Chemistry: analytical and immunological testing – Including sample preparation
Patent
1996-09-19
1998-06-30
Chin, Christopher L.
Chemistry: analytical and immunological testing
Including sample preparation
435 71, 435 78, 435 791, 435 11, 435 19, 435962, 436174, 436539, 436 13, 436 17, 436 63, 436 71, 436815, 436824, 436825, 436826, G01N 33533
Patent
active
057733040
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL FIELD
The present invention relates to a method for measuring cholesterol in high-density lipoproteins, eliminating the necessity of pretreatments such as centrifugal separation and effectively providing simple analytical method while requiring only small amounts of a sample.
BACKGROUND ART
Serum lipids such as cholesterols are bound to apoproteins and form lipoproteins. Lipoproteins are categorized into chylomicrons, very low-density lipoproteins, low-density lipoproteins (LDLs), and high-density lipoproteins (HDLs) based on differences in physical properties. Among these lipoproteins, LDLs are known to be atherogenic, and HDLs are known to have anti-atherogenic action.
As it has been proven epidemiologically that the cholesterol level in HDLs inversely correlates to the frequency of onset of arteriosclerotic diseases, measurement of cholesterol in HDLs is quite common today for the purposes of predicting and diagnosing ischemic diseases.
Several methods are known to measure HDL cholesterol. In one method, for example, cholesterol is measured after separating HDLs from other lipoproteins using an ultracentrifugation. In another method, lipids are stained after they are isolated by electrophoresis, and the strength of color development is measured. However, these methods all involve problems such as intricate procedures, inability to handle many samples, etc., and therefore, they have not been used routinely in clinical laboratories.
The most widely used method for measuring cholesterol in HDLs in clinical laboratories is a precipitation method. In this method, a precipitant is added to a sample to aggregate lipoproteins other than HDLs, the aggregated lipoproteins are precipitated by centrifugation, and cholesterol present in a supernatant containing only HDLs is measured.
Although this method is simpler than ultracentrifugation or electrophoresis, not all the analyzing steps can be completely automated. Since it involves a separation operation using a centrifugation. Manual processes for obtaining a supernatant containing only HDL in this method increases the risk of causing analytical error and demand a relatively great amount of sample when compared to other automated clinical tests.
On the other hand, methods using enzymes to fractionally determine cholesterol in HDLs are studied. For example, a method in which enzymatic reaction is carried out in the presence of a bile acid salt or a nonionic surfactant is known (Japanese Patent Application Laid-open (kokai) No. 63-126,498). This method is based on the finding that the enzymatic reaction rate in the initial stage of reaction is in proportion to LDL concentration, and thereafter, to the concentration of cholesterol in HDLs. This method cannot be said to be precise, since enzymatic reaction with cholesterol in HDLs cannot be completely separated from that with other lipoproteins. In other words, cholesterol subject to the enzymatic reaction shifts from cholesterol in LDLs to that in HDLs, not in steps manner but in a gradual manner involving partial overlap.
According to another known method, lipoproteins other than HDLs are aggregated in advance, and only the cholesterol in HDLs is enzymatically reacted. Thereafter, enzymes are inactivated, and the aggregated lipoprotein is simultaneously re-dissolved to measure absorption (Japanese Patent Application Laid-open (kokai) No. 6-242,110). This method requires addition of reagent at least three times. Therefore, it can be applied to limited autoanalyzers only, raising a drawback regarding range of use. In addition, in re-dissolving the precipitation, use of a high concentration salt is needed. Therefore, this method is not satisfactory from not only the view point of damage to the analytical apparatus, but also of difficulty in disposing the waste reagent.
Accordingly, an object of the present invention is to provide a method for quantitatively determining cholesterol in high-density lipoproteins which eliminates the necessity of pretreatments such as centrifugal separation, which effectively
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Hino Koichi
Manabe Mitsuhisa
Nakamura Mitsuhiro
Chin Christopher L.
Daiichi Pure Chemicals Co. Ltd.
Nguyen Bao-Thuy L.
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