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.
More particularly 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.
The enzyme sphingomyelinase (sphingomyelin phosphodiesterase, SMase) catalyzes the hydrolysis of sphingomyelin to ceramide and choline phosphate.
Three different types of SMase (acidic, neutral and alkaline) have been identified to-date, which occur as several isoforms, as follows:
lysosomal acidic SMase (A-SMase);
cytosolic Zn2+-dependent acidic SMase;
membrane neutral magnesium-dependent SMase (N—SMase);
cytosolic magnesium-independent N—SMase; and
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 signaling, atherogenesis, terminal differentiation, cell cycles arrest, apoptosis, inflammation, and the regulation of eukaryotic stress responses.
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 Mg2+-independent, bile salt-dependent and trypsin-resistant.
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.
First, the enzyme may be responsible for the hydrolysis of the dietary sphingomyelin occurring substantially in milk, eggs, meat and fish. Second, this enzyme may regulate cholesterol absorption. Third, 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.
Previous studies have also shown that, under physiological conditions, alkaline SMase is dissociated by bile salts from intestinal mucosal membrane to the lumen. However, under pathological conditions, whereby 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 feces or in biological fluids, i.e., bile. Consequently, the excess of 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.
In addition, some bacteria strains (e.g., Streptococcus htermophilus or Lactobacilli) contain high levels of SMase, and 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.
Previous methods for assaying alkaline SMase are already known. The activity of the SMases can be determined either in vivo through cell labeled with a radioactive precursor of SM and then determining the labeling product levels or in vitro using radiolabeled SM or a chromogenic analog of SM or colored and fluorescent derivatives of neutral SM.
These known commonly used assays are not entirely satisfactory since they are potentially very hazardous insofar as they are radioactive assays and less sensitive than a fluorometric assay.