Method and test strips for the measurement of fat loss during weight loss programs

Abstract

Disposable test strips and a wet chemistry method for measuring each of beta-hydroxybutyrate alone, combined beta-hydroxybutyrate and acetoacetate or total ketone bodies (i.e., beta-hydroxybutyrate, acetoacetate and acetone) in human bodily fluid samples, including but not limited to urine, saliva or sweat are described. The test strips need only be dipped in the sample and can be used by anyone in almost any milieu. Measurement can be made electrochemically, spectrophotometrically, fluorometrically or by comparision to a color standard. Combined acetoacetate and beta-hydroxybutyrate which account for 97-98% of total ketone bodies and may be measured in a cyclic reaction that occurs at pH about 7.0 to about 8.3 with beta-hydroxybutyrate dehydrogenase, (beta-HBD), nicotinamide adenine dinucleotide, a tetrazolium dye precursor and an electron mediator. Using this reaction, false positive results obtained from urine samples taken from patients on sulfhydryl drugs are avoided. beta-HBD from some sources was found to cause false negative results in samples (e.g. urine) containing high chloride content due to chloride inhibition of beta-HBD. Using a simple test for chloride inhibition, it was found that beta-HBD from Alcaligenes is not so inhibited. Using either beta-HBD that is not inhibited by chloride or using 10-20 times the normal concentration of this enzyme eliminates false negatives in samples having substantial chloride content, such as urine, both in the reaction described above and in other reactions disclosed for measuring each of beta-hydroxybutyrate alone, combined beta-hydroxybutyrate and acetoacetate and total ketone bodies, all of which reactions occur in the pH range of about 8.6 to about 9.5.

Description

Patent Application[0001] This application is a continuation-in-part of U.S. application Ser. No. 10 / 067,660 filed Feb. 4, 2002.[0002] The present invention affords a method and test strips for monitoring the effectiveness of a diet, or a diet plus exercise regimen (whether undertaken for a therapeutic purpose or for improving appearance), which specifically provides information on body fat loss and is usable by members of the general public on samples such as urine or saliva.[0003] Obesity of human beings is an ever-increasing source of medical concern, especially in the United States, today. It is estimated that over 60% of the U.S. population is obese. Studies have shown obesity to be the leading cause of diabetes, hypercholesterolemia and other disorders that lead to kidney and liver failure. A recently issued, widely commented upon, study attributes failure to diagnose many cancers at an early time, when effective treatment could be given, to serious obesity. Obesity likewise ac...

AI Technical Summary

Benefits of technology

0035] Ideally, the strips will be availed of on a daily basis by people on weight loss diets or persons with disease states in which such monitoring is advisable. The strips of this invention afford great advantages to people on weight loss diets because all embodiments described herein measure .beta.-hydroxybutyrate which typically comprises 75-80% of total ketone bodies. The embodiments which measure both acetoacetate and .beta.-hydroxybutyrate measure in the order of 97-98% of total ketone bodies, and of course, the embodiment which measures total ketone bodies gives what is essentially a 100% result, provided the measurement is made promptly after collection of the sample so that acetone content does not volatilize before measurement can occur. The ability to make these measurements of .beta.-hydroxybutyrate, with or without acetoacetate and acetone, in urine, saliva or sweat samples is likewise highly advantageous, especially when daily monitoring is desirable or indicated. Collecting blood samples, as has been required for .beta.-hydroxybutyrate measurement heretofore, is not only invasive but if done at frequent intervals is extremely uncomfortable for many people and certainly should not be a requisite for people seeking to monitor a weight loss diet. To patients whose diabetic, cardiovascular or epileptic conditions require daily or very frequent monitoring, the strips of the present invention with their ability to measure .beta.-hydroxybutyrate alone, or combined with acetoacetate, or further combined with acetone in urine rather than blood should offer a great boon.

0036] The present invention, in essence, provides the possibility of assaying for .beta.-hudroxybutyrate alone in a sample, using a test strip that provides .beta.-hydroxybutyrate dehydrogenase, NAD and a tetrazolium dye precursor, at a pH of about 8.6 or higher, up to about 9.5, plus a minor amount of an electron mediator capable of transferring an electron to the dye precursor to effect a color change. In this embodiment, the use of .beta.-hydroxybutyrate dehydrogenase (1) from an Alcaligenes source, or another source such that this enzyme is not inhibited by chloride ions in the sample, or alternatively, (2) the addition to the strip of an excess, in the order of at least 10 and up to about 20 times the amount required in strips designed primarily for use with blood samples, of .beta.-hydroxybutyrate dehydrogenase, obtained from a source that is inhibited by chloride ions in the sample, such as Pseudomonas, assures that the strip will measure .beta.-hydroxybutyrate in urine samples.

Problems solved by technology

Many of these diets are helpful in effecting weight loss, but the sad fact is that the majority of people who experience such weight loss gain it back, in many instances gaining even more than they lost, within a relatively short time period.

It has long been known, however, that for meaningful weight loss, body fat must be diminished on a steady basis and this cannot be measured by simple weighing because body fluid balance in particular, and perhaps other factors, play an ever-fluctuating and presently unmeasurable role in total body weight.

An overall drawback of this type of strip is that, when utilized to test urine of people on conventional "balanced" weight loss diets of 1000-1500 calories per day, it is generally insensitive, producing no reaction or a very faint one.

Both have methods have drawbacks which render them unsuitable for use by persons lacking laboratory training and / or availability of specialized instruments, in day-to-day monitoring of fat in weight loss regimens.

While the method is accurate, it is clearly not adaptable to use by persons lacking laboratory training.

It likewise cannot be converted to a dry chemistry format because the steps are incapable of being simultaneously performed because of their different pH requirements.

The color is hard to recognize with the naked eye and is easily interfered with if hemolysis occurs or bilirubin is present.

To sum up, the method, albeit accurate when conducted by a well-trained technician, is not amenable to being used in a dry chemistry solid phase test device operable by persons without laboratory training.

The system has the decided drawback that it is not cost effective for general use on an everyday basis, such as is desirable for monitoring weight loss programs.

This system is infeasible for home use because of its instrumentation and whole blood sample requirements.

Also, because it produces no color, its results are not as easy to follow as those that involve a color change.

The KetoSite.RTM. strips mentioned above measures .beta.-hydroxybutyrate in blood, but they have been found unsuitable for measuring .beta.-hydroxybutyrate in urine for two reasons, i.e.

b) the enzyme .beta.-hydroxybutyrate dehydrogenase present in these strips is inhibited by chloride ions, which abound in urine, with the result that false negative results may also be obtained.

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