SERUM ALKALINE PHOSPHATASE ACTIVITY AND ITS ISO-ENZYMES IN INFLAMMATORY

SYNDROMES Aurelian Udristioiu, Emergency County Hospital Targu Jiu, Clinical Laboratory, Romania.

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Different biochemical forms of alkaline phosphatase (ALP) are called iso-enzymes.  Neutrophils may be an important source of increased serum alkaline phosphatase, named neutrophil alkaline phosphatase (NAP). NAP in Leukemoid Reactions must be distinguished from other types of serum alkaline phosphatase enzyme, such as bone alkaline phosphatase (BAP) and bile alkaline phosphatase (bile-AP), increased in pathological conditions.

The present study describes the indirect evidence that high levels of ALP in serum may explain the high level of serum alkaline phosphatase by increasing BAP and NAP in patients with benign and malignant diseases of bone and inflammatory syndromes.    In the liver, hepatic alkaline phosphatase (HAP) is found on the edges of cells that join to form bile ducts that drain bile from the liver into the intestine, where it is necessary to help digest fats in the diet. HAP is increased in acute and chronic liver diseases.    When a person has high levels of serum ALP and the doctor is not sure why, for to discovery of causes, he can prescribes determination Semi-Quantitative Cyto-Chemical NAP.

• Total Serum ALP

• Normal values of serum for total ALP there are different in function of ages: 120-700 U/L for children 1-15 years, 90-190U/L for adult males and 85-165 U/L for adult females.

• Higher values, past limits of laboratory references, meet in pathology of liver as acute and chronic hepatitis, hepatic cirrhosis, biliary obstruction and stasis after 24 hours, malign processes, colangists, benign and malign diseases of bones, hyperparathyroidism, duodenal ulcer and in metastases.

• The children have higher ALP because of their bones which are growing. ALP is often very high during the “growth spurt" which occurs at different ages in males and females, eating some drugs, specially used in treat psychiatric problems.

Serum iso-enzymes ALP from Liver

• When a person has evidence of liver disease, very high ALP level can tell to doctor that the person `s bile ducts are somehow blocked. Often ALP is high to persons which have a cancer that has spread to liver or bone and doctors can further testing to see if this has happened.

• If other liver tests, such total bilirubin (TBIL), aspartate amino-transferase (AST), or alanine - amino-trasferase (ALT) are also high, usually the ALP is coming from the liver.

• In this case and enzyme gamma-glutamil-transferase (GGT), that is secreted in the same place with HAP, is high. HAP from liver represents 60% amount from total serum ALP.

• -Bile ALP• When the bile ducts are blocked, usually by gallstone or by

cancer, ALP and bilirubin may be increased more than AST or ALT. In the hepatitis, ALP usually much less elevated than AST and ALT. Also Bile-ALP is increased in cole-stasis, colangio-carcinoma, primary tumor of liver and metastases of liver.

• -Placentary ALP• The ALP levels can be increased in Pregnancy of three trimesters

and also in placentary malignancy, (ALP Reagan), by high level placentary ALP type.

• - Like placentary ALP, which can measured after warm of serum to 56 C*grade time of 10 minutes, is increased in more forms of cancers.

• The intestinal Izo-enzyme ALP• Intestinal ALP is increased in cirrhosis and blood group O or B

segregated .The “intestinal” form of ALP is determined by its phenylalanine sensitivity.

• -Duodenal ALP has normal value until 90 U/L. This type of ALP is higher in cirrhosis of liver, intra-liver colestasis, enteritis and chronic hemo-dialysis.

• Bone Alkaline Phosphatase in Serum (BAP).

• BAP is a glycoprotein that is found on the surface of osteoblast cells. BAP has been shown to be a sensitive and reliable indicator of bone metabolism. If calcium and phosphate measurements are abnormal, usually the ALP is confirmed as originally from the bones in some bone diseases such as a disorder called Paget`s disease or in cancer with bone metastasis.

Method of Measurements of BAP

• By an immune-enzymatic assay is utilized a mouse monoclonal antibody specific to BAP, paramagnetic particles coated with antibodies and complex Ag -Ac is measured with illuminometers. Normal values of BAP there are until 90 U/L, (30% of total ALP)

•

• Current Methods of Measurement of ALP from serum:

• 1. Cinetic-Colorimetric Method has as principle the activity of enzyme ALP in basic medium which p-nitrophenyl phosphate molecules. Finally results p-nitrophenol and an inorganic phosphate molecule. It use only fresh serum or plasma, delivered on heparin without hemolysis.

• Ions of magnesium accelerate activity of enzyme. Increased absorbance per units of time (yellow intense color) is proportionally with activity of alkaline phosphatase to 405 nanometers wavelength. For the type of Photometers with filters must accomplished the calibrations using a Standard.

• 2. The dry Chemistry uses the “Vitros ALKP Slide which is a multilayered analytical element coated on a polyester support.

• The p-nitrophenol which absorbs light of wavelengths in the region of 400 nm, diffuses into be underling layer of Vitros slide and it is monitored by reflectance of spectrophotometers. The rate of change in reflection density is converted to enzyme activity.

• • Reference interval of manufactures comprises the central 95% of

results from an interval study of 273 apparently healthy, adults from a working population, (154 females and 119 males) and has the normal range 36-126 U/L.ALP.

• Some metabolic components and Drugs that have significant light absorbance in the region 400 nanometers can cause a special interference (bilirubin, methotrexate, nitrofurantoin. A compilation of this information is available in the literature.

• 3. . Serum NAP ( neutrophil alkaline phosphatase) activity in serum can be measured by the method of Japanese Society of Clinical Chemistry, in which ALP its-enzymes are separated electrophoretically with Titan 3 supporting media (Helena Laboratories).

• Neutrophil Alkaline Phophatase (NAP), an enzyme closely associated with neutrophil granules, is ready measured by a quantitative chemical method or semi-quantitative histo-chemical method.

• NAP appears to be restrictive in bands of segmented neutrophil granulocytes. Its delivery from lysozome in cytoplasm is controlled by 2 genes of 21 chromosomes.

• On colored smears, in microscopic field, sites with enzymatic activity NAP will appear as dark-blue grants or black grants in cytoplasm of granulocytes.

• The interpret of reaction will be made in function of score to 100 segmented granulocytes after amounts and intensity of colored grants in scale 1-4.

• The score of Leukocyte Alkaline Phosphatase will be the product between number of counted cells and values of percent (%). Normal score are 60-100 values.

• Technical Method of Laboratory:

• Kit SIGMA: • -Sample-Fixation with solution alcohol-phenol 9/1

concentration,• -Incubation with glicero-phosphate substrate, • -Ad nitrate of cobalt 2% concentration,• -Wash slide,• -Ad solution sulfur ammonium acid Calcium black

sulfur, • -Coloration with May Grunwald Giemsa.

• DISCUSSIONS;

• In microscopy field, on smear, granulocytes display predominant toxic granulations, Dohle bodies, and in serum there is elevated NAP.

• -In mature granulocytes from CML, NAP decreases until “0”, and than appears after treatment with chemotherapy.

• -Permits the differentiation, in case of leukocytosis, between CML, where NAP is absent and PV where NAP is increased past normal interval.

• -Permits the differentiation of secondary poliglobuly, with NAP decreased or lower score with PV when NAP is very increased.

• -Can to differentiate an Acute Leukemia un-treated with cortyzol drugs:

• A) In Mieloblastic Acute Leukemia, in mature neutrophiles NAP zero or lowly.

• B) In Limphoblastic Acute Leukemia, in mature neutrophiles NAP is decreasd.

• C) In Hanry Cells Leukemia, with severe neutropenia, NAP is very high.

• In Non-Hodgkin Acute Lymphoma NAP is decreased but in Hodgkin Lymphoma has very high score.

• To children, the score LAP is increased in CML. In the Threesomya 21 –Mongolism, NAP is increased because of surplus of chromosomes.

• The enzyme NAP must be examined only in segmented and un-segmented neutrophils and eosinophils. The basophil is negatives NAP and not must be counted in the level of score ALP.

• The enzyme NAP permits the physical-pathological understanding of neutrophils which though in microscopy field appear normally but these have a modified chemical content and emphasis the functionality of leukocytes.

• CONCLUSION•

• -Direct evidence of iso-enzymes ALP is can be done by the biochemical and immunochemical properties of ALP, especially in the serum to patients with high numbers of leukocytes and we have proposed to assess laboratory results for to help clinicians in correct decisions to hospitalized patients, especially in the Point-of-Care Department.

• - We consider that this our objective must be in the attentions of all Laboratories.