Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving hydrolase
Reexamination Certificate
1999-10-04
2001-10-30
Leary, Louise N. (Department: 1623)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving hydrolase
C435S023000, C435S004000, C435S968000, C435S963000
Reexamination Certificate
active
06309850
ABSTRACT:
BACKGROUND OF THE INVENTION
Determination of altered enzyme levels by measurement of enzyme activity in biological samples is used routinely by clinicians to assist in the diagnosis of a multitude of diseases or conditions wherein physical symptoms alone may not be definitive. However, the usefulness of such assays is dependent upon the specificity of the enzyme to the disease or condition and the sensitivity and selectivity of the enzymatic assay.
For example, acute pancreatitis is defined clinically as a discrete episode of symptoms caused by intrapancreatic activation of digestive enzymes. The cause of this activation is unknown; however, premature activation of zymogen to active enzymes within the pancreas results in autodigestion and inflammation of the pancreas. Symptoms include a steady, dull or boring pain in the epigastrium or left upper abdominal quadrant which is poorly localized and reaches peak intensity within fifteen minutes to one hour. The incidence of acute pancreatitis is difficult to ascertain as uniform diagnostic criteria and effort have not been applied. However, there is an urgency in accurately diagnosing acute pancreatitis to exclude other acute conditions that require different, usually surgical, management such as perforated peptic ulcer, acute cholangitis, appendicitis and mesenteric infarction. In contrast, pancreatitis is best treated through a “hands off” approach of eliminating food intake and increasing hydration.
Determination of serum amylase activity is the test most frequently used for the diagnosis of acute pancreatitis. The frequent use of this test undoubtedly stems from the ease in obtaining substrate and performing the spectrophotometric analysis. In addition, the cost is significantly less as compared to an ultrasound or CT scan. However, results from this assay are difficult to interpret with any certainty due to the extensive distribution and background levels of amylase throughout the body. Pancreatic amylase only accounts for approximately 40% of the amylase found in serum. In fact, many individuals experience hyperamylasis for reasons unrelated to pancreatic pathology such as salivary diseases, gut diseases, liver diseases and other conditions such as renal failure, thermal burns, alcoholism, postoperative state, ketoacidosis, fallopian or ovarian cysts, pneumonia, anorexia and abdominal aortic aneurysm. (Pieper-Bigelow et al. (1990)
Gastroenterol. Clin. North Am
. 19:793-810).
Sensitivity of the amylase test is also suspect, in part, because of the short half-life of the enzyme relative to others produced in the pancreas. With a half-life of only two hours, amylase is the first enzyme to return to normal levels (Ventrucci et al. (1987)
Pancreas
2:506-509) resulting in a sensitivity of only 33% two days after an initial bout of pancreatitis (Winslet et al. (1992) Gut 33:982-986).
Further, even in cases where pancreatic disease is known to be present, there is no correlation between the severity of pancreatitis and the level of serum amylase.
Accordingly, a number of digestive enzymes produced by the pancreas have been considered as possible alternatives to amylase.
Carboxypeptidase A (CPA) is a digestive enzyme synthesized exclusively by the pancreas as a zymogen precursor, procarboxypeptidase A (PCPA). Significant levels of CPA have been detected in serum of those suffering from acute pancreatitis, while healthy individuals have little (Roth, M. and Rohner, A. (1983)
Clin. Chim. Acta
. 135:65-71; Kazmierczak, S. C. and Van Lente, F. (1989)
Clin. Chem
. 35:251-255) to no (Peterson et al. (1982)
Anal. Biochem
. 125:420-426; Brown et al. (1987)
Anal. Biochem
. 161:219-225) detectable amounts of the enzyme. Several substrates and a variety of assay procedures have been proposed for the determination of the pancreatic enzyme CPA. Such assays include UV spectrophotometry to directly monitor the cleavage of the peptide bond, and colorimetric and fluorometric methods to measure the amino acid released from the C-terminus. (Bergmeyer, H. U., Ed. (1974)
Methods of Enzymatic Analysis
, Vol. 2, 2nd ed., Academic Press, New York; Roth, M. and Rohner, A. (1983)
Clin. Chim. Acta
135:65-71). More frequently used substrates include N-benzyloxycarbonyl-glycyl-L-phenylalanine (Z-Gly-Phe) and hippuryl-phenylalanine (Bz-Gly-L-Phe). An assay involving the determination of the &agr;-naphthol released from the N-terminal blocking group in naphthoxycarbonyl-phenylalanine has also been disclosed. (Ravin, H. A. and Seligman, A. M. (1951)
J. Biol. Chem
. 190:391-402). In addition, a spectrophotometric assay employing N-(2-furanacryloyl)-L-Phe-L-Phe (FAPP) has been reported. (Peterson et al. (1982)
Anal. Biochem
. 125:420-426). However, while the FAPP substrate had the best kinetic constants of any CPA substrate to date, its modest change in absorbance at 330 nm (&egr;=2000) and the high initial absorbance at that wavelength (&egr;=9350) significantly reduce the sensitivity and precision of this assay.
A new class of synthetic peptides suitable for assaying peptidase activity was described by Kingsbury et al. (1984)
Proc. Nat'l Acad. Sci. USA
81:4573-4576. These peptides contain amino acid mimetics with nucleophilic substitutions at the &agr;-carbon of glycine residues. The amino acid mimetics are stable when the nitrogen lone pair electrons are delocalized, as they are in a peptide bond, and release of the amino acid mimetic results in its decomposition to generate the nucleophilic substituent. If the substituent is linked to the glycine residue through sulfur, decomposition yields a compound with a free sulfhydryl group. Its appearance can be monitored spectrophotometrically in the presence of Ellman's reagent which reacts rapidly and quantitatively with free sulfhydryl groups to form a highly colored anionic species that absorbs at 412 nm. (Ellman, G. L. (1959)
Arch. Biochem. Biophys
. 82:70-77). An assay for measuring CPA in serum with the N-blocked phenylalanine substrate, N-acetyl-phenylalanyl-L-3-thiaphenylalanine was developed. (Brown et al. (1987)
Analytical Biochemistry
161:219-225). However, use of an assay measuring CPA activity to diagnose acute pancreatitis has been debated.
Using p-OH Bz Gly Phe as a CPA substrate, Kazmierczak and Van Lente carried out an extensive study comparing CPA, amylase and lipase levels as indicators of acute pancreatitis. A major difficulty for CPA was their finding that patients with renal insufficiency, but without pancreatitis, appeared to have elevated levels of the enzyme. Kazmierczak and Van Lente also found the diagnostic sensitivity of the three assays to be comparable at cutoff values of 3 (23 &mgr;g/L), 185 and 300 U/L, respectively. They concluded that automated analysis for CPA activity, even in the absence of interferences, does not add to the diagnostic information provided by the widely available assays for amylase and lipase activity. (Kazmierczak, S. C. and Van Lente, F. (1989)
Clin. Chem
. 35(2):251-5). High levels of CPA were also reported to be present in normal serum by Roth, M. and Rohner, A. (1983)
Clin. Chim Acta
135:65-71. Both groups found the average value of putative CPA in normal sera to be approximately 3.9 &mgr;g/L.
Pancreatic cancer is even more difficult to diagnose than acute pancreatitis, resulting in an abysmal mortality rate since individuals frequently seek treatment only after the disease has reached advanced stages which are accompanied by pain, weight loss and jaundice. The cancer is rarely diagnosed in its initial stages, in part because no cost-effective, non-invasive diagnostic test exists to date. While ultrasonography, CT scans and endoscopic retrograde cholangiopancreatography can confirm the presence of pancreatic cancer, these procedures are too expensive to use for general screening and are normally not applied until too late. Attempts to discover a marker for pancreatic cancer have been hindered by the fact that little is known about risk factors which would predispose individuals to the cancer. However, a number of indiv
Leary Louise N.
Licata & Tyrrell P.C.
Princeton University
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