# US 2014/0193801 A1 · Schiff-base conjugate of n, n-dibutyl-p-phenylenediamine with pyridoxal 5'-phosphate for improved homocysteine assays using pyridoxal 5'-phosphate-dependent enzymes

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 2014/0193801 A1
- Document ID: US20140193801A1
- Status: Published application
- Kind: A1
- Application number: 14/152,784
- Filed: 2014-01-10
- Published: 2014-07-10
- Earliest claimed: 2013-01-10
- Assignees: AntiCancer, Inc.
- Applicants: Anticancer, Inc.
- Inventors: Yuying Tan; Shukuan Li
- Web page: https://patentgrep.com/patent/US20140193801A1

## CPC

- C12Q 1/527: involving lyase
- C07F 9/58: Pyridine rings
- C12Y 404/01001: Cystathionine gamma-lyase (4.4.1.1)
- G01N 2333/988: Lyases (4.), e.g. aldolases, heparinase, enolases, fumarase
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C07F: ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM (metal-containing porphyrins C07D487/22)
- C12Y: ENZYMES
- 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

A composition, method and kit for performing a two-reagent enzymatic homocysteine assay, wherein a single homocysteinase enzyme and a Schiff-based conjugate of N,N-dibutyl-p-phenyldiamine (DBPDA) with pyridoxal 5′-phosphate (PLP) are used to measure total homocysteine in plasma or serum. The assay measures a chromophore reaction product of H2S and the DBPDA released from the Schiff-base conjugate in the presence of a Fe+3 containing compound. The resulting chromophore may be measured absorbance or fluorescence spectrophotometry.

## Claims

### Claim 1

A method of measuring total homocysteine comprising the steps of:
providing homocysteine;
reducing the homocysteine with a reducing agent;
enzymatically reacting the homocysteine with homocysteinase (HCYase), producing hydrogen sulfide (H2S);
providing N,N-dibutyl-p-phenylenediamine-pyridoxal 5′-phosphate (DBPDA-PLP Schiff-base);
combining the DBPDA released from the DBPDA-PLP with the H2S in the presence of an Fe(III) containing compound producing at least one chromophore; and
measuring the at least one chromophore.

### Claim 2 (depends on 1)

The method of claim 1, wherein the homocysteinase is L-homocysteine α,γ-lyase (rHCYase).

### Claim 3 (depends on 1)

The method of claim 1, wherein the step of enzymatically reacting the homocysteine with HCYase, producing the H2S, occurs in approximately five minutes.

### Claim 4 (depends on 1)

The method of claim 1, wherein the step of combining the DBPDA released from the DBPDA-PLP with the H2S in the presence of the Fe(III) containing compound produces the at least one chromophore in approximately five minutes.

### Claim 5 (depends on 1)

The method of claim 1, wherein the Fe(III) containing compound is FeCl3.

### Claim 6 (depends on 1)

The method of claim 1, wherein the reducing agent is diothiothreitol (DTT).

### Claim 7 (depends on 1)

The method of claim 1, wherein the step of measuring the at least one chromophore is performed by measuring the chromophore's absorbance at approximately 660nm.

### Claim 8 (depends on 1)

The method of claim 1, wherein the step of measuring the at least one chromophore is performed by measuring an excitation spectrum of the chromophore's fluorescence at approximately 660 nm.

### Claim 9 (depends on 1)

The method of claim 1, wherein the step of measuring the at least one chromophore is performed by measuring an emission spectrum of the chromophore's fluorescence at approximately 700 nm.

### Claim 10

A method of producing N,N-dibutyl-p-phenyldiamine-pyridoxal 5′-phosphate comprising the steps of:
providing a solution of N,N-dibutyl-p-phenyldiamine (DBPDA) in an anhydrous alcohol;
mixing the solution with a pyridoxal 5′-phosphate (PLP) solution, producing a mixture;
forming a precipitate by adjusting a pH of the mixture to approximately 4.0; and
centrifuging the precipitate.

### Claim 11 (depends on 10)

The method of claim 10, comprising the additional steps of:
suspending the precipitate in acidic water;
centrifuging the precipitate a second time; and
drying the precipitate over P2O5, in a vacuum.

### Claim 12 (depends on 10)

The method of claim 10, wherein the anhydrous alcohol is an anhydrous ethyl alcohol.

### Claim 13 (depends on 11)

The method of claim 11, wherein the acidic water has a pH of approximately 4.5.

### Claim 14

A method of producing N,N-dibutyl-p-phenyldiamine-pyridoxal 5′-phosphate comprising the steps of:
providing a solution of N,N-dibutyl-p-phenyldiamine (DBPDA) in an anhydrous alcohol;
adding the solution to a pyridoxal 5′-phosphate (PLP) solution, producing a mixture; and
crystallizing the mixture.

### Claim 15 (depends on 14)

The method of claim 14, further comprising the steps of:
washing the crystallized mixture with an alcohol; and
lyophilizing the crystallized mixture.

### Claim 16 (depends on 14)

The method of claim 14, wherein the anhydrous alcohol is an anhydrous ethyl alcohol.

### Claim 17 (depends on 15)

The method of claim 15, wherein the alcohol is 75% ethyl alcohol.

### Claim 18

A kit comprising:
a first reagent, the first reagent including a mixture of a reducing agent, homocysteinase (HCYase) and N,N-dibutyl-p-phenyldiamine-pyridoxal 5′-phosphate;
a second reagent, the second reagent including an Fe(III) containing compound;
a first buffer solution; and
a second buffer solution.

### Claim 19 (depends on 18)

The kit of claim 18, wherein the HCYase is L-homocysteine α,γ-lyase.

### Claim 20 (depends on 18)

The kit of claim 18, wherein the Fe(III) containing compound is FeCl3.

### Claim 21 (depends on 18)

The kit of claim 18, wherein the first buffer solution is a sodium phosphate buffer.

### Claim 22 (depends on 21)

The kit of claim 21, wherein the first buffer solution further includes a non-ionic surfactant.

### Claim 23 (depends on 18)

The kit of claim 18, wherein the second buffer solution is a strong acid.

### Claim 24 (depends on 22)

The kit of claim 22 wherein the non-ionic surfactant is triton X-100.

### Claim 25 (depends on 18)

The kit of claim 18, further comprising at least one calibrator and at least one control.

### Claim 26 (depends on 25)

The kit of claim 25, wherein the at least one calibrator and the at least one control are each a matrix plasma containing total homocysteine of a known concentration.

### Claim 27

A method for detecting the risk of a total homocysteine (tHCY) dependent adverse condition comprising the steps of:
extracting plasma or serum containing homocysteine from a patient;
reducing the homocysteine with a reducing agent;
enzymatically reacting the homocysteine with homocysteinase (HCYase), producing hydrogen sulfide (H2S);
providing N,N-dibutyl-p-phenylenediamine-pyridoxal 5′-phosphate (DBPDA-PLP Schiff-base)
combining DBPDA released from the DBPDA-PLP with the H2S in the presence of an Fe(III) containing compound producing at least one chromophore; and
measuring the at least one chromophore wherein the step of measuring the at least one chromophore determines a tHCY concentration between 5-15 μmol/L or greater than 15 μmol/L.

### Claim 28 (depends on 27)

The method of claim 27, wherein the tHCY dependent adverse condition is cardiovascular disease, stroke, Alzheimer's disease, Parkinson's disease, diabetes, cancer, kidney failure, or other diseases.

### Claim 29

A composition comprising:
N,N-dibutyl-p-phenyldiamine conjugated to pyridoxal 5′-phosphate.