Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
  • C12Q1/44—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving esterase
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q2334/00—O-linked chromogens for determinations of hydrolase enzymes, e.g. glycosidases, phosphatases, esterases
  • C12Q2334/40—Triphenylmethane dye chromogens, e.g. fluorescein derivatives
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/90—Enzymes; Proenzymes
  • G01N2333/914—Hydrolases (3)
  • G01N2333/916—Hydrolases (3) acting on ester bonds (3.1), e.g. phosphatases (3.1.3), phospholipases C or phospholipases D (3.1.4)
  • G01N2333/918—Carboxylic ester hydrolases (3.1.1)

Definitions

• This new method for examining the exocrine pancreas function is carried out in that a predetermined amount of a fluorescein fatty acid monoester is introduced into and passed through the gastrointestinal tract, then the urine is collected or a portion of blood serum is withdrawn and the amount of released fluorescein contained in the urine or the blood serum is determined analytically.
• pancreas disorders The human abdominal salivary glands excrete a series of enzymes which are of great importance to digestion. Determination of the activity of these enzymes, which are excreted in the duodenum, gives information about the functioning of the pancreas and is important in the diagnosis of, in particular, pancreatitis, which is difficult to detect. In the diagnosis of pancreas disorders, it is important that the determination of the pancreas-enzyme concentrations in the duodenal fluid or blood serum can be carried out quickly and in the simplest way possible with good reliability, in order that chronic pancreatitis may be diagnosed.
• a specimen amount of duodenal fluid is sampled from the patient by means of a catheter, the sample is added to a substrate, such as triolein, and, after incubation, is examined for its content of free fatty acids.
• a substrate such as triolein
• a further object of the present invention is to provide a new and novel diagnostic composition
• a new and novel diagnostic composition comprising amonoester of fluorescein with a fatty acid containing 8 to 16 carbon atoms, preferably lauric acid, in a pharmaceutical carrier for determining pancreas enzymes in body fluids.
• the fluoroscein monoesters for use in this invention are colorless or slightly yellowish substances which are insoluble or only very slightly soluble in water and are not reabsorbable on its way through the human body. They are decomposed in the small intestine by the enzymes of the pancreatic secretion, in particular the esterases of pancreas, specifically into fatty acids and fluorescein. Unlike the fluorescein monoesters, the fluorescein resulting from the decomposition of the esters: is a readsorbable substance. The reabsorbed fluorescein is excreted in the urine; the concentration of fluorescein found there is a measure of the function of the pancreas.
• the mono fatty acid esters of fluorescein are suitable as substrates for enzymes, so that in the hydrolysis by the enzyme of the pancreas secretion, the quinonoid form of the dyestuff fluorescein is released in reabsorbable form.
• the reabsorbed fluorescein can be quantitively analyzed, photometrically or fluorimetrically or by any suitable analyzing method known in the art.
• the present invention providing a fluorescein mono fatty acid ester shows a number of improvements and advantages over the art, such as:
• the dosage units may be decreased of 25 percent without the fluorescein level in the organism decreasing.
• the fluorescein monoesters are preferably used in the conventional preparative forms, such as tablets, pills, capsules or drops. Moreover, it is preferred to incorporate in addition the usual pharmaceutical excipients, such as fillers, sweeteners and the other usual additives. Those forms are preferred which contain the esters in a finelydivided, e.g. micronized or suspended form.
• the solid preparative forms such as pills or coated tablets, can also be provided with coatings which have a retarding solubility and are soluble in the stomach or intestine. In order that the alkaline condition which prevails in the duodenum, is reliably controlled on dissolution of the diagnostic composition of the invention, it is also possible to add buffer substances, e.g. buffer salts which in solution adjust the pH value in the range of 7.5-8.5.
• the composition is administered to the patient in one of these preparative forms in dosage units with a content of fluorescein mono fatty acid esters with 8 to 16 carbon atoms in the range of about 0.1 mmol. (about 51 mg.) to about 1.0 mmol. (about 514 mg), preferably of about 0.5 mmol. (257 mg); after certain periods of time, the fluorescein, resulting from the hydrolysis, is analyzed in the combined portions of the urine, photometrically or fluorimetrically as described in the periodical Hoppe Seylers, Z. Phys. Chem. 349, pp. 1071-1072 (1968).
• pancreas enzymes only takes place when the pancreas is stimulated by the absorption of food; it is therefore relatively simple to determine, firstly in the fasting state, the mostly negligible basic readsorption value of fluorescein and compare this control value with the test value resulting after the pancreas having been excited, for example after a specimen breakfast.
• the value thus obtained stands in direct relation to the enzymatic activity of the pancreas.
• reduced fluorescein amounts are detected in the blood serum and urine compared to the normal value.
• tA further method according to the present invention is the n-vivo determination in the blood serum which is carried out as follows: Each 60 min., 90 min., and 120 min., resp. after the oral administration of a test capsule of the diagnostic agent according to the invention containing 0.5 mmol. of a fluorescein monoester, portions of blood serum are withdrawn from the patient. The portions are collected and the collected portions of the blood serum are diluted with a 0.1 N aqueous sodium hydroxide solution, 1n the ratio of about 1:5 (or, with low values of fluorescein, in the ratio of about 1:2). The diluted solutions are incubated for minutes at 90 C. and are then cooled down and centrifugated.
• the supernatant liquid is measured in a photometer at the wavelength of 492 m or fluorimetrically analyzed. Since the blood serum has a specific inherent color, a correction must be made with a zerocontrol figure obtained from measuring of a specimen of blood serum (which is free of fluorescein).
• the fluorescein mono esters used according to the invention are produced in that fluorescein is reacted in a molar proportion of 1:1 with a reactive derivative of a fatty acid containing 8 to 16 carbon atoms in an inert solvent, preferably in chloroform, if required under heating.
• the reaction can be promoted by the addition of tertiary or aromatic amines, such as for example triethylamine 0r pyridine.
• Acid chlorides or imidazolides may be used as the reactive derivatives.
• the fluorescein mono acld ester indicated in the following examples has a melting point of 109-1l1 C.
• EXAMPLE 1 A healthy test patient in the fasting state received orally a test sample containing 257 mg. (0.5 mmol.) of fluorescein mono lauric acid ester together with 700 ml. tea. The urine was then collected for 6 hours and examined for its content of fluorescein.
• EXAMPLE 2 A healthy test person received, together with as test breakfast consisting of 50 g. white bread, 20 g. butter and 700 ml. tea, which serves to stimulate the pancreas, 257 mg. (0.5 mmol.) of fluorescein mono lauric acid ester. The urine was then collected for 6 hours and examined for its content of fluorescein.
• pancreas was stimulated of the individual standard being extracted via the kidneys.
• a diagnostic composition for determining the activity of pancreas enzymes in the living human body comprising an oral dosage unit pharmaceutical carrier containing a predetermined oral dosage unit amount of about 51 mg. to about 514 mg. of a monoester of fluorescein with fatty acids having 8 to 16 carbon atoms.
• a diagnostic composition according to claim 1, comprising in addition to said fluorescein monoester a buffering agent adjusting the pH value to 7.5-8.5.
• a diagnostic composition according to claim 1 characterized in that said fluorescein monoester is incorporated in a pill, coated tablet or a capsule having a coating which has a retarding solubility and is soluble in the stomach or dissolves in the alkaline medium of the small intestine.
• a method for examining the exocrine pancreas function in the living human body characterized in that a predetermined oral dosage unit amount of about 51 mg. to about 514 mg. of a mono fatty acid ester of fluorescein is orally ingested and thereby is passed into the gastrointestinal tract and the released fluorescein found in the serum or excreted with the urine is analyzed in vitro photometrically or fluorimetrically.
• the pharmaceutical preparative form consists of a pill, a coated tablet or a capsule with a coating which only dis- References Cited UNITED STATES PATENTS 2,801,203 7/1957 Leb et a1 424-35 X 3,341,417 9/ 1967 Yaleko 424-2 2,996,431 8/ 1961 Barry 4247 2,770,571 11/1956 Vance et a1 20684 OTHER REFERENCES Schwartz et al., Advances in Clinical Chemistry, 13, 113, 126, 127, 128, 129, 130, 154, 155, 157 (1970),

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• Chemical & Material Sciences (AREA)
• Organic Chemistry (AREA)
• Life Sciences & Earth Sciences (AREA)
• Health & Medical Sciences (AREA)
• Wood Science & Technology (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Zoology (AREA)
• Engineering & Computer Science (AREA)
• Immunology (AREA)
• Biochemistry (AREA)
• Microbiology (AREA)
• Molecular Biology (AREA)
• Analytical Chemistry (AREA)
• Biotechnology (AREA)
• Physics & Mathematics (AREA)
• Biophysics (AREA)
• Bioinformatics & Cheminformatics (AREA)
• General Engineering & Computer Science (AREA)
• General Health & Medical Sciences (AREA)
• Genetics & Genomics (AREA)
• Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
• Investigating Or Analysing Biological Materials (AREA)
• Heterocyclic Carbon Compounds Containing A Hetero Ring Having Oxygen Or Sulfur (AREA)
• Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

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Cited By (10)

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• 1969
  • 1969-09-10 DE DE1945663A patent/DE1945663C3/de not_active Expired
• 1970
  • 1970-08-12 DE DE2039999A patent/DE2039999C3/de not_active Expired
  • 1970-09-03 FR FR7032085A patent/FR2070075B1/fr not_active Expired
  • 1970-09-03 US US00069425A patent/US3786140A/en not_active Expired - Lifetime
  • 1970-09-07 CH CH1330570A patent/CH542445A/de not_active IP Right Cessation
  • 1970-09-09 AT AT816070A patent/AT303268B/de not_active IP Right Cessation

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