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WO2009064843A1 - Procédé de mesure des concentrations en acides aminés - Google Patents

Procédé de mesure des concentrations en acides aminés Download PDF

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Publication number
WO2009064843A1
WO2009064843A1 PCT/US2008/083340 US2008083340W WO2009064843A1 WO 2009064843 A1 WO2009064843 A1 WO 2009064843A1 US 2008083340 W US2008083340 W US 2008083340W WO 2009064843 A1 WO2009064843 A1 WO 2009064843A1
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Prior art keywords
amino acid
trna
concentration
sample
cognate
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PCT/US2008/083340
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English (en)
Inventor
James A. Landro
Chris Forbes
Alex Szewczak
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Bayer Healthcare Llc
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Publication of WO2009064843A1 publication Critical patent/WO2009064843A1/fr

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    • 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
    • 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
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/60—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances involving radioactive labelled substances
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/9015—Ligases (6)

Definitions

  • the present invention relates generally to a method for measuring the concentration of one or more amino acids in a sample.
  • amino acids function as the basic building blocks of all polypeptides. Polypeptides, in turn, build cells and tissues, regulate metabolism, process genetic information, and function in our immune systems, neurological systems and endocrine/hormonal systems. Some forms of life can manufacture all the necessary amino acids themselves, e.g. certain microorganisms. Most other kinds of life must obtain certain amino acids from the diet. In humans, for example, eight of the 20 standard amino acids are 'essential,' i.e. cannot be manufactured by the body. The remaining amino acids are nonessential and can be synthesized with the proper nutrition.
  • amino acids Today, in many technology areas, the analysis of amino acids is an important requirement. Particularly in the rapidly expanding area of biotechnology, this analysis is a critical procedure in the characterization of proteins and peptides. In addition, amino acid analysis is required in medicine, where characterization of amino acids in biological materials can be useful in diagnostic procedures and the testing of drug or treatment efficacy. In the
  • Hereditary disorders of amino acid processing i.e. aminoacidopathies and other amino acid disorders, can be the result of defects either in the breakdown of amino acids or in the body's ability to transport the amino acids into cells. Because these disorders often produce symptoms early in life, newborns are routinely screened for aminoacidopathies. For example, newborns in the U.S. are commonly screened for phenylketonuria, maple syrup urine disease, homocystinuria, tyrosinemia, and a number of other inherited disorders. Phenylketonuria (PKU) is a disorder that causes a buildup of the amino acid phenylalanine, which is an essential amino acid that cannot be synthesized in the body but is present in food. More in
  • this condition is caused by an inherited deficiency of the enzyme phenylalanine hydroxylase — which converts phenylalanine to tyrosine.
  • Phenylalanine accumulates in the plasma and is oxidized by phenylalanine transaminase to phenylpyruvic acid, which is normally excreted in the urine.
  • Phyenylpyruvic acid is a ketoacid, hence the name phenylketonuria. Without the enzyme that converts it to tyrosine, phenylalanine builds up in the blood and is toxic to the brain, causing mental retardation. Children with maple syrup urine disease are unable to metabolize certain amino acids.
  • amino acid concentration can be measured using an ion- exchange chromatography followed by post-elution modification with the detection reagent ninhydrin.
  • chromatographic techniques require numerous steps and are time-
  • High throughput screening is an essential component in the repertoire of modern technologies that improve speed and efficiency in contemporary drug discovery. This process of screening large (e.g. > 10 6 ) diverse collections of low molecular weight compounds plays a critical role in providing novel chemical starting points for iterative optimization by medicinal chemistry. It is not uncommon to test more than 100,000 compounds per day in a given screening campaign.
  • a test or assay for HTS must measure a relevant activity of the target, produce a robust signal for that activity, be readily assembled from commercial and/or in-house prepared reagents, and entail the minimum possible sequence of physical steps involving addition or sampling of the test reagents/mixture. In this way, a large number of samples may be evaluated in a short period of time.
  • high throughput screening and other high throughput methods have become essential for identifying lead chemical matter for therapeutic drug discovery efforts (S. K. Chanda et al., Drug Discovery Today 8 (2003) 168-174; M.T. Veledo et al., Electrophoresis 27 (2006) 3101- 3107). No counterpart high throughput methodology is known for the analysis or detection of amino acids.
  • a viable high throughput method for the quantitative analysis of amino acids would be a significant advance in the art. Such a method would find use in the diagnosis and monitoring of aminocidopathies and other amino acid disorders, as well as in the evaluation of nutritional status. Quantitative analysis of amino acids, and especially, high- throughput analysis, would find use in assays for screening or evaluating compounds (e.g.
  • the present invention relates generally to a method to measure the concentration of individual amino acids is described which is efficient, effective, rapid and amendable to being carried out in a high throughput mode.
  • the method of the invention provides together for the first time (1) a scintillation proximity assay (SPA) to measure aminoacyl-tRNA synthetase (aaRS) activity and (2) the application of the enzymatic isotope dilution principle using aaRS activity to measure the concentration of amino acids.
  • SPA scintillation proximity assay
  • aaRS aminoacyl-tRNA synthetase
  • the inventive method takes advantage of competition between a fixed concentration of radiolabeled amino acid and an unknown concentration of the same non-radiolabeled amino acid for its cognate tRNA catalyzed by the aaRS specific for that amino acid at equilibrium. Under equilibrium conditions, preferably in the case of limiting tRNA, the rate of the enzyme-catalyzed reaction relative to substrate concentration (e.g.
  • the method of the present invention can be used to detect amino acids at a wide range of concentrations, for example, from about 25 nM to about 2500 nM, more preferably from about 50 nM to about 1250 nM, more preferably still from about 75 nM to about 625 nM, more preferably still from about 85 nM to about 300 nM, even more preferably from about 95 nM to about 200 nM, and
  • the sensitivity can be dependent upon cognate tRNA and radiolabeled amino acid concentrations.
  • This inventive competitive assay can be used as a general method to measure amino acid concentrations and enzyme activity in samples, is amenable to high throughput screening, and can be applied in a variety of ways, e.g. diagnosing an amino acid disorder, monitoring amino acid levels in a patient before, during, or after a therapy for an amino acid disorder, or in high throughput screening of compounds (e.g. therapeutic small molecules, inhibitory peptides, or antibodies or fragments thereof) which modulate (e.g. inhibit or promote) the activity or function of an enzyme involved in amino acid metabolism.
  • compounds e.g. therapeutic small molecules, inhibitory peptides, or antibodies or fragments thereof
  • the present invention provides a method of determining the concentration of a target amino acid in a sample, comprising (a) performing an aminoacyl- tRNA synthetase charging reaction under equilibrium conditions, said reaction comprising a cognate aminoacyl-tRNA synthetase, a cognate tRNA, a fixed quantity of radiolabeled target amino acid, and the sample comprising the target amino acid, (b) quantifying the signal emitted from the aminoacylated cognate tRNA by a scintillation proximity assay, and (c) calculating the concentration of the target amino acid in the sample by comparing the signal to a calibration curve, said calibration curve constructed under identical conditions as in (a) and (b) with the proviso that increasing known concentrations of target amino acid are substituted for the sample.
  • the sample can be a biological sample, e.g. blood, urine, cerebrospinal fluid, or amniotic fluid.
  • the sample can optionally be initially treated to generate or consume free amino acids, e.g. contacting the sample with an enzyme whose reaction products or substrates comprise free amino acids.
  • the enzyme can be a protease.
  • the method can be carried in a high-throughput manner.
  • cognate aminoacyl-tRNA synthetase can be obtained from any source, e.g. a recombinant or natural source. It can be in a purified form or provided as a mixture, e.g. a crude extract comprising the aminoacyl-tRNA synthetase.
  • the charging reaction can be carried out with other necessary or desired substrates or cofactors, e.g. ATP.
  • the radiolabeled target amino acid comprises a beta-particle emitting radioisotope.
  • the radioisotope can be, for example, 3 H, 33 P, 125 1, 35 S, or C 14 .
  • the scintillation proximity assay utilizes in the method of the invention a scintillant-impregnated surface having an affinity for aminoacylated tRNA
  • the surface can be, for example, a bead or a microtiter plate and can comprise yttrium silicate.
  • the charging reaction of the invention can be performed under tRNA-limiting conditions.
  • the conditions sufficient to establish simple isotopic dilution conditions can comprise a minimum of about 6 to 10-fold molar excess of radiolabeled target amino acid to cognate tRNA.
  • the step of comparing the signal against a calibration curve in the method of the invention further comprises the step of calculating the concentration of the target amino acid by the formula:
  • y the signal
  • x the concentration of the target amino acid
  • T maximum signal in the absence of any unlabeled target amino acid
  • B minimum signal with excess unlabeled target amino acid
  • IC 50 concentration of half maximal inhibition
  • n Hill Slope.
  • the method of the invention comprises the step of quantifying the signal by measuring the signal with a scintillation counting device.
  • the calibration curve of the present invention is created by performing a series of charging reactions in accordance with steps (a) and (b), but using increasing known concentrations of unlabeled target amino acid in place of the sample.
  • the method of the invention is carried out in a high-throughput manner and is performed in a well of a multi-well microtiter plate, e.g. a 96-well plate or 384- well plate.
  • a multi-well microtiter plate e.g. a 96-well plate or 384- well plate.
  • the present invention provides a method of diagnosing or monitoring an aminoacidopathy comprising (a) obtaining a biological sample suspected of having an abnormal level of a target amino acid, (b) performing an aminoacyl-tRNA synthetase charging reaction under equilibrium conditions, said reaction comprising a cognate aminoacyl-tRNA synthetase, a cognate tRNA, a fixed quantity of radiolabeled target amino acid, and the biological sample, (c) quantifying the signal emitted from the aminoacylated cognate tRNA by a scintillation proximity assay, (d) comparing the signal against a calibration curve, thereby revealing the concentration of the target amino acid in the biological sample, wherein the aminoacidopathy is detected if the concentration of the target amino acid is abnormal relative to the concentration of the target amino acid from healthy tissue.
  • the aminoacidopathy can be classified as an aminoacidemia or aminoaciduria.
  • aminoacidopathy can also be classified as a phenylketonuria, tyrosinemia, alcaptonuria, isovaleric academia, homocystinuria, urea cycle disorder or organic acid metabolic disorder.
  • the present invention provides a method for screening a plurality of compounds to identify a potential modulator of an enzyme that produces or consumes or modifies a target amino acid, comprising (a) obtaining a sample comprising the enzyme, (b) performing an aminoacyl-tRNA synthetase charging reaction under equilibrium conditions, said reaction comprising a cognate aminoacyl-tRNA synthetase, a cognate tRNA, a fixed quantity of radiolabeled target amino acid, and the sample, (c) quantifying the signal emitted from the aminoacylated cognate tRNA by a scintillation proximity assay, and (d) calculating the concentration of the target amino acid in the sample by comparing the signal to a calibration curve, said calibration curve constructed under identical conditions as in (b) and (c) with the proviso that increasing known concentrations of target amino acid are substituted for the sample, wherein a potential modulator of the enzyme is identified when the concentration of the amino acid is greater or lesser than the starting concentration of
  • the method of screening in accordance with the invention is carried out in a high-throughput manner.
  • the present invention also contemplates a method for measuring the concentration of a particular amino acid in a mixture, comprising the steps of: (a) combining a sample containing an unknown concentration of the particular amino acid with a second solution containing (i) a known concentration of the particular amino acid in radiolabeled form, (ii) ATP, and (iii) an aminoacyl tRNA synthetase and tRNA specific for the particular amino acid;
  • the amino acid sample can be derived from any biological sample, such as, for example blood, urine, cerebrospinal fluid or amniotic fluid obtained from an animal or patient.
  • the amino acid sample can also be derived from an in vitro source, e.g. protease- generated amino acids.
  • the aminoacyl tRNA synthetase can be derived from any suitable source, e.g. from an organism, microorganism, cell line, etc. It can also be obtained from a recombinant source, e.g.
  • aminoacyl tRNA synthetase can also be obtained, for example, as a crude cell extract derived from a natural or recombinant source, e.g. an organism, microorganism or cell line, or as a partially purified preparation made by fractionation of a cell extract to remove contaminating cellular components, or as a highly purified, homogeneous, enzyme complex.
  • the method of the present invention can be used in the evaluation and management of patients with urea cycle disorders, organic acidemias, amino acid
  • disorders aminoacidopathies, amino acid transport disorders, renal tubular dysfunction, and prognosis of seizures and brain damage.
  • the present invention provides a method for identifying compounds that modulate the activity of a protein or an enzyme that produces, consumes or modifies an amino acid, by measuring the change in amino acid concentration by
  • FIG. 1 is a graphical depiction of signal production by aminoacylation of a tRNA with a radiolabeled amino acid in a scintillation proximity assay format.
  • Specific aminoacylation of a cognate tRNA by amino acid can be catalyzed by the corresponding aminoacyl-tRNA synthetase in purified form or other suitable form, e.g. crude extract containing multiple synthetases from a bacterium such as E, coli or from some other organism or provided by recombinant means (e.g. see Example 1, which describes an aminoacyl-tRNA synthetase charging reaction).
  • aminoacylated tRNA has a high affinity for the yttrium silicate (YSi) scintillation proximity assay (SPA) bead. Binding of the aminoacylated tRNA to the SPA bead brings the radiolabeled amino acid into close proximity with the bead so that emitted beta particles excite bead scintillant, producing light flashes that are detected by laboratory instrumentation. In contrast, tRNA aminoacylated with nonradiolabeled amino acid will not give rise to a signal.
  • YSi yttrium silicate
  • SPA scintillation proximity assay
  • FIG. 2 is a graphical depiction of a aaRS-catalyzed tRNA aminoacylation reaction.
  • A shows the two step aminoacylation reaction catalyzed by an aaRS. In step 1, the amino acid and ATP react to form the aminoacyl-adenylate mixed anhydride and inorganic pyrophosphate. In step 2, the aminoacyl-adenylate reacts with its cognate tRNA to form the aminoacyl-tRNA. The aminoacyl-tRNA is referred to as a charged tRNA.
  • B shows the formation and hydrolysis of aa-tRNA. The equilibrium concentration of aminoacylated tRNA is a function of competing forward and reverse reactions, which reach equilibrium.
  • aaRS cognate synthetase
  • FIG. 3 is a graphical depiction of the relative signals (cpm) generated from tritiated amino acids using crude tRNA and aminoacyl-tRNA synthetases in an SPA charging reaction.
  • A shows the maximum cpm measured for nineteen amino acids (excluding cysteine). The data represent the maximum signal observed following optimization for each amino acid (see Example 2 for details).
  • B illustrates the specificity of the SPA charging reaction (see Example 3 for details). Black bars represent 0.2 ⁇ Ci cognate radiolabeled amino acid only; gray bars represent 0.2 ⁇ Ci cognate radiolabeled amino acid plus 30 ⁇ M each of 15 non- cognate non-radiolabeled amino acids (signal was normalized to cognate radiolabeled amino acid alone). Amino acids proline, glutamate, glutamine and asparagine were excluded from this analysis.
  • FIG. 4 is a graphical depiction of signal modified as a function of radiolabeled amino acid and tRNA concentrations.
  • Radiolabeled valine was titrated at 4 tRNA concentrations: 2 ⁇ g (o), 6 ⁇ g (D), 10 ⁇ g (•), 20 ⁇ g (x). The signal was plotted as a function of radiolabeled valine concentration.
  • concentration of tRNA at which the signal reached half- maximum value (EC 50; calculated by equation 7) increased as tRNA increased, and demonstrated a hyperbolic response. See Example 4 for details.
  • FIG. 5 depicts the construction of a standard curve to determine unknown amino acid concentrations.
  • A shows a series of non-radiolabeled valine competition curves constructed as a function of [ 3 H]valine at a fixed tRNA concentration. Valine radioactivity varied as follows: 0.025 ⁇ Ci ( ⁇ ), 0.05 ⁇ Ci (0), 0.1 ⁇ Ci (A), 0.15 ⁇ Ci (T), 0.2 ⁇ Ci ( ⁇ ), 0.3 ⁇ Ci (•), 0.4 ⁇ Ci (G), 0.5 ⁇ Ci ( ⁇ ). The amount of tRNA and synthetase was 10 ⁇ g and 10 units, respectively. The construction of a standard curve is further described in Example 5.
  • (B) demonstrates isotopic dilution conditions, as described in more detail in Example 6.
  • a family of competitive charging reactions was performed as in (A), with additional experimental conditions of 2 and 6 ⁇ g of tRNA.
  • the IC 50 value (calculated with equation 6) determined for each [ 3 H]valine concentration was plotted as a function of amount of tRNA used: 2 ⁇ g ( ⁇ ), 6 ⁇ g ( A), 10 ⁇ g ( ⁇ ).
  • the theoretical curve black line was generated by plotting a one-to-one correspondence between IC 5O and [ 3 H]valine concentration. For each experiment performed at a distinct [tRNA], the [ 3 H] valine concentration at which the curve intersected the theoretical curve was the condition at which simple isotopic dilution conditions were first met.
  • FIG. 6 is a graphical depiction of using the method of the invention to detect and quantify an unknown amount of an amino acid.
  • Standard curves for methionine (A) and AMC (B) were created as described in the text using the cSPA charging assay (40 Units aaRS, 10 ⁇ g tRNA) or a fluorescent detection assay, respectively.
  • Aminopeptidase reactions at 1, 10 and 100 mU in the fluorescent assay yielded identical results after complete hydrolysis of titrated Met- AMC ( ⁇ 1 hour) (ImU aminopeptidase data is shown).
  • Time and concentration dependence for aminopeptidase M activity was demonstrated using the cSPA charging assay (C) and the fluorescent detection assay (D).
  • Enzyme units in both assay formats were as follows: 0 mU ( ⁇ ), 0.01 mU ( ⁇ ), 0.025 mU (A), 0.05 mU (x). Concentration of product detected by both assay formats was calculated according to standard curves and plotted side by side (E). The IC 50 values determined for leuhistin (calculated using equation 5) using fluorescent detection ( ⁇ ) vs. the cSPA charging assay (o) were 1.39 ⁇ M and 1.18 ⁇ M respectively (F). See Example 7 for further detail.
  • FIG. 7 is a graphical depiction of the use of the method of the invention high throughput quantitative analysis of amino acid concentration.
  • FIG. 7 A represents a 384-well screening plate demonstrating utility of the invention to detect methionine produced by aminopeptidase M in high throughput mode.
  • the plate map was constructed as follows: methionine standard curve (wells Ia-Ip and 2a-2p) (A and B); enzyme titration (wells 3a-3h and 4a-4h, 0 aminopeptidase M; 3i-3n and 4i-4n, 0.025 mU aminopeptidase M; 3o-3p and 4o- 4p, 0.05 mU aminopeptidase M); test compounds in the presence of 0.025 mU aminopeptidase M (5a-5p to 24a-24p). Using 0.025 mU of uninhibited aminopeptidase M, 1064 ⁇ 129 nM of methionine was produced. The values of Z' and Z for the 0.025 mU
  • control wells and tests wells were 0.61 and 0.64, respectively (B). See Example 8 for further detail.
  • the invention presented here enables for the first time SPA technology to measure amino acid concentration by utilizing a tRNA charging reaction under equilibrium conditions in a competitive manner.
  • a single amino acid can be detected and quantified in a mixture of amino acids.
  • the assay of the present invention is not performed under initial rate conditions, but rather under equilibrium conditions, the K m of amino acid for cognate aaRS relative to the concentration of amino acid being tested does not contribute to the magnitude of the effect.
  • the assay may be performed under simple isotopic dilution conditions in which there is a molar excess of amino acid relative to tRNA, such that no further reaction is observed when additional amino acid is added.
  • aaRS aminoacyl tRNA synthetase
  • AMC 7-amino-4-methyl coumarin
  • cSPA competitive scintillation proximity assay
  • cpm counts per minute
  • HPES N-(2-hydroxyethyl)piperazine-N'- (2-ethanesulfonic acid).
  • HTS high throughput screening
  • metalAMC methionine 7-amido-4-methyl coumarin.
  • the abbreviation “RFU” means relative fluorescent units.
  • the abbreviation “SPA” means scintillation proximity assay.
  • tRNA transfer ribonucleic acid
  • YSi yttrium silicate
  • aminoacyl-tRNA synthetase charging reaction refers to a reaction whereby an amino acid is coupled to a cognate tRNA molecule.
  • aminoacidopathy refers to any of a group of disorders resulting from a defect in the metabolic pathway(s) of one or more amino acids or in the transport of certain amino acids into or out of cells or tissues.
  • the aminoacidopathies of the present invention can include, but are not limited to, phenylketonuria, tyrosinemia, alcaptonuria, isovaleric academia, and homocystinuria.
  • amino acid related disorder refers generally to any disorder, including an aminoacidopathy, having an etiology that includes abnormal levels of one of more amino acids in a cell or tissue as compared to the levels in a healthy cell or tissue.
  • amino acid related disorders include, but are not limited to, aminoacidopathies, aminoaciduria, urea cycle disorders, organic acid metabolic disorders, amino acid transport disorders, renal tubular dysfunction, brain damage, seizures, and organic acidemias.
  • the term “revealing” as in “thereby revealing the concentration of the target amino acid in the biological sample,” is meant to refer to the process of obtaining a
  • concentration of the target amino acid by calculation with reference to an appropriate standard curve and standard curve mathematical formula as described herein.
  • aminoaciduria or equivalently, “acidaminuria,” refers to a disorder of protein metabolism in which excessive amounts of amino acids are excreted in the urine.
  • cognate tRNA or "cognate tRNA molecule” refers to a tRNA molecule which corresponds to a specific cognate amino acid, which upon action by a cognate aminoacyl-tRNA synthetase would be covalently coupled to the cognate amino acid.
  • target amino acid refers to any one particular amino acid which is the subject of analysis by the method of the invention.
  • the target amino acid could be phenylalanine, especially in the case where the inventive method is used to monitor or evaluate or diagnose phenylketonuria.
  • the target amino acid is also the cognate amino acid of the particular aminoacyl-tRNA synthetase used in the inventive method.
  • the present invention provides a method that combines for the first time (1) a scintillation proximity assay (SPA) to measure aminoacyl-tRNA synthetase (aaRS) activity and (2) the application of the enzymatic isotope dilution principle using aaRS activity to measure the concentration of amino acids.
  • SPA scintillation proximity assay
  • aaRS aminoacyl-tRNA synthetase
  • the inventive method takes advantage of competition between a fixed concentration of radiolabeled amino acid and an unknown concentration of the same non-radiolabeled amino acid for its cognate tRNA catalyzed by the aaRS specific for that amino acid at equilibrium.
  • the method of the present invention allows one to determine the concentration of a target amino acid in a sample, comprising (a) performing an aminoacyl- tRNA synthetase charging reaction under equilibrium conditions, said reaction comprising a
  • cognate aminoacyl-tRNA synthetase a cognate tRNA, a fixed quantity of radiolabeled target amino acid, and the sample comprising the target amino acid
  • quantifying the signal emitted from the aminoacylated cognate tRNA by a scintillation proximity assay a scintillation proximity assay
  • comparing the signal against a calibration curve thereby revealing the concentration of the target amino acid in the sample.
  • the method can be carried out in a high- throughput manner.
  • the invention is guided by the principle that: (1) at equilibrium, the maximum signal obtained in an SPA aminoacylation reaction is determined by the amounts of radiolabeled amino acid, tRNA and aaRS present; (2) this signal is in turn modulated by the concentration of a competing non-radiolabeled cognate amino acid; (3) the sensitivity of the assay is determined by the amounts of tRNA and radiolabeled amino acid present in the reaction.
  • the procedure to determine unknown amino acid concentration is: (1) construct a standard curve using varying amounts of non- radiolabeled amino acid in the presence of a fixed amount of radiolabeled amino acid, tRNA and aaRS; (2) perform an aaRS charging reaction under identical conditions in the presence of an unknown amount of non-radiolabeled amino acid; (3) convert the signal in 2 into amino acid concentration using parameters derived from the standard curve in 1.
  • the charging reaction of the inventive method can be carried out under isotope dilution conditions.
  • Isotope dilution analysis is based on the principle that addition of a non-radiolabeled compound to a fixed amount of the radiolabeled form of the same compound decreases the specific activity (i.e., the radioactivity per unit amount of compound in the mixture) of that compound.
  • the enzyme-catalyzed reaction converts a radiolabeled substrate to product, and the product can be quantitatively distinguished from the substrate, so
  • an enzyme-produced product is expected to have a lower specific activity in the sample where the substrate specific activity has been decreased by the addition of non-radiolabeled compound.
  • the difference in product specific activity can be used to calculate the amount of non-radiolabeled substrate added to the radiolabeled substrate (A. Hagen et al., Methods of Enzymatic Analysis, Academic Press, New York, 1974, pp. 283-307).
  • the enzymatic isotope dilution technique is typically performed under initial rate conditions where a small fraction of substrate is converted to product ( ⁇ 10%).
  • concentration of substrate is below that required for saturation of the enzyme, the rate of the enzyme catalyzed reaction, and therefore product produced per unit time, will be lower than when enzyme is saturated with substrate and working at full capacity (A. Fersht, Enzyme structure and mechanism, second edition, W. H.. Freeman and Company, New York, 1985). Therefore, below enzyme saturation, as the concentration of substrate changes the amount of product formed per unit time changes, and this phenomenon will influence specific activity calculations (E.A. Newsholme, and K. Taylor, A new principle for the assay of metabolites involving the combined effects of isotope dilution and enzymatic catalysis. Biochimica et Biophysica Acta 158 (1968) 11-24).
  • tRNA-limiting conditions such as optionally in the present invention, if the enzyme-catalyzed reaction is allowed to reach equilibrium, the amount of product observed at a particular time becomes independent of substrate concentration. Therefore simple isotopic dilution conditions are met.
  • the present invention does not require performing the assay under tRNA- limiting conditions. As long as an amino acid standard curve is constructed under a specific set of conditions, these conditions can be used to accurately determine an unknown
  • the sensitivity of the assay can be adjusted to accommodate an unknown range of amino acid concentration in a sample by changing the concentration of added radiolabeled amino acid or tRNA, while the total signal is modulated by the concentrations of radiolabeled amino acid, tRNA, and aaRS, as well as by the amount of bead in the well.
  • AA-tRNA aminoacylated-tRNA
  • FIG. 2A The rate of formation of the AA-tRNA is determined by two sets of competing reactions (FIG. 2B).
  • the activated amino acid:tRNA: ATP:aaRS complex (ES) is converted to AA-tRNA product with an overall rate constant k cat (equation 1).
  • Parameters determined from the standard curve can be used to calculate the concentration of an unknown amount of amino acid in an assay done under similar conditions:
  • aminoacyl-tRNA synthetases are essential proteins found in all living organisms. They form a diverse group of enzymes that ensure the fidelity of transfer of genetic information from the DNA
  • aaRSs catalyze the attachment of amino acids to transfer RNAs (tRNA) and thereby establish the rules of the genetic code by virtue of matching the nucleotide triplet of the anticodon with its cognate amino acid.
  • the aminoacylation reaction i.e. the "charging reaction” is catalyzed by the family of aminoacyl-tRNA synthetases (AARS) each of which activates an amino acid by binding to ATP and transferring it to the end of the cognate tRNA.
  • AARS aminoacyl-tRNA synthetases
  • aaRS constitute a family of 20 cellular enzymes that are responsible for specific esterification of tRNAs with their cognate amino acids, and thus are essential in maintaining the fidelity of the protein biosynthesis process.
  • the aminoacylation reaction catalysed by aaRS is achieved in two steps.
  • the amino acid is activated by attaching a molecule of ATP at the alpha-phosphate, giving rise to a mixed anhydride intermediate, aminoacyl- adenylate, and inorganic pyrophosphate, hi the second step, the activated amino acid moiety is transferred to the 3 '-terminal ribose of the cognate tRNA, yielding the specific aminoacyl- tRNA (or "charged tRNA") and AMP.
  • tRNA binding to aaRS is thought to proceed through an initial broad specificity interaction that is followed by more precise recognition that involves conformational changes of both aaRS and cognate tRNA.
  • aminoacyl-tRNA synthetases catalyze the acylation, or charging, of tRNAs with their cognate amino acids.
  • the specificity of this set of reactions is critical for the fidelity of protein biosynthesis (P. Schimmel, Annual Review of Biochemistry 56 (1987) 125-158).
  • each of these synthetase enzymes must recognize a few select cognate tRNAs from approximately 80 distinct tRNA species.
  • the low frequency of errors found in synthesized proteins underscores the high degree of substrate discrimination (R.B. Loftfield, Biochemical Journal 89 (1963) 82-92; R.B. Loftfield et al., Biochemical Journal 128 (1972) 1353-1356).
  • a method such as the method of the present invention, which employs aaRS- catalyzed charging of a cognate tRNA to detect a particular amino acid will be highly specific for that amino acid, distinguished by its exact side chain.
  • Such a method can be used to detect the production, consumption, or modification of a given amino acid, including, but not limited to, methylation, deamination, acetylation (e.g. of the N-terminus), nitration, sulfation (e.g. of tyrosine hydroxyl), or isomerization.
  • the present invention further contemplates preparing a "standard curve" or
  • a calibration curve by which to compare the signal generated from an SPA assay of an unknown amino acid to reveal the concentration of the unknown amino acid of interest.
  • a standard curve can be prepared using the identical parameters for the aaRS cSPA charging assay described above, hi place of a sample containing an unknown concentration of one or more target amino acids, known concentrations of target amino acids are used in the cSPA charging reactions for standard curve preparations.
  • cpm level of signal
  • a calibration curve can be generated.
  • the range of known concentration of target amino acid can be generated.
  • concentrations of the target amino acid used to generate the standard curve encompasses the concentration of target amino acid in the sample (or the concentration of the target amino acid in a dilution of the sample, e.g. 1/10, 1/100, 1/1000, 1/10,000, 1/100,000 dilution or the like).
  • the signal generated in an SPA assay of the invention as a function of increasing non- radiolabeled amino acid concentration can be used to calculate or reveal (e.g. according to equation 5 or 6) the concentration of a target amino acid in a sample.
  • Example 5 is illustrative of preparing a standard curve.
  • the method of the invention further comprises a scintillation proximity assay.
  • SPAs are generally known in the art. See, e.g., U.S. Pat. No. 4,271,139; U.S. Pat. No. 4,382,074; U.S. Pat. No. 4,687,636; U.S. Pat. No. 4,568,649; U.S. Pat. No. 4,388,296; U.S. Pat. No.
  • SPAs quantify light energy produced by a radioactively labeled product, which is proportional to the amount of that radioactively labeled product (i.e. analyte) in the medium or sample or preferably that amount which is bound to the SPA
  • the light is produced by a scintillant that is incorporated or impregnated or otherwise a part of a support matrix or surface (e.g. a bead or multiwell plate).
  • the support matrix is coated with a receptor, ligand or other capture molecule (or has an innate affinity for the radiolabeled analyte) that can specifically bind to a radiolabeled analyte, such as a ligand.
  • the analyte of the present invention is preferably the charged tRNA molecule.
  • SPA uses fluomicrospheres, such as diphenyloxazole-latex, polyacrylamide-containing a fluophore, and polyvinyltoluene [PVT] plastic scintillator beads, and they are prepared for use by adsorbing compounds into the matrix. Also fluomicrospheres based on organic phosphors have been developed. Microplates made from scintillation plastic, such as PVT, have also been used [see, e.g., International PCT Application No. WO
  • the fluomicrospheres i.e. "beads” as used herein
  • the fluomicrospheres or plates are coated with acceptor molecules, such as receptors or antibodies to which ligand or analyte binds selectively and reversibly.
  • the charged tRNA analytes of interest have affinity for the fluomicrosphere beads under certain conditions, preferable acidic conditions, as described, for example, in Macarron et al., Analytical Biochemistry 284:183-190 (2000), which is incorporated herein by reference.
  • the beads or other SPA surfaces will have increased affinity for the charged tRNA at pH less than about 2.5, more preferable less than about 2.0, or lower.
  • the SPAs of the present invention can be performed using glass beads containing fluophores and functionalized with recognition groups for binding specific ligands [or receptors], such as organic molecules, proteins, antibodies, and other such molecules.
  • the support bodies used in these assays are prepared by forming a porous amorphous microscopic particle, referred to as a bead [see, e.g., European Patent Application No.O 154,734 and International PCT Application No. WO 91/08489, each of which are incorporated herein by reference].
  • the bead can be formed from a matrix material such as acrylamide, acrylic acid, polymers of styrene, agar, agarose, polystyrene, and other such materials, such as those set forth above. Cyanogen bromide can also be incorporated into the bead to provide moieties for linkage of desired capture molecules or biological particles to the surface.
  • Scintillant material can be impregnated or incorporated into the bead by precipitation or other suitable method.
  • the matrices are formed from scintillating material [see, e.g., International PCT Application No. WO 91/08489, which is based on U.S. application Ser. No. 07/444,297; see, also U.S. Pat. No. 5,198,670, each of which are incorporated by reference], such as, for example, yttrium silicates or other glasses, which when activated or doped respond as scintillators.
  • Dopants include Mn, Cu, Pb, Sn, Au, Ag, Sm, and Ce.
  • the beads of the present invention comprise yttrium silicate and under acidic conditions (e.g. pH of less than about 2.5), the analyte or target tRNA molecules will have affinity for the yttrium silicate beads.
  • acidic conditions e.g. pH of less than about 2.5
  • the tRNA molecules both charged and uncharged with amino acids, bind to the surface of the bead (or other SPA surface). Any radiolabeled amino acid that is not charged to tRNA as a result of the charging reaction does not come within close proximity to the bead relative to the pathlength of the ⁇ -emission of the radionuclide and therefore goes undetected.
  • tRNA molecules charged with the radiolabeled amino acid are brought into proximity with the
  • the SPAs of the present invention can be conducted in normal assay buffers and requires the use of a ligand labeled with an isotope, such as 3 H and 125 I, that emits low-energy radiation that is readily dissipated easily an aqueous medium. Because 3 H beta-particles and 125 I Auger electrons have average energies of 6 and 35 keV, respectively, their energies are absorbed by the aqueous solutions within very small distances. Thus, in a typical reaction of, for example, 0.1 ml to 0.4 ml the majority of unbound labeled ligands will be too far from the fluomicrosphere to activate the beads.
  • an isotope such as 3 H and 125 I
  • Bound ligands will be in sufficiently close proximity to the fluomicrospheres to allow the emitted energy to activate the fluophore and produce a light signal.
  • bound ligands e.g. tRNAs charged with radiolabeled amino acid
  • free ligands e.g. free non-radiolabeled or radiolabeled amino acids
  • assay beads emit light when they are exposed to the radioactive energy from the label bound to the beads,but the free radioactive amino acid species in solution are too far from the bead to elicit light.
  • the light from the beads can be measured in a liquid scintillation counter and can be a measure of the bound label.
  • the glasses can also include activators, such as terbium, europium or lithium.
  • the fiber matrix can be made from a scintillant loaded polymer, such as polyvinyltoluene.
  • SPAs are based on the principle that radioisotopes such as 3 H, 33 P, 5I and 35 S that emit weak ⁇ -particle radiation in solution must be in close proximity to scintillant molecules to produce light (FIG. 1) (N. Nelson, Analytical Biochemistry 165
  • Scintillation proximity assay beads are one type of surface that can be used in the present invention and are available commercially, e.g. from GE Healthcare (Piscataway, NJ). Such beads are impregnated with scintillant and have affinities for certain biomolecules, depending on the material of which they are made, or the coating which has been applied to their surface.
  • PDE bead cat # RPNQO 150
  • YSi yttrium silicate
  • Cerium atoms yttrium silicate
  • Scintillation proximity plates FlashPlates; available from PerkinElmer (Boston, MA
  • the plates are based on the same proximity principle as beads except that a well of a microtiter plate is impregnated with scintillant and the surface of the well can be coated with affinity tag.
  • Scintillation proximity membranes such as those described in Mattingly et al. (1995) J. Memb. Sci. 98:275-280 (incorporated herein by reference), can also be used with the present invention.
  • the method of the present invention can be used in diagnosing an amino acid related disorder, monitoring such disorders before, during or after treatment or nutritional support (e.g. administration of medical foods, e.g. U.S. Patent No. 6,355,612, incorporated herein by
  • Phenylketonuria The primary metabolic defect in PKU is the inability to convert excess dietary phenylalanine to tyrosine. As a result of this metabolic block, phenylalanine accumulates in the blood and cerebrospinal fluid and is excreted in excess in the urine. Abnormally high levels of phenylalanine are diverted to the formation of phenylpyruvic acid and its metabolic derivatives, phenylacetic, phenyllactic acid and orthohydroxyphenylacetic acids. There is excessive excretion in the urine of these acids. There is interference with the normal metabolism of tyrosine and tryptophan, and unusual intermediary products of these two amino acids appear in the urine.
  • Nutritional support is used to limit the intake of phenylalanine, in order to avoid any excess accumulation of this amino acid. A certain minimum phenylalanine requirement individual to each affected child, however, must be provided in the diet in order to facilitate
  • a phenylalanine intake of between 50-70 mg/kg/day is required for infants with PKU 2-4 months of age.
  • the symptoms of insufficient phenylalanine intake include apathy, anorexia, hypoglycemia, and vacuolization of the marrow erythroid and myeloid cytoplasm. Death after prolonged hypoglycemia due to insufficient phenylalanine intake has been observed.
  • the phenylalanine requirement in terms of body weight decreases rapidly during the first year of life. Readjustment of the phenylalanine intake must be made frequently during this year.
  • the effect of the restricted phenylalanine diet on mental development in PKU children is directly related to the age at which the diet is instituted.
  • Children with PKU seem to develop normally if they receive a low phenylalanine diet beginning very soon after birth.
  • the IQ of children with PKU fell linearly by about 4 IQ points for each month between birth and starting treatment, for each 300 umol/1 rise above normal in the average plasma phenylalanine concentrations, and for each five months within the first two years or life during which the phenylalanine concentration were below 120 umol/1.
  • Tyrosine is an essential amino acid and since it is the distal metabolic product of phenylalanine conversion it is necessary in such dietary formulations to include sufficient tyrosine in the diet to meet nutritional requirements.
  • These children are damaged in utero by the high maternal levels of phenylalanine. Plasma phenylalanine levels of PKU mothers must be controlled during pregnancy. Treatment with a phenylalanine restricted diet during pregnancy, particularly if initiated before conception, appears to offer some protection to the fetus from birth defects. When the blood phenylalanine levels are well controlled during the entire pregnancy the infant seems to be normal.
  • LofenalacTM manufactured by Mead Johnson Corporation, Evansville, Id., U.S.A.
  • LofenalacTM contains approximately 0.08% phenylalanine and is produced from an enzymatic hydrolysate of casein. Phenylalanine is removed from the casein hydrolysate by adsorption on activated charcoal columns. This formula is supplemented with carbohydrates, fats, minerals, vitamins and L-tyrosine, L- tryptophan, L-methionine and L-histidine dihydrochloride.
  • Three other protein hydrolysate- based products were developed in England: Albumaid XPTM, CymogranTM, and MinafenTM.
  • Albumaid XPTM (Powell and Scholefield, Ltd., England) is a bovine serum hydrolysate from which most of the phenylalanine is removed and which contains 40%
  • this product needs to be supplemented with fat, vitamin C, fat-soluble vitamins, and essential fatty acids.
  • CymogranTM (Alan and Hanbury's Ltd., London, England) contains 30% protein equivalent along with moderate levels of fat and carbohydrate. It requires supplementation with all vitamins and some minerals, as well as some dilution with low-protein foods.
  • MinafenTM (Cow & Gate, Trowbridge, England), a balanced infant formula-type product, contains about 8% of the energy requirements, but as a low phenylalanine protein hydrolysate, it is deficient in several vitamins.
  • PK Aid 1TM is an amino acid mixture free of phenylalanine.
  • the other amino acids are present in satisfactory amounts. This enables dietary supplementation to supply the minimum phenylalanine requirement. Supplementation with carbohydrate, fat, and all vitamins and minerals is necessary.
  • Phenyl-FreeTM (Mead Johnson Corp., Evansville, Id., U.S.A.), is another medical food used for the nutritional support of PKU.
  • This product is a mixture of L-amino acids excluding phenylalanine. It contains vitamins, minerals, carbohydrates and a small amount of fat. When this product is reconstituted with water, one pint of the product provides 400 kcal and contains the daily requirements of vitamins, minerals and essential amino acids. For a child two years of age or older, the additional energy and phenylalanine requirements can be met from conventional low protein foods given in prescribed amounts.
  • the product has the characteristic bitter taste of L-amino acid mixtures, but is palatable when flavored.
  • PKU- 1TM PKU- 2TM
  • PKU-3TM PKU- 1TM, PKU- 2TM and PKU-3TM (Milupa, Fredrichsdorf/Taunus, Germany) which comprise phenylalanine
  • the diagnosis and monitoring of PKU before, during and after treatment, e.g. with the above nutritional supports, can be advantageously achieved with the method of the present invention.
  • an infant suffering from PKU could be monitored in accordance with the method of the present invention to determine the levels of phenylalanine in the blood and/or tissues, e.g. during the course of one of the above nutritional therapies.
  • Tyrosinemia Type 1 is an inherited disorder of tyrosine metabolism, associated with deficient activity of fumarylacetoacetate hydrolase. Patients present with severe liver and renal disease in infancy and in later childhood develop hepatomas. Biochemically the disease is characterized by raised plasma levels of tyrosine and methionine and increased urinary excretion of tyrosine metabolites.
  • Tyrosinemia Type II is associated with autosomal recessive inheritance and has distinctive metabolic abnormalities, including increased levels of tyrosine in the plasma and urine, and increased levels of tyrosine metabolites in the urine.
  • the defect in oculocutaneous tyrosinemia is in the tyrosine aminotransferase of the hepatic cytosol, an enzyme that normally catalyzes the conversion of tyrosine to p-hydroxyphenylpyruvic acid.
  • Deficient enzyme activity results in tyrosine accumulation and blood tyrosine concentrations become elevated. This syndrome is often associated with a characteristic clinical syndrome of eye and skin lesions, permanent neurological damage, mental retardation, and blindness. Early diagnosis is of paramount importance for effective treatment of the disorder.
  • Treatment typically consists of a low-tyrosine, low-phenylalanine diet.
  • One such commercially available diet is the Mead Johnson Low Phe/Tyr Diet PowderTM (Mead Johnson Corp., Evansville, Id., U.S.A.). This product is generated from a casein hydrolysate with most of the tyrosine removed while still containing substantial phenylalanine. Rapid decreases of tyrosine plasma levels have been reported in response to restriction of the dietary intake of phenylalanine and tyrosine. Symptoms have been observed to respond quickly to changes in the concentration of tyrosine in body.
  • Other commercially available products for the treatment of tyrosinemia include T YPv-I TM and TYR-2TM (Milupa, Fredrichsdorf/Taunus, Germany)
  • alcaptonuria The symptoms of alcaptonuria first appear in adult life in the form of a discoloration in the connective tissue (ochronosis) and a characteristic arthritis transmitted as an autosomal recessive absence of homogentisic oxidase, which results in excretion of homogentisic acid in the urine.
  • a diet low in phenylalanine and tyrosine should reduce the formation of homogentisic acid, but there have not been any reported attempts with this therapy; however, a restriction of protein intake might have some beneficial effect.
  • tyrosinemia and alcaptonuria before, during and after treatment, e.g. with the above nutritional supports, can be advantageously achieved with the method of the present invention.
  • a patient suffering from either tyrosinemia or alcaptonuria could be monitored for the levels of phenylalanine and tyrosine in the blood to ensure that a proper diet low in these amino acids is being properly administered and maintained.
  • MSUD Maple Syrup Urine Disease
  • MSUD is another inborn error of metabolism that can be diagnosed and monitored by
  • MSUD The classical form of MSUD involves almost complete deficiency of branched-chain keto acid dehydrogenase complex.
  • the metabolic event that causes MSUD is a failure of the oxidative decarboxylation of the branched chain amino acids, leucine, isoleucine and valine.
  • the keto acid derivatives accumulate in excess in the blood and are excreted in the urine.
  • MSUD can be treated with a diet providing a limited intake of the branched chain amino acids. Following nutritional support as indicated, the characteristic MSUD odor disappears, neurologic manifestations gradually improve, the electroencephalogram returns to normal, and the abnormal plasma accumulation of the branched chain amino acids and their keto acid derivatives decreases.
  • valine and isoleucine become normal several days before the leucine level is in the normal range.
  • the intake of the branched chain amino acids can be provided in the form of prescribed amounts of infant formula, milk or low protein foods.
  • Commercial products available for the treatment of MSUD include MSUD-AidTM
  • MSUD Solid and Scholefield, Ltd., England
  • MSUD Diet PowderTM Mead Johnson Corporation
  • MSUD- 1TM MSUD-2TM
  • AnalogTM MaxamaidTM
  • Maxamum MSUDTM Scientific Hospital Supplies
  • BCKA Classic branched chain ketoaciduria
  • the diagnosis and monitoring of MSUD before, during and after treatment can be advantageously achieved with the method of the present invention.
  • the present invention could be used to measure and monitor the levels of the branched chain amino acids which are indicative of MSUD during the course of therapy.
  • Isovaleric acidemia is an inherited defect of leucine metabolism characterized by the presence of high levels of isovaleric acid (IVA) in the blood and urine.
  • IVA is a short chain fatty acid whose only known amino acid precursor is leucine.
  • the metabolic block is the failure to convert isovaleryl-CoA to beta-methylcrotonyl-CoA resulting in the large accumulation of IVA and metabolites, isovalerylglycine and beta-hydroxyisovaleric acid, in blood and urine. Even in remission these metabolites are present in increased quantities. Clinically this condition is accompanied by an odor very similar to that of sweaty feet.
  • IVA seems to be solely derived from leucine, which is an essential amino acid, reduction in dietary leucine is effective in controlling the abnormal accumulation of metabolites as well as sequelae.
  • glycine conjugation with isovaleryl-CoA dehydrogenase is instrumental in preventing IVA accumulation, it is advisable to restrict substances which compete for glycine conjugation, such as benzoic and salicylic acids.
  • the administration of glycine favors the formation of non-toxic isovalerylglycine (IVG) from precursor IVA and hence the consequent diminution of toxic levels of IVA in blood and tissue.
  • IVG non-toxic isovalerylglycine
  • Glycine therapy is particularly beneficial for the treatment of acute ketoacidotic episodes in older infants and children, and for the management of acute neonatal disease.
  • Glycine markedly reduces the rise in serum IVA produced by a leucine load.
  • Glycine administration is associated with a pronounced increase in excretion of IVG and in hippurate excretion— both IVG and hippurate excretion being increased significantly by glycine administration as compared with administration of leucine alone.
  • the diagnosis and monitoring of isovaleric acidemia before, during and after treatment, e.g. by the above approaches, can be advantageously achieved with the method of the present invention.
  • the level of glycine which is linked to the rise in serum IVA, could be monitored using the present invention.
  • the level of leucine in the blood could be monitored over time to assess whether dietary reduction of leucine is being achieved or maintained at a certain recommended level.
  • the basic metabolic defect in homocystinuria is a deficiency in the activity of the enzyme cystathionine synthetase which catalyzes an essential step in the trans-sulferation pathway associated with cystine synthesis. Typically this enzyme deficiency results in abnormal levels of homocystine in the urine.
  • ectopia lentis located lenses
  • a number of skeletal deformities Arterial and venous thromboses are frequent occurrences and are responsible for sudden death. These effects are secondary to the damage caused to the blood vessel walls by homocystine. Therapy should be attempted in all cases of homocystinuria in an effort to avoid the serious pathological sequelae described above.
  • homocystinuria caused by cystathione synthetase deficiency.
  • One form is amenable to therapy with large doses of pyroxidine, at least several hundred milligrams per day.
  • the other form requires a diet restricted in methionine and supplemented with cysteine. Both biochemical and clinical responses have been reported with diets low in methionine. These diets must be supplemented with cysteine, since the site of the metabolic block makes cysteine a dietary essential for these individuals.
  • MethionaidTM (Scientific Hospital Supplies Ltd., Liverpool, England) is a methionine-free synthetic mixture of L-amino acids, water soluble vitamins, fat soluble vitamins and minerals. High- fat and carbohydrate foods must be added as well as several vitamins and some minerals in order to provide a complete diet. Milupa (Fredrichsdorf/Taunus, Germany) has recently introduced HOM- 1TM and HOM-2TM which also provide methionine-free mixtures of amino acids, which contain mixtures of vitamins and minerals but no fat and little carbohydrate. Other medical foods available for the nutritional support of this disorder include Analog,
  • the diagnosis and monitoring of homocystinuria before, during and after treatment, e.g. by the above approaches, can be advantageously achieved with the method of the present invention.
  • the levels of methionine and cysteine be monitored using the present invention to ensure that a diet low in methionine and supplemented with cysteine is being administered.
  • Interruptions in the metabolic pathway for urea synthesis are caused by the deficiency or inactivity of any one of several enzymes involved in specific steps in the cascade.
  • the common pathologic sequelae of these clinical disorders is the extreme elevation of the plasma ammonia level.
  • Typically associated with this increase in ammonia buildup are acute episodes of vomiting, lethargy, convulsions and abnormal liver enzyme levels.
  • Protracted exposure to high levels of plasma ammonia leads to mental and physical retardation. If left untreated prolonged exposure to high levels of plasma ammonia is fatal typically following a period of lethargy, convulsions and coma.
  • N-acetyl glutamate synthetase deficiency which causes neurologic deterioration due to elevated blood ammonia
  • CPS carbamyl phosphate synthetase
  • OTD ornithine transcarbamylase deficiency
  • argininosuccinic acid synthetase deficiency which typically results in severe neurological impairment leading to mental retardation or death
  • argininosuccinate lyase deficiencies which result in clinical
  • the diagnosis and monitoring of some urea cycle disorders before, during and after treatment, e.g. by the above approaches, can be advantageously achieved with the method of the present invention.
  • the levels of arginine can be monitored using the present invention to ensure the appropriate level arginine in the body.
  • MMA methylmalonic acidemia
  • PA propionic acidemia
  • the present invention can be used to monitor the levels of isoleucine, methionine, threonine and valine in an infant to ensure the presence of safe and appropriate levels.
  • the present invention provides a method for screening a plurality of compounds for potential modulators (e.g. inhibitors) of amino acid metabolism. The method can be used, for example, in large-scale, high-throughput drug screening assays to identify potential therapeutic compounds for use in treating or ameliorating symptoms of an amino acid disorder or aminoacidopathy.
  • Modulators can include any small molecule compound, antibody, antibody fragment, peptide or the like, which binds to and affects the activity of an enzyme involved in the metabolism of one or more amino acids.
  • the screening method of the present invention is advantageously capable of detecting such compounds as a function of a changing amino acid concentration in the presence of one or more different amounts of a compound or a plurality of compounds.
  • the compounds screenable by the present invention can be capable of targeting one or more enzymes affecting the metabolism of one or more amino acids.
  • the present methods can be used to screen compound libraries for potential inhibitors of an enzyme that directly synthesizes or degrades an amino acid by measuring the amino acid concentration in the presence of the compound library.
  • Enzymes (i.e. target enzymes) against which potential useful compounds can be screened can be any enzyme involved in metabolism of an amino acid, including for example, enzymes whose direct product (e.g. enzymes which catalyze a final step in the biosynthesis of an amino acid or proteases which cleave amino acids from existing proteins) or substrate (e.g. enzymes controlling the degradation of an amino acid or the modification of an amino acid, such as, an enzyme catalyzing the acetylation or methylation of an amino acid) is an amino acid.
  • the enzymes against which potential targets are screened can be those enzymes which directly or indirectly produce or consume an amino acid being measured. In other words, potential inhibitors of enzymes catalyzing steps upstream of amino acid synthesis or downstream of amino acid degradation (or consumption or modification) which affect the level of amino acid can be screened by the present methods.
  • the screening method of the invention can also be utilized to screen for compounds that modulate a pathway affecting the concentration of a particular peptide of interest by first
  • Screenable compounds can be obtained by any suitable means known in the art.
  • the term "screenable compounds” refers to one or more different molecules that are tested for their ability to alter (e.g. increase or decrease or modulate) the concentration of a particular amino acid or set of amino acids in a reaction system that comprises at least one target enzyme.
  • the reaction system can comprise a target enzyme in a purified or substantially purified form, or as a mixture with other enzymes or cellular components, or as a component of a cellular extract.
  • the target enzyme can also be obtained naturally (i.e. as expressed or produced by a living cell from an endogenous gene) or by recombinant means (production by in vitro or in vivo expression system) or by any other suitable means.
  • reaction system refers to the vessel in which the charging reaction of the invention takes place and includes any appropriate buffers, co-factors, chelators and any other reaction components that would be suitable for any given reaction, e.g. addition of particular amino acids.
  • the target enzyme can be known or unknown. That is, in carrying out the method of screening for compounds that modulate amino acid concentrations in accordance with the invention, one does not have to have specific knowledge of what particular targeted enzyme is envisioned. Once a compound is identified, however, which has the effect of altering the amino acid concentration of a reaction system, the particular enzyme affected by the
  • target enzymes of interest may be a member of any of the major or minor classes of enzymes, including hydrolases, oxidoreductases, lyases, transferases, ligases, and isomerases, among others.
  • target enzymes may include kinases, phosphatases, carboxylases, phosphodiesterases, dehydrogenases, oxidases, peroxidases, metalloproteinases, proteases, saccharidases, nucleases, reverse transcriptases, polymerases, recombinases, isomerases, helicases, gyrases, etc., and preferably include those enzymes that are involved in the metabolism of one or more amino acids.
  • a target enzyme preferably can catalyze one or more anabolic, catabolic, or peripheral steps in the metabolism of an amino acid.
  • the target enzyme can be a component in the metabolism of one or more of amino acids glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, tyrosine, tryptophan, serine, threonine, cysteine, methionine, asparagines, glutamine, aspartate, glutamate, lysine, arginine or histidine.
  • a target enzyme can catalyze any metabolic step and are not meant to be limited to those enzymes which directly lead to the production of a product amino acid or those that directly consume a substrate amino acid.
  • the enzymes can catalyze any step in any pathway which can affect the concentration of an amino acid.
  • the target enzyme can function as a pharmaceutical target for the treatment of an amino acid disorder or aminoacidopathy, such as those described herein elsewhere.
  • the target enzyme may be obtained from any source.
  • the target enzyme may be substantially pure, with purity greater than about 50%, or more preferably with purity greater than about 60%, or 70%, or 80%, or even 90%, and may be a mixture of enzymes of the same or different classification, may share the same coenzyme, and/or may be a fraction from a
  • the target enzyme may be included in a sample supplied by a crude lysate, a lysate that has been fractionated or enriched for particular components, or the like. Enrichment may be achieved using chromatography, electrophoresis, density gradients, affinity columns, etc.
  • the particular source of the target enzyme generally is not critical to this invention, since the selected reagents may be robust, and diverse components may be present without interference with the assay. Components that change the binding affinity of the target enzyme(s) which would interfere with the assay may be removed prior to performing the assay.
  • Target enzymes may be selected based on known or predicted functional importance to an organism's growth, survival, health, availability of known inhibitors, and/or importance to a particular disease state, e.g. an aminoacidopathy.
  • targets enzymes may be selected based on structural similarity or distinctiveness between non-human and human homologs, uniqueness to a class or type of organism, and/or properties of mutants, among others.
  • the screenable compounds can include any kind of molecule, mixture of molecules or library of molecules, including, but not limited to, a library of small molecules, combinatorial library, peptide library, protein or protein fragment library, a library of vitamins & co-factors, an enzyme inhibitor library, a nucleic acid library, a carbohydrate library, a generic drug library, a natural product library, or antibody or antibody fragment library.
  • any means for obtaining or preparing such a combinatorial library known in the art can be used (e.g. U.S. Pat. No. 5,463,564, which is incorporated herein by reference).
  • a "combinatorial library” is a collection of compounds in which the compounds comprising the collection are composed of one or more types of sub-units.
  • the library will have at least 2 members, rarely less than about 5 members, usually at least about 10 members, frequently will have about 50 members or more, usually fewer than about 1,000 members, more usually fewer than about 500 members.
  • the sub-units may be selected from natural or unnatural moieties, including a variety of chemical moieties, such as synthetic compounds, naturally occurring compounds, e.g. amino acids, nucleotides, sugars, lipids, and carbohydrates, and synthetic analogs thereof, which are readily available commercially in a large variety of compounds.
  • the compounds of the combinatorial library differ in one or more ways with respect to the number, order, type or types of or modifications made to one or more of the sub-units comprising the compounds.
  • a combinatorial library may refer to a collection of "core molecular organization" which vary as to the conformation, size and charge distribution as a result of the presence of other moieties or differences in the way the core molecular organization is organized.
  • Numerous methods for producing combinatorial libraries are known in the art, including those involving biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the ⁇ one-bead one-compound” library method; and synthetic library methods using affinity chromatography selection.
  • the biological library approach is limited to polypeptide libraries, while the other four approaches are applicable to polypeptide, non-peptide oligomer, or small molecule libraries of compounds (K. S. Lam, 1997, Anticancer Drug Des. 12:145).
  • Non-limiting examples of small molecules, small molecule libraries, and combinatorial libraries are described in B. Seligmann, 1995, "Synthesis, Screening,
  • combinatorial chemical libraries that can be used in accordance with the invention include, but are not limited to: peptoids (PCT Publication No WO 91/19735, Dec. 26, 1991), random bio-oligomers (PCT Publication WO 92/00091, Jan. 6, 1992), benzodiazepines (U.S. Pat. No. 5,288,514), diversomers such as hydantoins, benzodiazepines and dipeptides (Hobbs et al., (1993) Proc. Nat. Acad. Sci. USA 90: 6909 6913), vinylogous polypeptides (Hagihara et al. (1992) J. Amer. Chem. Soc.
  • PCTIUS96/10287 carbohydrate libraries (see, e.g., Liang et al. (1996) Science, 274: 1520 1522, and U.S. Pat. No. 5,593,853), and small organic molecule libraries (see, e.g., benzodiazepines, Baum (1993) C&EN, Jan 18, page 33, isoprenoids U.S. Pat. No. 5,569,588, thiazolidinones and metathiazanones U.S. Pat. No. 5,549,974, pyrrolidines U.S. Pat. Nos. 5,525,735 and 5,519,134, morpholino compounds U.S. Pat. No. 5,506,337, benzodiazepines 5,288,514, pyrimidinediones (see, e.g., U.S. Pat. No. 6,025,371), and the like.
  • carbohydrate libraries see, e.g., Liang et al. (1996) Science, 274
  • libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts can be readily produced.
  • natural or synthetic compound libraries and compounds can be readily modified through conventional chemical, physical and biochemical means (see, e.g., Blondelle et al., 1996, Trends in Biotech. 14:60), and may be used to produce combinatorial libraries.
  • previously identified pharmacological agents can be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, and the analogs can be screened for targets and/or target-partner-modulating activity.
  • Examples of molecules that can comprise a screenable compound library in accordance with the invention are not limited to the following.
  • NADH/NAD NADPH/NADP, ATP/ADP, ATP- ⁇ -S, acetyl-CoA, biotin, S-adenosyl- methionine, thiamine pyrophosphate (TPP), sulfated oligosaccharides, heparin-like oligosaccharides, GTP, GTP- ⁇ -S, ⁇ -S, pyridoxal-5-phosphate, flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), folic acid, tetrahydrofolic acid, methotrexate, vitamin K.sub.l, vitamin E succinate salt, vitamin D 3 , vitamin D 3 25-hydroxy, vitamin D 3 -I- ⁇ -25-dihydroxy, vitamin Bi 2 , vitamin C, vitamin B 6 , coenzyme A, coenzyme A-n-butyryl, transretinoic acid, and heme.
  • Lipids Building Blocks Choline, Phosphoric acid, Glycerol, Palmitic acid, Oleic acid, Cholesterol and Higher Order Structures: Phosphatidylcholine.
  • Protease Inhibitors (Sigma Chemical Co., St. Louis, Mo.), PMSF, Leupeptin, Pepstatin A, Bestatin, Peptide aldehyde Cystatin (Cysteine protease inhibitors), Protein Tyrosine Kinase inhibitors (Calbiochem, San Diego, Calif.), Protein Phosphatase inhibitors (Calbiochem, San Diego, Calif.), Protein Kinase inhibitors (Calbiochem, San Diego, Calif.), Protein Kinase Activators (Calbiochem, San Diego, Calif.), Phosphodiesterase inhibitors (Calbiochem, San Diego, Calif.), Phospholipase Inhibitors, Transition State Analogs.
  • the present invention contemplates the using the method of determining amino acid concentration as a tool to screen and identify useful pharmaceutical compounds from any suitable pool or library of screening compounds, preferably those which contain compound candidates which modulate amino acid concentration and are advantageous in the therapeutic treatment of amino acid disorders and aminoacidopathies.
  • screening refers to the testing of a multiplicity of molecules or compounds for their ability to alter or modulate the concentration of one more amino acids.
  • the screening process is a repetitive, or iterative, process, in which molecules are tested for their ability to modulate amino acid concentration. If an entire compound library fails to contain any molecules that bring about a change in amino acid concentration, then the screening can be repeated using molecules from another compound library.
  • the screening method of the invention can be readily carried out in a high- throughput manner.
  • high-throughput or “high-throughput manner” refers broadly to investigations with a large number of tests such that formatting of each individual sample, minimizing preparation steps and complications, and measuring of the tests either in parallel or in rapid succession become important.
  • High-throughput tests generally do not include manual, one-at-a-time tests, such as tests by a single individual in which the preparation, execution, measurement, and data collection for one test are all completed before the test on the next compound (e.g., a second test ligand) is done.
  • High-throughput is meant to include, for example, any tests in which a plurality of reaction vessels are used, e.g.
  • microwell or microplate arrays of 24, 96, or 384, or more wells to carry out the method of measuring amino acid concentrations in accordance with the invention.
  • Any suitable technology that can be employed e.g. automation devices, robotics, multi-well dispensers, image capturing software etc., to facilitate the processing of a large number of reactions in accordance with the invention in parallel or in rapid succession is contemplated.
  • Aminoacyl-tRNA synthetase reactions were carried out at room temperature according to standard methods (R. Macarron et al., Analytical Biochemistry, 2000, 284:183-190; Jordan et al., Analytical Biochemistry, 2001, 298:133-136. Reactions were performed in white clear- bottom 384-well microtiter plates in charging buffer containing 35 mM HEPES (pH 7.5), 70 mM KCl, 10 mM MgOAc, 2 mM DTT and 0.01 % BSA in a final volume of 60 ⁇ L.
  • Aminoacyl-tRNA synthetase (Sigma Catalogue # A3646) and tRNA (Sigma Catalogue # R1753) concentrations varied according to the particular experiment, and are indicated in the figure legends.
  • radiolabeled amino acid was used at 0.15 ⁇ Ci per reaction (unless stated otherwise), with the concentration of amino acid varying according to specific activity. Reactions were initiated by addition of a mixture of tRNA, ATP (5 mM final concentration) and aaRS, incubated for 1 hour, and quenched with 40 ⁇ L of 0.5 mg YSi SPA beads in IM sodium citrate (pH 2.0).
  • the SPA beads were allowed to equilibrate for 1 hr, spun in a swinging bucket centrifuge at 350 x g for 1 min, and read on a Wallac 1450 Microbeta Plus liquid scintillation counter. Data were analyzed using GraphPad Prism (GraphPad Software, San Diego, CA). All reactions were run in duplicate and, where present, error bars represent ⁇ standard deviation. See FIG. 1, which depicts an aminoacylated tRNA bound to an SPA bead, and FIG. 2A and 2B, which depict the biochemical forward and reverse reactions constituting an aminoacylation charging reaction.
  • Example 2 Demonstration of charging activity for naturally occurring amino acids with the aaRS enzyme extracts and crude tRNA
  • Aminoacylation assays were performed for 19 of the 20 naturally-occurring amino acids. Accurate measurement of cysteine is not possible, due to the fact that the amino acid is present in a partially oxidized form (K. Beaucamp et al., Methods of Enzymatic Analysis 2nd Edition, Academic Press, New York, 1974, pp. 1656-1622). In each charging reaction, 0.15 ⁇ Ci of the indicated [3H]amino acid was used, with the molar concentration determined by the specific activity of each radiolabel.
  • Charging reactions were performed under conditions optimized for maximum signal. These conditions were determined for each amino acid by varying the amount of aaRS (2.5 - 50 units, where one unit will charge 1 pmol of radiolabeled arginine in 10 min at pH 7.6 at 37 C) and tRNA (4 - 30 ⁇ g) in a 1 hour reaction (1 hour being convenient for high throughput conditions). Control reactions for each amino acid included complete reaction mixture minus aaRS and complete reaction mixture minus radiolabeled amino acid. The results are shown in
  • FIG. 3 A A wide range in signal intensity was observed, but all amino acids showed some detectable level of charging above background.
  • Example 3 Demonstration of the specificity of the cSPA aaRS-catalyzed charging reaction
  • charging reactions were set up under the following conditions: radiolabeled cognate amino acid only, radiolabeled cognate amino acid plus non-radiolabeled cognate amino acid at 30 ⁇ M, radiolabeled cognate amino acid plus all noncognate non-radiolabeled amino acids at 30 ⁇ M each, radiolabeled amino acid plus non-radiolabeled cognate and noncognate amino acids at 30 ⁇ M each.
  • radiolabeled cognate amino acid only radiolabeled cognate amino acid plus non-radiolabeled cognate amino acid at 30 ⁇ M
  • radiolabeled cognate amino acid plus all noncognate non-radiolabeled amino acids at 30 ⁇ M each
  • radiolabeled amino acid plus non-radiolabeled cognate and noncognate amino acids at 30 ⁇ M each.
  • tRNA and radiolabeled amino acid could be titrated in a matrix format, and as shown in FIG. 4, a plot of observed signal vs. concentration of radiolabeled valine for a series of tRNA concentrations gave a family of hyperbolae.
  • the concentration of amino acid that gave half- maximal signal at a given tRNA concentration is defined as the EC 50 and was calculated using equation 7:
  • Bmax maximum theoretical signal at infinite radiolabeled cognate amino acid
  • EC 50 the amount of radiolabeled cognate amino acid needed to reach exactly half Bmax.
  • FIG. 4 below the EC 50 , there was a linear correspondence between the amount of radiolabeled amino acid added and signal observed; beyond this point, there was a non-linear response as the system approached saturation with respect to [tRNA] so that simple isotopic dilution conditions were achieved.
  • concentration of tRNA increases, the EC50 for amino acid increases. At a given concentration of tRNA simple isotopic dilution conditions are achieved when signal remains unchanged upon the addition of more radiolabeled amino acid.
  • Example 5 Construction of standard amino acid competition curves. In order to determine the usefulness and sensitivity of an aaRS cSPA charging assay for detection of non-radiolabeled amino acid in the presence of radiolabeled amino acid it was first necessary to perform charging reactions to create a standard curve. A standard curve was constructed by plotting observed cpm against log concentration of increasing non- radiolabeled amino acid at a fixed concentration of radiolabeled amino acid. The top of the curve was a plateau that corresponds to the amount of radiolabeled amino acid charged in the absence of the competing non-radiolabeled amino acid. The bottom of the curve was a plateau that corresponds to background signal of the experiment and in effect represented non-
  • radiolabled amino acid at infinite concentration.
  • concentration of non-radiolabeled amino acid that produced signal halfway between the upper and lower plateaus was defined as the IC50, and was calculated using equation 5.
  • Parameters determined from the standard curve could be used to calculate the concentration of an unknown amount of amino acid in an assay done under similar conditions using equation 6.
  • FIG. 5 A this method was used successfully to construct a family of eight standard curves for valine, each one at a different concentration of radiolabeled amino acid. Similar curves have been constructed for all amino acids tested (data not shown).
  • Example 6 Demonstration of dependence of isotopic dilution conditions on tRNA concentration
  • FIG. 5B a plot of IC 50 vs. radiolabeled amino acid concentration at three concentrations of tRNA depicts three curves that approach the theoretical curve where simple isotopic dilution conditions exist and, by definition, the IC 50 is the concentration of radiolabel.
  • the individual experimental curves intersected the theoretical curve at increasing concentrations of amino acid, and this concentration of amino acid was the point at which simple isotopic dilution conditions were first met.
  • the graph clearly shows that sensitivity depended upon tRNA concentration until simple isotopic conditions were established, at which time sensitivity was determined by the specific activity of the radiolabeled amino acid. Note that the competitive aaRS charging assay need not be performed under tRNA-limiting conditions. In either case, to
  • a standard curve is constructed under a specified set of conditions.
  • Example 7 Measurement of methionine concentrations and aminopeptidase M enzymatic activity
  • aminopeptidase M EC3.4.11.2; Roche catalogue # 10102768001
  • Aminopeptidase M hydrolyzes Met- AMC producing equal amounts of free methionine and free AMC (Met- AMC was used because valine-AMC was not hydrolyzed by aminopeptidase M).
  • standard curves were constructed (e.g. as shown in Example 5) for methionine and AMC using, respectively, the cSPA format and a conventional fluorescent detection assay format.
  • the methionine standard curve shown in FIG. 6A was created by performing aaRS- catalyzed charging reactions in the presence of a fixed concentration of radiolabeled methionine and varying concentrations of non-radiolabeled methionine. The data were then fitted using equation 6 to give methionine concentration.
  • 6B was created by subjecting varying amounts of Met- AMC to complete digestion in a reaction catalyzed by 1, 10 and 100 mU aminopeptidase M (one unit of aminopeptidase M hydrolyzes 1 ⁇ mol of leucine-4-nitranalide in 1 min at pH 7.5 and 25 0 C). Note that because the signals were overlapping for these data, only the 1 mU data are shown. Reaction progress was followed in real time, and endpoint readings were taken after the substrate was
  • FIGS. 6C and 6D illustrate the results using the cSPA and fluorescent assay formats, respectively.
  • the concentration of product was calculated based on the respective standard curves, and resulting data were plotted side-by-side (FIG. 6E).
  • FIG. 6E data demonstrated dose- and time-dependence of the aminopeptidase M catalyzed reaction, with good correlation between the results acquired in the two assay formats.
  • FIG. 6F illustrates the similarity of the leuhistin (Calbiochem Catalogue # 432077) dose curves that were obtained using the fluorescent assay format vs. the cSPA format, as evidenced by the near identical IC 50 values.
  • Example 8 Use of the invention in high throughput quantitative analysis of amino acid concentration
  • the invention was applied to a screen for aminopeptidase M inhibitors, utilizing a 384-well density microtiter plate assay.
  • the HTS assay required seven steps: (1) addition of assay buffer, (2) addition of DMSO or unknown compounds, (3) addition of aminopeptidase, and initiation of the assay by (4) the addition of Met-AMC.
  • the primary reaction was quenched by (5) addition of 50 ⁇ M leuhistin containing 0.15 ⁇ Ci [ 3 H]methionine, followed by (6) the addition of amino acid detection reagents: 40 units aaRS, 10 ⁇ g tRNA, and a final [ATP] of 5 mM.
  • the reaction was quenched by (7) addition of the acidic bead suspension. No special mixing or transfer steps
  • the calculated mean Z factor was 0.64 ⁇ 0.02 (mean ⁇ s.e.), with a range from 0.53 to 0.7.
  • the value of the Z factor is a useful parameter in evaluating, comparing and validating bioassay data. In general, a value of Z > 0.5 indicates that the assay is of high quality and suitable for HTS.
  • G. Pfleiderer, L-alanine Determination with glutamate-pyruvate transaminase and lactic dehydrogenase, in: H.U. Bergmeyer, (Ed.), Methods of Enzymatic Analysis, Academic Press, New York, 1965, pp. 378-380.
  • G. Pfleiderer, L-aspartic acid and L-asparagine Determination with glutamate- oxaloacetate transaminase and malic dehydrogenase, in: H.U. Bergmeyer, (Ed.),

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Abstract

La présente invention concerne un procédé de détermination de la concentration en acides aminés d'un échantillon, en effectuant une réaction de chargement d'aminoacyl-ARNt synthétase à l'équilibre, comprenant une aminoacyl-ARNt synthétase apparentée, un ARNt apparenté, une quantité fixe d'un acide aminé cible radiomarqué et l'échantillon contenant l'acide aminé cible. De plus, le procédé quantifie le signal émis par l'ARNt apparenté aminoacylé résultant par un essai de scintillation par proximité (SPA) et compare le signal à une courbe d'étalonnage afin de déterminer la concentration de l'acide aminé cible dans l'échantillon.
PCT/US2008/083340 2007-11-13 2008-11-13 Procédé de mesure des concentrations en acides aminés WO2009064843A1 (fr)

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Cited By (2)

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WO2014059025A1 (fr) * 2012-10-09 2014-04-17 Liposcience, Inc. Quantification par nmr d'acides aminés à chaîne ramifiée
WO2019198623A1 (fr) * 2018-04-12 2019-10-17 池田食研株式会社 Procédé de quantification d'acides aminés et kit de quantification d'acides aminés

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US20060228775A1 (en) * 2003-07-08 2006-10-12 Chemgenex Pharmaceuticals Llimited Differential expression of nucleic acid molecules
US20070037286A1 (en) * 2005-02-09 2007-02-15 Subhasish Purkayastha Thyroxine-containing compound analysis methods
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US20050164264A1 (en) * 2000-08-10 2005-07-28 Nanobiodynamcis Method and system for rapid biomolecular recognition of amino acids and protein sequencing
US20060228775A1 (en) * 2003-07-08 2006-10-12 Chemgenex Pharmaceuticals Llimited Differential expression of nucleic acid molecules
US20060040335A1 (en) * 2004-06-21 2006-02-23 Butt Tauseef R Diagnostic and screening methods and kits associated with proteolytic activity
US20070037286A1 (en) * 2005-02-09 2007-02-15 Subhasish Purkayastha Thyroxine-containing compound analysis methods

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014059025A1 (fr) * 2012-10-09 2014-04-17 Liposcience, Inc. Quantification par nmr d'acides aminés à chaîne ramifiée
EP2906956B1 (fr) 2012-10-09 2020-11-25 Liposcience, Inc. Quantification par nmr d'acides aminés à chaîne ramifiée
WO2019198623A1 (fr) * 2018-04-12 2019-10-17 池田食研株式会社 Procédé de quantification d'acides aminés et kit de quantification d'acides aminés

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