Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving hydrolase
Patent
1984-09-11
1987-07-21
Nucker, Christine M.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving hydrolase
435196, 435800, 435814, 435815, 436827, C12Q 142, C12N 916
Patent
active
046818421
DESCRIPTION:
BRIEF SUMMARY
Alkaline phosphatase (orthophosphoric monoester phosphohydrolase) catalyses the hydrolysis of phosphoric acid monoesters at alkaline pH values. This enzyme occurs in many tissues. There exist tissue-specific forms (isoenzymes) for example in the liver, in bones, in the small intestine, in the kidneys and in the placenta. From these organs, the isoenzymes are transmitted to the blood plasma. The characteristic properties of the particular isoenzymes are retained in the case of transfer from the producing organ into the blood plasma.
The blood plasma of healthy humans mainly contains the liver and bone isoenzymes. In the case of about one guarter of all persons, alkaline phosphatase is also found in the plasma which originates in the small intestine. In these cases, the proportion of this mall intestine isoenzyme lies at an average value of 10% of the total alkaline phosphatase content in the plasma. During pregnancy, especially during the last 3 months, alkaline phosphatase originating in the placenta is also transmitted to the blood plasma. The total alkaline phosphatase activity of the plasma is given by the sum of the activities of all tissue-specific individual components.
The determination in the plasma of the total activity of alkaline phosphatase, as well as especially of the activity of the liver and bone isoenzymes, is of especial diagnostic importance in the case of the investigation of diseases of the liver and of the bone system. The diseases bring about an increase of the alkaline phosphatase activity in the plasma since, under these conditions, alkaline phosphatase is transmitted from the liver or the bones to an increased extent. Consequently, the increase of the alkaline phosphatase activity of the liver or of the bone isoenzyme in the plasma can serve for the recognition of a disease of these tissues. In the case of a disease of the liver, in addition there arises a further enzyme variant of the alkaline phosphatase which results by complex formation of the liver isoenzyme with lipids or proteins and which is believed to be formed in the biliary tract. This so-called bile isoenzyme, which is also called high molecular, rapid or .alpha..sub.1 -alkaline phosphatase, can, in this case, appear in the plasma as an alkaline phosphatase component in a smaller proportion.
The differentiation of the alkaline phosphatase isoenzymes from small intestine, placenta, kidneys and bile from one another or also from the liver and the bone isoenzymes is readily possible since these tissue-specific forms clearly differ in their chemical, physical and immunological properties. Processes for the separation and determination of these isoenzymes are known and depend, for example, on the differing behaviour of the isoenzymes with regard to inhibitors, on differences in the electric charge, etc. Thus, for example, the differences in the electrophoretic mobility in alkaline buffer systems can be used for the separation of these isoenzymes.
However, only small differences are to be observed between the liver and the bone enzyme. In particular, the liver and the bone isoenzyme of the alkaline phosphatase differ only slightly with regard to the electric charge, the heat stability and in the response to inhibitors (for example with regard to urea). These differences do not suffice in order to achieve a usable differentiation between these two enzyme forms and a sufficient quantification of each form in a mixture of both forms.
It was the object of the invention to make available a new process with the help of which a differentiation and differentiated determination of the liver and bone isoenzme of alkaline phosphatase is made possible. This object is solved by the process according to the invention in which the sample, for example blood plasma or serum, is mixed with a lectin which is able to bind N-acetylglucosamine residues and incubated. Thereafter, there is carried out a separation of the lectin-bound from the free isoenzyme portions and the alkaline phosphatase activity is determined in one or in both separated me
REFERENCES:
Biochimica et Biophysica Acta, 616 (1980) 41-59 Lehmann, F. G.
Boehringer Mannheim GmbH
Krawczewicz Louanne C.
Nucker Christine M.
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