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Title:
METHOD FOR DETECTING APPENDICITIS
Document Type and Number:
WIPO Patent Application WO/1989/001471
Kind Code:
A1
Abstract:
Appendicitis can be detected by determining a threshold presence of a certain material, believed to be the urinary pigment uroerythrin, in the urine of persons suspected of having appendicitis. The threshold presence of this material can be determined by precipitation of a sediment having a pinkish to reddish color. Qualitative, semiquantitative, or quantitative methods such as HPLC, TLC, radioimmunoassay, colorimetric tests, nuclear magnetic resonance, mass spectrometry, electrophoresis, monoclonal antibody tests, enzymatic tests and zeolite tests may also be employed to detect the material.

Inventors:
Bar-or, David Greisman Stewart L.
Kastendieck, Jon G.
Application Number:
PCT/US1988/002715
Publication Date:
February 23, 1989
Filing Date:
August 10, 1988
Export Citation:
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Assignee:
Bar-or, David Greisman Stewart L.
Kastendieck, Jon G.
International Classes:
G01N33/72; (IPC1-7): C07D207/02; G01N25/14; G01N30/02; G01N33/493; G01N33/50
Other References:
BIOLOGICAL ABSTRACTS, Vol. 67, No. 40011, MIN Y., "Diagnosis and treatment of acute appendicitis from the point of view of internal medicine" entire document.
EUROPEAN JOURNAL OF BIOCHEMISTRY, Vol. 56, No. 1, 1975, BERUTER et al., "Isolation and Identification of the Urinary Pigment Uroerythrin", pages 239-244, (Note: Abstract page 239, col. 1, lines 11-17; page 240, col. 1, lines 39-40; page 241, Table 1).
GRAFF S.L., A HANDBOOK OF ROUTINE URINALYSIS, published in 1983 by J.B. LIPPINCOTT COMPANY, Philadelphia, Pa, pages 10-13, 83, 84, 90. (Note: page 10, lines 30-33; page 11, table 1-1; page 12, lines 4-7 and 15-16; page 83, lines 3-5, 8-10, 29-30 and 35-37; page 90, lines 6-9).
Editorial, LANCET, Vol. 1, No. 8526, 24 January 1987, "A Sound Approach of the Diagnosis of Acute Appendicitis", pages 198-200. Entire document.
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Claims:
Thus having disclosed our invention, we claim:
1. A method for diagnosing appendicitis comprising the steps of: (a) obtaining a urine sample from a human being suspected of having appendicitis; (b) inducing precipitation of sediment from the urine sample in less then about 30 minutes; and (c) noting whether or not a sediment of pinkish to reddish color precipitates from the urine sample and thereby indicating the presence of appendicitis in said human being.
2. A method for diagnosing appendicitis comprising the steps of: (a) obtaining a urine sample from a human being suspected of having appendicitis; (b) lowering the temperature of the urine sample to reach a temperature of less than about 65° Fahrenheit in less then about 30 minutes; and (c) noting whether or not a sediment of pinkish to reddish color precipitates from the urine sample and thereby indicating the presence of appendicitis in said human being.
3. A method for diagnosing appendicitis comprising the steps of: (a) obtaining a urine sample, from a human being suspected of having appendicitis; (b) lowering the temperature of the urine sample to reach a temperature of between about 35° and about 60° Fahrenheit in a period from about 5 to about 20 minutes; and (c) noting whether or not a sediment having a pinkish to reddish color precipitates from the urine sample, and thereby indicating the presence of appendicitis in said human being.
4. A method for diagnosing appendicitis comprising the steps of: (a) obtaining a urine sample from a human being suspected of having appendicitis; (b) determining that the concentration of a material capable of being precipitated from the urine sample as a pinkish to reddish precipitate is at least 2xl0~4 mg/cc.
5. A method for diagnosing appendicitis in human beings comprising the steps of: (a) obtaining a urine sample from a human being suspected of having appendicitis; (b) determining from detection determining means selected from the group consisting of temperature induced precipitation, acid induced precipitation, HPLC, TLC, colorimetric comparison, monoclonal antibody tests, radioimmunoassay tests, enzymatic tests, NMR, mass spectrometry, electrophoresis, and zeolite methods, " that a material capable of being precipitated from the urine sample as a pinkish to reddish precipitate is present in a concentration of at least 2xl0~4 mg/cc.
6. A method for diagnosing appendicitis comprising the steps of: (a) obtaining a urine sample from a human being suspected of having appendicitis; (b) subjecting the urine sample to a chemical process step for precipitating sediments from the urine step; and (c) noting the presence of a sediment having a pinkish to reddish color as being indicative of the presence of appendicitis in the human being.
7. The method of claim 6 wherein the chemical process step for precipitating the sediment is acidification.
8. The method of claim 6 where the chemical process steps for precipitating the sediment is crystallization.
9. A method for diagnosing appendicitis comprising the steps of: (a) obtaining a urine sample from a human being suspected of having appendicitis; (b) determining if the specific gravity of the urine sample is greater than 1.010; (c) refrigerating the urine sample for about 5 minutes to about 20 minutes in the temperature range from about 35° F. to about 65° F. ; and (d) noting whether or not a sediment having a pinkish to reddish color forms in the urine sample under the refrigeration conditions imposed upon the urine sample, and thereby determining the presence of appendicitis in said human being.
10. A method for diagnosing appendicitis comprising the steps of: (a) obtaining a urine sample from a human being suspected of having appendicitis; (b) refrigerating the urine sample for about 10 minutes to about 20 minutes in the temperature range of about 40° F. (c) noting whether or not a sediment having a pinkish to reddish color forms in the urine sample under the refrigeration conditions imposed upon the sample; and (d) comparing the color of the pink sediment to a preestablished colorimetric chart to indicate the presence of appendicitis.
11. A method for diagnosing appendicitis comprising the steps of: (a) obtaining a urine sample from a human being suspected of having appendicitis; (b) acidifying the urine sample; (c) refrigerating the urine sample for about 10 minutes to about 20 minutes in the temperature range from about 35° F. to about 55° F. ; and (d) noting whether or not a sediment having a pinkish to reddish color forms in the urine sample after refrigeration conditions imposed upon the sample; (e) separating the pink sediment from the remainder of the urine sample; and (f) comparing the color of the pink sediment to a colorimetric chart especially colored in shades of pink to red which have been predetermined to indicate the presence of appendicitis in said human being.
12. A method for diagnosing appendicitis comprising the steps of: (a) obtaining a urine sample from a human being suspected of having appendicitis; (b) acidifying the urine sample to precipitate a sediment from the sample; (c) noting that the sediment has a pinkish to reddish color as being indicative of the presence of appendicitis in said human being.
13. A method for diagnosing appendicitis comprising the steps of: (a) obtaining a urine sample from a human being suspected of having appendicitis; (b) mixing 1020 cc of a urine sample with sufficient l.OM HCl to bring sample to a pH of about 2.0; (c) adding about 0.10 g EDTA; (d) letting sample stand for about 5 minutes; and (e) noting whether or not a pinkish to reddish sediment forms and thereby indicating the presence of appendicitis in said human being.
Description:
"Method for Detecting Appendicitis"

BACKGROUND OF THE INVENTION

1. Field of the Invention.

This invention generally relates to methods for detecting appendicitis. The diagnosis of appendicitis often challenges a physician's ability to differentiate this disease from other abdominal or pelvic disorders such as abdominal aortic aneurysm, pelvic inflammatory disease, ectopic pregnancy, ruptured or perforated viscus, gastrointestinal bleeding, hemorrhage pancreatitis, perforated diverticulu , ovarian abscess, Crohn's disease, mesenteric adenitis and intestinal obstruction. Were it not for the fact that such diseases often simulate appendicitis, and the fact that a fully developed picture is seldom available to the physician, a diagnosis of appendicitis would be relatively straightforward: confirmation of periumbilical pain of less than 72 hours' duration, migration of such pain to the right lower quadrant in a patient with a temperature of 37.5 to 38.5 c, evidence of abdominal tenderness, rigidity, a right- lower-quadrant mass and the presence of a mass o rectal examination. In many respects, negative predictors of appendicitis are often more helpful in

excluding appendicitis from a diagnosis. The most commonly employed negative predictors include: symptoms lasting more than 72 hours, pain at locations other than those noted above, temperature below 37.5 or about 38.6 C and the absence of anorexia. Unfortunately, neither leukocyte counts nor roentgenograms are sensitive or specific to appendicitis; and no reliable chemical tests have heretofore been developed. Hence, diagnosis of appendicitis is made almost solely on those clinical grounds noted above. Consequently, a large number of patients are taken to the operating room for explorative operations, with an average false positive experience of about 20%. This rather high false positive experience is tolerated because prompt action is needed to prevent this relatively minor acute disease, curable by appendectomy, from advancing to one complicated by perforation, peritonitis, long-term sequelae, and even death.

2. Description of the Prior Art The prior art has recognized that the presence, or the absence, of certain bile pigments might be used as indicators of the existence of certain disease states. For example, the article, Bile Pigment Fate in Gastrointestinal Tract, Seminars in Hematology, Vol. 9, No. 1 (January) 1972, points out that the presence of fecal bile pigments, which can also be detected in the urine, can be used to help diagnose certain diseases, particularly those of the liver. Such diagnoses are based upon the- understanding that urobilinogen is produced in the gut from bilirubin excreted from the liver into the bile; and that when it is diseased, the liver's

excretory capacity for urobilinogen is greatly reduced. Consequently, more urobilinogen reaches systemic circulation. It is not generally believed that this, in turn, is due to increased anastomoses between the portal and systemic vessels as well as decreases in the number and function of heptocytes. There also appears to be evidence that bilirubin and urobilinogen share a mechanism for hepatic uptake. Hence, competition between increased amounts of bilirubin and urobilinogen for hepatic uptake are also thought to contribute to urobilinogenuria associated with hemolytic disorders. In any event, the above noted reference lists the following disease conditions which may lead to alterations of urinary urobilinogen concentrations:

Factors Influencing Conditions in Which Urinary Urobilinogen Urinary Urobilinogen Concentration Tends to Increase

Amount of bilirubin Increased e.g. , conjugate entering hemolytic states gut

Loss of bile pigment from gut

Alterations in Biliary tract gut flora infection; colonization of small bowel

Alteration in Constipation transmit time through gut

Excretion of Decreased due to urobilinogen hepatocellular disease, by liver transhepatic shunting of portal blood; competition with other substances; e.g. , bilirubin

Renal factors Decrease in urine volume

Conditions in Which Urinary Urobilinogen Tends to Decrease

Decreased e.g., obstruction of common bile duct

Biliary fistula; small bowel fistula; ileostomy; colostomy Newborn; suppression by antibiotics

Severe diarrhea

Decrease glomerular filtration rate; increase in urine volume

It is also known that a change in the color of feces is usually present in the case of biliary obstruction and that this color change also is related to urobilinogen concentration. Hence the daily excretion of urobilinogen has been used to assess the degree of obstruction. For example, it is known that the average daily fecal excretion of urobilinogen in normal individuals is about 140 mg (range 40-280 mg day) although there is wide variation in reported values. Males excrete significantly more urobilinogen than females even when expressed on a weight basis (females, 1.46 = 0.61 g/kg body weight/day; males 2.16 = 0.90 mg.kg body weight/day; mean SD) . Moreover, these values. fluctuate from day to day. However, lower levels (5 mg/24 hour in adults) are found when there is prolonged total obstruction of the biliary tract.

On the other hand, it is also known that in a variety of other disease states there is a surplus rather than a deficit of fecal urobilinogen in relation to the rate of destruction of mature circulating red blood cells and their hemoglobin. This surplus, at times quite large, is represented by what is now generally known as the "early labeled" bile pigment, that fraction of the total bile pigment, bilirubin, or urobilinogen, that exhibits isotopic labeling within a few days after adminstration of N 15 or C 14 labeled glycine. This is in contrast to the normally much larger fraction (85 to 90%) that exhibits its peak labeling in relation to the destruction of hemoglobin of mature circulating red blood cells in the period of 110 to 130 days after administration of labeled glycine. However, as noted in The Continuing Challenge of Hemoglobin and Bile Pigment Metabolism, Annals of Internal Medicine, Vol. 63, No. 6 (December) 1965, even though there is a presume relationship of bile bilirubin and fecal urobilinogen to the destruction of hemes, the amount of bilirubin formed from destroyed hemoglobin has not been represented quantitatively in the excreta by recognizable derivatives such as urobilinogen. Among the known pigments (e.g., bile pigments, porphyrines, hemoglobin, indole derivatives, falvines, melanines and pteridines) which may be excreted in human urine there are groups of yellow, brown, and red pigments, generally designated as urochrome, whose chemical structures are still not totally understood. This lack of understanding is due, at least in part, to. the highly labile nature of such pigments. Moreover, increased amounts of uroerythrin have been postulated in certain diseases. For example, an

article entitled, Isolation and Identification of the Urinary Pigment Uroervthrin, Eur. J. Biochem. 56, 239-244 (1975) teaches: (1) that the red pigment uroerythrin is absorbed by the amorphous urate sediments (sedimentum lateritium) , (2) that increased amounts of uroerythrin are observed in patients in certain pathological states (e.g., diseases of the liver) and (3) that uroerythrin most probably has a chemical structure which is based upon a tripyrrole system. This reference does not however correlate the presence of appendicitis with a threshold presence of uroerythrin or any other bile pigment.

Uroerythrin has been isolated from human urine and purified as its trimethyl derivative. For example, the previously cited article, Isolation and Identification of the Urinary Pigment Uroerythrin, Eur. J. Biochem, 56 (1975) , teaches a purification method based upon introduction of urine into a column of Amerlite XAD-2 resin which absorb the uroerythrin. Purification is then obtained through conversion of the uroerythrin into its trimethyl derivative and chromatography on silica gel thin- layer plates.

SUMMARY OF THE INVENTION The methods and apparatus of this patent disclosure are based upon Applicant's qualitative and quantitative findings regarding the presence of a certain material though to be uroerythrin, or a derivative thereof, in the urine of human beings suffering from appendicitis. Applicants have established a definite correlation between a threshold presence of uroerythrin found in human urine and the presence of appendicitis. This threshold presence at or above about 2xl0~ 4 mg/cc. This correlation has been clinically tested and proven valid. The material can be induced to precipitate from the urine as a sediment which is pinkish to reddish in color. Again, our tests give their most pronounced results if the uroerythrin is at a concentration of about 2xl0~ 4 mg/cc or higher. The precipitation will take place as the temperature of the urine sample drops from human body temperatures near e.g., 98.6° F., to temperatures below about 65°F. The precipitation is even more pronounced as the temperature falls into a range of about 35 to about 60°F. The material thought to be uroerythrin is itself very labile. However, the overall sediment material is reasonably stable. Apparently light and/or oxygen will change the material's chemical structure after about 30 minutes exposure. Hence the disclosed tests should be conducted in less than 30, and most preferably in less than 15, minutes after the urine sample is obtained. A temperature drop aided by use of refrigeration, is therefore a highly preferred version of our test. For example, placement of a urine sample under refrigeration at about 40°F. for about 10 to 20 minutes is a highly preferred

refrigeration aided test protocol. However, the urine sample generally should be sufficiently concentrated (e.g., specific gravity > 1.010) to produce a pronounced precipitate. Detection of the threshold presence, (e.g., above about 2xl0~ 4 mg/cc) of uroerythrin can be enhanced by various procedures hereinafter more fully described. For example, the pinkish to reddish ' precipitate can be produced through acidification of the urine sample, preferably to a pH of less than about 4, and most preferably to a pH of about 2. By way of further example, the acidification may employ i.OM HC1 and EDTA (or other salts) to produce a precipitation in less than about 5 minutes. Crystallization procedures can also be employed to enhance precipitation. It should be specifically noted that when such chemical precipitation procedures are employed, it is not necessary to refrigerate the sample. However, the chemical precipitation procedures (acidification, crystallization etc.) may be augmented by refrigeration in many instances. Furthermore, the presence of uroerythrin, and if need be, its concentration, can be confirmed by various other physical and/or chemical procedures. These other procedures include, but are not limited to: HPLC (high performance liquid chromatography) tests, TLC (thin layer chromatography) tests, color comparisons, monoclonal antibody techniques, radioimmunoassaying, enzymatic (biologic) procedures, electrophoresis, zeolite tests, NMR (nuclear magnetic resonanic) tests and mass spectrometry. Those skilled in the art will also appreciate that the readings of these tests can be related to predetermined reference curves showing

readings (numbers, colors etc.) which can be established as being indicative of a given uroerythrin level (e.g., 2xl0~ 4 mg/cc) and hence indicative of the presence of appendicitis.

DESCRIPTION OF THE PREFERRED EMBODIMENTS The following procedures and exemplary tests are presented as illustrations of our methods for testing for appendicitis in human beings. These tests are intended to illustrate the concept of this invention, but they should in no way be regarded as limitations upon the concept. Clinical Validation

One hundred twenty three consecutive uroerythrin tests were performed on urine samples of patients (most of these patients were seen at

Swedish Medical Center, Englewood, Colorado) who had urinalysis as part of their clinical evaluation of abdominal pain (where appendicitis was suspected) . Test results were reviewed for specificity and sensitivity. Precipitation tests without chemical enhancement, were employed. A test was considered positive when a pinkish to reddish sediment was identified in response to cooling. With respect to these patients:

28/123 were true positives (test positive and surgery positive) 1/123 were false negatives (test negative and surgery positive) 2/123 were false positives (test positive and surgery negative) 92/123 were true negatives (test negative and surgery negative or not required) true positives 28

Sensitivity = true positives & = 28 & 1 = 96.5% false negatives

Specificity = true negatives = 92 true negatives & 94 = 97.4% false positives

These results clearly support our discovery.

Precipitation Tests.

Precipitation is the easiest qualitative method of identifying uroerythrin in urine. Applicants have found that urine of persons with appendicitis will precipitate a pink or reddish sediment when the temperature of the urine sample is lowered, within about 30 minutes after the urine sample is taken. The temperature lowering process can be speeded up without loss of sensitivity by refrigerating the urine sample. Refrigeration temperatures in the range of about 35° to about 60°F. are preferred. Preferably the sample will be subjected to such temperatures within about 2.0 minutes to about 30 minutes from the time the sample is obtained. The refrigeration step can be most conveniently carried out at about 40°F. for from about 5 to about 15 minutes. The pinkish to reddish uroerythrin sediment is most readily identifiable from any other sediments in the precipitate when uroerythrin is present at or above the threshold level. In other words the threshold level is that level which will produce the pinkish to reddish precipitate when the urine sample's temperature is lowered to the range of about 35° to about 60°F. , within 30 minutes after the sample is taken. By virtue of its color alone, as seen by the naked eye, the sediment is identifiable without additional preparation of the urine sample. Again, however, the color can be related to a preestablished color chart which correlates the color of the sample to the existence of appendicitis. Analogous preestablished levels (numerical concentrations etc.) can be established for all the various detection methods, e.g., HPLC, TLC, NMR, monoclonal antibody etc. which can be employed to detect uroerythrin in a urine sample.

For example, the addition of about 0.4cc of buffer phosphate, at a pH of about 4.0, and 1 to 5 milligrams of NaCl and 1 to 5 milligrams of potassium uric acid, to about 5-10 cc of urine will greatly enhance precipitation of the pinkish sediment from the urine sample when appendicitis is in fact present. Refrigeration will further hasten this chemical precipitation. Another chemical technique for enhancing such a precipitation is to acidify the urine sample to a pH of about 2.0 with 1 molar HC1, then add 0.1 gram of EDTA, (ethanolamine diethyl tetraammonium) and finally stir and centrifuge; again no refrigeration is required; but it may likewise enhance this particular acid precipitation technique.

Again, those skilled in this art will appreciate that many other tests may be employed to detect the presence of and, if need be, the concentration of, uroerythrin in urine; NMR, HPLC, TLC, colorimetric, monoclonal antibody, radioimmunoassay enzyme, electrophoresis and mass spectrometry tests are the most readily employable tests. Liquid chromatography (e.g., HPLC and TLC are the more preferred methods for determining the concentration of uroerythrin in the urine sample. Specific representative techniques with respect to some of these possible techniques are given in later sections of this patent disclosure.

Some of the results and procedures used to establish the invention were as follows:

I. THE PRECIPITATE'S PHYSICAL PROPERTIES:

A. The pinkish to reddish precipitate is soluble in water.

B. It is insoluble in weak acids. C. It is slightly soluble in methanol.

D. It is slightly soluble in acetonitrile.

E. It is insoluble in hexane.

F. It is insoluble in methylene chloride. Conclusion: The pinkish to reddish sediment contains mainly polar compounds. Following the establishment of this observation, procedures were developed to isolate the material from the urine.] II. PROCEDURE

1. A C-18 disposable (Bond Elute, 3cc) column was used.

2. The column was primed with 2cc methanol and followed by 2cc water.

3. 5cc of urine were mixed with 5cc buffer phosphate (1M, pH 3.0). 4. The column was washed with 2cc water.

5. The wash contaminants and other bands were washed with lcc of 40% methanol and 60% 0.01M phosphate buffer, pH 2.5.

6. The material was eluted and collected in lcc of methanol.

7. The elution fluid was analyzed.

The eluted fluid has a reddish-orange color. Even though HPLC was employed, TLC could have been just as well utilized. III. CONDITION OF HPLC ANALYSIS:

Solvents: 60% Buffer Phosphate 0.01M, pH 2.5 30% Acetonitrile 10% Methanol Column: C-18 Micro Pack MCH-5 N CAP, 15CMX4MMID

Column Temperature: 35 C. Detector: UV/VIS at 490nm Flow Rate: 1.5cc/minute

The results of HPLC analysis under the above conditions reveal a peak at around 9 minutes. The

material was collected and its absorbance spectrum analyzed. The absorbance spectrum reveals 3 LAMBDA maximums at 269,325 and 490. These figures are in accordance with the LAMBDA maximums in the Eur. J. Biochem reference previously cited.

Since other peaks appeared in the chromatogram, and in order to verify the carboxylic groups of uroerythrin, the extraction procedure was repeated and HPLC run at a pH 7.0. since at this pH the carboxylic groups will not be ionized, uroerythrin will not be retained on the C-18 column and hence the suspect peak was eliminated at 9 minutes, but not the other peaks; this again suggests that the material is uroerythrin. By using the same extraction procedure on sediment that was dissolved in water, it was determined that the pinkish to reddish sediment contains uroerythrin but at a lesser concentration than in urine.

In the extraction process, a red band in the C- 18 column was observed. It was removed physically by cutting it from the column. When analyzed by HPLC it revealed uroerythrin.

After repeated injections of the same extract containing uroerythrin to the HPLC, a marked decrease in uroerythrin concentration was observed. It was also determined that the material is most probably very liable in the presence of light. IV. PREPARATION OF STANDARD FOR HPLC

(1) Pass 20CC of urine of a patient with appendicitis through a C-18 column. (Bond Elute)

(2) Wash in 10CC water.

(3) Wash with 10CC 20% methanol 80% buffer.

(4) Wash with 10CC 40% methanol 60% buffer.

( 5 ) Cut red band .

(6) Dissolve in methanol.

(7) Filter out the C-18 material.

(8) Dilute with water and reconcentrate on C-18.

(9) Elute with methanol. (10) Inject in HPLC.

(11) Determine purity.

(12) Evaporate to dryness and weigh. (13) Purity x weight = amount of uroerythrin in that peak.

V . PR E C I P I T AT I O N P RO CE DURE WI THOUT REFRIGERATION :

10-20CC of urine Acidify to pH 2.0 with 1.0M HCL

Add 0.1GM EDTA (or possibly any other salt) Let stand for 5 minutes, preferably with stirring or centrifuging.

VI. COLOR TEST OF C-18 COLUMN: After elimination of all other contaminants by a purification process, but before elution, a color reaction with uroerythrin carboxylic group can be done. This can be done either by a pH indicator or by substituting the carboxyl group with other groups to generate a color reaction proportionate to the amount of uroerythrin. The semiquantitative TFA (Trifluoroacetic acid) technique can be used for this purpose.

Those skilled in the art will appreciate that the conditions employed in these various tests will be those appropriate to the particular precipitation agents being used. Thus, while the invention has been described by specific examples and preferred embodiments, there is no intent to limit the inventive concept which is set forth in the

following claims.