# US 5,346,599 A · Capillary zone electrophoretic detection of biological thiols and their S-nitrosated and oxidized derivatives

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- Number: US 5,346,599 A
- Document ID: US5346599A
- Status: Granted
- Kind: A
- Application number: 08/009,469
- Filed: 1993-01-26
- Granted: 1994-09-13
- Earliest claimed: 1993-01-26
- Inventors: Jonathan Stamler; Joseph Loscalzo
- Web page: https://patentgrep.com/patent/US5346599A

## CPC

- G01N 27/447: using electrophoresis
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

Individual thiols present in a biological sample are separated and identified using capillary electrophoresis. S-nitrosated and oxidized derivatives of thiols are also detected using capillary electrophoresis. In addition, capillary electrophoresis may be performed on samples which have been treated with an acid, so as to achieve a pH between 2.0 and 6.8, in order to further enhance detection. Separation and detection of S-nitrosated derivatives of thiols provides a means for monitoring the extent of a disease state associated with abnormal levels of nitric oxide. Such disease states include septic shock, cardiogenic shock, hypovolemic shock, atherosclerosis, hyperhomocysteinemia, venous thrombosis, arterial thrombosis, coronary occlusion, pulmonary embolism, cerebrovascular accidents, vascular fibrosis, ectopia lentis, osteoporosis, mental retardation, skeletal deformities, pulmonary hypertension, malignancy, infections and central nervous system disorders.

## Claims

### Claim 1

A method for separating and detecting individual thiol compounds in a biological sample comprising subjecting said sample to capillary electrophoresis.

### Claim 2

A method for separating and detecting individual thiol compounds in a biological sample comprising the steps:
(a) determining the elution time of known individual thiols to establish thiol standards:
(b) subjecting said biological sample to capillary electrophoresis; and
(c) comparing the elution times obtained for each component present in the sample, to the known elution times of the thiol standards prepared in step (a), to determine the identity of each individual thiol compound present in the sample.

### Claim 3

A method tbr separating and detecting individual S-nitrosothiol compounds in a biological sample comprising subjecting said sample to capillary electrophoresis.

### Claim 4

A method for separating and detecting individual S-nitrosothiol compounds in a biological sample comprising the steps:
(a) determining the elution time of known individual S-nitrosothiols to establish S-nitrosothiol standards;
(b) subjecting said biological sample to capillary electrophoresis; and
(c) comparing the elution times obtained for each component present in the sample to the known elution times of the S-nitrosothiol standards prepared in step (a), to determine the identity of each individual thiol compound present in the sample.

### Claim 5

A method for separating and detecting oxidized derivatives of individual S-nitrosothiol compounds in a biological ample comprising subjecting said sample to capillary electrophoresis.

### Claim 6

A method for separating and detecting oxidized derivatives of individual S-nitrosothiol compounds in a biological sample comprising the steps:
(a) determining the elution time of known oxidized derivatives of individual thiol compounds to establish oxidized derivative standards;
(b) subjecting said biological sample to capillary electrophoresis; and
(c) comparing the elution times obtained for each compound present in the sample to the known elution times of the oxidized derivative standards prepared in step (a), to determine the identity of each individual oxidized thiol derivative present in the sample.

### Claim 7 (depends on 1, 2, 3, 4, 5, 6)

The method of any one of claims 1-6 wherein the sample is treated with an acid just prior to subjecting it to capillary electrophoresis.

### Claim 8 (depends on 7)

The method of claim 7 wherein treatment of said sample with acid results in a sample pH between 2.0 and 6.8.

### Claim 9 (depends on 1, 2, 3, 4, 5, 6)

The method of any one of claims 1-6 wherein said biological sample is selected from the group consisting of blood, serum, urine, cerebrospinal fluid, semen, synovial fluid, peritoneal fluid, sputum, intestinal secretions, corneal fluid, amniotic fluid, stool, saliva and sweat.

### Claim 10 (depends on 1, 2, 3, 4, 5, 6)

The method of any one of claims 1-6 wherein said capillary electrophoresis is conducted at a temperature of 4.degree. C.-37.degree. C.

### Claim 11 (depends on 3)

A method for monitoring the extent of a disease state associated with abnormal levels of nitric oxide, nitrosonium or nitroxyl, comprising the method of claim 3 to monitor S-nitrosothiol levels in the biological fluids of a patient.

### Claim 12 (depends on 11)

The method of claim 11 wherein said disease state is selected from the group comprised of septic shock, cardiogenic shock, hypovolemic shock, atherosclerosis, hyperhomocysteinemia, venous thrombosis, arterial thrombosis, coronary occlusion, pulmonary embolism, cerebrovascular accidents, vascular fibrosis, ectopia lentis, osteoporosis, mental retardation, skeletal deformities, pulmonary hypertension, malignancy, infections and central nervous system disorders, renal disorders, respiratory disorders and gastrointestinal diseases.