Reverse isotope dilution assay and lactose intolerance assay
View Patent ↗A “reverse isotope dilution assay” herein, wherein a pathway that produces a given metabolite is assayed by diluting a labelled metabolite produced by a second constitutive pathway. In one aspect, the invention relates to a method for monitoring lactose maldigestion or lactose intolerance in humans. Specifically, the method requires administering a reverse tracer of labeled glucose and unlabeled lactose to an individual and assessing labeled carbon dioxide in breath or blood. If the lactose is digested, the labeled CO 2 produced by the labeled glucose is diluted by the metabolism of the lactose.
1. A method of assaying enzyme activity in a subject, said method comprising:
a) administering to a subject an effective amount of a reverse tracer wherein said reverse tracer is a carbon labeled molecule that is constitutively metabolized by the subject to produce a labeled metabolite wherein said carbon labeled molecule is selected from the group consisting of acetate, glucose, bicarbonate, glycine, octanoate, palmitate, formate, propionate, and urea;
b) administering to said subject an effective amount of an unlabeled substrate, wherein said substrate is specifically metabolized by an enzyme to be assayed and wherein said substrate is metabolized by said enzyme to produce and unlabeled metabolite that is the same as the metabolite from step a);
c) collecting a specimen from said subject; and
d) measuring the amount of labeled metabolite in said specimen to determine the activity of said enzyme in said subject wherein the dilution of labeled metabolite indicates enzyme activity.
2. The method according to claim 1 , wherein said carbon-labeled molecule is selected from the group consisting of a 13 C labeled molecule, a 14 C labeled molecules, and mixtures thereof.
3. The method according to claim 1 , wherein said carbon-labeled molecule is labeled at the 1-position.
4. The method according to claim 1 , wherein said carbon-labeled compound comprises a plurality of labeled carbons.
5. The method according to claim 1 , wherein said metabolite is carbon dioxide.
6. The method according to claim 5 wherein said labeled metabolite is 13 C carbon dioxide.
7. The method according to claim 1 , further comprising comparing said amount of labeled metabolite with a standard, whereby said comparing yields a measure of enzyme activity, and whereby said standard is the mean amount of labelled metabolite produced by a control population of healthy subjects.
8. A method according to claim 1 wherein said enzyme is lactase, said reverse tracer is selected from the group consisting of 13 C labeled glucose, 13 C labeled glucose, and mixtures thereof said unlabeled substrate is lactose; and said labeled metabolite is selected from the group consisting of 13 C labeled CO 2 , 14 C labeled CO 2 , and mixtures thereof.
9. A method according to claim 1 wherein:
a) said enzyme is lactase, said reverse tracer is 13 C labeled glucose, said unlabeled substrate is lactose, and said labeled metabolite is 13 CO 2 ;
b) said enzyme is lactase, said reverse tracer is 14 C labeled glucose, said unlabeled substrate is lactose, and said labeled metabolite is 14 CO 2 ;
c) said enzyme is lactase, said reverse tracer is 13 C labeled acetate, said unlabeled substrate is lactose, and said labeled metabolite is 13 CO 2 ;
d) said enzyme is lactase, said reverse tracer is 14 C labeled acetate, said unlabeled substrate is lactose, and said labeled metabolite is 14 CO 2 ;
e) said enzyme is lactase, said reverse tracer is 13 C labeled bicarbonate, said unlabeled substrate is lactose, and said labeled metabolite is 13 CO 2 ;
f) said enzyme is lactase, said reverse tracer is 14 C labeled bicarbonate, said unlabeled substrate is lactose, and said labeled metabolite is 14 CO 2 ;
g) said enzyme is a fructose transporter protein, said reverse tracer is 13 C labeled glucose, said unlabeled substrate is fructose, and said labeled metabolite is 13 CO 2 ;
h) said enzyme is a fructose transporter protein, said reverse tracer is 14 C labeled glucose, said unlabeled substrate is fructose, and said labeled metabolite is 14 CO 2 ;
i) said enzyme is a fructose transporter protein, said reverse tracer is 13 C labeled acetate, said unlabeled substrate is fructose, and said labeled metabolite is 13 CO 2 ;
j) said enzyme is a fructose transporter protein, said reverse tracer is 14 C labeled acetate, said unlabeled substrate is fructose, and said labeled metabolite is 14 CO 2 ;
k) said enzyme is a fructose transporter protein, said reverse tracer is 13 C labeled bicarbonate, said unlabeled substrate is fructose, and said labeled metabolite is 13 CO 2 ; and
l) said enzyme is a fructose transporter protein, said reverse tracer is 14 C labeled bicarbonate, said unlabeled substrate is fructose, and said labeled metabolite is 14 CO 2 .