Analysis of amino acids in body fluid by liquid chromotography-mass spectrometry

Abstract

This disclosure provides methods for quantifying individual amino acids in various bodily fluids obtained from a human patient. Also provided are reference ranges for normal amino acid levels in the various bodily fluids (e.g., blood plasma, urine, cerebrospinal fluid, and saliva) and for various age groups (e.g., neonates, infants, children, and adults).

Claims

1. A method for determining the amount of one or more amino acids in a sample by tandem mass spectrometry, the method comprising: (a) purifying the one or more amino acids by liquid chromatography (LC); and (b) ionizing the one or more amino acids under conditions suitable to produce one or more ions detectable by mass spectrometry; (c) detecting the amount of ions of the one or more amino acids by tandem mass spectrometry (MS/MS); (d) determining the amount of the one or more amino acids in the sample from the amount of ions determined in step (c); wherein the one or more amino acids comprise at least one amino acid selected from allo-isoleucine and phosphoserine.

2. The method of claim 1, wherein the one or more amino acids comprise allo-isoleucine.

3. The method of claim 1, wherein the one or more amino acids comprise phosphoserine.

4. The method of claim 1, wherein the one or more amino acids comprise phosphoserine and allo-isoleucine.

5. The method of claim 2, wherein the one or more amino acids further comprise leucine, isoleucine, and valine.

6. The method of claim 4, wherein the one or more amino acids further comprise alanine, alpha-amino-adipic acid, alpha-amino-butyric acid, anserine, arginine, arginosuccinic acid, asparagine, aspartic acid, beta-alanine, beta-amino-isobutyric acid, citrulline, carnosine, cystathionine, cystine, ethanolamine, gamma-amino-butyric acid, glutamic acid, glutamine, glycine, histidine, hydroxylysine, hydroxyproline, isoleucine, leucine, lysine, methionine, 1-methylhistidine, 3-methylhistidine, ornithine, phenylalanine, phosphoethanolamine, proline, serine, sarcosine, taurine, threonine, tryptophan, tyrosine, and valine.

7. The method of claim 2, wherein the one or more amino acids further comprise taurine, aspartic acid, hydroxyproline, threonine, serine, asparagine, glutamic acid, glutamine, sarcosine, alpha-amino-adipic acid, proline, glycine, alanine, citrulline, alpha-amino-butyric acid, valine, cysteine, methionine, cystathionine, isoleucine, leucine, tyrosine, phenylalanine, arginosuccinic acid, beta-alanine, beta-amino-isobutyric acid, homocystine, gamma-amino-butyric acid, tryptophan, hydroxylysine, ornithine, lysine, histidine, and arginine.

8. The method of claim 2, wherein the one or more amino acids further comprise taurine, aspartic acid, threonine, serine, asparagine, glutamic acid, glutamine, glycine, alanine, citrulline, valine, cystine, methionine, isoleucine, leucine, tyrosine, phenylalanine, beta-alanine, gamma-amino-butyric acid, ornithine, lysine, histidine, and arginine.

9. The method of claim 1, wherein the sample is selected from the group consisting of plasma, urine, cerebrospinal fluid (CSF), and saliva.

10. The method of claim 1, wherein the sample is plasma and the amount of the amino acid is compared to the reference ranges of the corresponding amino acid provided in Table 1.

11. The method of claim 1, wherein the sample is urine and the amount of the amino acid is compared to the reference ranges of the corresponding amino acid provided in Table 2.

12. The method of claim 1, wherein the sample is cerebrospinal fluid (CSF) and the amount of the amino acids is compared to the reference ranges of the corresponding amino acid provided in Table 3.

13. The method of claim 1, wherein the sample is saliva and the amount of the amino acid is compared to the reference ranges of the corresponding amino acid provided in Table 4.

14. The method of claim 1, wherein the amount of the one or more amino acids is used to diagnose or monitor one or more diseases selected from the group consisting of Maple Syrup Urine Disease, kidney failure, Crohn's disease, ulcerative colitis, chronic fatigue syndrome, Wilson's disease, Cushing's disease, gout, and hyperactivity disorders.

15. The method of claim 1, wherein the method comprises using one or more internal standards.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1 is a table showing various amino acids detected and quantifying by the methods described herein. Third column indicates MW molecular weight; fourth column indicates PITC molecular weight; fifth column indicates molecular weight of each PITC derivatized amino acids; six column is the LC retention time; seventh column indicates mass spectrometry ions observed.

(2) FIG. 2 shows the results of an MS analysis of a single sample containing the indicated amino acids, previously subject to LC. The ion size characteristic of each amino acid is indicated.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

(3) As used herein, amino acid means any molecule that includes an alpha-carbon atom covalently bonded to an amino group and an acid group. The acid group may include a carboxyl group. Amino acid may include molecules having one of the formulas:

(4) ##STR00001##
wherein R is a side group and Z includes at least 3 carbon atoms. Amino acid includes, but is not limited to, the twenty endogenous human amino acids and their derivatives such as lysine, asparagine, threonine, serine, isoleucine, methionine, proline, histidine, glutamine, arginine, glycine, aspartic acid, glutamic acid, alanine, valine, phenylalanine, leucine, tyrosine, cysteine, tryptophan, phosphoserine (PSER), sulfo-cysteine, arginosuccinic acid (ASA), hydroxyproline, phosphoethanolamine (PEA), sarcosine (SARC), taurine (TAU), carnosine (CARN), citrulline (CIT), anserine (ANS), 1,3-methyl-histidine (ME-HIS), alpha-amino-adipic acid (AAA), beta-alanine (BALA), ethanolamine (ETN), gamma-amino-butyric acid (GABA), beta-amino-isobutyric acid (BAIA), alpha-amino-butyric acid (BABA), L-allo-cystathionine (cystathionine-A; CYSTA-A), L-cystathionine (cystathionine-B; CYSTA-B), cystine, allo-isoleucine (ALLO-ILE), DL-hydroxylysine (hydroxylysine (1)), DL-allo-hydroxylysine (hydroxylysine (2)), ornithine (ORN), homocystine (HCY), and derivatives thereof. Amino acids also includes stereoisomers such as the D-amino acid and L-amino acid forms. Unless specifically indicated otherwise, the term amino acid refers to the individual (i.e., free) amino acid molecules apart from amino acids present in dipeptides, polypeptides, and proteins.

(5) As used herein, body fluid means any fluid that can be isolated from the body of an individual. For example, body fluid may include blood, plasma, serum, bile, saliva, urine, tears, perspiration, cerebrospinal fluid (CSF), and the like. Preferably the body fluid is plasma, serum, cerebrospinal fluid, urine, or saliva, with plasma being the most preferred.

(6) As used herein, derivatizing means reacting two molecules to form a new molecule. For example, an amino acid may be derivatized by reacting the amino acid with a derivatizing agent to form a derivatized amino acid. Derivatizing may include reacting the alpha amino group of the amino acid with an electrophilic atom of a derivatizing agent to form a covalent bond. Derivatizing agents may include isothiocyanate groups, dinitro-fluorophenyl groups, nitrophenoxycarbonyl groups, and/or phthalaldehyde groups.

(7) As used herein, liquid chromatography (LC) means a process of selective retardation of one or more components of a fluid solution as the fluid uniformly percolates through a column of a finely divided substance, or through capillary passageways. The retardation results from the distribution of the components of the mixture between one or more stationery phases and the bulk fluid, (i.e., mobile phase), as this fluid moves countercurrent to the stationery phases. The process is used for analysis and separation of mixtures of two or more substances. Liquid chromatography includes reverse phase liquid chromatography (RPLC) and high pressure liquid chromatography (HPLC).

(8) As used herein, mass spectroscopic analysis or mass spectrometry (MS analysis) means an analytical technique to identify unknown compounds including: (1) ionizing the compounds and potentially fractionating the compounds to form charged compounds; and (2) detecting the molecular weight of the charged compound and calculating a mass-to-charge ratio (m/z). The compound may be ionized and detected by any suitable means. A mass spectrometer includes means for ionizing compounds and detecting charged compounds.

(9) As used herein, electrospray ionization means a technique used in mass spectrometry to ionize macromolecules and to overcome the propensity of macromolecules to fragment. In electrospray ionization a liquid is pushed through a very small charged metal capillary by a carrier gas. The liquid contains the substance which is to be studied, the analyte, as well as a large amount of solvent, which is usually much more volatile then the analyte. The charge contained in the capillary transfers to the liquid which charges the analyte molecule. As like charges repel, the liquid pushes itself out of the capillary and forms a mist or an aerosol of small droplets about 10 m across, to increase the distance between the similarly charged molecules. A neutral carrier gas is used to evaporate the neutral solvent in the small droplets, this in turn brings the charged analyte molecules closer together. The proximity of the molecules becomes unstable, however, and as the similarly charges molecules come closer together, the droplets once again explode. This process repeats itself until the analyte is free of solvent and a lone ion is formed. The lone ion is transported to a mass analyzer.

(10) As used herein, a quadrupole analyzer is a mass analyzer composed of quads (i.e., two pairs of metallic rods aligned in parallel), wherein one pair of rods is at a positive electrical potential and the other set of rods is at a negative potential. To be detected, an ion must pass through the center of a trajectory path bordered and parallel to the aligned rods. When the quads are operated at a given amplitude of direct current and radio frequency voltages, only ions of a given m/z ratio will resonate and have a stable trajectory to pass through the quadrupole and be detected. Positive ion mode means a mode wherein positively charged ions are detected by the mass analyzer. Negative ion mode means a mode wherein negatively charged ions are detected by the mass analyzer. For single ion monitoring or selected ion monitoring (i.e., SIM), the amplitude of the direct current and the radio frequency voltages are set to observe only a specific mass.

(11) As used herein, a low molecular weight fraction is a fraction that is enriched in one or more low molecular weight molecules. A low molecular weight molecule typically has a molecular weight of less than about 1000 daltons, and more typically less than about 500 daltons.

(12) As used herein, hydrophobic means not dissolving or dissolving poorly in water. Hydrophobic compounds include long chain alkanes. A hydrophobic solvent is a solvent that is capable of dissolving a hydrophobic compound.

(13) As used herein about when used in the context of a number means the number plus or minus 10%.

(14) Disclosed is a method for identifying and/or quantifying amino acids in a body fluid. The body fluid can be blood, plasma, serum, bile, saliva, urine, cerebrospinal fluid, and the like. A preferred body fluids are plasma, serum, CSF, urine, or saliva, with plasma being the most preferred.

(15) A set of individual amino acid standards representing the types of amino acids that might be present in a particular body fluid is preferably added to the body fluid sample before any processing. The set of amino acid standards preferably contains a known amount of each individual amino acid present in the set. A set of amino acid standards may include one or more amino acids from the group consisting of lysine, asparagine, threonine, serine, isoleucine, methionine, proline, histidine, glutamine, arginine, glycine, aspartic acid, glutamic acid, alanine, valine, phenylalanine, leucine, tyrosine, cysteine, tryptophan, phosphoserine, sulfo-cysteine, arginosuccinic acid, hydroxyproline, phosphoethanolamine, sarcosine, taurine, carnosine, citrulline, anserine, 1,3-methyl-histidine, alpha-amino-adipic acid, beta-alanine, ethanolamine, gamma-amino-butyric acid, beta-amino-isobutyric acid, alpha-amino-butyric acid, L-allo-cystathionine (cystathionine-A), L-cystathionine (cystathionine-B), cystine, allo-isoleucine, leucine, DL-hydroxylysine (hydroxylysine (1)), DL-allo-hydroxylysine (hydroxylysine (2)), ornithine, tryptophan, homocystine, and isomers thereof (e.g., stereoisomers).

(16) The amino acids of the set of amino acid standards may be modified so that they can be easily discriminated from the corresponding amino acids found in the body fluid. The internal standard amino acid preferably behaves closest to the amino acid that it is chosen to represent chemically and physically but fragments to ions of a different mass upon mass spectrometric analysis. Thus, a preferred set of amino acids standards is deuterated.

(17) Body fluid may be processed prior to derivatization to obtain an enriched preparation of the amino acids. Various procedures may be used for this purpose depending on the type of body fluid. These include filtration, precipitation, centrifugation, combinations thereof and the like. Separation of a low molecular weight fraction is a preferred approach. Size separation on small volumes of sample is preferably performed by filtering using a low molecular weight cutoff filter. The filtered body fluid sample (i.e., permeate) will include free amino acids and the retained components (i.e., retentate) will include high molecular weight components such as proteins. Suitable filters for generating a filtrate include 45 micron, 22 micron and 100,000, 50,000 and 10,000 dalton cutoff filters. In addition, high molecular weight components may be precipitated from the plasma sample by adding alcohol (e.g., methanol) or acid to the sample. High molecular weight components may also be removed from the sample by high speed centrifugation.

(18) Derivatization of amino acids is performed following any necessary processing of the body fluid sample. The amino acids in the sample typically are derivatized to facilitate separation and/or detection of free amino acids in the sample during LC-MS (e.g., pre-column derivatization is first performed where LC is subsequently performed). The derivatizing agent may include substituents that facilitate detection of the derivatized amino acids during or after chromatography (e.g., fluorophores or chromophores). In addition, the derivatizing agent may include substituents that facilitate ionization of the derivatized amino acids during mass spectrometry. Typical derivatizing agents include isothiocyanates (e.g., phenyl isothiocynate (PITC)), o-phthaldialdehyde (OPA), 2,4-dinitrofluorobenzene (DNFB), and N-(2,4-dinitro-5-fluorophenyl)-L-alainamide (FDAA). In a preferred embodiment, the derivatizing agent is PITC.

(19) After the amino acids in the sample have been derivatized, the sample is subjected to chromatographic separation, preferably high pressure liquid chromatographic separation, and mass spectrometry (i.e., LC-MS).

(20) Liquid chromatography and mass spectrometry may be performed by placing the derivatized sample in an instrument that includes a chromatographic column in flow communication with a mass spectrometer. The chromatographic column typically includes a medium (i.e., a packing material) to facilitate separation of the derivatized amino acids (i.e., fractionation). The medium may include minute particles that have a diameter of approximately 2-6 m, preferably about 3 m. For example, the particles may be silica particles. The particles may have pores that have a diameter of approximately 50-300 angstroms, preferably 150 angstroms. Additionally, the particles may have a surface area of approximately 50-600 m.sup.2/g, preferably 100 m.sup.2/g.

(21) The particles include a bonded surface that interacts with the derivatized amino acids to facilitate separation of the amino acids. One suitable bonded surface is a hydrophobic bonded surface such as an alkyl bonded surface. Alkyl bonded surfaces may include C-4, C-8, or C-18 bonded alkyl groups, preferably C-18 bonded groups.

(22) The column may have any suitable dimensions. In particular, the column may have a diameter of about 0.5-5 mm and a length of about 15-300 mm. Preferably, the column has a diameter of about 2 mm and length of about 50 mm.

(23) Suitable media for preparing a chromatographic column and/or prepared columns may be obtained from commercial sources. In particular, suitable columns may be obtained from Thermo Electron Corporation (e.g., 2502.1 mm, 5 m, BetaBasic C18 column).

(24) The chromatographic column includes an inlet port for receiving a sample and an outlet port for discharging an effluent that includes the fractionated sample. In the method, the derivatized sample is applied to the column at the inlet port, eluted with a solvent or solvent mixture, and discharged at the outlet port. Different solvent modes may be selected for eluting the amino acids. For example, liquid chromatography may be performed using a gradient mode, an isocratic mode, or a polytyptic (i.e. mixed) mode. Preferably, liquid chromatography is performed using a gradient mode. In the gradient mode, the derivatized sample is applied to the column and a mixture of two solvents (i.e., the mobile phase) is passed through the column to elute the amino acids. Generally, as known in the art, one of the solvents will tend to be relatively hydrophilic, and the other solvent will tend to be relatively hydrophobic. As a specific example of a solvent combination found to be suitable in the practice of the present method, the hydrophilic solvent may be 95% H.sub.2O, 5% acetonitrile and the hydrophobic solvent may be 100% acetonitrile. Optionally, the solvent combination may include one or more reagents to facilitate separation and/or detection of the derivatized amino acids (e.g., 20 mM ammonium acetate). Some reagents may be added to the mobile phase to improve the shape of the chromatographic peak and/or to provide a source of ions for LC-MS.

(25) In most cases, to perform liquid chromatography with a gradient solvent, two pumps are used that mix the two solvents. Initially, as the solvents are mixed, the solvent mixture that is passed through the column (i.e., mobile phase) includes mostly hydrophilic solvent. Gradually, the amount of hydrophilic solvent in the mixture is decreased and the amount of hydrophobic solvent in the mixture is increased to create a solvent gradient. Ultimately, the solvent mixture that is passed through the column includes mostly hydrophobic solvent. In this manner, hydrophilic amino acids will be eluted before hydrophobic amino acids.

(26) The mass spectrometer includes an inlet port for receiving the fractionated sample that is in flow communication with the outlet port of the chromatographic column. The mass spectrometer is capable of generating one or more mass spectroscopic data sets for identifying one or more amino acids in the sample. Suitable instruments for performing LC-MS may be obtained from commercial sources. In particular, suitable instruments for performing LC-MS may be obtained from Agilent Technologies (e.g., Agilent 1100 Series LC/MSD).

(27) The mass spectrometer will include an ion source for ionizing the fractionated sample and creating charged molecules for further analysis. Ionization of the sample may be performed by electrospray ionization (ESI), atmospheric pressure chemical ionization (ACPI), photoinonization, electron ionization, fast atom bombardment (FAB)/liquid secondary ionization (LSIMS), matrix assisted laser desorption ionization (MALDI), field ionization, field desorption, thermospray/plasmaspray ionization, and particle beam ionization. Electrospray ionization is preferred.

(28) After the sample has been ionized, the positively charged or negatively charged ions thereby created may be analyzed to determine a mass-to-charge ratio (i.e., mlz). Preferably, the negatively charged ions are analyzed. Suitable analyzers for determining mass-to-charge ratios include quadropole analyzers, ion traps analyzers, and time-of-flight analyzers. Preferably, the mass-to-charge ratio is determined using a quadropole analyzer. The ions may be detected by using several detection modes. For example, selected ions may be detected (i.e., using a selective ion monitoring mode (SIM)), or alternatively, ions may be detected using a scanning mode. Preferably, the ions are detected by using SIM.

Example 1Analytical Procedures

(29) Urine, plasma, and cerebrospinal fluid (CSF) samples were obtained from normal individuals. Heparinized plasma samples were collected from patients after an overnight fast. Non-fasting samples for pediatric patients were used. The following procedure was used for amino acid quantification in urine, plasma, and CSF.

(30) Deuterated internal standards were added to about 100 l of the test material (i.e., plasma, urine, or CSF) to form a test mixture. The test mixture then was passed through a 10,000 molecular weight filter to provide a low molecular weight filtrate fraction as a test sample and then dried under nitrogen at 40-75 C. The dried sample was dissolved in about 25 l of a redry solution (equal volumes of methanol, 1M sodium acetate, and triethylamine) and dried under nitrogen at 40 C. The sample was dissolved in 50 l of a derivatizing solution (1.12 l of 100% methanol, 1.60 l water, 1.60 l of 100% triethylamine, and 1.60 l of 100 phenylisothiocyanate (PITC)). The dissolved sample was then heated at about 40 C. for about 15-20 minutes and then dried under liquid nitrogen at 50-60 C. The dried sample was dissolved in 100 l of a reconstitution solution (95% H.sub.2O, 5% acetonitrile), vortexed, and transferred to vials for LC/MS.

(31) LC/MS determination of the sample amino acids was performed using an Agilent 1100 Series LC/MSD with a Thermo Beta-Basic C-18 (2502.1 mm) HPLC column. The mobile phase consisted of (A) 20 mM ammonium acetate and (B) 100% acetonitrile, heated to 50 C. Sample amino acids were eluted from the column using a step-gradient as follows:

(32) TABLE-US-00001 Maximum Time Mobile Phase Flow Pressure Step (minutes) % B (mL/Minute) (bar) 1 0.00 97.0 0.550 350 2 1.40 97.0 0.550 350 3 4.50 92.0 0.550 350 4 6.50 90.0 0.550 350 5 10.50 82.0 0.550 350 6 13.50 80.0 0.650 350 7 16.50 73.0 0.650 350 8 17.50 55.0 0.650 350 9 18.50 50.0 0.650 350 10 19.70 20.0 0.550 350 11 20.70 20.0 0.550 350 12 21.00 97.0 0.550 350

(33) As the separated amino acids exit the HPLC column, they were introduced into the spray chamber where the effluent is sprayed and de-solvated by the electrospray ion source. The PITC derivatives were negatively charged during the electrospray process and then further separated through the quadrupole mass filter. Amino acid ions were detected and their abundances measured. The ratio of the area abundances of each amino acid ion to its internal standard was plotted against a six-point calibration curve. FIGS. 1 and 2 show the results of a typical run.

Example 2Quantification of Amino Acid Content in Plasma

(34) Amino acid analysis was performed on heparinized plasma samples obtained from neonates (30 days), infants (1-23.9 months), children (2-17.9 years), and adults 18 years). All individuals were assessed as clinically normal or obtained from samples submitted for infectious disease determinations. All subjects were fully ambulatory, community dwelling, healthy, and on no medications. The demographics of the test groups is as follows:

(35) TABLE-US-00002 # of subjects # of # of Test Group (n) males females Neonates 55 33 22 (30 days) Infants 115 59 56 (1-23.9 months) Children 134 76 58 (2-17.9 years) Adults 134 64 70 (18 years

(36) Based on the analysis of these plasma samples, normal Reference Ranges were constructed using standard parametric and non-parametric statistical procedures. If the data was determined to be Gaussian, the appropriate mean2 SD range was chosen. If the data was non-gaussian, the non-parametric 95 percentile range or observed range was chosen. Table 1 provides normal reference ranges for each amino acid assayed, along with the limit of quantification (LOQ). Reference ranges are provided in M (micromoles per liter). The normal range for amino acids which are undetectable should be taken to be less than or equal to the LOQ.

(37) TABLE-US-00003 TABLE 1 Adult and Pediatric Amino Acid Levels in Plasma Amino Acid LOQ Neonates Infants Children Adults Pserine N/A 0.67 0.56 Undetectable 0.90 Aspartic Acid 1.0 2.4-19.5 2.3-14.3 1.3-8.2 0.9-3.9 Sulfo-Cysteine UNK 0.13-1.60 1.66 1.62 0.44-3.95 Glutamic Acid 1.0 51-277 32-185 9-109 10-97 Arginosuccinic Acid 1.0 1.15 1.10 1.00 1.00 (ASA) Hydroxyproline 1.0 13-72 7-63 6-32 4-27 (OH-Proline) Hawkinsin 1.0 Undetectable Undetectable Undetectable Undetectable Serine UNK 87-241 83-212 85-185 65-138 Asparagine 1.0 12-70 20-77 23-70 31-64 -Amino Adipic 1.0 2.8 3.6 2.1 2.4 Acid (AAD) Glycine 4.0 133-409 103-386 138-349 122-322 Glutamine UNK 240-1194 303-1459 405-923 428-747 Sarcosine 1.0 4.5 4.0 3.9 3.7 Phospho- 2.0 Undetectable Undetectable Undetectable Undetectable Ethanolamine (PEA) -Alanine 2.0 8.3 7.8 4.7 4.8 (BALA) Taurine UNK 29-161 26-130 32-114 31-102 Histidine UNK 40-143 42-125 54-113 60-109 Citrulline 1.0 3-35 4-50 9-52 16-51 Carnosine 1.0 10.0 6.1 Undetectable Undetectable Arginine UNK 14-135 30-147 38-122 43-407 Threonine UNK 56-392 40-428 59-195 67-198 Alanine UNK 83-447 119-523 157-481 200-483 1-Methylhistidine 1.0 4.2 8.6 27.4 47.1 (1-Me-His) Anserine 1.0 Undetectable Undetectable Undetectable Undetectable -Amino-Butyric 1.0 Undetectable Undetectable 2.2 3.1 Acid (GABA) 3-Methylhistidine 1.0 9.9 8.2 1.4-6.3 2.1-8.6 (3-Me-His) -Amino-Isobutyric 3.0 9 8 6.sup. Undetectable Acid (BAIB) Proline 1.0 87-375 104-348 99-351 104-383 Ethanolamine UNK 8-106 5-19 5-15 5-13 -Amino-Butyric 1.0 1-20 4-30 6-30 7-32 Acid (AAB) Cysteine 1.0 5.3 16.3 34.6 6.6-73.5 Tyrosine UNK 33-160 24-125 31-108 38-96 Valine UNK 57-250 84-354 130-307 132-313 Methionine 1.0 13-45 12-50 14-37 16-34 Cystathionine-A 1.0 Undetectable Undetectable Undetectable Undetectable (Cysta-A) Cystathionine-B 1.0 Undetectable Undetectable Undetectable Undetectable (Cysta-B) Cystine 1.0 25 19 39 8-52 Isoleucine 1.0 12-92 10-109 33-97 34-98 Allo-Isoleucine UNK Undetectable Undetectable Undetectable Undetectable (allo-Ile) Leucine UNK 23-172 43-181 65-179 73-182 Hydroxy-Lysine-1 1.0 3-10 1-8 1-4 1-7 (OH-Lysine-1) Hydroxy-Lysine-2 1.0 Undetectable Undetectable Undetectable 2.34 or less (OH-Lysine-2) Homocystine 1.0 Undetectable Undetectable Undetectable Undetectable Phenylalanine UNK 30-79 31-92 38-86 40-74 Tryptophan 1.0 17-85 16-92 30-94 40-91 Ornithine 2.0 29-168 19-139 33-103 27-83 Lysine UNK 66-226 70-258 98-231 119-233

Example 3Quantification of Amino Acid Content in Urine

(38) Amino acid analysis was performed on urine samples obtained from neonates (30 days), infants (1-23.9 months), children (2-17 years), and adults (>17 years). All individuals were assessed as clinically normal or obtained from samples submitted for infectious disease determinations. All subjects were fully ambulatory, community dwelling, healthy, and on no medications. The demographics of the test groups is as follows:

(39) TABLE-US-00004 # of subjects # of # of Test Group (n) males females Neonates 17 14 3 (30 days) Infants 40 23 17 (1-23.9 months) Children 80 42 38 (2-17 years) Adults 111 55 56 (>17 years)

(40) The amino acid content of urine is initially measured on a concentration (mol amino acid per liter of urine; M) basis. The amino acid concentrations were then normalized based on the urinary creatinine levels and normal Reference Ranges were constructed using standard parametric and non-parametric statistical procedures. If the data was determined to be Gaussian, the appropriate mean2 SD range was chosen. If the data was non-gaussian, the non-parametric 95 percentile range or observed range was chosen. Table 2 provides normal reference ranges (mmol/mol creatinine) for each amino acid assayed. The limit of quantification (LOQ) is based on the amino acid determination in urine, unadjusted for creatinine. The normal range for amino acids which are undetectable should be taken to be less than or equal to the LOQ.

(41) TABLE-US-00005 TABLE 2 Adult and Pediatric Amino Acid Levels in Urine (mmol/mol creatinine) LOQ Amino Acid (M) Neonates Infants Children Adults Pserine 1 8 3 Undetectable 247 Aspartic Acid 1 7 11 2 2 Sulfo-Cysteine 1 50 45 30 11 Glutamic Acid 1 4-19 3-30 10 3 Arginosuccinic Acid 1 15 9 6 4 (ASA) Hydroxyproline 1 30-485 2-345 4 2 (OH-Proline) Hawkinsin 1 Undetectable Undetectable Undetectable Undetectable Serine 1 44-454 39-422 13-127 10-71 Asparagine 1 8-42 5-132 3-42 2-37 -Amino Adipic UNK 10 36 34 11 Acid (AAD) Glycine 1 215-2053 105-413 23-413 330 Glutamine UNK 355 41-396 18-188 21-182 Sarcosine 1 18 19 2 69 Phospho- 2 23 32 15 4 Ethanolamine (PEA) -Alanine 2 9 15 5 10 (BALA) Taurine 1 650 670 255 232 Histidine 1 40-301 56-543 9-425 17-266 Citrulline 1 4 13 4 2 Carnosine 1 9-72 15-65 23 8 Arginine 2 30 35 8 5 Threonine 1 112 9-158 4-60 4-46 Alanine UNK 45-264 16-294 8-156 9-67 1-Methylhistidine 1 16 4-71 5-400 204 (1-Me-His) Anserine 1 8 30 87 411 -Amino-Butyric 1 1.4 1.5 1.6 1.6 Acid (GABA) 3-Methylhistidine UNK 9-45 14-35 11-40 10-35 (3-Me-His) -Amino-Isobutyric 3 269 309 133 88 Acid (BAIB) Proline 1 219 216 11 2 Ethanolamine 1 87-490 54-176 27-114 21-65 -Amino-Butyric 1 7 7 5 2 Acid (AAB) Cysteine 1 Undetectable Undetectable 4 5 Tyrosine UNK 4-59 10-69 3-48 3-19 Valine 2 2-20 4-21 2-20 2-5 Methionine 1 7 7 5 2 Cystathionine-A 1 2 1 2 3 (Cysta-A) Cystathionine-B 1 20 29 8 5 (Cysta-B) Cystine UNK 15-58 6-28 3-20 3-13 Isoleucine 3 9 12 5 3 Allo-Isoleucine 1 Undetectable Undetectable Undetectable Undetectable (allo-Ile) Leucine 2 23 24 13 6 Hydroxy-Lysine-1 1 83 71 8 8 (OH-Lysine-1) Hydroxy-Lysine-2 1 3 3 Undetectable Undetectable (OH-Lysine-2) Homocystine 1 Undetectable 4 Undetectable Undetectable Phenylalanine UNK 3-24 6-39 2-22 2-9 Tryptophan 1 2-21 5-46 2-27 2-14 Ornithine 2 39 11 5 4 Lysine UNK 13-284 4-239 3-112 3-59 Hydroxy-Lysine, Total 1 5-117 2-72 8 8 Cystathionine, Total 1 2-20 29 8 9

Example 4Quantification of Amino Acid Content in Cerebrospinal Fluid (CSF)

(42) Amino acid analysis was performed on CSF samples obtained from neonates (3 months), infants (3-23.9 months), children (2-10 years), and adults (>10 years). All individuals were assessed as clinically normal or obtained from samples submitted for infectious disease determinations. All subjects were fully ambulatory, community dwelling, healthy, and on no medications. The demographics of the test groups is as follows:

(43) TABLE-US-00006 # of subjects # of # of Test Group (n) males females Neonates 27 23 4 (3 months) Infants 22 16 6 (3-23.9 months) Children 21 9 12 (2-10 years) Adults 57 21 36 (>10 years)

(44) Based on the analysis of these CSF samples, normal Reference Ranges were constructed using standard parametric and non-parametric statistical procedures. If the data was determined to be Gaussian, the appropriate mean2 SD range was chosen. If the data was non-gaussian, the non-parametric 95 percentile range or observed range was chosen. Table 3 provides normal reference ranges for each amino acid assayed, along with the limit of quantification (LOQ). Reference ranges are provided in M (micromoles per liter). The normal range for amino acids which are undetectable should be taken to be less than or equal to the LOQ.

(45) TABLE-US-00007 TABLE 3 Adult and Pediatric Amino Acid Levels in CSF Amino Acid LOQ Neonates Infants Children Adults Pserine N/A 4.62 2.39 3.85 4.19 Aspartic Acid 1.0 2.7 Undetectable Undetectable 2.0 Sulfo-Cysteine UNK 0-1 0-1 0-1 0-1 Glutamic Acid 1.0 1-9 5.1 10.6 1.1-13.2 Arginosuccinic 1.0 4.3 2.4 3.0 2.5 Acid (ASA) Hydroxyproline 1.0 0.9-3.9 1.6 Undetectable 1.7 (OH-Proline) Hawkinsin 1.0 Undetectable Undetectable Undetectable Undetectable Serine 30-88 22-61 15-62 9-41 Asparagine 1.0 27 13 25 24 -Amino Adipic 1.0 Undetectable Undetectable Undetectable Undetectable Acid (AAD) Glycine 4.0 3-26 12 13 10 Glutamine UNK 525-1583 386-742 377-1738 361-1175 Sarcosine 1.0 Undetectable Undetectable Undetectable Undetectable Phospho- 2.0 Undetectable Undetectable 4.2 4.8 Ethanolamine (PEA) -Alanine 2.0 Undetectable Undetectable Undetectable Undetectable (BALA) Taurine UNK 0-18 8 1-8 1-8 Histidine UNK 8-32 4-25 7-25 7-22 Citrulline 1.0 1-4 3 1-2 2 Carnosine 1.0 Undetectable Undetectable Undetectable Undetectable Arginine UNK 2-27 7-32 9-31 10-32 Threonine UNK 23-104 10-55 8-85 12-64 Alanine UNK 13-50 8-48 5-62 1-107 1-Methylhistidine 1.0 6.4 9.0 3.8 4.2 (1-Me-His) Anserine 1.0 26 27 19 28 -Amino-Butyric 1.0 Undetectable Undetectable 2.2 3.1 Acid (GABA) 3-Methylhistidine 1.0 3.3 1.8 2.5 2.7 (3-Me-His) -Amino-Isobutyric 3.0 Undetectable Undetectable Undetectable Undetectable Acid (BAIB) Proline 1.0 3.9 2.3 1.7 5.9 Ethanolamine UNK 1-46 3-19 5-40 4-23 -Amino-Butyric 1.0 6 6 1-11 1-11 Acid (AAB) Cysteine 1.0 Undetectable Undetectable Undetectable Undetectable Tyrosine UNK 9-41 5-20 5-32 5-18 Valine UNK 11-31 8-19 2-37 7-42 Methionine 1.0 2-14 1-7 9 1-8 Cystathionine-A 1.0 Undetectable Undetectable Undetectable Undetectable (Cysta-A) Cystathionine-B 1.0 Undetectable Undetectable Undetectable Undetectable (Cysta-B) Cystine 1.0 3.7 3.2 1.6 2.2 Isoleucine 1.0 3-11 3-7 2-13 3-10 Allo-Isoleucine UNK Undetectable Undetectable Undetectable Undetectable (allo-Ile) Leucine UNK 7-22 7-12 8-27 9-32 Hydroxy-Lysine-1 1.0 Undetectable Undetectable Undetectable Undetectable (OH-Lysine-1) Hydroxy-Lysine-2 1.0 Undetectable Undetectable Undetectable Undetectable (OH-Lysine-2) Homocystine 1.0 Undetectable Undetectable 2.5 2.1 Phenylalanine UNK 4-31 4-14 2.5 6-31 Tryptophan 1.0 5.9 7.7 0.6-4.6 9.3 Ornithine 2.0 25.7 4.5 4.7 14.2 Lysine UNK 6-38 3-29 9-58 19-60

Example 5Quantification of Amino Acid Content in Saliva

(46) Amino acid analysis was performed on saliva samples of nine (9) adults (3 males and 6 females). All individuals were assessed as clinically normal and were fully ambulatory, community dwelling, healthy, and on no medications. Table 4 provides the actual measured ranges of amino acid levels. For diagnostic purposes, these ranges may be considered normal ranges.). The normal range for amino acids which are undetectable should be taken to be less than or equal to the LOQ.

(47) TABLE-US-00008 TABLE 4 Adult Amino Acid Levels in Saliva Amino Acid Normal Range Aspartic Acid 2.6-9.2 Arginosuccinic Acid (ASA) Undetectable Sulfo-Cysteine 1.3 Glutamic Acid 1.5-26.0 Hydroxyproline (OH-Proline) 1.6 Serine 0.9-4.9 Asparagine Undetectable Phospho-Ethanolamine (PEA) 133.8 -Amino Adipic Acid (AAD) 0.4-2.8 Glycine 24.5-425.9 Glutamine 0.7-20.2 Sarcosine 0.4-11.0 Histidine 4.3-59.8 -Alanine (BALA) 6.0 Taurine 26.5-177.5 Citrulline 1.5-21.7 Carnosine 0.0 Threonine 0.1-0.4 Arginine 4.9-26.4 Anserine 0.1 1-Methylhistidine (1-Me-His) 0.8 3-Methylhistidine (3-Me-His) 0.4 Alanine 7.6-39.6 -Amino-Butyric Acid (GABA) 0.4-2.4 -Amino-Isobutyric Acid (BAIB) 1.0 Proline 10.5-264.1 Ethanolamine 5.1-42.3 -Amino-Butyric Acid (AAB) 0.6 Tyrosine 8.5-63.1 Valine 0.3-7.9 Methionine 0.4-0.9 Cystathionine-A (Cysta-A) Undetectable Cystathionine-B (Cysta-B) Undetectable Cystine 3.7 Isoleucine 0.4-1.7 Allo-Isoleucine (allo-Ile) Undetectable Leucine 0.2-6.1 Hydroxy-Lysine-1 (OH-Lysine-1) 0.1 Hydroxy-Lysine-2 (OH-Lysine-2) Undetectable Homocystine Undetectable Phenylalanine 3.8-31.5 Tryptophan 1.2 Ornithine 4.8-72.0 Lysine 6.2-81.0

(48) All patents and other references cited in the specification are indicative of the level of skill of those skilled in the art to which the invention pertains, and are incorporated by reference in their entireties, including any tables and figures, to the same extent as if each reference had been incorporated by reference in its entirety individually.

(49) One skilled in the art would readily appreciate that the present invention is well adapted to obtain the ends and advantages mentioned, as well as those inherent therein. The methods, variances, and compositions described herein as presently representative of preferred embodiments are exemplary and are not intended as limitations on the scope of the invention. Changes therein and other uses which will occur to those skilled in the art, which are encompassed within the spirit of the invention, are defined by the scope of the claims.

(50) It will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. Thus, such additional embodiments are within the scope of the present invention and the following claims.

(51) The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein. Thus, for example, in each instance herein any of the terms comprising, consisting essentially of and consisting of may be replaced with either of the other two terms. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.

(52) In addition, where features or aspects of the invention are described in terms of Markush groups or other grouping of alternatives, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group or other group.

(53) Also, unless indicated to the contrary, where various numerical values are provided for embodiments, additional embodiments are described by taking any two different values as the endpoints of a range. Such ranges are also within the scope of the described invention.

(54) Thus, additional embodiments are within the scope of the invention and within the following claims.