Enzymes: Phosphatases Flashcards
What are the general products formed when phosphatase catalyzes a reaction?
An alcohol and a phosphate ion is formed.
What reaction do phosphatases catalyze?
The hydrolysis of organic phosphate esters
Why do phosphatases have a low substrate specificity?
They can split a phosphate ion from any # of phosphate containing compounds.
What are the two phosphatases?
Alkaline phosphatase (ALP) and Acid phosphatase (ACP)
What ion is a required component of ALP?
Zinc
What is the pH optimum for ALP?
~10 but it depends on the buffer and substrate
What ions can be activators of ALP?
Magnesium, Manganese, Cobalt
What is ALP inhibited by?
Cyanide, borate, excess Zinc, and phosphate
Why is phosphate an inhibitor of ALP?
Phosphate ions are also formed in the reaction involving ALP. They act as a competitive inhibitor and can bind to the active site. (expand on this?)
How can phosphate inhibition be minimized?
Use buffers that will accept free phosphate ions
What are the buffers that can be used to stop phosphate inhibition?
DEA (Diethanolamine), TRIS (Trihydroxymethyl-aminomethane), and AMP (2-amino 2-methyl propanol)
Why can’t inert buffers be used in ALP assays?
They cannot accept phosphate ions and therefore, ALP activity is decreased.
Why can’t glycine (common enzyme assay buffer) be used for ALP assays?
It complexes with Magnesium, which is an ion needed for ALP to catalyze the reaction.
Where is ALP found?
On surfaces of most cells in the body
Where is ALP found in high concentrations?
Bone osteoblasts, liver, intestinal epithelium, kidney tubules, placenta
How do ALP isoenzymes differ from each other?
They differ depending on what tissue they originate from.
Elevations of ALP are seen in:
Hepatobiliary diseases, bone disease, malignancies
In hepatitis, amyloidosis, sarcoidosis and tuberculosis, by how much is the ALP URL increased?
<3x URL
In intrahepatic obstruction, by how much is the ALP URL increased?
2.5x the URL
In extrahepatic obstruction, by how much is the ALP URL increased?
3-10x URL
Why is ALP increased in the case of hepatic obstruction?
ALP is normally excreted by the liver and biliary system. If there is a blockage of any kind, the ALP will start backing up into the serum. The liver will also start synthesizing ALP in high amounts.
Why is ALP increased in Paget’s disease?
Bone tissue is being resorbed and in an attempt to rebuild bone, osteoblasts will increase activity, which results in increased ALP.
In the case of Paget’s disease, by how much will ALP be increased?
10-25x the URL
Why is ALP increased in hyperparathyroidism?
Increased PTH will stimulate bone resorption. If hypersecretion continues, there is a secondary increase in osteoblastic activity in an attempt to have bone reformation catch up to resorption. This results in increased ALP.
How much of an ALP increase will there be if a bone fracture is healing?
Only a transient increase
How much of an effect will hyperparathyroidism have on ALP levels?
Slight to moderate increase
What are the names of the carcinoplacental ALP isoenzymes?
Regan and Nagao
How many (percentage) cancer patients will have an increase in carcinoplacental ALP isoenzymes?
3-15% of patients
How does the Nagao ALP isoenzymes differ from the Regan isoenzyme?
Nagao is only a variant. Detected in metastatic carcinomas of pleural surfaces and adenocarcinoma of pancreas and bile duct.
In what type of cancer will ALP carcinoplacental enzymes be the highest?
Gynecological cancer
What is the preferred specimen for ALP analysis?
Serum or heparinized plasma
Why must only heparin be used for samples in ALP analysis?
Other anticoagulants will inhibit ALP activity.
How does hemolysis affect ALP analysis?
It will falsely elevate the ALP activity.
Should a patient be fasting when doing ALP analysis?
Yes
Why must a patient be fasting for ALP analysis?
Patients who have Type O or B blood and are intestinal secretors will have falsely increased ALP following a meal.
How must samples be stored if ALP analysis is delayed?
Analyze within 4 hours of collection. Levels will increase at room temp or when refrigerated.
What is the most common substrate used for ALP analysis?
4-Nitrophenylphosphate (PNPP)
What is the optimum pH for ALP analysis?
~10
At what absorbance will ALP be measured during analysis?
405 nm
What color will the end product be during ALP analysis?
Yellow
Can ALP isoenzymes be separated by electrophoresis?
Yes
What ALP isoenzyme will migrate furthest during electrophoresis?
Liver isoenzyme, followed by the bone isoenzyme and then the intestinal isoenzyme(if present).
What other ALP isoenzyme fraction may be present in metastatic liver cancer and obstructive jaundice?
Fast liver fraction, which migrates further than the normal liver isoenzyme fraction.
Why would further ALP isoenzyme differentiation be needed?
Differences in net charge can be very small and lead to a visual overlap.
What process can be done if ALP isoenzymes need to be differentiated after electrophoresis?
Differential inhibition
What needs to be done in ALP differential inhibition?
Incubate sample with neuraminidase
Why does a sample need to be incubated with neuraminidase in ALP differential inhibition?
Bone ALP is more susceptible to hydrolysis of its carbohydrate chains by neuraminidase than liver ALP is. Its net charge is decreased and mobility is less than liver ALP.
What effect does neuraminidase have on bone ALP after incubation?
Causes it to lose more sialic acid than ALP
Why would differential thermal inactivation (ALP analysis) need to be done?
To determine what type of ALP tissue isoenzyme is the most abundant
How is differential thermal inactivation (for ALP analysis) performed?
Measure ALP activity initially, incubate serum at 56 degrees Celsius for 10 minutes and measure activity again.
In differential thermal inactivation for ALP isoenzyme analysis, if <20% activity remains after incubation, what isoenzyme is predominant?
Bone ALP
In differential thermal inactivation for ALP isoenzyme analysis, if 25-55% activity remains after incubation, what isoenzyme is predominant?
Liver ALP
In differential thermal inactivation for ALP isoenzyme analysis, if >55% activity remains, what isoenzyme is predominant?
Placental ALP (Regan/Nagao) isoenzyme
Why would differential chemical inactivation need to be done? (ALP)
To differentiate between the types of ALP tissue isoenzymes
What sort of chemicals can be used to differentiate ALP isoenzymes?
phenylalanine, urea, wheat germ lectin
In differential chemical inactivation, what effect does phenylalanine have on the ALP isoenzymes?
Intestinal/placental ALP are more susceptible to inhibition by phenylalanine than bone/liver ALP
In differential chemical inactivation, what effect does urea have on ALP isoenzymes?
Bone ALP most susceptible to denaturation by urea
In differential chemical inactivation, what effect does wheat germ lectin have on ALP isoenzymes?
Wheat germ lectin has a higher affinity for bone ALP than for liver ALP, which will slow down its migration during electrophoresis.
What assay is most promising for ALP analysis?
Immunoassay, which has replaced electrophoresis in most cases
What reaction does Acid Phosphatase catalyze?
The hydrolysis of organic phosphate esters at an acidic pH
At what pH is acid phosphate catalytic activity lost?
Greater than 7.0
What is the pH optimum for acid phosphatase activity in prostate tissue?
~5.0
What is the pH optimum for acid phosphatase activity in red blood cells?
5.5-6.0
In what tissues is acid phosphatase found in?
Prostate gland, RBC, liver, spleen, platelets, bone osteoclasts
Which type of ACP is inhibited by L-tartrate? (tissue source)
Prostate gland
In what diseases is ACP elevated in?
Prostate cancer, Paget’s disease, hyperparathyroidism, bone malignancy, Gaucher’s disease, thrombocytopenia
Serum ACP activity can also be increased in what situations not related to disease?
Following prostatic massage or a biopsy
Why would elevated ACP be found in vaginal secretions?
ACP is found in high concentrations in semen, so testing for ACP can be indicative of sexual activity or rape.
Should hemolysis be avoided when collecting a sample for serum ACP analysis?
Yes, ACP is contained within RBCs normally, so hemolysis will falsely elevate the results.
At what temperature will serum ACP activity become unstable?
> 37 degrees Celsius
If kept at room temperature, what will happen to serum ACP?
It will decrease due to Carbon Dioxide being lost into the air, which will increase the pH of the sample.
What are the recommendations for serum ACP collection?
Separate serum immediately from the cells. Acidify the sample.
If separated immediately and acidified, what is the stability for serum ACP?
Stable for several hours at room temp and for up to a week when refrigerated.
What is the only difference between serum ALP and ACP measurement?
Reaction done at a different pH (Alkaline for ALP, Acidic for ACP)
What reaction does 5’-Nucleotidase (NTP) catalyze?
The hydrolysis of nucleoside-5’-phosphates (AMP)
Elevations of NTP are seen in:
Hepatobiliary obstruction and hepatitis
