ACID PHOSPHATASE

ACP

it promotes the hydrolysis of a number of orthophosphate esters

there are number of substrate used but at present it has no clear and specific physiological substrate used for the enzyme

5.0 (4.8-6.0)

In ACP it has the same reactions like the ALP/Alk P by its hydrolase catalase but of different pH approximately ?

lysosomes

erythrocytes and platelets

1/3 to half of ACP

prostate gland

Seminal fluid

CSF

almost everyday body tissue contains this enzyme located at the ?

also seen in ? and most significantly seen in serum or plasma in healthy individuals from platelet turnover

MALES; ? measured in plasma comes from ? (richest source in ACP)

also seen in significant levels in ? and an important finding in rape cases

also released from specific bone cells and leukocytes but absent in ?

plasma

sample choice is ? for the reason that ACP is seen in erythrocytes (high ACP content), platelets and therefore it requires immediate separation

labile enzyme

citrate buffered

pH 6.2 - 6.6

room temperature

in a week

frozen (-20°C)

immediate assay

hemolysis

SAMPLE COLLECTION

ACP is also a ? which loses its activity if not stored at the appropriate ph and temperature

anticoagulant of choice is ? to a pH of ?

ACP is stable for a few hours at ? if left in the clot but loses its activity as soon it is separated

in refrigerator condition, little activity is lost in a ?

if ? , sample is stable for a couple of months

to avoid problems, ? should be done and ? must be avoided since ACP in erythrocytes is quite high

Prostatic carcinoma (particularly metastatic CA of the prostate gland)

important indicator for the detection of ?

Prostate Specific Cancer (PSA)

a new marker has evolved in the detection of prostatic CA such as ?

a more useful in the screening and diagnosing prostatic cancer

forensic clinical chemistry

rape cases investigation

it has become a usefool tool for ? particularly in ?

it is elevated if present in the vaginal secretions of women after any ejaculation or seminal fluid present

Substrate used

Product measured

Time of reaction

Temperature

Buffer

pH of the reaction

the units also differ in the ? (6)

P-nitrophenylphosphate

most commonly employed substrate

Hudson

sample size (0.1 ml or more)

at least 30 minutes

by ? this is rapid and nonspecific

major drawback is the ?, incubation period of ?

(yellow)

What is the color of p-nitrophenol ?

410 nm

end product read at ?

acid buffer (acetate buffer, pH 4.5-5.5)

this reaction takes place in an ? , kinetic reading would be impossible

maximum intensity at alkaline pH (10)

since the production of the yellow p-nitrophenolate will appear at ?

substrate blank

what substrate is recommended with each assay since p-nitophenolphosphate hydrolyzes slowly at acid pH, may result to elevated results

Thymolphthalein monophosphate

major reason of assaying ACP is to determine prostatic CA and thus deriving to find a specific substrate

this substrate should react only to the particular isoenzyme found in patients with the disease and not with the other ACP isoenzymes

Use of this substrate which is also used for ALP was later used in ACP

Thymolphthalein monophosphate

using this substrate shows high specificity for prostatic ACP with little hydrolysis with the other isoenzyme

hold true by comparing serum ACP activities pre and post prostatectomy

citrate buffer (pH 6.0) thymolphthalein monophosphate

0.2 ml of serum

30 min

addition of base

reaction is carried out in a ? added with a ? will be incubated for ?

addition of ? will stop the reaction and enhances the color reaction

blue chromogen

590 nm

product thymolphthalein (colorless) will be converted to a ? which has a strong absorbance of at ?

ammonium salts

stable at RT in dry form

sodium salt

stable only at refrigerated temp

fluoride, oxolates, heparin

anticoagulant inhibits the enzyme activitys

change in pH

prolonged storage at RT

low activity

storage conditions are critical since it causes ?

hemolysis

methodologies that uses the wavelength at 410 nm

HIGH LEVELS OF ACP:

age

2-3x

childhood

adolescence

adulthood

ACP levels vary according to ?

newborns has ? as that of adults

? is higher than adult levels, ? - serum or plasma ACP begins to decline, stable and stationary ACP values at ?

0.5 -1.9 U/L

using p-nitrophenylphosphate 1-12 U/L

RR

Thymolphthalein Monophosphate -

Bessey - Lowry - Brock

Electrophoresis

Column Chromatography

Isoenzymes can be identified by ? (will characterize the disease better)

approx. 100,000 - 125,000 mol wt.

native form of ACP is a dimer of ?

scialic acid (complex carbohydrates)

attached to the enzyme are a number of molecules of ?

Chemical Methods

Electrophoretic Separation

Immunoassay Method

What are the 3 assays ?

Chemical Methods

use of tartrate (ionized form of tartaric acid) wherein prostatic ACP is inhibited whereas other fractions show full activity

however this method is not specific ACP in thrombocytes, wbc's and platelets are also inhibited by tartrate

therefore this method is not specific and is also cumbersome

starch gel, polyacrylamide gel and other separation media

what media are used in the electrophoretic separation that shows up to 8 isoenzyme forms

rbc

no movement in mobility

prostate, kidney, bone and from leukemic cells

great mobility in movement

Electrophoretic Separation

on electrophoretic studies, 2 isoenzymes of prostate exist, separation through does show a clear cut fraction for good quantitation

Disadvantage includes being not easily produced and too complicated for routine clinical use

Immunoassay method

RIA and EIA

reliable methods were developed for this isoenzyme determination

both ? approaches are the best approach for prostatic ACP isoenzyme

prostatic CA

is one of the most common type of CA that develops among elderly males

stage 3

40 - 70%

involves the nearby areas in pelvic regions

survival drops at ?

stage 4

16 - 25%

metastasis to other tissues mainly lymph nodes and bone

? after 5 years

stage 1

equal

small islands of CA within the prostate

survival rate is ? to someone in normal population

stage 2

70 - 90%

nodule formation in the prostate and does not spread to other tissues

5 year survival rate is ?

12%

15%

approx 70%

60%

stage 1 shows ? elevation of serum ACP and stage 2 only ?

stage 3 ? of px studied demonstrate a normal ACP by enzyme activity measurement

Stage 4 only at this stage where significant or majority of patients ? Shows an elevated ACP

immunoassay technique

30-35%

using ?, stage 2 and 3 between 70-80% of pxs shows increase ACP, over 90% of pxs with stage 4 have elevated ACP

the difficulty lies in the result of for stage 1 ca, only ? of px are detected by immunoassay techniquee

between 5 - 10%

? of px having non prostatic CA demonstrate increased ACP with immunoassay

Vaginal swab

since ACP is seen at high concentration in seminal fluid, it is then established that ? having an elevated ACP will tell of having a recent sexual intercourse

Prostatic Cancer (Progresses as the condition progresses)

Neoplasm in Women (Breast)

Bone Diseases (Osteoporosis)

Multiple Myeloma

Pagets Disease

Gauchers Disease

Kidney Disorders (Urine ACP is elevated)

Liver Diseases

Biliary Obstruction

Elevated Enzyme Seen in Certain Conditions

Elevated in: