Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving oxidoreductase
Reexamination Certificate
2001-05-31
2002-08-06
Gitomer, Ralph (Department: 1623)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving oxidoreductase
C435S188000
Reexamination Certificate
active
06428972
ABSTRACT:
TECHNICAL FIELD
The present invention relates to a biochemical methods, and kits to measure the niacin status of an individual, particularly to a colorimetric method for the detection of intracellular pyridine nucleotide content or concentration in a biological sample such as whole blood or tissues.
BACKGROUND OF THE INVENTION
Niacin, also called nicotinic acid, pyridine 3-carboxylic acid, vitamin B3 or vitamin PP, is a water soluble vitamin. The known biological roles of niacin are attributable to the function of its active metabolites, nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP).
1
As used herein, pyridine coenzymes or pyridine nucleotides refer to NAD and NADP. NAD and NADP represent the total oxidized and reduced pools of each nucleotide, respectively. Thus, NAD represents the sum of NAD
+
and NADH while NADP represents the sum of NADP
+
and NADPH.
1
′ NAD is also called diphosphopyridine nucleotide (DNP) and conenzyme I or cozymase. NADP is also called triphosphopyridine nucleotide (TPN) or coenzyme II. These alternative names are not popular or are no longer used.
In humans, niacin equivalents can be obtained from dietary nicotinate, nicotinamide, and tryptophan. Consequently, niacin status depends on the amount of these in the diet and on factors that influence uptake, distribution, efficiency of conversion to tissue NAD and NADP, and excretion or reutilization of the nicotinamide moiety formed by the turnover of NAD and NADP. The term niacin number has been chosen as a convenient way to represent niacin status and is defined as the ratio of the concentration of NAD to the concentration of NADP multiplied by 100 (100·[NAD]/[NADP]) in whole blood. For example, if the relative concentration of NADP([NADP]) is 1 and the relative concentration of NAD([NAD]) is 0.72, the niacin number would be 72 (from the formula 100·[NAD]/[NADP]=72 wherein [NAD] is 0.72 and [NADP] is 1). Expressing the values in this way yields a whole number that is linearly related to intracellular NAD content of red blood cells.
Attempts have been made to measure niacin status in a subject. Previous attempts have involved the determination of urinary metabolites of nicotinamide (R. A. Jacob et al.,
J. Nutr.
119, 591 (1989)). In these tests the urinary excretion of niacin or niacin metabolites are measured to infer the niacin status in a subject. These attempts involve multiple biochemical steps that are labor intensive, and slow and do not measure niacin bioavailability or intracellular niacin active metabolites directly. Thus, current methods for measuring niacin status are not suitable to wide-scale screening and the relationship of the nicotinamide metabolites to niacin status is still poorly understood.
A metabolic ward study has shown that the NAD content of erythrocytes is a sensitive marker for niacin status in humans (C. S. Fu et al.,
J. Nutr.
119, 1949 (1989)). Test subjects restricted to a niacin intake of approximately 50% of the recommended daily allowance showed a 70% decrease in NAD content after five weeks. In contrast, NADP content remains relatively constant throughout the niacin restricted diet period (C. S. Fu et al.,
J. Nutr.
119, 1949 (1989)). Thus, because the NADP concentration remains constant while the NAD concentration is affected by niacin intake, the ratio of NAD to NADP reflects niacin status. Niacin status refers to the bioavailability of niacin and niacin derivatives such as NAD and NADP inside a cell and is an indication of the bioavailability of NAD. Because more than 98% of the total pyridine nucleotide pool of whole blood is in the erythrocyte fraction, (E. L. Jacobson and M. K. Jacobson,
J. Int. Med.
233, 59 (1993)) niacin status can be obtained from a few microliters of whole blood. One aspect of the invention is directed to a method of assessment of niacin status which is inexpensive, relatively accurate and rapid, and suitable to wide scale-application in the human population.
Niacin status derived from erythrocytes or whole blood from humans varies over a wide range. The data of Table I show the mean niacin number and the range of values measured in several populations. Using data from a nonrandom population of free living healthy adults and metabolic ward subjects on controlled niacin intake (C. S. Fu et al.,
J. Nutr.
119, 1949 (1989)), the mean niacin number is found to be approximately 175 and from the standard deviation it is predicted that 95% of the population would have values between 127 and 223. In a separate study of a large population of 46- to 64-year-old individuals in Malmö, Sweden a range of 28 to 337 was seen, with a mean of 160. The effect of dietary niacin intake on niacin status was shown in a study of individuals undergoing niacin therapy where the average pretherapy value of 175 was increased to 665 by niacin supplements. Taken together these data illustrate that niacin status varies widely in the human population and can be modulated by niacin supplementation.
TABLE I
NIACIN STATUS IN HUMAN POPULATIONS
Mean
Range
Population studied
n
niacin number
observed
Metabolic ward controls
a
7
178 ± 36
N/A
b
(100% of RDA for niacin)
Metabolic ward subjects
a
7
62 ± 11
N/A
(50% RDA, 5 weeks)
Healthy adults (United States)
c
30
175 ± 24
132-211
Healthy adults, 46-64 years old
687
160 ± 37
28-337
(Malmö , Sweden)
d
Hypercholesterolemia subjects
e
Pretherapy
5
175 ± 45
131-242
Two months of niacin therapy
10
665 ± 115
517-746
a
Calculated from the data in Ref. 3. RDA, U.S. recommended dietary allowance
b
N/A, Not available.
c
A nonrandom population of health -conscious adults, most of whom supplement their diet with a multiple vitamin containing the U.S. recommended dietary allowance for niacin.
d
Blood samples were provided by the Malmö Diet and Cancer Study via a grant from the Texas Higher Education Coordinating Board Advanced Research Grant 009768-025.
e
Blood samples from hypercholesterolemia subjects were provided by H. I. Robins, University of Wisconsin Clinical Cancer Center and the University of Wisconsin Lipid Clinic. Subjects received 750 mg of niacin twice daily for two months.
The assay of the invention is useful for determining the optimal amounts of dietary niacin to obtain an optimal level of intracellular niacin metabolites (niacin number). NAD is involved with ADP-ribose transfer reactions and these reactions have been implicated in a number of metabolic signaling processes (M. K. Jacobson, et al., in ADP-Ribosylating Toxins and G Proteins: Insights into Signal Transduction, J. Moss and M. Vaughan, eds., p. 479 American Society for Microbiology, Washington, DC 1990; K. C. Williamson and J. Moss, in ADP-Ribosylating Toxins and G Proteins: Insights into Signal Transduction, J. Moss and M. Vaughan, eds., p. 493. American Society for Microbiology, Washington, D.C., 1990; M. A. De Matteis et al.,
Proc. Natl. Acad. Sci. U.S.A.
91, 1114 (1994); H. C. Lee et al.,
Vitam. Horm.
48, 199 (1994); F.-J. Zhang et al.,
Bioorg. Med. Chem. Lett.
5,2267 (1995); C. Q. Vu et al.,
J. Biol. Chem.
271, 4747 (1996)) and in cellular recovery from DNA damage (F. R. Althaus and C. Richter, “ADP-Ribosylation of Proteins: Enzymology and Biological Significance.” Springer-Verlag, Berlin, 1987). For example, studies of the conversion of NAD to ADP-ribose polymers in response to DNA damage indicate that an optimal cellular content of NAD may be a preventive factor in cancer (E. L. Jacobson and M. K. Jacobson,
J. Int. Med.
233, 59 (1993); E. L. Jacobson et al., in ADP-Ribosylating Reactions, G. G. Poirier and P. Moreau, eds., p. 153. Springer-Verlag, New York, 1992). Such studies have shown a need for a rapid and accurate method for measuring niacin content based on tissue NAD in humans.
There is a need for a highly sensitive, accurate and reliable method to determine niacin status as measured by intracellular NAD content relative t
Jacobson Elaine L.
Jacobson Myron K.
Fulbright & Jaworski LLP.
Gitomer Ralph
Niadyne, Inc.
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