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Clinical Chemistry > Enzymes: Phosphatases > Flashcards

Enzymes: Phosphatases Flashcards

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0
Q

What are the general products formed when phosphatase catalyzes a reaction?

A

An alcohol and a phosphate ion is formed.

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1
Q

What reaction do phosphatases catalyze?

A

The hydrolysis of organic phosphate esters

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2
Q

Why do phosphatases have a low substrate specificity?

A

They can split a phosphate ion from any # of phosphate containing compounds.

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3
Q

What are the two phosphatases?

A

Alkaline phosphatase (ALP) and Acid phosphatase (ACP)

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4
Q

What ion is a required component of ALP?

A

Zinc

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5
Q

What is the pH optimum for ALP?

A

~10 but it depends on the buffer and substrate

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6
Q

What ions can be activators of ALP?

A

Magnesium, Manganese, Cobalt

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7
Q

What is ALP inhibited by?

A

Cyanide, borate, excess Zinc, and phosphate

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8
Q

Why is phosphate an inhibitor of ALP?

A

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?)

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9
Q

How can phosphate inhibition be minimized?

A

Use buffers that will accept free phosphate ions

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10
Q

What are the buffers that can be used to stop phosphate inhibition?

A

DEA (Diethanolamine), TRIS (Trihydroxymethyl-aminomethane), and AMP (2-amino 2-methyl propanol)

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11
Q

Why can’t inert buffers be used in ALP assays?

A

They cannot accept phosphate ions and therefore, ALP activity is decreased.

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12
Q

Why can’t glycine (common enzyme assay buffer) be used for ALP assays?

A

It complexes with Magnesium, which is an ion needed for ALP to catalyze the reaction.

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13
Q

Where is ALP found?

A

On surfaces of most cells in the body

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14
Q

Where is ALP found in high concentrations?

A

Bone osteoblasts, liver, intestinal epithelium, kidney tubules, placenta

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15
Q

How do ALP isoenzymes differ from each other?

A

They differ depending on what tissue they originate from.

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16
Q

Elevations of ALP are seen in:

A

Hepatobiliary diseases, bone disease, malignancies

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17
Q

In hepatitis, amyloidosis, sarcoidosis and tuberculosis, by how much is the ALP URL increased?

A

<3x URL

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18
Q

In intrahepatic obstruction, by how much is the ALP URL increased?

A

2.5x the URL

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19
Q

In extrahepatic obstruction, by how much is the ALP URL increased?

A

3-10x URL

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20
Q

Why is ALP increased in the case of hepatic obstruction?

A

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.

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21
Q

Why is ALP increased in Paget’s disease?

A

Bone tissue is being resorbed and in an attempt to rebuild bone, osteoblasts will increase activity, which results in increased ALP.

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22
Q

In the case of Paget’s disease, by how much will ALP be increased?

A

10-25x the URL

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23
Q

Why is ALP increased in hyperparathyroidism?

A

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.

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24
Q

How much of an ALP increase will there be if a bone fracture is healing?

A

Only a transient increase

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25
Q

How much of an effect will hyperparathyroidism have on ALP levels?

A

Slight to moderate increase

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26
Q

What are the names of the carcinoplacental ALP isoenzymes?

A

Regan and Nagao

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27
Q

How many (percentage) cancer patients will have an increase in carcinoplacental ALP isoenzymes?

A

3-15% of patients

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28
Q

How does the Nagao ALP isoenzymes differ from the Regan isoenzyme?

A

Nagao is only a variant. Detected in metastatic carcinomas of pleural surfaces and adenocarcinoma of pancreas and bile duct.

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29
Q

In what type of cancer will ALP carcinoplacental enzymes be the highest?

A

Gynecological cancer

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30
Q

What is the preferred specimen for ALP analysis?

A

Serum or heparinized plasma

31
Q

Why must only heparin be used for samples in ALP analysis?

A

Other anticoagulants will inhibit ALP activity.

32
Q

How does hemolysis affect ALP analysis?

A

It will falsely elevate the ALP activity.

33
Q

Should a patient be fasting when doing ALP analysis?

A

Yes

34
Q

Why must a patient be fasting for ALP analysis?

A

Patients who have Type O or B blood and are intestinal secretors will have falsely increased ALP following a meal.

35
Q

How must samples be stored if ALP analysis is delayed?

A

Analyze within 4 hours of collection. Levels will increase at room temp or when refrigerated.

36
Q

What is the most common substrate used for ALP analysis?

A

4-Nitrophenylphosphate (PNPP)

37
Q

What is the optimum pH for ALP analysis?

A

~10

38
Q

At what absorbance will ALP be measured during analysis?

A

405 nm

39
Q

What color will the end product be during ALP analysis?

A

Yellow

40
Q

Can ALP isoenzymes be separated by electrophoresis?

A

Yes

41
Q

What ALP isoenzyme will migrate furthest during electrophoresis?

A

Liver isoenzyme, followed by the bone isoenzyme and then the intestinal isoenzyme(if present).

42
Q

What other ALP isoenzyme fraction may be present in metastatic liver cancer and obstructive jaundice?

A

Fast liver fraction, which migrates further than the normal liver isoenzyme fraction.

43
Q

Why would further ALP isoenzyme differentiation be needed?

A

Differences in net charge can be very small and lead to a visual overlap.

44
Q

What process can be done if ALP isoenzymes need to be differentiated after electrophoresis?

A

Differential inhibition

45
Q

What needs to be done in ALP differential inhibition?

A

Incubate sample with neuraminidase

46
Q

Why does a sample need to be incubated with neuraminidase in ALP differential inhibition?

A

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.

47
Q

What effect does neuraminidase have on bone ALP after incubation?

A

Causes it to lose more sialic acid than ALP

48
Q

Why would differential thermal inactivation (ALP analysis) need to be done?

A

To determine what type of ALP tissue isoenzyme is the most abundant

49
Q

How is differential thermal inactivation (for ALP analysis) performed?

A

Measure ALP activity initially, incubate serum at 56 degrees Celsius for 10 minutes and measure activity again.

50
Q

In differential thermal inactivation for ALP isoenzyme analysis, if <20% activity remains after incubation, what isoenzyme is predominant?

A

Bone ALP

51
Q

In differential thermal inactivation for ALP isoenzyme analysis, if 25-55% activity remains after incubation, what isoenzyme is predominant?

A

Liver ALP

52
Q

In differential thermal inactivation for ALP isoenzyme analysis, if >55% activity remains, what isoenzyme is predominant?

A

Placental ALP (Regan/Nagao) isoenzyme

53
Q

Why would differential chemical inactivation need to be done? (ALP)

A

To differentiate between the types of ALP tissue isoenzymes

54
Q

What sort of chemicals can be used to differentiate ALP isoenzymes?

A

phenylalanine, urea, wheat germ lectin

55
Q

In differential chemical inactivation, what effect does phenylalanine have on the ALP isoenzymes?

A

Intestinal/placental ALP are more susceptible to inhibition by phenylalanine than bone/liver ALP

56
Q

In differential chemical inactivation, what effect does urea have on ALP isoenzymes?

A

Bone ALP most susceptible to denaturation by urea

57
Q

In differential chemical inactivation, what effect does wheat germ lectin have on ALP isoenzymes?

A

Wheat germ lectin has a higher affinity for bone ALP than for liver ALP, which will slow down its migration during electrophoresis.

58
Q

What assay is most promising for ALP analysis?

A

Immunoassay, which has replaced electrophoresis in most cases

59
Q

What reaction does Acid Phosphatase catalyze?

A

The hydrolysis of organic phosphate esters at an acidic pH

60
Q

At what pH is acid phosphate catalytic activity lost?

A

Greater than 7.0

61
Q

What is the pH optimum for acid phosphatase activity in prostate tissue?

A

~5.0

62
Q

What is the pH optimum for acid phosphatase activity in red blood cells?

A

5.5-6.0

63
Q

In what tissues is acid phosphatase found in?

A

Prostate gland, RBC, liver, spleen, platelets, bone osteoclasts

64
Q

Which type of ACP is inhibited by L-tartrate? (tissue source)

A

Prostate gland

65
Q

In what diseases is ACP elevated in?

A

Prostate cancer, Paget’s disease, hyperparathyroidism, bone malignancy, Gaucher’s disease, thrombocytopenia

66
Q

Serum ACP activity can also be increased in what situations not related to disease?

A

Following prostatic massage or a biopsy

67
Q

Why would elevated ACP be found in vaginal secretions?

A

ACP is found in high concentrations in semen, so testing for ACP can be indicative of sexual activity or rape.

68
Q

Should hemolysis be avoided when collecting a sample for serum ACP analysis?

A

Yes, ACP is contained within RBCs normally, so hemolysis will falsely elevate the results.

69
Q

At what temperature will serum ACP activity become unstable?

A

> 37 degrees Celsius

70
Q

If kept at room temperature, what will happen to serum ACP?

A

It will decrease due to Carbon Dioxide being lost into the air, which will increase the pH of the sample.

71
Q

What are the recommendations for serum ACP collection?

A

Separate serum immediately from the cells. Acidify the sample.

72
Q

If separated immediately and acidified, what is the stability for serum ACP?

A

Stable for several hours at room temp and for up to a week when refrigerated.

73
Q

What is the only difference between serum ALP and ACP measurement?

A

Reaction done at a different pH (Alkaline for ALP, Acidic for ACP)

74
Q

What reaction does 5’-Nucleotidase (NTP) catalyze?

A

The hydrolysis of nucleoside-5’-phosphates (AMP)

75
Q

Elevations of NTP are seen in:

A

Hepatobiliary obstruction and hepatitis

Clinical Chemistry (6 decks)

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