BIOCHIMIE CLINICA - 7

ENZIME UTILIZATE IN DIAGNOSTIC

Sl dr Georgeta Irinel Verman

Disciplina Biochimie
Facultatea de Medicina
Universitatea “Ovidius” din Constanta
BONE DISEASES: ALP
The phosphatases, which are analysed in the clinical laboratory, are generally
phosphomonoesterases, catalyzing the hydrolysis reaction of the phosphoric esters (-
glycerophosphate, p-nitrophenylphosphate) and production of ortophosphoric acid.
R - O - PO3H2 + H2O  R - OH + H3PO4

The phosphatases are not very specific but their optimum pH for the activity is varying with the
organ of origin. From this point of view, they can be classified in:
1) Phosphomonoesterases type I (alkaline phosphatases):
the optimum pH is 9.0-10.4;
they are activated by the presence of Mg2+;
they are widely distributed in the body, being present in high concentration in bones
(osteoblasts-cells of growing bone), intestinal mucosa, renal tubule cells, and in lower
concentration in the liver (cells lining the sinusoids, bile canaliculi), leukocytes, placenta,
mammary gland; normal serum contains a mixture of isoenzymes derivated primarily from liver,
intestines, bones; the one from the liver is predominant excepting the cases of rapid skeletal
growth.
2) Phosphomonoesterases type II (acidic phosphatases):
the optimum pH is 5.0-5.5;
they are not activated by Mg2+, and are inhibited by fluorides and oxalates;
they exist in the prostate gland, kidney, spleen, platelets and erythrocytes; they have a low
concentration in plasma.
Diagnostic significance:

The values that are obtained represent the activity of more isoenzymes. The
alkaline phosphatases are eliminated by bile, except the bone fraction (it has a high
molecular mass and is catabolized by the reticulo-endothelial system cells).

Reference values:
 adults: 20 - 48 IU/L
 children: 38 - 138 IU/L
 third trimester of pregnancy: 28 - 115 IU/L

The children high values reflect the increased osteoblastic activity that occurs
during periods of rapid skeletal growth.
The physiological increased value in the third trimester of pregnancy is due to the
elaboration of a placental isoenzyme that is absorbed into the maternal
bloodstream.
After a meal rich in carbohydrates and lipids the intestinal isoenzyme is increased,
especially in the persons with B or O blood groups.
Pathological significance:

Increased activity can be noticed after treatment with drugs for epilepsy,
anticoagulant, antidiabetic, especially in women.
 In order to determine the organ origin of the increased values the isoenzymes
can be separated by electrophoresis.
 High values due to the bone isoenzyme are present in:

 bone disorders with increased osteoblastic activity - Paget’s disease

(osteitis deformans), osteoblastic tumours with metastases;
 hyperparathyroidism (mobilization of calcium and phosphorus from bone);

 deficiency of vitamin D3: rickets, osteomalacia.

Decreased values exist in:

 hypophosphatasemia, rare congenital defect;

 depressed osteoblastic activity - dwarfs;

 hypothyroidism - deficiency of thyroid hormone;

 pernicious anemia (deficiency of vitamin B12).

MUSCLE DISEASES: CK-MM, LDH, ALD, AST,
CREATINEKINASE (CPK, CK)
Creatine phosphokinase (creatinekinase) catalyzes the phosphorylation of creatine
by ATP in muscle cells and brain tissue.
Because of its role closely linked to energy production, CK reflects normal tissue
catabolism. A rise above normal in serum indicates an alteration of cells containing
a high amount of enzyme.

CK isoenzymes can be separated into three distinct molecular structures:

 CK-BB (CPK1) located mainly in brain tissue

 CK-MB (CPK2) located mainly in cardiac muscle and less in skeletal muscle

 CK-MM (CPK3) located in skeletal muscle.

Elevated CK due to skeletal muscle cell damage prevents detection of myocardial

infarction by detection of total CK. Fractionation and measurement of CK
isoenzymes replace the use of total CK for a more precise location of tissue
damage.
Reference values ​may vary depending on the method of determination
used:

 Total CK men 40-175 U/l women 25-140 U/l

 CK-BB undetectable
 CK-MB undetectable - 7 U/l
 CK-MM 5-70 U/l

Clinical implications:
 Elevated CK-MM follows the skeletal muscle damage after an injury or
muscle disease. Moderate increase is observed in hypothyroidism, a
marked increase in muscle activity caused by intense agitation.
 Total CK elevations may be due alcoholic cardiomyopathy, carbon
monoxide poisoning, severe hyperthermia, post seizure, severe
hypokalemia.
LACTATE DEHYDROGENASE (LD, LDH)

LDH catalyzes the reversible conversion of pyruvic acid into lactic acid during
anaerobic glycolysis.

Because LDH exists in almost all tissues (in high concentrations in the liver,
myocardium, kidney, skeletal muscle, erythrocytes) specific cell damage cause a
general increase in total serum LDH. This limits the diagnostic usefulness of LDH.
However, inactivation by heat or electrophoresis can identify and measure five
isoforms in particular tissues
 LDH1 and

 LDH2 occur mainly in the heart, kidney and erythrocytes

 LDH3 occurs mainly in the lungs

 LDH4 and

 LDH5 exist in liver and skeletal muscle.

Reference values:

Total LDH 48-115 U/L with the following distribution of the isoenzymes:
 LDH1 17,5-28,3 %
 LDH2 30,4-36,4 %
 LDH3 19,2-24,8 %
 LDH4 9,6-15,6 %
 LDH5 5,5-12,7 %
CARDIOVASCULAR DISEASES: CK, AST, ALT, LDH

CK
 CK-MB> 5% of total CK (or more than 10 U / l) suggest an acute
myocardial infarction (AMI).
 In IMA and after cardiac surgery CK-MB
 begins to rise after 2-4 hours,

 reaches maximum values in 12-24 hours and

 usually returns to normal within 24-48 hours. The persistence of high

values ​indicates the evolution of myocardial damage.
 Total CK following much the same pattern as CK-MB but grows a little
later. Total CK elevations may be due alcoholic cardiomyopathy, carbon
monoxide poisoning, severe hyperthermia, post seizure, severe
hypokalemia.
LDH
LDH1 and LDH2 occur mainly in the heart, kidney and erythrocytes

The wide clinical application (in combination with other cardiac enzymes) is in
the diagnosis of acute myocardial infarction (AMI). It is useful when creatine
phosphokinase (CK) was not measured in the first 24 hours of evolution of
IMA. Myocardial LDH level increases later than CK (12-48 hours after
infarction) peaking after 2-5 days and returns to normal in 7-10 days, if not
persistent tissue necrosis. The test is useful in the diagnosis of liver, lung,
erythrocyte diseases.
The increase in myocardial infarction occurs later than the one of CK and
SGOT, and is less intense; the clinical value lies in the longer period of time
(7-10 days) of persistent growth, long after the CK and SGOT returned to
normal
The isoenzyme electrophoresis is required for the diagnosis; some conditions
can maintain normal levels of total LDH but isoenzyme distribution may
change. This indicates a specific organ damage. For example, in AMI LDH1
concentration becomes higher than LDH2 in 12-48 hours of onset. This
reversal of the normal distribution is typical for AMI.
SERUM TRANSAMINASES

 The serum transaminases act at intracellular level, catalysing the transfer

reaction of the amino group (-NH2) from one -amino acid to an -keto
acid. Their activity does not manifest in the serum, so they may be
considered as non functional plasmatic enzymes.
 The transamination reaction is important in the intermediate metabolism
for the synthesis of the own amino acids using the -amino acids and -
keto acids in excess in the metabolic “pool”. Through this reaction, the
connection between the protein and carbohydrate metabolisms is
established, using the -keto acids produced in Krebs Cycle as
intermediates.
 Glutamate oxalylacetate aminotransferase (GOT) or aspartate
aminotransferase (AST) catalyzes the transfer of amino group from L-aspartic
acid to -ketoglutaric acid (2-oxoglutaric acid) with the production of
oxalylacetic acid and glutamic acid:

COOH COOH
│ │
COOH CH2 COOH CH2
│ │ GOT(AST) │ │
CH2 + CH2 CH2 + CH2
│ │ │ │
CH-NH2 C=O C=O CH-NH2
│ │ │ │
COOH COOH COOH COOH
aspartic -ketoglutaric oxalylacetic glutamic
acid acid acid acid

 GOT (AST) exists in high concentration in the myocardium and liver and in low
concentration in the skeletal muscles.
 Glutamate pyruvate aminotransferase (GPT) or alanine aminotransferase (ALT)
catalyzes the transfer of amino group from L-alanine to -ketoglutaric acid with
the production of pyruvic acid and glutamic acid.

COOH COOH
│ │
CH2 CH2
│ GPT (ALT) │
CH3 + CH2 CH3 + CH2
│ │ │ │
CH-NH2 C=O C=O CH-NH2
│ │ │ │
COOH COOH COOH COOH
L-alanine -ketoglutaric pyruvic glutamic
acid acid acid

 GPT (ALT) is predominant in the liver; its concentration is low in the myocardium
and skeletal muscles.
The coenzyme of aminotransferases is pyridoxal-5-phosphate (PALP) a derivative
of pyridoxine (vitamine B6) that acts as intermediate acceptor of amino group,
transforming into pyridoxamine-5-phosphate (PAMP).
Diagnostic significance

Reference values:
 GOT (AST):

 children younger than 3 months maximum : 40 IU

 children 3 months-5 years old: 2 - 28 IU
 adults: 2 - 20 IU
 GPT (ALT):

 children younger than 5 years: 0.2 – 13.0 IU

 adults: 2.0 - 16.5 IU
 GOT/GPT (AST/ALT) ratio (De Ritis) 1.3
Pathological significance:

 The aminotransferases are cellular enzymes; their activity in the serum is

normally decreased.
 When the tissue cells containing large amounts of these enzymes are injured or
killed, the enzymes diffuse into the blood stream, where a temporary high degree
of enzyme activity occurs. The degree of the activity depends on the extent of
the tissue damage, the prior concentration of the enzyme in the tissue and the
time course following the tissue injury.
GOT(AST) exists in high concentration in myocardium and hepatic tissue and
reduced in muscles. In the liver 60% of AST is in cytoplasm and 40% in
mitochondria. Reduced amount also exist in kidney, pancreas, lungs,
erythrocytes.
GPT(ALT) predominates in the cytoplasm of hepatocytes and a reduced
concentration in myocardium, muscles, lungs, lungs, kidneys, pancreas.
AST(GOT) serum activity is increased in:

 myocardial infarction - the activity begins to rise about 6 to 12 hours

after the infarction and usually reaches its maximum value in about 24-
48 hours; it usually returns to normal 4-6 days after the infarction; it is a
much less specific indication of the myocardial infarction than the rise in
creatinephosphokinase (CK) because many other conditions can cause
a rise of GOT (liver, muscle, hemolytic diseases);
 prolonged myocardial ischemia;
 congestive heart failure (hepatic ischemia and anoxia are produced).
ALT (GPT) is used to resolve some ambiguous increases in serum GOT in
cases of suspected myocardial infarction, when CK, CK-MB or LD
isoenzymes are not available or electrocardiogram signs are not
characteristic:
 when both GOT and GPT are elevated in serum, the liver is the primary
source of the enzymes (liver ischemia because of congestive heart failure
or other sources of liver cells injury);
 if the serum GOT (AST) is elevated while the GPT (ALT) remains within
normal limits in a case of suspected myocardial infarction, the results are
compatible with myocardial infarction.

GOT/GPT ratio (de Ritis) in myocardial infarction:

GOT is increased and GPT normal. Thus, GOT/GPT  1.3
HEMATOLOGIC DISEASES: LDH

LDH activity elevation is noticed in

 Acute myeloblastic leukemia (AML)
 Acute lymphoblastic leukemia (ALL) – markedly elevated
serum levels are more sugestive
 Chronic lymphoblastic leukemia (CLL)
 Hodgkin’s and non-Hodgkin’s disease
The elevation of LDH3 is often observed
 AFECTIUNI HEPATICE
SINDROMUL CITOLITIC
1.TRANSAMINAZELE SERICE
Numeroase studii au demonstrat ca nivelul si durata cresterii enzimelor (ALT, AST) sunt
paralele cu extinderea leziunii (adica cu modificarea permeabilitatii celulare si necroza),
corelatiile exacte nu pot fi facute in cele mai multe cazuri clinice, considerandu-se totusi ca:
- in absenta unei necroze acute sau leziuni ischemice care sa afecteze miocardul,
cresterile AST si ALT pot sugera gradul leziunii hepatice:
¨ in hepatitele virale severe (cu necroza intensa) nivelul enzimelor atinge chiar valori de
25-75 ori mai mari decat valorile normale, iar in cazul necrozelor mai putin severe
cresterea tranzitorie este de 10-25 ori
¨ in hepatitele cronice, hepatite subclinice, anicterice virale, ciroza Laennec, infiltratii
granulomatoase si invazii tumorale, cresterea transaminazelor este mai mica (de1-5 ori)
¨ in colestaza intra sau extrahepatica (in absenta necrozei celulare), nivelul AST si ALT nu
este semnificativ crescut, doar exceptional se ating valori de 7 ori mai mari decat valorile
de referinta
- determinarile repetate in (dinamica) ale AST si ALT se dovedesc deosebit de utile in
urmarirea evolutiei hepatitei cronice sau acute (pentru a se vedea eventual cronicizarea
acestora); dar interpretarea nivelului anormal trebuie sa se faca cu prudenta si in contextul
clinic.
In corpul unor bolnavi cu hepatita cu evolutie fulminanta se poate ca nivelul AST si ALT sa
scada din cauza eliminarii excesive anterioare a enzimelor din ficat. Ciroza Laennec si
postnecrotica poate fi asociata numai cu cresteri usoare sau chiar valori normale ale
transaminazelor.
Cresterile ALT (mai mult decat ale AST) reprezinta un semn precoce in hepatitele virale, aceste
determinari putand fi utilizate ca un martor al leziunii hepatocitelor si astfel, sa se poata face
o diferentiere intre afectarea hepatica si cea produsa prin colestaza.
Cresteri de 100 ori peste valoarea normala apar in :
¨ afectiuni hepatice
¨ hepatita acuta virala (inaintea aparitiei icterului);
¨ hepatita toxica (cloroform, CCl4, compusi ai fosforului).
Cresterea este precoce in hepatita acuta, deseori inainte chiar de aparitia icterului si poate fi
uneori, singurul semn al unei hepatite anicterice sau un semn precoce al unei noi perioade
de acutizare a bolii. Cresteri impresionante sunt observate in serul bolnavilor cu necroze
hepatice acute.
¨ afectiuni pancreatice
¨ pancreatita acuta,
¨ unele tipuri de tumori
Crestere moderata de 1-9 ori valoarea normala apare in:
¨ colestaza hepatica cronica
¨ ciroza (la 60-70% se constata cresteri ale AST),
¨ icterele mecanice (pana la 7-10 ori valorile normale),
¨ tumori hepatice,
¨ mononucleoza infectioasa cu interesare si hepatica.
¨ staza hepatica din insuficienta cardiaca acuta
¨ leziuni ale musculaturii (cresterile ALT sunt mai putin exprimate decat cresterea AST)
Cand ambele transaminaze au valori crescute ficatul este sursa primara de
enzima (ficatul ischemic datorita insuficientei cardiace congestive,
afectare hepatocelulara).
Bolnavii cu ictere posthepatice si colestaza intrahepatica prezinta cresteri
mai moderate ale transaminazelor.
 Un semnal biologic interesant in afectiunile hepatice, este si raportul de
Ritis, AST/ALT<1,3.
 Cand AST prezinta valori crescute iar ALT are valori normale este
posibila suspectarea prezentei unui infarct miocardic, in care raportul de
Ritis este AST/ALT>1,3
2. LACTAT DEHIDROGENAZA (LD, LDH)
 LDH catalizeaza conversia reversibila a acidului piruvic in acid
lactic. Deoarece LDH exista in aproape toate tesuturile (in
concentratii mari in ficat, miocard, rinichi, muschi scheletici,
eritrocite) lezarea specifica a celulelor determina o crestere generala
a LDH total in ser. Aceasta limiteaza utilitatea diagnostica a LDH.
Totusi, inactivarea prin caldura sau electroforeza poate identifica si
masura cinci izoenzime in anumite tesuturi:
 LDH1 si LDH2 apar in special in inima, eritrocite si rinichi
 LDH3 apare mai ales in plamani
 LDH4 si LDH5 exista in ficat si muschii scheletici.
 Cea mai larga aplicatie clinica (in asociere cu alte enzime cu origine
cardiaca) este in diagnosticul infarctului miocardic acut (IMA).
 Testul este util si in diagnosticul afectarii hepatice, pulmonare,
eritrocitare.
 3. 5'-NUCLEOTIDAZA (5'NT)
5' Nucleotidaza este o fosfomonoesteraza microzomala formata aproape in
intregime la nivelul tesuturilor hepatobiliare. Spre deosebire de fosfataza alcalina,
(enzima nespecifica), 5'NT hidrolizeaza numai gruparile 5'fosfat ale nucleozidelor
(5'- adenozin monofosfat=AMP).
Activitatea 5'NT, FA si leucin aminopeptidazei (LAP) este crescuta la pacientii cu
obstructie a tractului biliar, metastaze hepatice, hepatocarcinom.
FA si LAP cresc deasemenea la pacientii cu afectiuni osoase si la gravide.
La acestia 5'NT nu se modifica. Deci 5'NT este mai specifica pentru detectarea
disfunctiei hepatice decat FA si LAP si este utila pentru a determina daca
cresterea valorilor FA se datoreaza unei afectiuni hepatice sau osoase.

Scopul determinarii:
- pentru diagnosticul diferential intre o afectiune osoasa si una hepatobiliara daca nu
se poate detecta sursa certa a cresterii FA.
- diagnosticul diferential intre obstructia biliara si afectarea hepatocelulara acuta,
- detectarea metastazelor hepatice in absenta icterului.
Implicatii diagnostice ale 5’NT
 Valori de referinta

adulti: 2-17 UI/l (0.3-3.2 unitati Bodansky)

copii: valori mai mici
 Variatii patologice

• Cele mai inalte valori apar la pacienti cu obstructie a ductului biliar prin calculi
sau tumori. Acesti calculi sau tumori apar secundar unei cauze ce determina
colestaza intrahepatica severa, de exemplu in infiltrarea neoplazica a ficatului.
Metastazele hepatice in absenta icterului determina cresterea GGT, 5'NT si FA.
GGT este cea mai sensibila dar 5'NT confirma diagnosticul.
• Cresteri usoare sau moderate pot indica afectare acuta hepatocitara sau ciroza
hepatica activa. Atat GGT cat si 5'NT cresc in hepatita infectioasa si scad odata
cu vindecarea; o crestere ulteriara indica o exacerbare. Ambele teste sunt
sensibile si dau aceleasi informatii.
• Cresterea simultana a 5'NT si FA confirma afectarea biliara ca determinanta a
cresterii FA. Valori normale ale 5'NT cu valori mari ale FA sustin diagnosticul de
afectare osoasa (boala Paget, rahitism).
4. GLUTAMAT DEHIDROGENAZA (GLDH)

Glutamat dehidrogenaza (GLDH) este o enzima exclusiv mitocondriala.

Este utila in diferentierea intre hepatotoxicitatea acuta si hepatita virala.
Necroza celulara produsa de un toxic de exemplu halotanul la
indivizi sensibilizati, determina o crestere mult mai accentuata a
GLDH serice decat a AST sau ALT. Raspunsul invers este specific
pentru hepatita virala.

Valori de referinta: maximum 7,5 U/l la 37oC.

 5. ALDOLAZA SERICA (ALD)
Aldolaza este o enzima glicolitica care catalizeaza catabolizarea fructozei-1,6-
difosfatului in trioze-fosfat. Aceasta este una dintre reactiile importante ale
catabolismul glucozei.
Aceasta enzima este larg raspandita in cele mai multe tesuturi. ale organismului.
Testul este util pentru situatiile patologice in care exista distrugere tisulara si necroza
sau permeabilitate crescuta a membranelor, de exemplu in hepatita, atrofie
musculara progresiva, infarct miocardic, cancer.
Se recolteaza sange venos in conditii "a jeun".

Valori de referinta 1,5-12,0 /L la 37oC

Semnificatie diagnostica:
Valori crescute:
• cele mai inalte valori se intalnesc in distrofiile musculare; cresteri mai reduse in
dermatomioliza, polimiozite;
• cresteri moderate (sau valori normale) in hepatita cronica, ciroza portala, icter
obstructiv;
• se pot asocia cu: discrazii sanguine, trichineloza, delirium tremens, arsuri,
gangrena, tumori prostatice, carcinoame hepatice metastatice, 20% din pacientii
cu tumori maligne (mai frecvent cu interesare hepatica)
6. ORNITIN-CARBAMIL TRANSFERAZA (OCT)
OCT transfera o grupare carbamil de la carbamil fosfat la ornitina intr-un stadiu initial al
ciclului de formare a ureei. Aceasta reactie are loc in ficat, unde se afla localizata
aceasta enzima. Cu exceptia unei concentratii scazute in intestin, enzima este
practic absenta in alte tesuturi. Normal exista numai urme in ser astfel incat valorile
usor crescute ale activitatii OCT serice sunt specifice afectarii hepatice.
Este cel mai sensibil si cel mai specific test. Desi este mai sensibil decat GOT sau
GPT nu este un test practicat de rutina. Chiar cand este necesara o analiza de o
foarte inalta specificitate se solicita GGT sau acizii biliari.
 Scopul determinarii:
• este o metoda de electie pentru diagnosticul obstructiei acute, intermitente a ductului biliar
comun.
• este utilizat pentru diagnosticul diferential intre afectarea hepatocelulara si afectarea altor
organe deoarece exista in cantitati infime in alte organe
 Valori de referinta 1-6 U/l
 Implicatii clinice
• Valorile crescute reflecta aproape intotdeauna necroza hepatocelulara.
• Cresteri de 10-200 ori peste valoarea normala apar in hepatita virala acuta
• Cresteri moderate sau mici apar in colecistita, ciroza, icter obstructiv, carcinom metastatic
• Hepatotoxicitatea alcoolului este demonstrata de valori crescute ale OCT
• Rar, valorile crescute pot semnifica infarct intestinal intins si eliberarea masiva a enzimelor din
tesutul necrozat.
 SINDROMUL COLESTATIC
1. FOSFATAZA ALCALINA (ALP, FA)
Fosfatazele sunt monoesteraze nespecifice care catalizeaza reactia de hidroliza a esterelor fosforici (-
glicerofosfati, fenil-fosfat disodic, para-nitrofenilfosfat etc.), eliberand acidul fosforic si fenolul sau alcoolul
respectiv.
R-O-PO3H2 + H2O  R-OH + H3PO4
Desi practic manifesta aceeasi specificitate de substrat, fosfomonoesterazele se clasifica dupa pH-ul optim
de actiune in:
- fosfataze alcaline (pH=9-10,4);
- fosfataze acide (pH=5,0-6,0) sau fosfataze acide (pH=3,4-4,2)

Fosfataza alcalina:
 prezinta maximum de activitate la pH 9-10,4
 este activata de ionii de Mg2+,
 este necesara pentru hidroliza fosfatilor organici,
 este larg raspandita in oase (in osteoblasti), mucoasa intestinala, celulele tubilor renali, ficat (celulele ce
delimiteaza canaliculele biliare), celulele glandei mamare si placentei (in timpul sarcinii),
 detine un rol important in formarea tesutului osos, regenerarea tesutului hepatic, participand in diferite
reactii biochimice (metabolismul glucidelor, lipidelor, nucleoproteidelor-nucleaze), in procesele de
contractie musculara, absorbtie intestinala etc.
Determinarile de rutina efectuate in clinica masoara de fapt rezultatul activitatii mai multor izoenzime de
provenienta hepatica, osoasa, renala precum si cele eliberate din peretele intestinal sau placenta.
Izoenzima hepatica este predominanta, cu exceptia perioadelor de crestere marcata a scheletului.
Testul este un indicator al proceselor hepatice inlocuitoare de spatiu. Sunt necesare si alte teste ale functiei
hepatice pentru identificarea afectiunii.
Valori de referinta: depind de metoda de determinare.
Cand se masoara inhibarea chimica valorile depind de varsta si sex:
barbati 90-239 U/l
femei sub 45 ani 76-196 U/l
femei peste 45 ani 87-250 U/l
Metoda Bodansky: 1,5-4 unitati Bodansky/dl
Metoda King-Armstrong: 4-13,5 unitati King- Armstrong/dl
Metoda Bessey-Lowry-Brock: 0,8-2,5 unitati Bessey-Lowry-Brock/dl
Metoda SMA 1260: 30-110 U/l (0,5-1,8 Kat/l)
Sugarii, copiii si adolescentii au valori de pana la 3 ori mai mari decat adultii datorita formarii si
cresterii osoase accelerate.
In cursul sarcinii cresc valorile datorita izoenzimei placentare.
Dupa un pranz bogat in glucide sau lipide creste izoenzima intestinala, mai ales la persoanele
cu grupa sanguina B sau O
Implicatii clinice:
Valori crescute pot indica o
¨ afectiune scheletica cu activitate osteoblastica intensa (boala Paget, metastaze osoase), hiperparatiroidism,
rahitism , osteomalacie
¨ obstructie biliara intra sau extrahepatica, afectiune hepatica acuta, neoplasm hepatic, granulomatoza
hepatica, leucemie
¨ hiperfosfatazemie genetica, extrem de rara.
Cresteri moderate pot reflecta:
- obstructie biliara acuta prin inflamatie hepatocelulara in cadrul unei ciroze active, mononucleoze, hepatite
virale (util in diagnosticul diferential intre icterul obstructiv si cel hemolitic - valori normale)
- osteomalacie, rahitism carential.
Cresteri marcate pot aparea in
- obstructie biliara completa prin infiltrare maligna, inflamatorie sau fibroza,
- in boala Paget si
- ocazional in obstructie biliara, metastaze osoase intinse, hiperparatiroidism.
Metastazele osoase ale cancerului pancreatic pot determina cresterea FA fara crestere concomitenta a AST
Valori scazute apar in
- hipofosfatazemia - congenitala
- activitate osteoblastica scazuta (nanici, hipoparatiroidism, deficienta vitaminei B12 )

Valorile crescute apar in colestaza intra- si extrahepatica (creste sinteza hepatica, nu retentie); determinarea
activitatii fosfatazei alcaline este cea care permite diagnosticul diferential între icterul obstructiv si cel
hemolitic (în care valorile sunt normale); forma colestazica a hepatitei virale acute; granulomatoza
hepatica; neoplasm; ciroza; in absenta sarcinii sau unei boli osoase, cresterea nivelului seric al fosfatazei
alcaline reflecta lezarea functiei excretorii hepatice.
2. GAMA-GLUTAMILTRANSFERAZA
(GAMA-GLUTAMILTRANSPEPTIDAZA)
(-GT, GGT)

GGT catalizeaza transferul gruparii -glutamil de pe un peptid pe un alt peptid sau pe un

aminoacid:
-glutamil-peptid A + peptid B  peptid A + -glutamil-peptid B
GGT exista in special in ficat, caile biliare, pancreas, splina. rinichi, prostata si veziculele
seminale. Ficatul este sursa primara pentru activitatea serica a enzimei, chiar daca
cea mai mare concentratie a sa exista in rinichi.
Se considera ca are rol in metabolismul glutationului si in transportul aminoacizilor si
peptidelor din celula prin membrana celulara. Prin acest mecanism enzima intervine
in retroresorbtia tubulara a aminoacizilor din filtratul glomerular.
Nivelul normal este mai crescut la barbati datorita cantitatii crescute care exista in
prostata.
 Scopul determinarii:
- in evaluarea disfunctiei hepatice;
- in detectarea afectiunilor hepatice induse de consumul de alcool (GGT este foarte
sensibila la cantitatea de alcool consumata de alcoolicii cronici).
- testul (evaluarea activitatii GGT) este mult mai sensibil in detectarea icterului obstructiv,
a colecistitei decat determinarea activitatii fosfatazei alcaline sau a TGP, TGO.
 Valori normale:
Barbati: 6-28 U/L la 25oC
Femei: 4-18 U/L la 25oC
 Valoarea diagnostica a GGT consta in special in cresterea impresionanta a activitatii sale in serul
bolnavilor cu retentie biliara.
De o deosebita importanta este cresterea GGT la alcoolici (chiar atunci cand transaminazele sunt normale) sau
dupa administrarea de barbiturice. Se considera ca aceste modificari ale GGT in ser nu sunt consecinte ale
unei leziuni a membranei celulare neevoluand paralel cu modificari ale transaminazelor ci reprezinta un
rezultat al inducerii microzomale de enzima; staza biliara, alcoolul sau barbituricele stimuleaza sinteza de
enzima in ficat crescand permeabilitatea membranelor microzomale.
GGT este usor crescuta (1,5-2 ori limita superioara a normalului) si in serul subiectilor cu hipertrigliceridemie
sugerand o stimulare a proteosintezelor hepatice la acesti subiecti.
 Implicatii clinice:
Valori crescute
¨ colecistita, colelitiaza, cancer metastatic al ficatului, ciroza hepatica,
¨ pancreatita acuta, cancer al canalului biliar, alcoolism cronic;
¨ nefroza lipoidica, obstructii ale tractului biliar; consumul de barbiturice;
¨ drogurile hepatotoxice utilizate in terapia cancerului cresc nivelurile GGT mai mult decat cancerul insusi
¨ in IM nivelul GGT este de obicei normal; cu toate acestea, poate aparea o crestere la 4 zile dupa IM,
implicand eventual existenta unei disfunctii hepatice secundare unei insuficiente cardiace.
Valorile activitatii GGT sunt normale in:
¨ sarcina, hepatita neonatala
¨ afectiuni osoase
¨ afectiuni ale musculaturii scheletice
¨ afectiuni renale
PANCREATIC DISEASES: AMY, LIP
THE SERUM -AMYLASE

The amylase catalyses the specific hydrolysis of the  1-4 glycosidic bonds in the
starch and glycogen, with the formation of a series of intermediates - olygoglucides
with reducing properties.
Depending on the position of the broken bond in the glycosidic chain, the amylases
are:
 -amylase (endoamylase) -  1-4 bonds are in the middle of the chain;

 -amylase (exoamylase) - splits off the maltose units from the free nonreducing
end of the chain.
 Amylases that act in the human digestive tract are -amylases. They are
secreted by the salivary glands and pancreas and reach the gastro-
intestinal tract. They are important for the digestion of the ingested starch
(especially the pancreatic isoenzyme, because the salivary one is
inactivated in the acidic medium of the stomach). The amylose chains are
broken down into smaller fragments until the maltose is obtained. The
intermediates are called dextrines and give specific coloured products in
reaction with iodine, depending on their molecular weight:
 blue-violet colour: amylodextrines;
 red colour: erythrodextrines;
 yellow colour: flavodextrines;
 colourless: acrodextrines.
 The serum -amylase has its origin in the salivary glands, pancreas, liver (3/4 of
normal serum amylase), intestine, fat tissue. To specify the origin of the
amylases, it is necessary to determine the different isoenzymes.
 The catalytic activity of the enzymes depends on a series of factors: pH,
temperature (30-400 C), the presence or absence of specific ions.
 The optimal conditions for -amylase are: pH 7.1; 380C; the presence of Cl-
(NaCl).

To determine the serum amylase activity, the serum is reacted with the starch in
standard conditions of pH, temperature, ionic force, time. One can determine either
the amount of starch that is transformed (observing the decrease of the intensity of
the blue colour which appears normally in reaction with iodine) or the amount of the
reducing oses that appear by starch hydrolysis (slower method, influenced by the
hyperglycemia).
Diagnostic significance:
Reference values depend on the method used for the determination:
 60 - 200 Amylase Units/100 ml serum (Smith & Roe method);

 10 - 32 Wohlgemuth Units/100 ml serum (amyloclastic);

 60 - 80 Somogyi Units/100 ml serum (saccharogenic).

Pathological significance:
 Increased activity:

 considerably raised (6 - 10 times the normal) values in acute pancreatitis,

obstruction of the pancreatic ducts (stones, inflammation, cancer of the
head of pancreas) are rapid and temporary, reaching a maximum value in
about 24 hours with a return to normal in 2-3 days; in hemorrhagic
pancreatitis the values may be higher;
 mildly increased values can be noticed in the obstruction of the parotide
glands (stones) or mumps (parotiditis);
 less than 4 times the normal values:

 acute abdominal affections;peptic ulcer that has perforated the

duodenum wall;high intestinal occlusion (reabsorption);acute hepatitis
and cholecystitis;tubal pregnancy (tubal izoenzyme);after the
administration of morphine, analgetic narcotics, the values can be high
for 24 hours; the ingestion of alcohol, Azathioprin, Chlorthalidone,
Chlorothiazide, oral contraceptives can lead to falsely increased
values;renal failure may decrease the excretion;after medication with
heparin and hemodialysis the concentration is increased if the method
is with iodine-starch reaction (Pasternack-Stenman method).
 Decreased values:pancreatic atrophy;acute or chronic hepatocellular damage
(but is not a sensitive liver function test).
 In urine only those enzymes whose molecular weight is smaller than 60,000
Daltons are normally found. They are filtered through the glomerular
membrane and are not reabsorbed in the tubules.
 Examples: lysosyme (MW 11,450-14,500), amylase (MW 45,000).

Amylase, with its two isoenzymes, salivary and pancreatic is the most analysed
in the clinical laboratory.
 The increase of amylasemia determines the increase of amylasuria.

 The last one persists longer than the elevation in serum amylase activity
and can help to establish the diagnosis of acute pancreatitis.
 The urinary amylase may be elevated for 7 to 10 days, whereas the serum
amylase returns to normal in 2 or 3 days after attack.
Reference values:
8 -64 WU.
Pathological significance:
 increased values appear in:
 acute pancreatitis and persist for as long as a week after the serum amylase has returned to
normal;
 obstruction of the pancreatic ducts (stone, inflammation, compression of the common bile
duct by a cancer of the head of the pancreas);
 obstruction by a stone in the parotid duct, mumps (parotiditis).
 decreased excretion is present in:
 chronic renal disease with decreased glomerulus’s filtration;
 severe damage of hepatic cells.
LIPASE

The lipases or carboxyester hydrolases are enzymes that catalyse the

hydrolysis of carboxylic esters with the production of one molecule of
fatty acid and one of alcohol, as in the following reaction:
R - CO - O -R’ + HOH  R - COOH + R’ - OH
The lipases that act on the simple lipids exist in the pancreatic juice,
blood, lungs, kidneys, stomach.
The pancreatic lipases breakdown the triacylglycerides of the
unsaturated fatty acids with a high rate. The hydrolysis takes place in
steps with the production of diacylglycerides and monoacylglycerides and
finally simple compounds such as glycerol and fatty acids.
The activity of lipases is stimulated by the presence of activators: Ca2+
salts, bile acids, albumins.
They are inhibited by heavy metals.
Pancreatic lipase has optimum pH 8.0 and is inhibited by quinine.
Diagnostic importance:
Reference values:
0 - 0.05 IU

Pathological significance:
The concentration of serum lipase is related with the activity of the pancreas being
more specific than the amylase. In all the pancreatitis that have been verified by
surgical or necroptical diagnosis the lipasemia have been increased even if the
amylasemia could have shown normal values.
In acute pancreatitis the increase of lipase is shown but after few hours the serum
level is high for longer time, while its excretion is delayed. Its determination is
useful especially after the first 3 days when amylasemia becomes normal. That is
the reason why for the monitoring the evolution of an acute pancreatitis is indicated
to dose both enzymes.
In pancreatic cancer the hyperlipasemia is more frequently noticed than
hyperamylasemia.
Lipase and amylase may be tested in the pleural or peritoneal liquid. In acute
pancreatitis their concentration is higher than that in the serum, helping the
diagnosis.
PROSTATE CANCER: ACP

Reference values:
Total ACP Prostatic ACP
- serum male 4.7 - 13.5 IU/L maximum 3.6 IU/L
female 5.0 - 11.0 IU/L
- plasma male 1.5 - 8.6 IU/L maximum 1.0 IU/L
female 3.0 - 16.0 IU/L
Diagnostic significance
Increased concentration:
Total ACP and Prostatic ACP are increased in metastasizing carcinoma of the
prostate (more than 3-15 times the upper level of the normal); the carcinoma
has to invade blood capillaries, lymph channels, other tissues before the
elevation in the serum of ACP occurs; a discrete prostatic cancer that has not
penetrated beyond the capsule does not cause the rise in serum ACP; the
decrease of values after therapy indicates its efficiency. Massage of the
prostate increases ACP activity for 1 or 2 days.
Nonprostatic ACP is increased in sphingolipidoses (Gaucher’s disease).
Occasionally, the tartrate-inhibitable (prostatic) ACP may be elevated in some
bone diseases: Paget’s disease, female brest cancer that has metastasized to
bone.
Decreased concentration has no pathologic significance.
 Various immunotechniques (RIA, counterimmunoelectrophoresis, enzyme
immunoassay) based on a monoclonal antibody against a prostate-specific
ACP isoenzyme (PAP) are used in attempt to detect a prostatic carcinoma
before it is detectable by touch (palpable) in a rectal examination (early
prostatic carcinoma). These tests are about 1,000-fold more sensitive than
conventional activity measurements.