US20050118152A1 - Analytical method for detecting alkaline sphingomyelinase and kit for use in such method - Google Patents

Analytical method for detecting alkaline sphingomyelinase and kit for use in such method Download PDF

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Publication number
US20050118152A1
US20050118152A1 US10/499,336 US49933604A US2005118152A1 US 20050118152 A1 US20050118152 A1 US 20050118152A1 US 49933604 A US49933604 A US 49933604A US 2005118152 A1 US2005118152 A1 US 2005118152A1
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United States
Prior art keywords
sample
sphingomyelinase
alkaline
fluorescence
sphingomyelin
Prior art date
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Abandoned
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US10/499,336
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English (en)
Inventor
Claudio De Simone
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VSL Pharmaceuticals Inc USA
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Individual
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Assigned to VSL PHARMACEUTICALS, INC. reassignment VSL PHARMACEUTICALS, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: DE SIMONE, CLAUDIO
Publication of US20050118152A1 publication Critical patent/US20050118152A1/en
Priority to US11/359,619 priority Critical patent/US7211410B2/en
Priority to US11/712,417 priority patent/US7323317B2/en
Abandoned legal-status Critical Current

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    • 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
    • G01N33/57407—Specifically defined cancers
    • G01N33/57419—Specifically defined cancers of colon
    • G01N33/575—
    • G01N33/57535—
    • 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.
  • 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.
  • sphingomyelin 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 ).
  • This latter compound in the presence of horse-radish peroxidase, is caused to react with 10-acetyl-3.7-dihidroxyphenoxazine, a sensitive fluorogenic probe for H 2 O 2 (hereinbelow referred to as “Amplex Red Reagent”) yelding the highly fluorescent compound resorufin. Fluorescence is measured with a fluorocount microplate fluorometer using excitation at 530-560 nm and fluorescence detection at 590 nm.
  • 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,
  • 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:
  • 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.
  • FIG. 1 the standard curve is shown for demonstration only. A standard curve should be generated for each set of samples assayed.
  • 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:

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Immunology (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Molecular Biology (AREA)
  • Physics & Mathematics (AREA)
  • Biotechnology (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Genetics & Genomics (AREA)
  • General Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biophysics (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Biomedical Technology (AREA)
  • Pathology (AREA)
  • General Physics & Mathematics (AREA)
  • Cell Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Food Science & Technology (AREA)
  • Oncology (AREA)
  • 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)
US10/499,336 2001-12-21 2002-12-19 Analytical method for detecting alkaline sphingomyelinase and kit for use in such method Abandoned US20050118152A1 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
US11/359,619 US7211410B2 (en) 2001-12-21 2006-02-23 Analytical method for detecting alkaline sphingomyelinase and kit for use in such method
US11/712,417 US7323317B2 (en) 2001-12-21 2007-03-01 Analytical method for detecting alkaline sphingomyelinase and kit for use in such method

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
IE011100 2001-12-21
IE20011100A IE20011100A1 (en) 2001-12-21 2001-12-21 Analytical Method for Detecting Alkaline Sphingomyelinase and Kit for Use in Such Method
PCT/IT2002/000811 WO2003056031A2 (en) 2001-12-21 2002-12-19 Method and kit for detecting alkaline sphingomyelinase

Related Parent Applications (1)

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PCT/IT2002/000811 Continuation WO2003056031A2 (en) 2001-12-21 2002-12-19 Method and kit for detecting alkaline sphingomyelinase

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US11/359,619 Division US7211410B2 (en) 2001-12-21 2006-02-23 Analytical method for detecting alkaline sphingomyelinase and kit for use in such method

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US10/499,336 Abandoned US20050118152A1 (en) 2001-12-21 2002-12-19 Analytical method for detecting alkaline sphingomyelinase and kit for use in such method
US11/359,619 Expired - Fee Related US7211410B2 (en) 2001-12-21 2006-02-23 Analytical method for detecting alkaline sphingomyelinase and kit for use in such method
US11/712,417 Expired - Fee Related US7323317B2 (en) 2001-12-21 2007-03-01 Analytical method for detecting alkaline sphingomyelinase and kit for use in such method

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US11/359,619 Expired - Fee Related US7211410B2 (en) 2001-12-21 2006-02-23 Analytical method for detecting alkaline sphingomyelinase and kit for use in such method
US11/712,417 Expired - Fee Related US7323317B2 (en) 2001-12-21 2007-03-01 Analytical method for detecting alkaline sphingomyelinase and kit for use in such method

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US (3) US20050118152A1 (enExample)
EP (1) EP1456405B1 (enExample)
JP (2) JP2005512601A (enExample)
KR (1) KR101002068B1 (enExample)
CN (1) CN101126716A (enExample)
AR (1) AR038037A1 (enExample)
AT (1) ATE408707T1 (enExample)
AU (1) AU2002367123B2 (enExample)
BR (1) BRPI0215045B1 (enExample)
CA (1) CA2469796C (enExample)
CY (1) CY1108512T1 (enExample)
DE (1) DE60228993D1 (enExample)
DK (1) DK1456405T3 (enExample)
EG (1) EG25804A (enExample)
ES (1) ES2312666T3 (enExample)
HR (1) HRP20040632B1 (enExample)
HU (1) HU228812B1 (enExample)
IE (1) IE20011100A1 (enExample)
IL (2) IL162513A0 (enExample)
MX (1) MXPA04005919A (enExample)
NO (1) NO331806B1 (enExample)
NZ (1) NZ533806A (enExample)
PL (1) PL207876B1 (enExample)
PT (1) PT1456405E (enExample)
RU (1) RU2316002C2 (enExample)
SI (1) SI1456405T1 (enExample)
WO (1) WO2003056031A2 (enExample)
ZA (1) ZA200405762B (enExample)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070141041A1 (en) * 2005-12-15 2007-06-21 Chemgen Corporation Enzymes for reduced immunological stress
CN103760348A (zh) * 2014-02-11 2014-04-30 苏州博源医疗科技有限公司 一种甘胆酸免疫检测试剂及其制备和检测方法

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
IE20011100A1 (en) 2001-12-21 2003-07-09 Vsl Pharma Ltd Analytical Method for Detecting Alkaline Sphingomyelinase and Kit for Use in Such Method
CN104614371A (zh) * 2015-02-15 2015-05-13 史春龙 一种在便器中检测排泄物生化指标的方法及使用该方法的检测设备
CN116106281A (zh) * 2023-02-03 2023-05-12 大连医科大学 一种粪便样本中肠道菌脂肪酶活性的检测方法
CN117630364B (zh) * 2023-12-05 2024-07-30 临沂大学 一种双酶型免疫荧光系统检测痕量黄曲霉毒素b1的方法

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6265179B1 (en) * 2000-02-01 2001-07-24 Molecular Probes, Inc. Detection of phosphate using coupled enzymatic reactions

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
IT1311495B1 (it) * 1999-06-09 2002-03-13 Mendes S U R L Composizione comprendente sfingomielinasi alcalina, utilizzabile qualeprodotto dietetico, integratore alimentare o medicamento.
IE20011100A1 (en) 2001-12-21 2003-07-09 Vsl Pharma Ltd Analytical Method for Detecting Alkaline Sphingomyelinase and Kit for Use in Such Method

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6265179B1 (en) * 2000-02-01 2001-07-24 Molecular Probes, Inc. Detection of phosphate using coupled enzymatic reactions

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070141041A1 (en) * 2005-12-15 2007-06-21 Chemgen Corporation Enzymes for reduced immunological stress
US7914782B2 (en) 2005-12-15 2011-03-29 Chemgen Corporation Enzymes for reduced immunological stress
EP2263685A3 (en) * 2005-12-15 2011-04-06 Chemgen Corporation Alkaline phosphatase for reducing immunological stress
US20110171344A1 (en) * 2005-12-15 2011-07-14 Chemgen Corporation Enzymes for reduced immunological stress
US20110171345A1 (en) * 2005-12-15 2011-07-14 Chemgen Corporation Enzymes for reduced immunological stress
US20110177195A1 (en) * 2005-12-15 2011-07-21 Chemgen Corporation Enzymes for reduced immunological stress
US8778648B2 (en) 2005-12-15 2014-07-15 Eli Lilly And Company Enzymes for reduced immunological stress
CN103760348A (zh) * 2014-02-11 2014-04-30 苏州博源医疗科技有限公司 一种甘胆酸免疫检测试剂及其制备和检测方法

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HRP20040632A2 (en) 2005-04-30
BRPI0215045B1 (pt) 2015-09-29
EG25804A (en) 2012-08-06
BR0215045A (pt) 2004-11-03
SI1456405T1 (sl) 2008-12-31
US7211410B2 (en) 2007-05-01
JP2009195239A (ja) 2009-09-03
WO2003056031A3 (en) 2003-12-18
JP5009332B2 (ja) 2012-08-22
NZ533806A (en) 2006-05-26
HU228812B1 (hu) 2013-05-28
MXPA04005919A (es) 2004-11-01
CA2469796A1 (en) 2003-07-10
DE60228993D1 (de) 2008-10-30
HRP20040632B1 (hr) 2012-10-31
HUP0402542A2 (hu) 2005-03-29
IL162513A (en) 2009-12-24
KR20040068209A (ko) 2004-07-30
JP2005512601A (ja) 2005-05-12
AR038037A1 (es) 2004-12-22
US20060141551A1 (en) 2006-06-29
US20070154974A1 (en) 2007-07-05
US7323317B2 (en) 2008-01-29
HUP0402542A3 (en) 2010-03-29
RU2316002C2 (ru) 2008-01-27
PL374085A1 (en) 2005-09-19
IE20011100A1 (en) 2003-07-09
IL162513A0 (en) 2005-11-20
EP1456405B1 (en) 2008-09-17
AU2002367123A1 (en) 2003-07-15
NO331806B1 (no) 2012-04-10
WO2003056031A2 (en) 2003-07-10
CN101126716A (zh) 2008-02-20
AU2002367123B2 (en) 2008-01-10
EP1456405A2 (en) 2004-09-15
RU2004122426A (ru) 2005-03-27
DK1456405T3 (da) 2009-01-05
NO20042992L (no) 2004-09-20
HK1075069A1 (zh) 2005-12-02
PL207876B1 (pl) 2011-02-28
ZA200405762B (en) 2006-06-28
CY1108512T1 (el) 2014-04-09
ATE408707T1 (de) 2008-10-15
PT1456405E (pt) 2014-01-09
CA2469796C (en) 2010-09-14
KR101002068B1 (ko) 2010-12-17
ES2312666T3 (es) 2009-03-01

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