample containing the homocysteinase provide the concentration of tHcy.
  • the difference in these concentrations represents the total cysteine concentration in the biological fluid.
  • Samples to be assayed are divided into two portions: One portion is treated with non-specific desulfurase and the other with homocysteinase.
  • the samples are then treated with 10 ⁇ l of recombinant homocysteinase or nonspecific desulfurase (0.275 mg).
  • the desulfurase is recombinantly produced and is that described by Tan, et al. (supra), isolated from P. putida. the homocysteinase is that described by Han, et al. (supra). Both samples are incubated at 37°C for ten minutes.
  • reaction is then stopped by adding 50 ⁇ l of chromogen (40 mM N,N- dibutyl-phenylenediamine hydrochloride in 6 M HC1) followed by addition of 50 ⁇ l oxidizing agent (40 mM potassium ferricyanide in 20 mM potassium phosphate, pH 8.3).
  • chromogen 40 mM N,N- dibutyl-phenylenediamine hydrochloride in 6 M HC1
  • oxidizing agent 40 mM potassium ferricyanide in 20 mM potassium phosphate, pH 8.3

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Abstract

A method to determine a total cysteine in biological fluids utilizes similarly treated portions of the fluid with a homocysteinase and a non-specific desulfurase.

Description

TOTAL CYSTEINE ASSAY
Cross-Reference to Related Applications
[0001] This application claims priority under 35 U.S.C. § 119 to provisional application 60/333532 filed 20 November 2001. The contents of that application are incorporated herein by reference.
Technical Field
[0002] The invention relates to an enzymatic assay for total cysteine in blood or plasma.
Background Art
[0003] It has become apparent that the total cysteine content in plasma (tCys) is an important parameter in evaluating the risk of cardiovascular disease. It has been reported that low risk of cardiovascular disease is associated with tCys levels at 250-275 μM whereas a higher risk is associated both with lower levels (<225 μM) and higher levels (>300 μM). El-Khairy, et al, Circulation (2001) 2544-2549. The levels of the cysteine homolog, homocysteine (tHcy) are also relevant as the risk of venous thrombosis and myocardial infarction are increased at high levels of homocysteine. Marcucci, et al, M. J. Clin. Pathol (2001) 116:56-60. It appears that the levels of cysteine and homocysteine are interrelated since cysteine is, along with albumin, a covalent carrier of the homocysteine in circulation and cysteine is also a competitor of homocysteine for cellular uptake as well as a homocysteine metabolite via the transsulfuration pathway. Hortin, et al, J. Clin. Chem. (2001) 47:1121-1124.
[0004] It is also known that individuals with very high levels of homocysteine due to defects in the transsulfuration pathway have low levels of total cysteine. It is estimated that one percent of the population is defective in the enzyme responsible; homozygous deficiency results in homocysteinuria. The ratio of tCys/tHcy will assist in identifying heterozygotes. Boddie, et al, Metabolism (1998) 47:207-211.
[0005] In addition, total cysteine levels are correlated with age, total cholesterol concentration, diastolic blood pressure and coffee consumption, but unlike homocysteine levels, not smoking status, folate and vitamin intake, heart rate and physical activity. El-Khairy, et al, Am. J.'Clin. Nutr. (1999) 70:1016-1024. Both tCys and tHcy are associated with renal failure. Mansoor, et al, Clin. Chem. (1993) 39:980-985.
[0006] Methods to determine total homocysteine enzymatically have also been described, for example, in U.S. patent 6,468,762 issued 22 October 2002 as well as U.S. patents 6,066,467; 5,998,191; and 5,985,540, the disclosures of which are incorporated herein by reference.
[0007] An alternative method of measuring total cysteine is also described in the above mentioned '762 patent wherein the sample to be assayed is treated with a non-specific desulfurase. To measure cysteine directly, S-adenosyl homocysteine hydrolase (SAHH) and adenosine are added to the sample containing a non-specific desulfurase, where the SAHH is present in sufficient quantity to catalyze the conversion of all of the homocysteine in the sample to SAH, thus protecting it from the action of the desulfurase. Thus, only cysteine levels in the sample are measured.
[0008] The present invention provides an alternative enzymatic based assay for total cysteine. The availability of both assays permits not only each total homocysteine and total cysteine to be determined, but also the tCys/tHcy ratio.
Disclosure of the Invention
[0009] The invention is directed to determination of total cysteine in biological fluids, preferably plasma, by concomitant or otherwise controlled determination of both total homocysteine and the sum of the concentrations of homocysteine and cysteine. By utilizing an enzyme which is reactive with both homocysteine and cysteine to produce ammonia, hydrogen sulfide, and the respective α-keto-carboxylic acids (α-ketoglutarate or pyruvate) in one reaction vessel and an enzyme which- carries out this desulfurase (lyase) reaction only with homocysteine in another reaction vessel, the total cysteine level can be determined by the difference in concentrations. The results are linear in the range of 2 μM- 1,000 μM in plasma.
[0010] Thus, in one aspect, the invention is directed to a method to determine the total cysteine concentration (tCys) in a biological fluid which method comprises a) treating a first sample of said fluid with a desulfurase which utilizes both homocysteine and cysteine as substrates and measuring the level of at least one product of said desulfurase to determine the sum of the concentrations of cysteine (tCys) and of homocysteine (tHcy); b) treating a second sample of said biological fluid with a desulfurase that uses homocysteine specifically as a substrate and measuring the level of at least one product to determine the total concentration of homocysteine (tHcy) and c) subtracting the tHcy obtained in step b) from the tCys plus tHcy obtained in step a) to obtain total cysteine concentration.
[0011] In another aspect, the invention is directed to a kit for determining total cysteine concentration which comprises, in suitable containers, a desulfurase which utilizes both cysteine and homocysteine as a substrate, a desulfurase which utilizes homocysteine specifically as a substrate, and optionally, a reducing agent effective to reduce disulfide bonds, and optionally reagents for detection of at least one product, along with instructions for conducting the assay.
Brief Description of the Drawings
[0012] Figure 1 is a graph showing a typical standard curve of the assay of the invention.
Modes of Carrying Out the Invention
[0013] The invention takes advantage of the specificity characteristics of desulfurase enzymes such that the levels of both homocysteine and cysteine can be determined in a biological fluid. Suitable biological fluids include, for example, urine, cerebrospinal fluid, blood and plasma or serum. Much of the literature concerning the significance of these levels relates to plasma or serum. It is usually necessary to treat the sample with a reducing agent that is effective to reduce disulfide bonds (such as dithioerythritol) before the assay is conducted in order to liberate cysteine and homocysteine in their sulfhydryl form so as to be subject to the action of a desulfurase.
[0014] The desulfurases useful in the invention convert homocysteine to α-ketoglutarate, ammonia and hydrogen sulfide and may convert cysteine to pyruvate, ammonia and hydrogen sulfide. The assays may involve measurement of any of these products. However, measurement of ammonia or hydrogen sulfide may be more convenient since the same methodology can be employed with respect to the products of both amino acids. The 'measurement of hydrogen sulfide is particularly preferred, as methods for this measurement are relatively easy. However, means are well known in the art for measurement of α-ketoglutarate, pyruvate, and ammonia and measurement of these products' concentrations could be utilized as well.
[0015] One method to conveniently measure the levels of hydrogen sulfide produced is described in the above-referenced U.S. 6,468,762. Briefly, this method involves a first chromogenic reagent which contains an oxidizing agent such as potassium ferricyanide and a second chromogenic reagent which comprises an N,N-dialkyl phenylenediamine. These reagents react with the resulting hydrogen sulfide to form a colored complex which can be measured either spectraphotometrically by absorbance, or, with more sensitivity, by utilizing the fluorescence of the complex. A preferred dialkylphenylenediamine is N,N-dipropyl phenylenediamine (DPPDA) or N,N-dibutyl phenylenediamine (DBPDA).
[0016] However, any method of measuring the products of the reaction may be used.
[0017] As stated above, one of the desulfurases will utilize both cysteine and homocysteine as a substrate so that when a biological fluid is treated with this desulfurase, after a suitable reaction time, such as 10 minutes or 20 minutes, the desulfurase will have converted both the homocysteine and the cysteine in the biological sample to the above described products. Such enzymes can suitably be prepared from microorganisms. One example is the recombinantly prepared desulfurase from Pseudomonas putida. This enzyme is described by Tan, et al, Protein Purification & Expression (1997) 9:233-245.
[0018] A desulfurase specific for homocysteine, referred to herein as a "homocysteinase" is defined as an enzyme having desulfurase activity with respect to homocysteine as a substrate in preference to cysteine as a substrate such that the amount of hydrogen sulfide liberated from treatment of a sample of blood, urine, tissue fluid, serum, or plasma of a subject with said enzyme is substantially generated from the homocysteine and not from the cysteine in said sample, even when the concentration of homocysteine is ten fold less than the concentration of cysteine in said sample. Alternatively, the homocysteinase has the property that at least about 90% of the hydrogen sulfide produced by action of said homocysteinase upon contacting a biological fluid is contributed by homocysteine, when the concentrations of homocysteine and cysteine in said fluid are, respectively, about 5-15 μM and about 100-300 μM, respectively or wherein at least about 90%) of the hydrogen sulfide produced by action of said homocysteinase upon contacting a biological fluid is contributed by homocysteine when the fluid contains 5 μM homocysteine 1 ,000 μM cysteine. Such homocysteinase enzymes are described in the above-referenced patent; and by Han, et al, Protein Expression & Purification (1998) 14:267-274.
[0019] As set forth above, the biological fluid is divided into two samples preferably treated identically. Typically, the desulfurase is added to a sample in an amount needed to achieve a final concentration of 0.01-1,000 μg/ml; and reagents are added in amounts needed to achieve final concentrations of 1-100 mM of buffer, more preferably 5-50 mM; 0.01-100 mM reducing agent for disulfide bonds, even more preferably 0.1-10 mM. The nature of the remaining reagents depends on the detection system chosen for the assay.
[0020] In one particularly preferred method of detection, the level of products is determined by measuring the concentration or amount of hydrogen sulfide produced. Particularly preferred is the formation of a chromophore which also has the property of being fluorescent. The complex, which is able to absorb light and to fluoresce, is obtained by reaction with N,N-dialkyl phenylenediamine, preferably N,N-dipropyl phenylenediamine (DPPDA) or N,N-dibutyl phenylene diamine (DBPDA). The complex is then oxidized with a suitable oxidizing agent, such as ferric ion in the form of, for example, ferricyanide. While the level of the colored reagent can be measured by absorbance, typically at approximately 670-680 nm, the fluorescence of this complex could be measured by using an excitation wavelength in the range of approximately 665 nm or 640 nm and the measuring corresponding emission at lower wavelengths. The use of fluorescence detection as opposed to absorbance is illustrated below and is shown to enhance the sensitivity of the assay.
[0021] The results in the sample containing the non-specific desulfurase provide concentration levels of tHcy plus tCys. The results in the sample containing the homocysteinase provide the concentration of tHcy. Thus, the difference in these concentrations represents the total cysteine concentration in the biological fluid.
[0022] The following example is offered to illustrate but not to limit the invention. Example 1 Determination of Cysteine and Homocysteine
[0023] Samples to be assayed are divided into two portions: One portion is treated with non-specific desulfurase and the other with homocysteinase.
[0024] In each case, 10-20 μl of sample and correspondingly 980-990 μl of Conversion Buffer to a sample total of 1 ml is incubated for 30 minutes at 37°C. The Conversion Buffer is 20 mM potassium phosphate, pH 8.3, 150 mM NaCl, 30 μg/ml, 0.2% Triton X- 100, and l mM DTT.
[0025] The samples are then treated with 10 μl of recombinant homocysteinase or nonspecific desulfurase (0.275 mg). The desulfurase is recombinantly produced and is that described by Tan, et al. (supra), isolated from P. putida. the homocysteinase is that described by Han, et al. (supra). Both samples are incubated at 37°C for ten minutes.
[0026] The reaction is then stopped by adding 50 μl of chromogen (40 mM N,N- dibutyl-phenylenediamine hydrochloride in 6 M HC1) followed by addition of 50 μl oxidizing agent (40 mM potassium ferricyanide in 20 mM potassium phosphate, pH 8.3). The portions are then incubated for 10 minutes at 37°C and read by fluorescence with an excitation wavelength of 665 nm and an emission wavelength of 690 nm or by absorbance at 675 nm.

Claims

Claims
1. A method to determine the total cysteine concentration (tCys) in a biological fluid which method comprises a) treating a first sample of said fluid with a non-specific desulfurase which utilizes both homocysteine and cysteine as substrates and measuring the level of at least one product of said desulfurase to determine the sum of the concentration of cysteine (tCys) and of homocysteine (tHcy); b) treating a second sample of said biological fluid with a homocysteinase that uses homocysteine specifically as a substrate and measuring the level of at least one product to determine the total concentration of homocysteine (tHcy); and c) subtracting the tHcy obtained in step b) from the (tCys + tHcy) obtained in step a) to obtain total cysteine concentration.
2. The method of claim 1, wherein the product measured in step a) and step b) is hydrogen sulfide.
3. The method of claim 2, wherein the hydrogen sulfide is measured by treating with an oxidizing agent and a dialkyl phenylenediamine.
4. The method of claim 3, wherein the oxidizing agent is ferric ion.
5. The method of claim 1, wherein the non-specific desulfurase is isolatable from P. putida.
6. The method of claim 1, wherein the homocysteinase is isolatable from T. vaginalis.
7. The method of claim 1 , wherein said biological fluid is serum or plasma.
8. A kit for determining total cysteine concentration which comprises, in suitable containers, a non-specific desulfurase which utilizes both cysteine and homocysteine as a substrate, a homocysteinase which utilizes homocysteine specifically as a substrate, along with instructions for conducting the assay.
9. The kit of claim 8, which further includes a reducing agent effective to reduce disulfide bonds.
10. The kit of claim 8, which further includes reagents for detection of at least one product.
PCT/US2002/037420 2001-11-20 2002-11-20 Total cysteine assay WO2003044220A1 (en)

Priority Applications (5)

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JP2003545841A JP4319545B2 (en) 2001-11-20 2002-11-20 Total cysteine assay
CA2466503A CA2466503C (en) 2001-11-20 2002-11-20 Total cysteine assay
AU2002346474A AU2002346474B2 (en) 2001-11-20 2002-11-20 Total cysteine assay
DE60212998T DE60212998T2 (en) 2001-11-20 2002-11-20 GESAMTCYSTEIN ASSAY
EP02784538A EP1456399B1 (en) 2001-11-20 2002-11-20 Total cysteine assay

Applications Claiming Priority (2)

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US33353201P 2001-11-20 2001-11-20
US60/333,532 2001-11-20

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AT (1) ATE332394T1 (en)
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CA (1) CA2466503C (en)
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EP1555530A1 (en) * 2004-01-13 2005-07-20 Daiichi Pure Chemicals Co., Ltd. Method for quantitatively determining homocysteine and a reagent for quantitative determination of homocysteine
US9365625B1 (en) 2011-03-31 2016-06-14 David Gordon Bermudes Bacterial methionine analogue and methionine synthesis inhibitor anticancer, antiinfective and coronary heart disease protective microcins and methods of treatment therewith

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EP1539988A4 (en) * 2002-09-03 2007-11-07 Anticancer Inc Homocysteine assay adaptable to screening
JP5862373B2 (en) * 2012-03-05 2016-02-16 ニプロ株式会社 Method for measuring the concentrations of hippuric acid and methylhippuric acid in biological samples
JP5862374B2 (en) * 2012-03-05 2016-02-16 ニプロ株式会社 Method for measuring the concentration of hippuric acid in a biological sample
JP5978658B2 (en) * 2012-03-05 2016-08-24 ニプロ株式会社 Method for measuring the total concentration of hippuric acid and methylhippuric acid in biological samples

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Publication number Priority date Publication date Assignee Title
EP1555530A1 (en) * 2004-01-13 2005-07-20 Daiichi Pure Chemicals Co., Ltd. Method for quantitatively determining homocysteine and a reagent for quantitative determination of homocysteine
US7198890B2 (en) 2004-01-13 2007-04-03 Daiichi Pure Chemicals Co., Ltd. Method for quantitatively determining homocysteine and a reagent for quantitative determination of homocysteine
US9365625B1 (en) 2011-03-31 2016-06-14 David Gordon Bermudes Bacterial methionine analogue and methionine synthesis inhibitor anticancer, antiinfective and coronary heart disease protective microcins and methods of treatment therewith

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CN1289688C (en) 2006-12-13
AU2002346474B2 (en) 2008-01-17
JP4319545B2 (en) 2009-08-26
KR20050044561A (en) 2005-05-12
ATE332394T1 (en) 2006-07-15
US6927038B2 (en) 2005-08-09
AU2002346474A1 (en) 2003-06-10
DE60212998T2 (en) 2006-12-21
CN1589329A (en) 2005-03-02
KR100976967B1 (en) 2010-08-23
JP2005509441A (en) 2005-04-14
EP1456399A1 (en) 2004-09-15
EP1456399A4 (en) 2004-12-29
CA2466503A1 (en) 2003-05-30
US20030162239A1 (en) 2003-08-28
EP1456399B1 (en) 2006-07-05
CA2466503C (en) 2010-06-01
DE60212998D1 (en) 2006-08-17

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