Clin. Biochem. 8, 11-17 (1975) OBSERVATIONS ON THE HEAT STABILITY AND RETIC PATTERN OF ALKALINE PHOSPHATASES FROM VARIOUS TISSUES

ELECTROPHO. EXTRACTED

M. L. L E R O U X and W. F. P E R R Y 1

Dep~rtment of Clinical Chemistry, Health Sciences Centre, and Department of Biochemistry, University of Manitoba, Winnipeg, Manitoba. (Received March 18; accepted for publication May 28, 197~)

CLBIA, 8, (1) 11-17 (1975)

Cli~. Biochcm.

Leroux, M. L. and P e r r y , W. F.

Department of Clinical Chemistry, Health Sciences Centre, and Dzpartment , f Biochemistry, University of Manitoba, Winnipeg, Manitoba O B S E R V A T I O N S ON T H E H E A T S T A B I L I T Y A N D E L E C T R O P H O R E T I C PATTERN OF A L K A L I N E PHOSPHATASES EXTRACTED FI~OM VARIOUS TISSUES Various tissues were extracted with either normal saline or heat inactivated s e r u m ( H I S ) and the h e a t stability and electrophoretic migration of the alkaline phosphatase enzymes ( A P ) of the extracts were compared to the heat stability and electrophoretic properties of serum AP. The electroFhoretic p a t t e r n of H I S extracts of liver and bone differed f r o m t h a t of saline extracts but the p a t t e r n was unaffected if H I S was added to the saline extracts. The h e a t stabilities of the tissue AP also differed depending on whether t h e y were extracted with saline or H I S . However, serum A P h e a t stability can help differentiate between liver and bone disease. I t is concluded t h a t the comparison of serum and tissue A P heat stabilities or the comparison of serum and tissue A P electrophoretic p a t t e r n s as criteria for identification of the tissue source of the serum enzyme m a y be misleading since bo~h these p a r a m e t e r s v a r y , depending on the medium used for e x t r a c t i n g the tissue and the e x t r a c t ( s ) m a y contain a m i x t u r e of enzymes different f r o m t h a t in serum. I t is f u r t h e r concluded f r o m the electrophoretic studies on tissue AP t h a t the increased serum AP in patients with hepatobiliary disease w a s unlikely to be due to r e g u r g i t a t i o n of bile but due to increased synthesis and release of alpha 1 and alpha 2 A P isoenzymes f r o m liver, bile ducts or gall bladder. In patients with bone disease the increased s e r u m A P is derived f r o m bone. The source of the serum A P of " n o r m a l " subjects m a y be either liver or vascular tissue or both.

I N A PREVIOUS PUBLICATION 1 w e r e p o r t e d o u r f i n d i n g s r e g a r d i n g t h e h e a t stability and electrophoretic pattern of alkaline phosphatase in the serum of patients with bone disease and diseases of the hepatobiliary system.

1Present address: D e p a r t m e n t of Clinical Laboratories, Brandon Brandon, Manitoba. Correspondence: M. L. Leroux

General Hospital,

12

L E R O U X aT~d F E R R Y

With these techniques it was possibie to differentiate bone from liver d i s e a s e and some hepatobiliary diseases f r o m one another; however, it was not shown in this work that AP enzymes appearing in the serum actually originated in the tissues involved in the disease process. In the present communication are reported dat~ on the heat stability and electrophoretic patterns of A P enzymes extracted from various tissues together with an attempt to relate these data to those previously reported for serum. MATERIALS AND METHODS

Alkaline vhosphatase ( A P ) of serum and tissue extracts w a s determined by the method of Babson ~. The techniques used to study the A P heat stability and electrophoretic mobility h a v e been described previously 1. Tissues, (fresh a u t o p s y material, accidental death), were extracted with 0.85% sodium chlorida H u m a n adult sternum w a s cut into small pieces and rinsed with 0.85% sodium chloride to remove blood. The bone pieces w e r e 'homogenized for app r o x i m a t e l y seven m i n u t e s in 1½ vol. 0.85% NaC1 using a Virtis homogenizer. The p r e p a r a t i o n w a s spun f o r 20 minutes at 12000 g in a r e f r i g e r a t e d Sorvall ultracentrifuge. The A P of the supernatant w a s investigated as to h e a t stability and electrophoretic mobility. Adult h u m a n liver, bile ducts, gall bladder and vascular tissue (portal vein and v e n a cava) were cut into pieces and rinsed wibh 0.~5% sodium chloride to remove blood. The p r e p a r a t i o n s w e r e then homogenized for 3 minutes in 3 volumes 0.85% sodium chloride using the Virtis homogenizer and centrif u g e d f o r 20 minutes a t 1'2000 g. The A P in the supernatant w a s investigated as to heat stability and electrophoretic mobility. Tissue extracts were also which had been heated f o r w a s checked f o r residual A P to as heat-inactivated-serum

similarly p r e p a r e d using as extractants pooled serum 1 hour a t 55.5 ° to inactivate the AP. The serum pool activity p r i o r to being used. This pool will be r e f e r r e d (HIS).

Bile was used directly or diluted 1:1 with H I S . RESULTS

The heat stability and electrophoretic patterns of various tissue A P are shown in Tables 1 and 2. The use of HIS instead of saline for extraction of tissues resulted in a decrease in mean A P heat stability for extracts of bone and gall bladder, an increase in heat stability for extracts of liver and extrahepatic ducts but had little effect on bile AP. Vascular tissue extracted with HIS had a m e a n A P heat stability of 21.6±5.6%. The effect of replacing saline with HIS as extractant on the tissue A P electrophoretic pattern can be seen in Table 2. In all saline extracts of bone the A P was present at the origin and in 8 of 12 extracts as a broad band of A P between the ~2 and ~ globulin regions and in 3 of the extracts the pattern was a wide 2 globulin A P and a discrete/~ globulin AP, plus the origin band. W h e n HIS was used in place of isotonic saline the distinct band of A F in the fl globulin region, together with the a2 globulin band of AP, appeared more frequently (4 of 6 extracts).

OBSERVATIONS OF AP

13

TABLE 1 TISSUE ALKALINEPHOSPHATASEHEAT STABILITYIN SALINEANDHIS EXTRACTS Mean % enzyme actvity ( + 1 S.D.) remaining after 15 min at-55°C. The number in indicates the number of specimens tested. "" Tissue

Saline Extract i0.0 ±

Bone .............................. Liver ............................

1.9% (12)

7.0 :i: 3.7% (15)

Hepatic Ducts .......................

27.0 +

10.0% (19)

Gall Bladder ........................

57.0 +

9.0% (11)

UNTREATED

Bile. . . . . . . . . . . . . . . . . . . . . . . . . . . . .

61.0 :l: 16.6% (12)

Vascular Tissue. . . . . . . . . . . . . . . . . .

parent~sis

H I S Extract

6.7 ~

1.2% (6)

26.5 4-

7.4% (7)

30.0 ±

12.3% (8)

HIS (1:1)

58.0 -4- 13.2% (9) 21.6 ± 5.6% (5)

Saline extracts of liver when electrophoresed had, in addition to a band at the origin, only one other band of activity in the a2 globulin region. However, HIS extract of liver showed, in most cases, 2 bands of AP in the a2 globulin region and in all cases, a band of A F w a s also present in the a l globulin region. The electrophoretic p a t t e r n of AP of gall bladder, and bile were similar when either saline or HIS was used as extractant. When HIS was added to saline extracts of tissues (1:1 dilution) the electrophoretic pattern of the tissue A P was unchanged; however, the A P heat stability was altered ~ v i n g results similar to those noted with HIS extracts of tissue. DISCUSSION

In a previous paper 1 we reported heat stabilities of serum AP of 2 1 ± 5.6% for "normal" subjects, of 3 2 ± 3 . 5 % for patients with hepatic malignancy, of 1 7 ± 2 . 7 % for patients with infectious hepatitis and of 1 3 ± 5.7% for patients with bone disease. Of these the heat stability for "norreal" serum A P most closely resembles t h a t of HIS extracts of liver or vascular tissue and t h e heat stability of hepatic malignancy serum AP most closely resembles t h a t of HIS extract of gall bladder or saline extract of bile ducts. The heat stability of serum AP from infectious hepatitis and that f r o m bone disease do not appear to resemble those of any of the tissue HIS extracts; however in the case of bone disease t h e heat stability of the serum AP and the AP in saline extract of bone are similar. A t t e m p t i n g to identify tissue source of serum AF, using heat stability

14

L E R O U X a~d P E R R Y TABLE

2

ELECTR0?HORESIS OF TISSUE ALY~LINE PHOSPHATASE SALINE EXTRACT L o c a t i o n of APActivity

Tissue

BO~

LIVER

HIS EXTRACT

!

N,,mber oF Speeinene

L o c a t i o n of AF A c t i v i t y

or

1

o r , a=-B

o r , a2-B

8

o r , broad a=, B

or, broad a2, B

3

o r , a2

ii

or, al, orp a l l

~= al,

a2

or, al, GALL BLADDER

al,

az

a2

3

a2

o r , a2

1

a2

1 1

a=, B

2

or, a=, B

or, az, ~

i

al,

a2, B

1

az, 8, post B no v i s i b l e

a=l B

1

a = , B, p o s t B

1

1

bands

1

1

or

1

aI , a2

Or, a2 orl al, B~PA~C DUCT

2

1

or, al,

al,

2

a21G2*

1 1

Ntmber OF Specimens

1

a2

a~, B

a2

az

or, al,

1

1

a=

1

or, a=, B a2, B or~ al,

PORTAL VEIN Ir£NA CAVA

a2,

2 B

3

a2

1

a2

1

a=, B

1

a2, B

1

a2, B

1

az

1

no b a n d s

1

a2, B

1

UNTREATED

a l j G2

1

G I s GZ

1

or, a2, B

1

or, a2, B

1

or, a|, BILE

a2, ~

6 1

or, alp B or, al,

or, al,

a=, B

or, al,

1

post B

1

post B

1

or, ah

or, an, post B

3

or, a2, post B

or, al,

an, post ~

3

or, al,

6

B

a2, post B

h 2

or = orlgJ~ a2-B = b a n d o f AP a c t i v i t y

e x t e n d i n g fl-om t h e a2 g l o b u l i n r e g i o n

into the 8 globulin region b r o a d a2 = b r o a d b a n d o f AP a c t i v i t y

i n t h e a= g l o b u l i n r e g i o n

B = band o f AP i n t h e B globtLlin r e g i o n p o s t B = band o f AP i n t h e r e g i o n b e t v e e n t h e ~ & 7 g l o b u l i n s a= = band o f AP i n t h e a2 g l o b u l i n r e g i o n a 2 ' = s e c o n d band o1" AP i n t h e a2 g l o b u l i n r e g i o n

O B S E R V A T I O N S OF A P

15

of the serum enzyme as a criterion m a y be misleading unless the type of e x t r a c t a n t used in p r e p a r i n g the tissue extract is known and even here caution must be used. It would seem more logical to compare the serum A P heat stability to the heat stability of A P f r o m tissues extracted with HIS but even so it is difficult to relate the serum heat stabilities'to the heat stability of A P f r o m such tissue extracts. I t is probable t h a t tissue extracts contain a m i xt ur e of AP isoenzymes, in m a n y cases d i f f e r e n t in composition f r o m t h a t of serum, and the heat stability value determined will be t h a t of the enzyme mixture. The finding t h a t the addition of HIS to saline extracts of tissue had the same effect on the A P heat stability as extracting the tissue with HIS indicates t h a t the effect of the H I S is due to the constituents in it, some of t h e m being probably ions and not to HIS h a v i n g extracted f r o m the tissue either a different isoenzyme or some factor affecting the enzymes' heat stability. Addition of H I S to saline extracts of tissues did not affect the A P electrophoretic pattern. However, extraction of liver and bone with HIS, compared with saline extracts, resulted in the case of bone of a more frequent f i n d i n g of a separately defined band of A P in the fl glibulin region and in the case of liver to appearance of ~1 AP in all extracts and, as well, usually to the appearance of 2 b a n d s of AP' activity i n the ~2 globulin region. This m a y be a solubilizing effect of HIS or an enzyme stabilizing effect. All tissues studied irrespective of type of extract showed a band of n on-mi gra t i ng A P activity at the origin. On a few occasions a fain t origi.n band of A P has been seen in electrophoresed serum. The significance of this A P it not known. Likewise the significance of a distinct band of A P in the fl globulin region in some extracts of bone is not k n o w n ; we have not seen this enzyme in sera f r o m bone disease patients in which the A P occurs as a broad band between the a2 and fl globulins. We reported previously 1 t he presence of ~1 A P in the serum of some patients with bone malignancy. Since no ~1 A P was found in bone extracts, with either saline or HIS, but was present in the sera of most patients with hepatobiliary disease, it was considered t h a t the presence of this enzyme m i g h t indicate some hepatobiliary as well as bone involvement of the malignancy. Howe ve r we have recently found ~1 A P in the sera of two patients with non-malignant bone disease and with no evidence of liver disease which would suggest t h a t ~1 A P in s e r um is not necessarily an indicator of malignancy. The significance of the ~ A F in some bone disease sera remains unclear. Since we have shown ~ A P is pr e s en t in all HIS extracts of liver, liver can be r e g a r d e d as a possible ~ource of the ~1 A P seen in certain patholog-

16

L E R O U X and P E R R Y

ical sera. However, an al A P can also-be extracted (but not consistently) from gall bladder, bile ducts and bile and therefore these tissues are also other possible sources of this enzyme. Bile, bile ducts and gall bladder extra?ts showed A P in the fl globulin region as frequdntly as in the al globulin origin. Thus if the increased serum A P in patients with hepatobiliary disease is the result of bile duct obstruction with regurgitation of bile and its enzymes into the blood s, one would expect to see serum A P in the fl globulin region just as frequently as in the al globulin region. This is not the case, suggesting that the increased serum A P is not due to regurgitation of bile into the blood. The A P in the fl globulin region of tissue extracts of bone, bile ducts, gall bladder and venous tissue either does not get into the blood or is present in blood in too small an amount to be detected by our method. In patients with bone disease one would expect that the most likely source of the increased serum A P would be bone and since a relatively heat-labile AP m i g r a t i n g electrophoretically in the a2-~ globulin region is present both in bone extracts and in the sera of patients with bone disease, it would seem t h a t this is the case. Similarly in patients with hepatobiliary disease one would expect t h a t the most likely source of increased AP is the hepatobiliary system. The serum of patients with hepatobiliary disease show A P in the a2 and _al globulin regions and occasionally in the fl globulin region. The a2 and c~1 globulin AF's are likely of liver, bile duct or gall bladder source athough vascular tissue may also be a source of some a2 globulin AP. The A P in the fl globulin region may be from gall bladder, hepatic ducts, or venous tissue although some workers 4 believe that the A P in the fi globulin region is of intestinal origin. Since regurgitation of bile does not seem to be the cause of increased serum A P it would seem probable, as others 5, 6 have claimed, t h a t the increased serum A P in patients with hepatobiliary disease is due to increased synthesis and release of AP. The exact mechanism(s) for " t u r n i n g on" the a2 a n d / o r a1 A P enzymes remains to be determined. There are of course other possibilities; there may be other tissue sources, contributing A P to serum, there may be activation of inactive enzyme already present in the blood, or, a2 and a l A P m a y be so situated in or on the cell structure such that they are released into the blood but the fl A P is not. The source of the a~ globulin A P seen in normal subjects could be either liver or vascular tissue or both on the basis of the electrophoretic mobility and the enzyme heat stability in the presence of HIS. It is possible t h a t there is some a l AP present in normal serum but the activity is too low to detect using our current methodology. We are modifying our present electrophoretic technique to increase the sensitivity and have found two a2 AP bands of activity in the sera of some patients with

OBSERVATIONS OF AP

17

h e p a t o b i l i a r y disease. W e will i n v e s t i g a t e m o r e p a t h o l o g i c a l s e r a a n d also s e r u m f r o m " n o r m a l s " to see i f we a r e able to d e t e c t a l A P o r t w o b a n d s of A P a c t i v i t y in t h e u2 g l o b u l i n r e g i o n .

ACKNOWLEDGEMENT T h e w o r k r e p o r t e d in t h i s p a p e r w a s p a r t of a p r o j e c t s u p p o r t e d b y a g r a n t f r o m t h e Seller's F o u n d a t i o n , W i n n i p e g , M a n i t o b a .

REFERENCES

1. Leroux, M. & Perry, W. F. (1972). Clin. B~ochem. 5, 201-207. 2. Babson, A. L., Greely, J. J., Coleman, C. M. & Phi]lips, G. E. (1966). Clan. Chem. 12, 4~2-490. 3. Hill, P. G. & Sammons, H. G. (1967). J. Clan. Path. 20, 654-659. 4. Posen, S., Neale, F. C., Birkett, D J. & Brundenell-Woods, J. (1967). Amer. J. Clan. Path. 20, 654-659. 5. Yong, J. M. (1967). J. Cliz~. Path. 20, 647-653. 6. Kaplan, M. M. (1972). New Engl. J. Med. 286, 200-202.