Determination of small-molecule thiols and disulfides: protein bound cys and total cysteine as biomarkers of oxidative stress

Abstract

Compositions and methods for determining the level of thiol and disulfide containing molecules in a sample are provided. The compositions and methods can be used to determine the level of oxidative stress in a subject with or without antioxidant treatment. Also provided are biomarkers of oxidative stress.

What is claimed: 
     
         1 . A method for determining the level of thiol and disulfide containing molecules in a sample, the method comprising:
 a) treating a sample with a reagent to prevent free thiol oxidation; (ii) adding an isotopically labeled analogue of each molecule of interest; and (iii) adding methanol to extract the molecules;   b) subjecting the sample to liquid chromatography-tandem mass spectrometry (LC-MS/MS) with Multiple Reaction Monitoring (MRM) to determine the levels of reduced and oxidized forms of thiol and disulfide containing molecules in the sample;   wherein the thiol and disulfide containing molecules are at least one of: GSH, GSSG, cysteine, cystine, N-acetyl-cysteine (NAC), N-acetyl-cystine (NACss), CysGly (CG), gamma-GluCys (γEC), homocysteine (Hcy), homocytine, Cys-ss-GSH, Cys-ss-NAC, Cys-ss-Hcy, Cys-ss-CG, Cys-ss-γEC, GSH-ss-NAC, CGss, γECss, Protein-ss-Cys (p-ss-Cys), Protein-ss-GSH (p-ss-GSH), Protein-ss-NAC (p-ss-NAC), Protein-ss-Hcy (p-ss-Hcy), or Protein-ss-CG.   
     
     
         2 . The method of  claim 1 , wherein the sample is blood or a fraction thereof, cells, or tissue. 
     
     
         3 . The method of  claim 1 , wherein the sample is from a subject with a disease that results in oxidative stress. 
     
     
         4 . The method of  claim 3 , wherein the disease that results in oxidative stress is sickle cell disease (SCD), acute respiratory distress syndrome (ARDS), or thrombotic thrombocytopenic purpura (TTP). 
     
     
         5 . The method of  claim 1 , wherein the levels of thiol and disulfide containing molecules in the sample are compared to a control sample from a normal healthy donor or an average value derived from samples of healthy donors. 
     
     
         6 . The method of  claim 5 , wherein a greater amount of oxidized forms of thiol and disulfide containing molecules in the sample as compared to the control is indicative of oxidative stress in the subject. 
     
     
         7 . The method of  claim 1 , wherein the isotopically-labeled analogue is at least one of GSH* (Glutathione-(glycine- 13 C2,  15 N), Cys*(L-Cysteine- 13 C3, 15 N) Cys** (L-Cysteine- 13 C3, D3,  15 N), Cystine* (L-Cystine- 13 C6,  15 N2), NAC* (L-Cysteine- 13 C3, 15 N, N-acetyl), Hcy-d4, NEMd5: N-ethylmaleimide (ethyl-D5), GSSG*(disulfide bound Glutathione-(glycine- 13 C2,  15 N), NAC*ss, Hcy*ss, Cys*-ss-GSH* (Cys* disulfide bound to GSH*), Cys*-ss-NAC*, Cys**-ss-Hcyd4, Cys*-ss-CG, Cys*-ss-γEC, CG-NEMd5 (CysGly alkylated with N-ethylmaleimide (ethyl-D5)), γEC-NEMd5. 
     
     
         8 . The method of  claim 1 , wherein the reagent to prevent free thiol oxidation sample is N-ethylmaleimide (NEM). 
     
     
         9 . The method of  claim 1  or  7 , wherein the reagent to prevent free thiol oxidation sample is supplied in a dried form in the sample collection container. 
     
     
         10 . The method of  claim 1 , wherein the amount of the oxidized and reduced forms of thiols: GSH, GSSG, cysteine, cystine, N-acetyl-cysteine (NAC), N-acetyl-cystine, CysGly, gamma-GluCys, homocysteine, Cys-ss-GSH, Cys-ss-NAC, Cys-ss-CysGly, Cys-ss-γGluGys, and Cys-ss-Hcy are measured simultaneously in one assay run. 
     
     
         11 . The method of  claim 1 , wherein the optimized MRM transitions are as shown in Table 1. 
     
     
         12 . The method of  claim 5 , wherein the level of the oxidized forms of thiol and disulfide containing molecules in a sample is at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0 times the level in the control sample. 
     
     
         13 . The method of  claim 1 , wherein the level of protein-ss-Cys is determined, and wherein a greater amount of protein-ss-Cys in the sample as compared to a control is indicative of oxidative stress in a subject from which the sample was obtained. 
     
     
         14 . The method of  claim 1 , wherein the level of total cysteine is determined, and wherein a greater amount of total cysteine in the sample as compared to a control is indicative of oxidative stress in a subject from which the sample was obtained. 
     
     
         15 . The method of  claim 1 ,  13 , or  14 , wherein if a greater amount of oxidized forms of thiol and disulfide containing molecules as compared to a control is determined in the sample, the subject is administered an antioxidant treatment. 
     
     
         16 . The method of  claim 15 , wherein the antioxidant treatment is the administration of N-acetyl cysteine. 
     
     
         17 . The method of  claim 16 , the administration of N-acetyl cysteine is for 1, 2, 3, or more hours at 75 mg/kg, 150 mg/kg or 300 mg/kg by i.v. infusion. 
     
     
         18 . The method of  claim 16 , the administration of N-acetyl cysteine is by oral administration. 
     
     
         19 . A kit for performing the method of  claims 1 - 18 . 
     
     
         20 . A method for determining oxidative stress in a subject, the method comprising:
 a) treating a sample from a subject with a reagent to prevent free thiol oxidation; (ii) adding an isotopically labeled analogue of each molecule of interest; and (iii) adding methanol to extract the molecules;   b) subjecting the sample to liquid chromatography-tandem mass spectrometry (LC-MS/MS) with Multiple Reaction Monitoring (MRM) to determine the levels of reduced and oxidized forms of thiol and disulfide containing molecules in the sample;   wherein the thiol and disulfide containing molecules are at least one of: GSH, GSSG, cysteine, cystine, N-acetyl-cysteine (NAC), N-acetyl-cystine (NACss), CysGly(CG), gamma-GluCys (γEC), homocysteine (Hcy), homocytine, Cys-ss-GSH, Cys-ss-NAC, Cys-ss-Hcy, Cys-ss-CG, Cys-ss-γEC, GSH-ss-NAC, CGss, γECss, Protein-ss-Cys (p-ss-Cys), Protein-ss-GSH (p-ss-GSH), Protein-ss-NAC (p-ss-NAC), Protein-ss-Hcy (p-ss-Hcy), or Protein-ss-CG; and   wherein a greater amount of oxidized forms of thiol and disulfide containing molecules as compared to a control is indicative of oxidative stress in the subject.   
     
     
         21 . The method of  claim 20 , wherein the sample is blood or a fraction thereof, cells, or tissue. 
     
     
         22 . The method of  claim 20 , wherein the subject is a patient with a disease that results in oxidative stress. 
     
     
         23 . The method of  claim 22 , wherein the disease that results in oxidative stress is sickle cell disease (SCD), acute respiratory distress syndrome (ARDS), or thrombotic thrombocytopenic purpura (TTP). 
     
     
         24 . The method of  claim 20 , wherein the control is a sample from a normal healthy donor or an average value derived from a sample of healthy donors. 
     
     
         25 . The method of  claim 20 , wherein the isotopically-labeled analogue is at least one of GSH* (Glutathione-(glycine- 13 C2,  15 N), Cys*(L-Cysteine- 13 C3, 15 N) Cys** (L-Cysteine- 13 C3, D3,  15 N), Cystine* (L-Cystine- 13 C6,  15 N2), NAC* (L-Cysteine- 13 C3, 15 N, N-acetyl), Hcy-d4, NEMd5: N-ethylmaleimide (ethyl-D5), GSSG*(disulfide bound Glutathione-(glycine- 13 C2,  15 N), NAC*ss, Hcy*ss, Cys*-ss-GSH* (Cys* disulfide bound to GSH*), Cys*-ss-NAC*, Cys**-ss-Hcyd4, Cys*-ss-CG, Cys*-ss-γEC, CG-NEMd5 (CysGly alkylated with N-ethylmaleimide (ethyl-D5)), γEC-NEMd5. 
     
     
         26 . The method of  claim 20 , wherein the reagent to prevent free thiol oxidation sample is N-ethylmaleimide (NEM). 
     
     
         27 . The method of  claim 20  or  26 , wherein the reagent to prevent free thiol oxidation sample is supplied in a dried form in the sample collection container. 
     
     
         28 . The method of  claim 20 , wherein the amount of the oxidized and reduced forms of thiols: GSH, GSSG, cysteine, cystine, N-acetyl-cysteine (NAC), N-acetyl-cystine, CysGly, gamma-GluCys, homocysteine, Cys-ss-GSH, Cys-ss-NAC, Cys-ss-CysGly, Cys-ss-γGluGys, and Cys-ss-Hcy are measured simultaneously in one assay run. 
     
     
         29 . The method of  claim 20 , wherein the optimized MRM transitions are as shown in Table 1. 
     
     
         30 . The method of  claim 20 , wherein the level of the oxidized forms of thiol and disulfide containing molecules in a sample is at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0 times the level in the control sample. 
     
     
         31 . The method of  claim 20 , wherein the level of protein-ss-Cys is determined, and wherein a greater amount of protein-ss-Cys in the sample as compared to a control is indicative of oxidative stress in the subject. 
     
     
         32 . The method of  claim 20 , wherein the level of total cysteine is determined, and wherein a greater amount of total cysteine in the sample as compared to a control is indicative of oxidative stress in the subject. 
     
     
         33 . The method of  claim 20 , wherein if a greater amount of oxidized forms of thiol and disulfide containing molecules as compared to a control is determined in the sample, the subject is administered an antioxidant treatment. 
     
     
         34 . The method of  claim 33 , wherein the antioxidant treatment is the administration of N-acetyl cysteine. 
     
     
         35 . The method of  claim 34 , the administration of N-acetyl cysteine is for 1, 2, 3, or more hours at 75 mg/kg, 150 mg/kg or 300 mg/kg by i.v. infusion. 
     
     
         36 . The method of  claim 34 , the administration of N-acetyl cysteine is by oral administration. 
     
     
         37 . A kit for performing the method of  claims 20 - 36 . 
     
     
         38 . A sample collection container comprising a dried reagent to prevent free thiol oxidation. 
     
     
         39 . The sample collection container of  claim 38 , wherein the container is a vacutainer tube. 
     
     
         40 . The sample collection container of  claim 38 , wherein the reagent to prevent free thiol oxidation is NEM. 
     
     
         41 . The sample collection container of  claim 40 , wherein the NEM is an evenly coated amount of 2-4 mg on the walls of a 1 ml tube.