Comparative Study of Serum 5' Nucleotidase,Alkaline Phosphatase , Aminotransferases and Bilirubin in Hepatpbilrary Diseases

Dr.Anil Batta

A R T I C L E I N F O A B S T R A C T

Keywords:

Original article

CholecystitisHepatitisMalignancyAlcoholc cirrhosis

1. Introduction

The liver is one of the largest and most complex organs in the body.

It stores vital energy and nutrients, manufactures proteins and

enzymes necessary for good health, protects the body from

disease, and breaks down (or metabolizes) and helps remove

harmful toxins, like alcohol, from the body. Because the liver is the

chief organ responsible for metabolizing alcohol, it is especially

vulnerable to hepatobiliary diseases related injury. Even as few as

three drinks at one time may have toxic effects on the liver when

combined with certain over–the–counter medications, such as

those containing acetaminophen. This issue of Alcohol Alert

examines the diagnosis and treatment of alcoholic liver disease (ALD), a serious and potentially fatal consequence of drinking

alcohol. Another disorder, hepatitis C, also featured here, often is

found in patients with ALD. Heavy drinking for as little as a fewdays

can lead to “fatty” liver, if drinking continues, in some patients this

inflammation eventually leads to alcoholic cirrhosis, in which

healthy liver cells are replaced by scar tissue (fibrosis), leaving the

liver unable to perform its vital functions. This finding has lead the

researchers to single out an enzyme & metabolite which is specific

& undoubtedly an early index of hepatobiliary disease [1-5].

Secondary liver cancer is a cancer that did not originate in the

liver, but originated in some other part of the body and eventually

spread to the liver. It is important to realize, however, that even

though the tumor spread to the liver, secondary liver cancer will

behave according to its origin. Prostate cancer involving the liver

will behave like prostate cancer. Secondary liver cancer is always

the result of primary cancer elsewhere in the body. Primary cancer

is caused by the body's cells continuously dividing and growing

into a lump called a tumor. If the cancer cells that make up this

tumor escape via the bloodstream and settle in the liver, it becomes

secondary liver cancer [6-9].

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International Journal of Current Biomedical and Pharmaceutical Research

Int J Cur Biomed Phar Res. 2011; 1(3): 93 -97

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Associate Professor, Baba Farid Univ. of Health Sciences, Faridkot, India.

* Corresponding Author : Dr.Anil Batta

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Differential diagnosis of liver and biliary tract depends a lot on study of various enzymes assay. The development of serum enzymology has improved a lot the ability to diagnose & to monitor the clinical course of patients & treatment of patients with Symptomatic & asymptomatic hepatobiliary diseases.For this a huge repository of enzyme assays is now available. The application of serum enzymes to the diagnosis of alcoholic hepatitis was introduced a way back in 1960s with the demonstrated usefulness of Alkaline Phosphatase assay in differential diagnosis of jaundice. A few years after that Ladue, Wroblewski & karmen showed that higher level of AST & ALT were characterized of some forms of hepatic disease led to an explosive increase of interest in serum enzyme assay as a diagnostic tool. Serum enzymology has proved to be a particular welcome addition to clinical hepatology. Of the more than 100 tests of hepatic function that have been devised none by itself has been reliable in the distinction of hepatobiliary diseases. The addition of selected enzyme tests to the roster of other procedures has enhanced the precision diagnosis. Amino transferases value is often useful in monitoring the course of acute & chronic parenchymal liver disease, though they are misleading certain circumstances (Wrobewski & La Dua, 1985). ALT is relatively specific for hepatobiliary disease.ALP activity may be increased in many diseases not associated with hepatic disease like bone, pregnancy, intestine, kidney diseases. Enzyme 5' NT was selected to observe its impact as a key enzyme for diagnosis of Hepatobiliary diseases.

Associate ProfessorBaba Farid Univ. of Health SciencesFaridkot IndiaE mail: akbattafarid@yahoo.co.in

There are several primary cancer sites from which metastatic

tumors in the liver may originate, including lung, kidney, breast,

stomach and colon. Even though there are many possible origins,

secondary liver cancer results from the spread of primary

colorectal cancer up to 50 percent of the time. In some cases, the

origin of secondary liver cancer cannot be found, even with

medical testing. The reason is that the primary tumor may be too

small to detect and is not causing any symptoms. This is

occasionally referred to as unknown primary cancer. Secondary

liver cancer has many possible origins because of the important

functions the liver performs. All of the body's blood passes

through the liver, leading to early metastasis Because the liver

filters the body's blood, it is often the first site of metastatic spread.

As a result, prognosis for secondary liver cancer does not always

mean the cancer cells have spread to other organs of the body,

making early discovery is even more important.. In malignancy the

cancer cells detection in ascitic fluid becomes significant. So there

is parenchymal damage raising the AST/ALT. Again both 5'NT and

ALP rise due to proximity of the lesion and cholestasis. Having a

liver metastasis is the most important indicator of disease

progression. Monitoring the elevation of ALP levels at each

patient's follow-up visit may be economically used as an indicator

of subsequent liver metastasis. Furthermore, a change in ALP

levels of greater than 120 U/L over a period of 4 to 6 months may

be indicative of advanced disease progression, which warrants a

more aggressive treatment or a change in regimen. ALP is a simple,

low cost, relatively sensitive screening tool for detecting liver

metastasis. Future studies aiming at better defining the role of ALP

in colon cancer is significant. Here again 5'NT has an edge due to

its relative specificity and sensitivity [10-15].

Transaminases are present in most of the tissues of the body.

They catalyze the inter conversions of the amino acids and 2-

oxacids by transfer of amino groups.Transaminases are specific

for the amino acid from which the amino group has to be

transferred to a keto acid. 2-oxoglutarate and glutamate couple

serves as one amino group acceptor and donor pair in all amino

transfer reactions. In both the reactions pyridoxal-5'-phosphate

functions as a prosthetic group in the amino transfer reactions.

Normal serum values: AST (SGOT) - 0-41 IU/L and ALT (SGPT) - 0-

45 IU/L. In newborns value up to 120 units for AST and 90 units for

ALT is considered normal.

It may increase only up to 5 fold of the normal activities. Up to 10

fold increase is seen in carcinoma of the liver. In both cirrhosis and

carcinoma activity of AST is found to be higher than the ALT. Even

though the activities of both AST and ALT are elevated in the serum

of the patients with liver diseases, ALT is more liver specific

enzyme as increased ALT activity in serum is hardly seen in tissues

other than liver cell damage [11-14].

Serum 5'NT activity is generally elevated in hepatobiliary

diseases, especially with intrahepatic obstruction, but, unlike

serum alkaline phosphatase, serum 5'NT activity is not increased

in infancy, childhood, pregnancy, or osteoblastic disorders. It can

help confirm the hepatic origin of an elevated ALP. Genetic

deficiency of erythrocyte pyrimidine 5'NT activity is a common

cause of hereditary non-spherocytic hemolytic anemia. Acquired

deficiency of erythrocyte pyrimidine 5'NT activity occurs in

patients with beta-thalassemia and lead poisoning. 5'NT activity is

low in circulating monocytes, increases markedly upon their

differentiation to tissue macrophages, and subsequently

diminishes during macrophage activation. Lymphocyte ecto-5'NT

activity, a plasma membrane marker of cell maturation, is

generally low in immunodeficiency states, and undergoes

characteristic changes in patients with certain lymphomas and

leukemias. In cancer patients, elevated serum 5'NT activity does

not always indicate hepatobiliary involvement; in some cases,

5'NT may be released into serum from the primary tumor or local

metastases [3-5].

Alkaline phosphatases are present in almost all tissues of the

body. They are membrane bound and are zinc containing

metalloenzymes. Alkaline phosphatases are a family of

isoenzymes. They hydrolyze a variety of organic phosphate esters

transferring phosphate groups from a donor substrate to an

acceptor containing a hydroxyl group. The isoforms derived from

the tissue non-specific isoenzyme by post translational

modification include the variants of the enzyme found in the liver,

bone, kidney and the placenta. Some malignant tumors can

produce a placental form of the enzyme called the Regan's

isoenzymes.

Physiological bone growth elevates ALP in serum and hence in

the sera of growing children enzyme activity is 1.5-2.5 times that

in normal adult serum. The level of ALP in the serum of women in

the third trimester of pregnancy is 2-3 times more than that of

normal level. Biliary obstruction due to any cause may elevate

ALP level by increasing its synthesis from the hepatocytes

adjacent to the biliary canaliculi. This newly synthesized ALP

enters the circulation and elevates the enzyme level in the serum.

Elevation of ALP in the serum is more with extra hepatic

obstruction by stones or by carcinoma head of pancreas than in

intrahepatic obstruction. The enzyme level may return to normal

on removal of the obstruction. Liver diseases affecting

parenchymal cells like infectious hepatitis show only moderate

elevation or normal serum ALP levels. Bone diseases with

increased osteoblastic activity shows increased ALP level in the

serum. Serum ALP activity may be increased in many diseases not

Clinical Significance

Liver diseases: Determinations of activities of AST and ALT in

serum in patients with liver diseases like viral hepatitis and other

forms of liver diseases with necrosis, give high valueseven before

the appearance of clinical signs and symptoms like jaundice.

Activity levels of 20 to 50 fold higher than normal are frequently

seen in liver cells damage but it may reach as high as 100 times in

severe damage to cells. Highest serum activities are seen between

7th and 12th days and return to normal levels by the 3rd to 5th

week. In sevre tissue damage ALTactivity is higher than AST and

the ALT:AST ratio becomes ≥1 (normally <1). Some increase in

the activities of ALT and AST are seen in extra hepatic cholestasis.

In cirrhosis the level of activities vary with the severity of the

disease.

Clinical significance

Anil Batta / Int J Cur Biomed Phar Res. 2011; 1(3): 93 -9794

associated with liver like bone diseases, pregnancy, normal growth

and occasionally the presence of malignancy not involving either

bone or liver. For this reason the tissue, the organ of tissues of origin

must be identified. Serum hepatic ALP is usually increased in the

bile duct obstruction but this increased enzyme reflects more of

enzyme synthesis rather than decreased biliary excretions or

leakage from damaged cells. On the other hand 5'Nucleotidase

(5'NT) is increased in hepatobiliary diseases doesn't usually rise in

bone diseases.

The serum activity of 5'NT is increased in cholestasis & its major

clinical value is that it may be of help in identifying the source of

elevated serum ALP activity.

But we are concerned about a marker which is significant in all

hepatobiliary diseases. In this work we explored the significance of

5'NT,ALP,AST,ALT and serum bilirubin. 5'NT is the enzyme which is

increased in hepatobiliary diseases. It is phosphatase which is

involved in release of inorganic phosphate only from the nucleoside

5' –phosphate, e.g. it acts on adenosine 5'-monophosphate (AMP) to

form adenosine and release inorganic phosphate. It is ubiquitously

present in all the tissues and is localized in the plasma membrane of

the cells in which it is present. Serum 5'NT activity is generally

elevated in hepatobiliary diseases, especially with intrahepatic

obstruction. But, unlike serum alkaline phosphatase, serum 5'NT

activity is not increased in infancy, childhood, pregnancy, or

osteoblastic disorders. It can help confirm the hepatic origin of an

elevated ALP [15-20].

years. Out of these 40 served as control taking good diet, without

H\O smoking & drinking & taking healthy diet. While 60 patients of

different diseases hepatobiliary diseases on the bases of clinical

findings were selected as follows:

1) Group 1) 21 patients suffering from Cholecystitis with

cholelithiasis.2) Group 2) 19 patents of hepatic malignancy 3) Group 3) 33 Infective Hepatitis cases 4) Group4) 27 cases suffered from alcoholic liver disease (ALD).

Jaundice : 27 cases showed as a clinical finding.

Keeping in mind the Helsinki guidelines all patients were asked

for permission. All patients attending the OPD & Indoor of Rajindra

Hospital, Patiala were selected. Study was conducted on 100

patients of different hepatobiliary disease .Age group was 20 to 60

Are expressed (Table-1& Figures) as Mean ±SEM. Stastical

significance was determined by students'' test for unpaired data.

The value significance was evaluated with 'p' values. The differences

were considered significant at p<0.001.

3. Results 2. Materials & Methods

Table 1. Statistical analysis of serum levels in study Group

About 20 ml. of blood was collected by venepuncture using all

aseptic method & allowed to clot at room temperature. It was

centrifuged at 3000 rpm for 10 minutes to separate serum.

Following methods were adopted:

Estimation of following enzymes was done by following

colorimetric methods:

1. Serum 5'—Nucleotidase—Method of Campbell 1962.2. Serum Alkaline Phosphatase –Method of kind & King 1954

using aminoantipyrine.3. Serum ALT | AST by method of Reitman & Frankel, 1957.4. Serum bilirubin by method of Malloy & Evelyn, 1937. However all the results were counterchecked so as to remove

problem of any discrepancy. These were done by fully automated

device.

2.1 Collection of Serum

95

Group and Diagnosis

I. Cholecystitis with

cholelithiasis

2.hepatic

malignancy

3.Infective

hepatitis

4.Alcoholic

liver disease

Control

Hepatobiliary

diseases

Cholecystitis

21

Hepatic

malignancy

19

Viral hepatitis

33

Alcohol

liver disease

27

40

100

2.27±4.43

(0.4--19)

5.95±6.55

(0.6—22)

8.46±4.5

(4-16)

(2.54±1.3)

4.66±5.77

(0.6-22)

52.9±5.91

(22-48)

66.88±7.16

(41-97)

53.87±13.20

(43-88)

57.53±7.32

(42-65)

19.98±2.87

(20-24)

62.66±25.87

(46-165)

50.45±12.96

(33-64)

69.34±10.98

(45-76)

65.87±9.87

(49-88)

51.12±7.8

(38-52)

20.2±3.5

16-28

67.45±8.8

(59-121)

276±34.88

(147-287)

287±26.18

(159-323)

76.90±12.98

(65-87)

198±69.5

(198-298)

130±25.12

(121-145)

(198±287)

120.9±19.1

(46-187)

52.28±13.45

(28-121)

59.34±2.98

(50-76)

50.28±13.76

(34-98)

12.9±2..12

(18-20)

49.09±87.54

(43-97)

6.34±1.01

(6.2-6.7)

5.50±0.19

(5-6.01)

7.23±1.98

(7.8-5.6)

6.34±2.56

(5.9-6.8)

7.67±0.17

(7.32)

6.12±2.5

(6.32)

2.3±0.09

(1.8-2.5)

1.23±0.65

(1.32-0.98)

1.87±0.43

(1.76-2.09)

0.98±0.02

(0.87-0.99)

2.34±0.87

(0.17-0.21)

1.12

(1.87)

Number of patients studied

Total Bilirubin mg%

Mean (Range)

AST IU/LMeanRange

ALT IU/LMeanRange

ALP KAU/LMeanRange

5'NT IU/LMeanRange

Total Protein G%Mean Range

AlbuminG%

Mean Range

Anil Batta / Int J Cur Biomed Phar Res. 2011; 1(3): 93 -97

Figure 1.

4. Discussion

5. Conclusion

Figure 2.

more helpful in ALD. Raised bilirubin is insignificant when

compared with Serum ALT| AST & 5'NT in cirrhosis or chronic ALD.

Contrary to other workers we found 5'NT is markedly significant

than ALP. There is markedly low level of serum proteins as they are

produced by liver as it is major synthesizer of proteins. Because of

this there is low blood volume which causes hypotension due to

hemodynamic disturbance. In Cholecystitis with cholelithiasis real

time Ultrasound allowed distinction between extra hepatic causes

with dilated bile ducts within the liver from intrahepatic cause.

ALT|AST were markedly raised but insignificant.

ALP is significantly increased than 5'NT as bile salts is increased

in hepatocytes & solubilize the plasma membrane.5'NT is

significantly raised in extra & intra hepatic cholestasis. Hepatic

malignancy is fourth big cause of liver cancer & is third for mortality

(Parkin 2005). In India, hepatitis B is major risk factor in third world

countries. More than 80% are due to background of cirrhosis which

causes angiogenesis & is characteristic sinusoidal capiliarization.

Increased expression of enzymes in endothelial cells along with

deposition of extra cellular matrix & micro vessel formation there

Liver is the most common site of distant metastasis due to unique

vascular architecture liver allows metastasis as it is a storehouse of

nutrients, oxygen through various mechanism like angiogenesis.

According to Barcelona clinical liver classification carcinomas are

grade 'c',tumour, stage is advanced, there are large multiple nodules

with vascular invasion & extra hepatic secondaries. Total bilirubin

& aminotransferases levels were not significantly raised. '5' NT

levels were raised significantly due to angiogenesis, total proteins &

serum albumin level were significantly decreased.

By all practical purposes liver biopsy is the best test to study

chronic liver disease as part of management. This explains severity

of liver damage & fibrosis (Bravo A Aet al, 2001).but this is hurting,

complicated, costly, invasive & full of error.Mortality rate among

them is 0.3%.

As a result of those problems & reluctances of patients to go for it

biochemical biomarkers carry an upper hand to precisely predict

the severity of liver disease (Carl.A lehmann, 2005) So this warrants

a cautious approach in interpreting results of the tests. But if taken

together the data provides reason that 5'NT is so widely raised but it

is not confined to be a marker of ethanol consumption, it's very

much useful in deciding the cause of raised ALP in liver either from

bone/liver, very much useful in differentiation of mechanical biliary

obstruction & intra hepatic cholestasis represents one of the classic

diagnostic challenge in clinical medicine. Most of clinicians consider

5' NT as a significant indicator in obstructive choleststasis

conditions: However the rise in serum 5'NT increases several days

after obstruction of biliary ductile system & may lag behind the

elevations in serum ALP.

So ultimately based on our study we conclude 5'NT is a

significant, non-invasive biochemical tool in the differential

diagnosis of all hepatobiliary diseases when it is juxtaposed with

ALP, AST, ALT and bilirubin level.

Patients were scanned of all 4 groups. There is an intra

comparison between patients with Alcoholic Liver Disease & in

cholecystitis.

The patients with ALD & cirrhosis (Group 4) compared with

patients of hepatic malignancy (Group 2) show loss of protein

causing significant loss of weight. This is explained due to reduced

adipose tissue .In viral Hepatitis (Group 3) hyperbilirubinemia is

common symptom like in many other groups of patients but it

separated hepatic malignancy (Group 3) from other Groups.

Severity of rise in ALT is more significant than serum AST in this

group. 5' NT & ALP were also somewhat significant (p<0.01).Ratio

of AST/ALT was lower than in normal individuals. In acute hepatitis

AST & ALT were more significant than 5'NT.It was also observed that

it was more significant in hepatitis B & hepatitis C as compared to

biliary canaliculi enzymes. Toxicity of alcohol is common in Indians

3.5% of global burden is due to this (Racial & Ethnic 2002) Ethanol

induced endotoxaemia & acetaldehyde which is produced by

oxidation of ethanol is more often a cause of liver damage. This is

due to free radical or sometimes by not. Conjugated levels of

bilirubin are raised much more in ALD. Deficiency of pyidoxal

phosphate is common in ALD..Ratio of AST/ALT is more in heavy

drinkers. It is a good marker of ALD. Level of 5'NT with ALP was

96Anil Batta / Int J Cur Biomed Phar Res. 2011; 1(3): 93 -97

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97

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