Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving transferase
Reexamination Certificate
2000-04-17
2001-12-11
Witz, Jean C. (Department: 1651)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving transferase
C435S004000
Reexamination Certificate
active
06329162
ABSTRACT:
TECHNICAL FIELD
The invention relates to diagnostic methods for conditions which are characterized by abnormal levels of certain metabolites, in particular, folates, and to simplified methods of detection. The improved methods of the invention provide rapid and accurate assessment of the concentrations of folate and other analytes.
1. Background Art
Folates are critical co-factors in methyl transfer reactions. Abnormalities of folate levels in biological fluids such as blood and plasma are indicative of conditions that are characterized by inappropriate or inadequate transfer of methyl groups. Thus, it is important to be able to provide an accurate measure of folate levels in such fluids and to compare them to those expected in normal individuals. Low folate concentrations are associated with megaloblastic anemia and reduced DNA synthesis, as well as cardiovascular disease. Birth defects may also result.
Folate can be measured efficiently using the methods of the invention. The invention also provides an improved method to detect H
2
O
2
in the presence of peroxidase
2. Disclosure of the Invention
The invention method for determining levels of folate in biological fluids takes advantage of the ability of folate to inhibit the methyl transfer reaction whereby glycine is converted to sarcosine. The enzyme which carries out this conversion, glycine N-methyltransferase (GMT) can be isolated from liver or pancreas. In the assay systems of the invention, glycine and S-adenosyl methionine (SAM) are the reactants and sarcosine and S-adenosyl homocysteine (SAH) are the products. The effectiveness of GMT in converting these reactants is dependent on folate concentration in vivo.
The folate in the sample can be converted to a form (to the extent it does already exist in said form) whereby it inhibits the ability of GMT to form the foregoing products. The folate in the sample is first converted to folypoly glutamate (FPG) which is an inhibitor of the GMT. Yeo, E-J, et al.,
J. Biol. Chem.
(1999) 274:37556-37564. Under these conditions, the greater the amount of folate in the sample, the less products formed.
In the invention method, the levels of product are typically measured. As the levels of reactants are provided in excess, although it is theoretically possible to measure the diminution in reactants, this is less desirable as it is, of course, more difficult to detect small differences in large concentrations than to measure a difference from zero. However, it is not impossible, and such measurements are within the scope of the invention as well.
Either the levels of SAH or of sarcosine can be measured or both. A variety of methods is available in each case. Preferred embodiments for the measurement of SAH, however, include its conversion to homocysteine and subsequent lysis of homocysteine to obtain readily detectable products as further described below. In the case of sarcosine, a convenient method is the use of a specific oxidase which, in the presence of this amino acid, generates hydrogen peroxide which can also be readily detected by a variety of methods known in the art, preferred embodiments of which are illustrated below. Thus, in one aspect, the invention is directed to:
A method to assess the level of folate in a biological fluid sample which method comprises
providing said sample with glycine N-methyltransferase (GMT) and with an excess of S-adenosyl methionine (SAM) and of glycine;
providing a control which contains no folate with said GMT and excess SAM and glycine in comparable amounts to those provided to the sample; and
comparing the concentration of at least one product formed in the sample with the concentrations of said product formed in the control,
whereby the difference in levels of said product in the sample as compared to the control is directly proportional to the level of folate in the sample.
In the foregoing method, either sarcosine or SAH levels may be measured. The concentrations of products will be diminished in the sample as compared to the control. Pretreatment of the sample with an enzyme which converts folate to an inhibitor of GMT, FPG, set forth above, is desirable. This enzyme, folypoly glutamate synthetase (FPGS) is readily available in the art.
In another aspect, the invention is directed to a particularly convenient method for the endpoint of sarcosine measurement. It has been found that a particularly convenient method to measure hydrogen peroxide in analytical samples, including biological fluids, is to treat the sample containing the peroxide with both peroxidase and a dialkyl phenylene diamine. The resultant is a colored compound which can be measured spectrophotomctrically. As will be evident, since a number of protocols are available which result in the generation of hydrogen peroxide, for example the treatment of substrates with their pertinent corresponding and specific oxidases, the combination of peroxidase and dialkyl phenylene diamines has a broad range of utility.
MODES OF CARRYING OUT THE INVENTION
The invention is directed, in one embodiment, to the measurement of folates in biological fluids. Suitable biological fluids are most commonly blood, serum and plasma, although other fluids may be of interest as well, such as cerebral spinal fluid, urine, and other blood fractions. Measurement in plasma or serum is preferred.
By “folates” is meant folic acid and its salts as well as the dihydro and teterahydro form. In order to ensure uniformity of results, it may be desirable to add a reducing agent to the reaction mixtures in order to ensure that all of the folates are in the same oxidation state—i.e., the teterahydro folate (THF) form. However, it is believed that the predominant form, almost to the exclusion of the others, is biological systems is tetrahydrofolate, and in particular, 5-methyltetrahydrofolate. It is this form, specifically, in combination with glutamate which acts as an inhibitor of GMT endogenously.
By way of background, it is believed that the function of GMT in the liver and pancreas is to diminish undesirably high concentrations of SAM by creating a “sink” in the form of sarcosine, which has no known function. However, this enzyme is regulated by folate; when high levels of methylated THF are present, combination with glutamate occurs and the resulting compound is a powerful GMT inhibitor. The present invention takes advantage of the ability of folates to inhibit GMT using this mechanism.
For use in the present invention, GMT can be isolated from pancreas or liver or theoretically could be produced recombinantly. If the enzyme is isolated from native sources, it should first be treated to disassociate it from any endogenous folate. This can be effected by treating the enzyme with an anion exchange column and effecting a separation using, for example, a gel exclusion method.
In the methods of the invention, glycine N-methyltransferase (GMT) catalyzes the reaction:
glycine+SAM→sarcosine+SAH.
The GMT is purified from any convenient source. A method for purification of to the enzyme from rat liver is described in the examples below; also described is a method to obtain the enzyme free of inhibitor by treating the isolated GMT on an anion exchange column and separating the dissociated folate by gel filtration.
To carry out the method, both control and sample are incubated with folate-free GMT after treatment with glutamic acid in the presence of the enzyme FPGS which converts any folate into an inhibitor of GMT, FPG. Either sarcosine or SAH can be measured, the amounts or concentrations of product present in the sample will be less than those of the control in a degree proportional to the concentration of folate in the sample.
Thus, the sample and the control are treated with glutamine and the synthetase FPGS to convert the folate to an inhibitor (FPG) of the GMT. In this embodiment, the sample is incubated with the glutamine and FPGS in suitable amounts prior to or concomitant with, treatment with GMT. The remainder of the assay is conducted according to the alternatives outlined below. Measurement
Han Qinghong
Tan Yuying
Tang Li
Xu Mingxu
Yagi Shigeo
Anticancer Inc.
Morrison & Foerster / LLP
Ozga Brett
Witz Jean C.
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