HK1075069B - Kit for detecting alkaline sphingomyelinase - Google Patents

Description

Kit for detecting alkaline sphingomyelinase

The present invention relates to an analytical method for the assessment of alkaline sphingomyelinase in the stool or biological fluid of a patient in need of such assessment. The invention also relates to a kit for use in the method.

More specifically, the method of the invention is an in vitro fluorometric method for detecting alkaline sphingomyelinase, which (as described below) is a marker for severe pathological conditions such as colon cancer and familial adenomatous polyposis.

Sphingomyelinase (sphingomyelin phosphodiesterase, SMase) catalyzes the hydrolysis of sphingomyelin to ceramide and choline phosphate.

To date, three different smases (acidic, neutral and basic) have been identified, which exist in several isoforms as follows:

-lysosomal acidic SMase (A-SMase);

-cytoplasmic Zn²⁺Dependent acidic SMase;

membrane neutral magnesium dependent SMase (N-SMase);

-cytoplasmic magnesium independent N-SMase; and

alkaline SMase.

SMase has been shown to play a role in many physiological and pathological processes, including: lysosomal hydrolysis of endocytic SM, ceramide-mediated cell signaling, atherogenesis, terminal differentiation, cell cycle inhibition, apoptosis, inflammation formation, and regulation of eukaryotic stress responses.

In contrast to acidic and neutral SMases, which are commonly present in cells and act as lysosomes and membrane-immobilized enzymes, respectively, alkaline SMase shows tissue and species variability. In humans, alkaline SMase is present in the intestinal mucosa and bile. Alkaline SMase is first produced in the duodenum, is present in higher amounts in the intestine, particularly in the distal jejunum, and is present in high amounts in the colon and rectum. The SMase has an optimum alkaline pH of 9.0 and is Mg²⁺Independent, bile salt dependent and trypsin resistant.

The pathological importance of alkaline SMase has only recently been recognized, promoting the development of certain studies for the following reasons.

First, the enzyme may be responsible for the hydrolysis of dietary sphingomyelin, which is mainly present in milk, eggs, meat and fish. Second, the enzyme may regulate cholesterol absorption. Third, the presence of alkaline SMase in the gut and its detected reduction in selectivity in colorectal cancer indicate that the enzyme plays a role in gut carcinogenesis, as under physiological conditions, the enzyme stimulates apoptosis, protecting the intestinal mucosa from carcinogenesis.

Previous studies have shown that under physiological conditions, alkaline SMase can be dissociated by bile salts from the intestinal mucosa into the lumen. However, under pathological conditions, due to an abnormal increase in the concentration of bile salts, the dissociation of alkaline SMase by bile salts exceeds the normal enzyme biosynthesis, resulting in a low level of activity of alkaline SMase in the mucosa and an abnormal increase in the excretion of enzymes in the feces or biological fluid, i.e. bile. Thus, excretion of excess alkaline SMase above normal basal levels in stool or biological fluids can serve as a valuable diagnostic marker for colorectal carcinogenesis and familial adenomatous polyposis; therefore, there is a need to obtain reliable detection methods to detect alkaline SMase in the stool or biological fluids of patients who may be in the aforementioned intestinal pathological conditions.

In addition, some bacteria (e.g.Streptococcus thermophilus Lactobacillus) contain high levels of SMase, and the assessment of alkaline SMase may provide a means of assessing the variation in the number of said bacteria, i.e.after treatment with a micro-ecological regulator or/and a micro-ecological regulator-based product.

Previous methods for evaluating alkaline SMase are well known. The activity of SMase can be determined in vivo by labeling cells with a radioactive precursor of SM and then determining the level of the labeled product, or in vitro using radiolabeled SM or a chromogenic analog of SM, or a colored or fluorescent derivative of neutral SM.

These known methods are potentially hazardous due to radioactivity and are less sensitive than fluorescent detection and are not entirely satisfactory.

It is an object of the present invention to provide a reliable, inexpensive method for detecting alkaline SMase in the stool or biological fluid of a patient who may suffer from colorectal cancer and familial adenomatous polyposis, or gallbladder or liver diseases, which method overcomes the drawbacks of the known methods.

It is a further object of the present invention to provide a detection kit for use in the above detection method.

Another object of the invention is to assess bacterial proliferation under different health conditions, or after disease or after treatment with drugs or probiotics or food supplements.

The indirect fluorescence analysis method of the present invention is based on the following sequence of reactions.

Under the action of alkaline SMase present in feces or other biological fluidsSphingomyelin is hydrolyzed to ceramide and phosphorylcholine, which is hydrolyzed by alkaline phosphatase to produce choline. In the presence of choline oxidase, choline generates hydrogen peroxide (H)₂O₂)。

Reacting hydrogen peroxide with 10-acetyl-3, 7-dihydroxyphenoxazine (H) in the presence of horseradish peroxidase₂O₂The sensitive fluorescent probe (hereinafter referred to as "Amplex Red reagent") reacts to produce highly fluorescent resorufin. Fluorescence was measured using a fluorometric microplate fluorometer, with excitation at 530-560nm, and detection at 590 nm.

According to the aforementioned reaction sequence and fluorescence detection method, the detection method for detecting alkaline SMase in feces according to the present invention comprises the following steps. However, it will be apparent to those of ordinary skill in the art that the present method can be readily adapted to apply to biological fluids, such as bile, with some routine modification.

1) Collecting a fecal sample from the patient and drying;

2) approximately 3-4 grams of the dried sample was weighed and suspended in 20ml of a suspension containing 0.24-0.26M sucrose, 0.14-0.16M KCl, 45-55mM KH₂PO₄pH 7.4 in the homogenization buffer;

3) centrifuging the sample at +4 ℃ for 60 minutes at 4000 rpm;

4) recovering the supernatant, and centrifuging at +4 ℃ and 4000rpm for 15 minutes;

5) determining the Protein content of the supernatant using a Pierce Protein Assay using bovine serum albumin as a standard reference, the Protein concentration used being between 32mg/ml and 40mg/ml for each sample, 25. mu.l of sample being aspirated into each well;

6) adding 65. mu.l of buffer solution with pH 8.9-9.1 containing 45-55mM Tris/HCl, 1.9-2.2mM EDTA, 0.14-0.16M NaCl and 10. mu.l of sphingomyelin 28-30. mu.M to 25. mu.l of sample, and adding bile salts (TC, TDC, GC, GCDC) at a concentration of 2.9-3.1 mM;

7) incubation at 37 ℃ for 1 hour;

8) pipettes 100. mu.l and 10. mu.l each of sphingomyelin (28-31. mu.M) for each standard reference, incubated for 1 hour at 37 ℃ as the samples;

9) after 1 hour, 100. mu.l of a reaction buffer containing 45-55mM Tris/HCl (pH7.3-7.5), 9-11mM β -phosphoglycerate, 745. mu.M ATP, 4-6mM EDTA, 4-6mM EGTA, 95-105. mu.M Amplex Red, 7-9U/ml alkaline phosphatase, 0.1-0.3U/ml choline oxidase, 1.5-2.5U/ml horseradish peroxidase were added;

10) incubating at 37 ℃ for 1 hour or more in the absence of light;

11) detecting and emitting fluorescence at 590nm by using an excitation light source with the range of 530-560nm and a fluorescence microplate reader;

12) for each value, background fluorescence was corrected by subtracting the non-sphingomyelinase control value.

The invention also relates to a test kit for detecting alkaline sphingomyelinase in a patient's stool or biological fluid according to the aforementioned detection method, comprising test tubes containing the following reagent samples, respectively:

a) sphingomyelin that will be hydrolyzed by alkaline sphingomyelinase present in feces or biological fluids to produce phosphorylcholine;

b) alkaline phosphatase for catalyzing the hydrolysis of phosphorylcholine to choline;

c) choline oxidase for oxidizing choline to hydrogen peroxide;

d) horseradish peroxidase for assisting hydrogen peroxide reaction;

e) ampler Red reagent (10-acetyl-3, 7-dihydroxyphenoxazine) for obtaining the fluorescent compound resorufin, the fluorescence of which is a marker for the alkaline SMase present in feces or biological fluids; and

f) lyophilized bacterial sphingomyelinase was used as the standard concentration.

For proper application of the assay method of the present invention, in addition to the above-described kit components, the following materials and equipment are also required:

sucrose;

potassium chloride (KCl);

potassium dihydrogen phosphate (KH)₂PO₄)；

Trizma base；

EDTA；

Sodium chloride;

taurocholic acid (TC);

taurodeoxycholic acid (TDC);

glycocholic acid (GC);

hydroxycodeoxycholic acid (GCDC);

beta-glycerophosphate;

ATP disodium salt;

EGTA；

BCA Protein Assay Reagent；

bovine serum albumin;

freezing the centrifuge;

a microplate reader capable of detection at 550-.

To complete the quantification of SMase activity, the following assays will be performed.

Preparation of Standard Curve

The kit contains a standard preparation of SMase consisting of a bacterial extract containing SMase acting at pH 9. The following operations were performed.

Preparing an SMase calibration curve: the standard concentrate was diluted to give a series of dilutions.

The SMase standard was reconstituted with 1ml of assay buffer (pH 9.0) to give a 96mU/ml stock solution.

0.500ml of assay buffer was pipetted into each tube. Stock solutions were used to obtain a series of dilutions. Each tube was mixed thoroughly before the next transfer. Undiluted standards were used as high concentration standards (96mU/ml) and the standard curve included the following concentration values (mU/ml): 96-48-24-12-6-3. The buffer was used as a 0 concentration standard (0 mU/ml).

Typical standard curve

The standard curve in fig. 1 is for exemplary purposes only. A standard curve was made for each test sample.

Calculation results

The two readings for each standard and sample were averaged and the mean fluorescence for the zero concentration standard was subtracted.

The fluorescence of the standards was plotted against the activity of the standards (mU/ml) for the best curve. To determine the SMase activity of each sample, the fluorescence on the y-axis was first found and then extended horizontally to the standard curve. Extending perpendicularly to the x-axis at the intersection, the corresponding SMase activity can be read.

The method can detect the activity of SMase in vitro; methods have been developed for detecting alkaline SMase in organic samples.

To specifically detect alkaline SMase, the method employs conditions for detection of acidic and neutral SMase activity. The practice is that:

the homogenization buffer is at neutral pH, but is free from protease and phosphatase inhibitors so that neutral SMase can be excluded, since the latter are sensitive to and therefore inhibited by the activity of proteases and phosphatases;

absence of MgCl in the homogenization buffer₂Thereby blocking the activity of Mg-dependent neutral SMase；

Reaction buffer containing beta-phosphoglycerol and ATP to prevent excessive activity of the acidic SMase at neutral pH, in which both EDTA and EGTA are present in high concentrations to inhibit the neutral SMase.

Claims (1)

1. A kit for detecting alkaline sphingomyelinase in a sample of stool or biological fluid from a patient comprising test tubes containing samples of the following reagents, respectively:

a) sphingomyelin, which is hydrolyzed by alkaline sphingomyelinase present in feces or biological fluids, to produce phosphorylcholine, and EDTA;

b) alkaline phosphatase for catalyzing the hydrolysis of phosphorylcholine to choline;

c) choline oxidase for oxidizing choline to hydrogen peroxide;

d) horseradish peroxidase for assisting hydrogen peroxide reaction;

e) ampler Red reagent (10-acetyl-3, 7-dihydroxyphenoxazine) for obtaining the fluorescent compound resorufin, the fluorescence of which is a marker for alkaline sphingomyelinase present in faeces or biological fluids;

f) lyophilized bacterial sphingomyelinase used as a standard concentration; and

g) EGTA, taurocholic acid (TC), taurodeoxycholic acid (TDC), glycocholic acid (GC), hydroxycodeoxycholic acid (GCDC) for enhancing alkaline sphingomyelinase activity, beta-glycerophosphate, and ATP for inhibiting neutral and acidic sphingomyelinase activity.