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
  • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
• • 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
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
  • G01N33/57407—Specifically defined cancers
  • G01N33/57419—Specifically defined cancers of colon
• • 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
  • G01N2333/902—Oxidoreductases (1.)
  • G01N2333/90206—Oxidoreductases (1.) acting on the CH-CH group of donors (1.3)
• • 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
  • G01N2333/902—Oxidoreductases (1.)
  • G01N2333/908—Oxidoreductases (1.) acting on hydrogen peroxide as acceptor (1.11)
• • 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)

Definitions

• the present invention relates to an analytical method for assessing the presence of alkaline sphingomyelinase in the stools or biological fluids of patients in need of such an assessment.
• the invention also relates to a kit for carrying out the analytical method.
• the method of the present invention is an in vitro fluorometric method for detecting alkaline sphingomyelinase which, as will be described in detail hereinbelow, is a marker of serious pathological states such as colon cancer and familial adenomatous polyposis.
• sphingomyelinase sphingomyelin phosphodiesterase, SMase catalyzes the hydrolysis of sphingomyelin to ceramide and choline phosphate.
• A-SMase lysosomal acidic SMase
• N-SMase membrane neutral magnesium-dependent SMase
• cytosolic magnesium-independent N-SMase alkaline SMase.
• SMases have been shown to play a role in a wide variety of physiologic and pathological processes, including: lysosomal hydrolysis of endocytosed SM, ceramide mediated cell signalling, atherogenesis, terminal differentiation, cell cycles arrest, apoptosis, inflammation, and the regulation of eukaryotic stress responses.
• alkaline SMase In contrast to acidic and neutral SMase, which are currently present in cells as lysosomal and membrane-bound enzymes, respectively, alkaline SMase exhibits tissue and species difference. In human beings, the alkaline SMase is found in intestinal mucosa and bile. Alkaline SMase starts to appear in the duodenum, reaches a high level in the intestine, especially in the distal part of the jejunum, and occurs in considerable amounts in the colon and rectum. This SMase presents optimal alkaline pH at 9.0, is Mg 2+ -independent, bile salt-dependent and trypsin-resistant.
• alkaline SMase The pathological importance of alkaline SMase has only recently been recognized and this has prompted several studies to be carried out, mainly for the following reasons.
• the enzyme may be responsible for the hydrolysis of the dietary sphingomyelin occurring substantially in milk, eggs, meat and fish.
• this enzyme may regulate cholesterol absorption.
• the presence of alkaline SMase along the intestinal tract and its selective decrease detected in colorectal carcinoma suggests that this enzyme plays a role in intestinal carcinogenesis, since under physiological conditions, it stimulates apoptosis and protects the intestinal mucosa against carcinogenesis.
• alkaline SMase is dissociated by bile salts from intestinal mucosal membrane to the lumen.
• bile salt concentration is abnormally increased
• the dissociation of alkaline SMase by bile salts may exceed the biosynthesis of the enzyme, resulting in a low level of activity of alkaline SMase in the mucosa, and an abnormally increased excretion of the enzyme in the faeces or in biological fluids, i.e. bile.
• alkaline SMase excreted in the stools or in biological fluids over normal, basal values, may be interpreted as a valuable diagnostic marker for colon rectal carcinoma and familial adenomatous polyposis, hence; the need of a reliable assay for detecting alkaline SMase in the stools or in biological fluids of patients likely to be suffering from the aforesaid pathologies of the intestinal tract.
• bacteria strains e.g. Streptococcus termophilus Lactobacilli
• the assessment of alkaline SMase may provide a method to evaluate changes in the number of said bacteria, i.e. after a treatment with probiotics or/and probiotic-based products.
• SMases Previous methods for assaying alkaline SMase are already known.
• the activity of the SMases can be determined either in vivo through cell labelled with a radioactive precursor of SM and then determining the labelling product levels or in vitro using radiolabelled SM or a chromogenic analog of SM or colored and fluorescent derivatives of neutral SM.
• An object of the present invention is to provide a reliable, unexpensive assay for alkaline SMase in the stools or biological fluids of patients likely to suffer from colorectal carcinoma and familial adenomatous polyposis, or gall bladder or liver diseases, which overcomes the drawbacks of the known methods.
• a further object of the present invention is to provide an analytical kit for use in the aforesaid assay.
• Another object of the present invention is the assessment of bacterial colonization in different health conditions or following diseases or treatment with drugs or probiotics or food supplements.
• the fluorometric, indirect assay method of the present invention is grounded on the following sequence of reactions. Under the action of alkaline SMase, present in faeces or other biological fluids, sphingomyelin is hydrolyzed to ceramide and phosphorylcholine which, under the action of alkaline phosphatase, is hydrolyzed yelding choline. In the presence of choline oxidase, choline produces hydrogen peroxide (H 2 O 2 ).
• the assay method of the present invention for assaying alcaline SMase comprises the following steps which refers to stools.
• this method can be easily applied also to biological fluids such as bile with appropriate routine variations,
• reaction buffer containing 50 mM Tris/HCl pH 7.4, 10 mM ⁇ -glycerophosphate, 750 ⁇ M ATP, 5 mM EDTA, 5 mM EGTA, 100 ⁇ M Amplex Red, 8 U/ml alkaline phosphatase, 0.2 U/ml choline oxidase, 2 U/ml horseradish peroxidase;
• the invention also relates to a kit for detecting alkaline sphingomyelinase in a patient's stools or biological fluids according to the previously disclosed method, which comprises test tubes separately containing samples of the following reagents:
• sphingomyelin to be hydrolized by alkaline sphingomyelinase present in the stools or biological fluids, to give phosphorylcholine;
• alkaline phosphatase for catalyzing the hydrolysis of phosphorylcholine to choline
• choline oxidase for oxidizing choline to hydrogen peroxide
• TDC Taurodeoxycholate
• GCDC Glycochenodeoxycholate
• a microplate reader capable of measurement at 550-562 nm
• a fluorocount microplate fluorometer In order to accomplish the quantification of SMase activity, the following measures should be taken.
• the kit is supplied with a standard preparation of SMase, it consists of bacterial extract containing a type of SMase that works at pH 9. The following operations should be performed.
• the described method is able to assay SMase activity in vitro; it has been developed with the intent to detect alkaline SMase in an organic sample.
• the method uses conditions that detect the acid and neutral SMases activity. In fact:
• the homogenization buffer is at neutral pH, but it have not protease and phosphatase inhibitors to exclude the neutral SMase since the latter is sensitive to activities of proteases and phosphatases and is consequently inhibited by these enzymes;
• the MgC is absent to block the activity of Mg dependent neutral SMase
• the reaction buffer contains ⁇ -glycerophosphate and ATP to preclude acid SMase moreover activity at neutral pH, in this buffer EDTA and EGTA are present in high concentration to inhibit neutral SMase.

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• General Health & Medical Sciences (AREA)
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• Hospice & Palliative Care (AREA)
• Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
• Investigating Or Analysing Biological Materials (AREA)
• Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
• Enzymes And Modification Thereof (AREA)

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