Serum 5’-nucleotidase

Bertrand A, Buret J. A one-step determination of serum 5 -nucleotidase using a centrifugal analyzer. Clin Chim Acta 1982 119 275-84. 

P5. Parsons, V., Walker, R. ]., and Howarth, P. J. N., Serum 5 nucleotidase and alkaline phosphatase isoenzymes in diagnosis, with special reference to haemochromato-sis. Ann. Clin. Biochem. 10, 179-184 (1973). 

Carakostas, M. C., R. J. Power, and A. K. Banerjee.1990. Serum 5 -nucleotidase activity in rats A method for automated analysis and criteria for interpretation. Veterinary Clinical Pathology 19 109-113. 

Dooley, J. R, and L. Racich. 1980. A new kinetic determination of serum 5 -nucleotidase activity with modifications for a centrifugal analyzer. Clinical Chemistry 26 1291-1297. 

Brydon and Roberts- added hemolyzed blood to unhemolyzed plasma, analyzed the specimens for a variety of constituents and then compared the values with those in the unhemolyzed plasma (B28). The following procedures were considered unaffected by hemolysis (up to 1 g/100 ml hemoglobin) urea (diacetyl monoxime) carbon dioxide content (phe-nolphthalein complex) iron binding capacity cholesterol (ferric chloride) creatinine (alkaline picrate) uric acid (phosphotungstate reduction) alkaline phosphatase (4-nitrophenyl phosphate) 5 -nucleotidase (adenosine monophosphate-nickel) and tartrate-labile acid phosphatase (phenyl phosphate). In Table 2 are shown those assays where increases were observed. The hemolysis used in these studies was equivalent to that produced by the breakdown of about 15 X 10 erythrocytes. In the bromocresol green albumin method it has been reported that for every 100 mg of hemoglobin/100 ml serum, the apparent albumin concentration is increased by 100 mg/100 ml (D12). Hemolysis releases some amino acids, such as histidine, into the plasma (Alb). 

Serum ALP and total bilirubin (unconjugated and conjugated fractions) are traditionally used to monitor cholestatic injury. The ALP families of enzymes are zinc metalloproteases that are present in nearly all tissues. In the liver, ALP is immu-nolocalized to the microvili of the bile canaliculus [124]. Increased synthesis of ALP and its release into the circulation occurs within hours of cholestatic injury [129]. Serum assays of 5 -nucleotidase (5 -NT) or y-glutamyltransferase activity (GGT) are used to confirm the liver as the specific origin for the elevation of ALP. Increases in serum bilirubin or bile acids are usually the result of bile retention subsequent to impaired bile flow, increased production associated with accelerated erythrocyte destruction, or altered bilirubin metabolism [129]. 

ACS = anunonium (-l-)-DlO-camphorsulfonate BSA = bovine serum albumin CD = circular dichroism CT = charge transfer hCCS = human copper chaperone for superoxide dismutase hSCAN = human soluble calcium-activated nucleotidase-1 LMCT = ligand-to-metal charge transfer METP = miniaturized electron transfer protein MECT = metal-ligand charge transfer ORD = optical rotatory dispersion SODs = superoxide dismutases tCCS = tomato copper chaperone for superoxide dismutase UTP = uracil triphosphate. 

The principal biochemical symptom of cholestasis is the rise in bile acids in serum (as well as changes in its spectrum) in combination with an increase in enzymatic markers of cholestasis (AP, LAP, y-GT, 5 -nucleotidase). Cholestasis is directly related to the metabolism of bile acids. In clinical terms, the subsequent rise in activity of enzymatic markers of cholestasis may be attributed to cholestasis, yet these enzymes are not necessarily specific to this condition, (s. p. 89)... 

Enzymes in this category include alanine and aspartate aminotransferases, glutamate dehydrogenase (GLD), ATP, 5 -nucleotidase (NTP), y-glutamyl transferase (GGT), glutathione S-transferase (GST), and serum cholinesterase (CHE). The aminotransferases and ALP are widely used. They have long been mistakenly called, as a group, liver function tests. They are not, of course, but the habit persists. GGT is widely available in the United States and on automated analyzers. The others have not been adopted as widely. 

Pagani F, Panteghini M. 5 -nucleotidase in the detection of increased activity of the liver form of alkaline phosphatase in serum. Clin Chem 2001 47 2046-8. 

In liver metastases, the serum alkaline phosphatase level shows a better correlation with the extent of liver involvement than those of other liver tests. To differentiate the origin of elevated alkaline phosphatase levels, tests of other liver enzymes may be performed, such as that for 5 -nucleotidase or y-glutamyltransferase. Determination of alkaline phosphatase isoenzymes may provide additional specificity. The liver isoenzyme is thermally more stable than the bone isoenzyme (see Chapter 21 for a more detailed discussion). Other malignancies, such as leuicemia, sarcoma, and lymphoma complicated with hepatic infiltration, may also show elevated ailcaline phosphatase levels. 

Measurement of serum y-GT activity has clinical significance. The enzyme is present in all tissues, but the highest level is in the kidney however, the serum enzyme originates primarily from the hepatobiliary system. Elevated levels of serum y-GT are found in the following disorders intra- and posthepatic biliary obstruction (elevated serum y-GT indicates cholestasis, as do leucine aminopeptidase, 5 -nucleotidase, and alkaline phosphatase) primary or disseminated neoplasms some pancreatic cancers, especially when associated with hepatobiliary obstruction alcohol-induced liver disease (serum y-GT may be exquisitely sensitive to alcohol-induced liver injury) and some prostatic carcinomas (serum from normal males has 50% higher activity than that of females). Increased activity is also found in patients receiving phenobarbital or phenytoin, possibly due to induction of y-GT in liver cells by these drugs. 

Previous work on human alkaline phosphatases has utilized chromatography (ElO) and starch-gel electrophoresis. Thus in 1956 Boman and Westlund (B34) reported the purification and separation of serum phosphatases by Dowex-2 column chromatography. Moss (M34) used gel filtration on Sephadex G-200 and DEAE-celluIose chromatography for separating 5 -nucleotidase and nonspecific alkaline phosphatase activities in human sera. In most of the studies of alkaline phosphatases in human tissues of liver (M33), intestine (M34, M35), bone (M36), kidney (B46), and urine (B44, B46, B47), crude extracts of these tissues were used and... 

K23. Kowlessar, O. D., Haeffner, L. J., and Riley, E. M., Localization of serum leucine aminopeptidase, 5-nucleotidase and nonspecific alkaline phosphatase by starch-gel electrophoresis. Clinical and biochemical significance in disease states. Ann. N.Y. Acad. Sci. 94, 836-843 (1961). 

Often there is no good clinical test available to determine the exact type of hepatic lesion, short of liver biopsy. There are certain patterns of enzyme elevation that have been identified and can be helpful (Table 38-3). ° The specificity of any serum enzyme depends on the distribution of that enzyme in the body. Alkaline phosphatase is found in the bile duct epithelium, bone, and intestinal and kidney cells. 5-Nucleotidase is more specific for hepatic disease than alkaline phosphatase, because most of the body s store of 5 -nucleotidase is in the liver. Glutamate dehydrogenase is a good indicator of centrolobular necrosis because it is found primarily in centrolobular mitochondria. Most hepatic cells have extremely high concentrations of transaminases. Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) are commonly measured. Because of their high concentrations and easy liberation from the hepato-cyte cytoplasm, AST and ALT are very sensitive indicators of necrotic lesions within the liver. After an acute hepatic lesion is established, it may take weeks for these concentrations to return to normal. ... 

Patients with partial obstruction have elevated serum alkaline phosphatase and gamma-glutamyl transpeptidase. The serum of patients with clinically apparent jaundice shows increases in total and conjugated bilirubin. Alkaline phosphatase levels are increased to more than three times normal. Elevated alkaline phosphatase levels are accompanied by increases in gamma-glutamyl transpeptidase and 5 nucleotidase, usually disproportionate to serum transaminase levels. Serum aminotransferase levels usually are less than 300 lU/ml. 

At regular intervals the immune status of the patient was assessed as well as the serum content and the urinary excretion of purine- and pyrimidine metabolites according to methods described in detail previously (6,8,10). The (deoxy)ribonucleotide content of the erythrocytes was analyzed by HPLC Ul)- Perchloric acid extracts of freshly withdrawn blood were made according to Cohen et al. (2) with minor modifications. In order to analyze deoxyribo-nucleotides the neutralized perchloric acid extracts were treated with sodium periodate according to Garret and Santi (12). 2,3 Di-phosphoglycerate (2,3-DPG) in the erythrocytes was determined as previously described (13). Ecto-5 -nucleotidase on intact lymphocytes was determined as described(14). Adenosine deaminase activity of the lymphocytes was determined essentially according to van Laar-hoven et al. (15). 

Since ATP normally inhibits 5 -nucleotidase and inorganic phosphate inhibits AMP deaminase, these changes would be expected to stimulate the catabolism of AMP to inosine. This hypothesis would most readily account for the rapid rise in serum urate concentration, the increased urinary excretion of inosine, oxypurines and uric acid, and the lack of an increase in intracellular PP-ribose-P levels following fructose infusion. 

A phosphatase enzyme which catalyses the hydrolysis of phosphate from nucleoside 5 -phosphates. Increased serum levels are found in hepatobiliary diseases, together with increased alkaline phosphatase activity. The increase in 5 -nucleotidase activity is usually greater than that of alkaline phosphatase and the higher levels persist for longer. 5 -Nucleoti-dase measurements can be made in situations where the origin of a raised serum alkaline phosphatase level is sought. 

Adenosine S -monophosphate (AMP) is used as a substrate for S -nucleotidase assay. However this substrate can also be hydrolysed by nonspecific phosphatases. Nickel ions inhibit S -nucleotidase but not the nonspecific phosphatases. Serum is therefore incubated with AMP, with and without nickel ions and the amounts of inorganic phosphate liberated by the reactions are measured. The difference between the two values corresponds to the activity of 5 NT. 

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