Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving hydrolase
Reexamination Certificate
2000-02-01
2002-07-30
Leary, Louise N. (Department: 1623)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving hydrolase
C435S004000, C435S968000, C435S015000, C435S016000, C435S975000
Reexamination Certificate
active
06426194
ABSTRACT:
TECHNICAL FIELD
The invention concerns an assay for Vitamin B
6
, the active form of which is pyridoxal 5′-phosphate and to improvements in detection of H
2
S by using fluorescence. More specifically, the invention concerns kits and methods for determining pyridoxal phosphate concentrations in biological fluids using the apoenzyme of a homocysteinase. It also concerns improving the sensitivity of such assays and assays for homocysteine as well by measuring the fluorescence of a complex generated by the reaction of H
2
S with a dialkyl phenylene diamine and an oxidizing agent.
BACKGROUND ART
PCT publication WO 99/05311 describes high specificity homocysteine assays in biological samples using a homocysteinase enzyme with high specificity for homocysteine in comparison to cysteine sufficient for biological fluid levels. This permits the measurement of the common product, H
2
S, generated by these enzymes both from cysteine and homocysteine as a valid measure of homocysteine per se in physiological fluids. The H
2
S generated can be measured in a variety of ways as described in that application. It has now been found that the sensitivity of this assay can be improved by measuring the fluorescence of the complex formed by hydrogen sulfide with the chromogenic reagents and an N,N-dialkyl p-phenylene diamine and an oxidizing agent such as potassium ferricyanide. The resultant, 3,7-(bis dialkylamino)phenothiazine-5 chloride can be measured by absorbance or by excitation and measurement of fluorescence.
This measurement of fluorescence is also useful in the assay herein described for pyridoxal 5′-phosphate.
Pyridoxal 5′-phosphate (PLP) is the biologically active form of Vitamin B
6
. PLP is a cofactor for many essential enzymes involved in amino acid metabolism and fatty acid metabolism, including methioninase and homocysteinase. Additional enzymes for which PLP is the prosthetic group include glycogen phosphorylase as well as all aminotransferases. PLP is also involved in decarboxylations, deaminations, racemizations, transaminations and aldol cleavages at the &agr;-carbon atom of amino acids. These enzymes depend on PLP in order to be active, so PLP is an important metabolic factor. PLP is derived from Vitamin B
6
; Vitamin B6 is not synthesized by most mammals, including humans. Therefore, this vitamin is most commonly supplied in the diet. Epidemiological studies have shown that PLP deficiency is the strongest nutritional correlate to mortality from cardiovascular diseases. Deficiency of Vitamin B
6
results in symptoms such as dermatitis and nervous disorders.
Given the involvement of PLP in metabolism and its deficiency in various disease states, multiple methods for the determination of Vitamin B
6
levels and B
6
status are known in the art.
Microbiological assays using
Saccharomyces carlsbergensis
(
S. uvarum
),
Streptococcus faecium,
and
Lactobacillus casei
have been used to measure the various forms of B
6
in blood and urine. Fluorometric assays of urinary 4′-pyridoxic acid and blood PLP after conversion to a cyanide complex or condensation with a fluorophore, such as methyl anthranilate followed by reduction are also used. 4′-Pyridoxic acid can also be determined by HPLC. The PLP concentration in plasma is also measured by determining the formation of radioactively labeled tyramine from labeled tyrosine using the apoenzyme form of tyrosine decarboxylase. The reference interval is 5 to 30 ng/ml of plasma (Reynolds, R. D.,
Fed. Proc.
(Abst. No. 2185) (1983) 42:665). This form of assay is also available commercially as an “ALPCO” kit from American Laboratory Products Company, Ltd. (Windham, N.H. 03087).
Blood transaminases have been used as an indicator of Vitamin B
6
status. The enzyme activity in serum is depressed in B
6
deficiency. However, release of these enzymes reflects cell death, and breakdown in various tissues causes variability. Erythrocyte levels of aspartate and alanine aminotransferases provide a better indication of Vitamin B
6
status (Briggs, M., Ed.
Vitamins in Human Biology and Medicine,
Boca Raton, Fla., CRC Press, Inc., 1981).
Measurement of urinary tryptophan metabolites, such as xanthurenic acid, following an oral loading (2-5 g) of L-tryptophan have also been used to indicate B
6
status. Amounts of xanthurenate above the normal (25 mg/d) level indicate Vitamin B
6
deficiency. A methionine loading test has also been utilized (Briggs, M., op. cit., Brown, M. L., Ed.
Present Knowledge in Nutrition.
6
th
ed. Washington, D.C., International Life Sciences Institute-Nutrition Foundation, 1990). The ratio of cystathionine to cysteine sulfinic acid measured by amino acid analysis is elevated in a 24-h urine of B
6
-deficient patients after a 3-g methionine load.
Given the increasing awareness of the role of Vitamin B
6
in cardiovascular disease, and the public health goal to screen patients for risk from cardiovascular disease, there is a critical need for more convenient and reliable analytical procedures that can be used to determine PLP/Vitamin B
6
levels in patients. Such procedures are expected to provide considerable medical benefit both in those cases where decreased PLP/Vitamin B
6
concentrations place an individual at risk for a particular disease state, and in cases where decreased PLP/vitamin B
6
concentrations are a detectable byproduct of an existing disease state.
Such analytical procedures would provide great benefit by predicting a patient's susceptibility to cardiovascular disease before onset can be detected by other procedures. In this regard, great benefit would be achieved by adapting such procedures to the widespread screening of the general population, and in particular, to patients otherwise suspected of being at risk for cardiovascular disease. It is therefore critically important that the assay be convenient to use, simple, and inexpensive. The present invention provides such methods, including diagnostic kits for use in the clinical setting.
DISCLOSURE OF THE INVENTION
The invention provides assays capable of detecting PLP levels in a biological sample fluids such as urine, tissue fluid, blood, whole blood, blood serum or blood plasma from a subject. The methods of the invention are thus useful to assess risk for cardiovascular disease. The invention methods also include methods of determining results which are useful not only in methods to determine PLP/B
6
, but also in assay methods which assess the production of H
2
S as a product and measure of the desired analyte using chromogenic reagents.
In one aspect, the invention is directed to a method for determining the amount of PLP in a biological sample which method comprises contacting said sample with the apoenzyme form of a PLP-requiring enzyme which can generate a product when PLP is present preferably one that is determinable by color or fluorescence. Thus, preferred apoenzymes of the invention are homocysteine/methionine alpha-gamma lyases which have been depleted of the normally associated PLP. Also preferred are such enzymes which generate hydrogen sulfide from cysteine. These enzymes generate products which are readily detectable by colorometric or fluorometric means when the enzymes are restored/activated to the holoenzyme form. The sensitivity of the assay is greatly multiplied by the function of the PLP as a co-factor; high levels of substrate can therefore be used and high levels of product generated.
In another aspect, the invention is directed to a method to improve the sensitivity of detection of H
2
S generated in assays, such as assays for homocysteine, which generate hydrogen sulfide, which method comprises measuring the fluorescence of the product of hydrogen sulfide with a dialkyl p-phenylene diamine and an oxidizing agent such as a ferricyanide. This improvement is especially important in assays for homocysteine or cysteine per se as the sensitivity of the assay is enhanced over one hundred fold. However, this method may also be used in assays for PLP where, because of the multiplying effect of the co-
Han Qinghong
Tan Yuying
Xu Mingxu
Anti-Cancer, Inc.
Leary Louise N.
Morrison & Foerster / LLP
LandOfFree
Homogeneous enzymatic assay for vitamin B6 and improvements... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Homogeneous enzymatic assay for vitamin B6 and improvements..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Homogeneous enzymatic assay for vitamin B6 and improvements... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2894010