Polarographic analysis of cholesterol and other macromolecular substances
First Claim
1. A method for quantitative polarographic determination of a substance which is convertible by at least one enzyme to produce ultimately hydrogen peroxide comprising the steps ofproviding a polarographic cell including at least one electrode sensitive to hydrogen peroxide positioned behind a membrane permeable to hydrogen peroxide and impermeable to said substance being measured, said cell containing an electrolyte,at least one enzyme contained in said cell on the side of the membrane opposite said electrode for enzymatic reaction with said substance to produce ultimately hydrogen peroxide,establishing a potential across said cell such that current is produced which is proportional to the amount of hydrogen peroxide produced,adding a quantity of material containing said substance for said enzymatic reaction on the side of the membrane opposite said electrode and to effect diffusion of at least a portion of said hydrogen peroxide into said membrane and into contact with said electrode, anddetermining the current flowing across said cell as a function of the amount of hydrogen peroxide formed and as an indication of the amount of said substance in said material.
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Abstract
A micro-method for measurement of sterols such as cholesterol, and other macromolecular substances, is disclosed utilizing enzymes for the conversion of such substances to produce ultimately hydrogen peroxide and measurement of the generated hydrogen peroxide with a membrane covered polarographic anode. The polarographic anode is set at a voltage so as to produce current proportional to hydrogen peroxide concentration. According to the method of this invention, the macromolecular substance under analysis enters into an enzymatic reaction in a sample chamber on the side of the membrane opposite the anode and the membrane is impermeable to such substances, but senses hydrogen peroxide. The method is adapted to measure free and total blood cholesterol in a precise, rapid, sensitive and specific manner. Other substances of a macromolecular or conventional membrane impermeable nature such as high molecular weight starches or proteins, which undergo enzymatic reaction to produce ultimately hydrogen peroxide, may be analyzed by employing this polarographic technique.
387 Citations
23 Claims
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1. A method for quantitative polarographic determination of a substance which is convertible by at least one enzyme to produce ultimately hydrogen peroxide comprising the steps of
providing a polarographic cell including at least one electrode sensitive to hydrogen peroxide positioned behind a membrane permeable to hydrogen peroxide and impermeable to said substance being measured, said cell containing an electrolyte, at least one enzyme contained in said cell on the side of the membrane opposite said electrode for enzymatic reaction with said substance to produce ultimately hydrogen peroxide, establishing a potential across said cell such that current is produced which is proportional to the amount of hydrogen peroxide produced, adding a quantity of material containing said substance for said enzymatic reaction on the side of the membrane opposite said electrode and to effect diffusion of at least a portion of said hydrogen peroxide into said membrane and into contact with said electrode, and determining the current flowing across said cell as a function of the amount of hydrogen peroxide formed and as an indication of the amount of said substance in said material.
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15. A method for the quantitative determination of cholesterol in blood which comprises the steps of
providing a polarographic cell including at least one electrode sensitive to hydrogen peroxide positioned behind a membrane permeable to hydrogen peroxide and impermeable to cholesterol, separating from said blood the component selected from the group consisting of blood plasma and blood serum, introducing cholesterol oxidase into said cell on the side of the membrane opposite said electrode for enzymatic reaction with cholesterol to produce hydrogen peroxide, providing oxygen in said cell for said enzymatic reaction, establishing a potential across said cell such that current is produced which is proportional to the amount of hydrogen peroxide produced, adding a quantity of said component for reaction with said enzyme and to effect diffusion of at least a portion of said hydrogen peroxide into said membrane and into contact with said electrode, and determining the current flowing across said cell as a function of the amount of hydrogen peroxide formed and as an indication of the amount of cholesterol in said blood.
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22. A method for the quantitative determination of cholesterol in whole blood which comprises the steps of
providing a polarographic cell including at least one electrode sensitive to hydrogen peroxide positioned behind a membrane permeable to hydrogen peroxide and impermeable to cholesterol, immobilizing cholesterol oxidase on the membrane on the side opposite said electrode for enzymatic reaction with cholesterol to produce hydrogen peroxide, providing oxygen in said cell for said enzymatic reaction, establishing a potential across said cell such that current is produced which is proportional to the amount of hydrogen peroxide produced, adding a quantity of said blood for reaction with said enzyme and to effect diffusion of at least a portion of said hydrogen peroxide into said membrane and into contact with said electrode, and determining the current flowing across said cell as a function of the amount of hydrogen peroxide formed and as an indication of the amount of cholesterol in said blood.
Specification