C haracterization of Serum Alkaline Phosphatase o f Hepatobiliary an d O steoblastic O rigin *

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1 A n n a l s o f C l i n i c a l L a b o r a t o r y S c i e n c e, Vol. 3, N o. 3 Copyright 1 973, Institute for Clinical Science C haracterization of Serum Alkaline Phosphatase o f Hepatobiliary an d O steoblastic O rigin * DONALD T. FORMAN, Ph.D., C. RAYMOND ZEISS, M.D., C. LAURENCE ETHERIDGE, M.D., AND SAMUEL H. GRAYSON, B.A. Departments of Pathology and Medicine, Evanston Hospital, Evanston, IL 60201, and Departments of Biochemistry, Pathology and Medicine, Northwestern University Medical School, Chicago, IL ABSTRACT A rapid and reproducible method for the electrophoretic separation and quantitation of serum alkaline phosphatase isoenzymes is described. Sera from patients with biopsy proven parenchymal liver diseases and diseases related to increased osteoblastic activity were subjected to electrophoresis using a continuous tris-borate buffer on a five percent polyacrylamide gel. Following electrophoresis, bands of alkaline phosphatase activity were located on the gels using a substrate of a-napthyl phosphate and a diazonium salt as activator. The stained gels were subsequently photographed, the relative mobility of each isoenzyme fraction measured and the transparencies scanned using an integrating, recording densitometer. This method coupled with heat inactivation of serum alkaline phosphatase provides a reliable indication of the tissue origin of this enzyme in patients with elevations of serum alkaline phosphatase of osteoblastic and/or hepatobiliary origin. In trod u ction Determinations of serum alkaline phosphatase activity are of practical clinical importance in the investigation and management of hepatobiliary and bone diseases. Considerable interest has focused on the nature and properties of the enzyme and on * Reprint requests should be sent to Donald T. Forman, Ph.D., Department of Pathology, Evanston, Hospital, Evanston, IL the possible existence of tissue specific variants or isoenzymes. In 1968, Ivor Smith et al4 showed that acrylamide gel electrophoresis could differentiate alkaline phosphatase isoenzymes which were obtained from extracts of liver, bone, kidney, and intestine. This electrophoretic method has been coupled with the selective difference in the rates of inactivation of human alkaline phosphatases on 20 1

2 202 FO R M A N, Z E IS S, E T H E R ID G E AND GRAYSON heating at 56 C1 3 in order to define better the nature of the cellular damage in a given disease process. M aterials and M ethods S p e c i m e n s Serum from patients with biopsy proven, parenchymal liver disease and serum from patients with proven osteoblastic disease were used in this study. Pooled control serum was used as a laboratory reference control in daily alkaline phosphatase determinations. Serum alkaline phosphatase activity was determined by a modification of the method of Kind and King5 and expressed as units per dl. Procedure Two tenths of a ml of serum was diluted in 9.8 ml of 15 percent sucrose. This was used as a stock sample for each patient and kept frozen until ready for use. The ideal range for measuring isoenzyme activity by gel electrophoresis was between 6 to 35 King Armstrong units per dl, and 200 /xl of this stock sample was applied onto the running gel. For serum enzyme activity greater than 35 King Armstrong units, the stock sample was further diluted (usually 1 :4 ) with 15 percent sucrose, and 100 /*1 or 200 jtil used for electrophoresis. Dilution of high activity sera was necessary because the bands of alkaline phosphatase activity which developed on the gel after electrophoresis were too opaque for accurate densitometry. H e a t I n a c t iv a t io n An aliquot of the stock sample was incubated at 56 C ± 0.1 for exactly thirty minutes in a constant temperature waterbath. Heated and unheated samples were then subjected to gel electrophoresis and their activities compared. A c r y l a m id e G e l E l e c t r o p h o r e s is The method of Smith4 was followed and Canalco Model 12 electrophoresis apparatus was employed. Siliconized glass tubes 6 cm in length and 0.6 mm I.D. were used as gel holders. One ml of running gel was added to each tube. A sample or spacer gel was not used. The sample in 15 percent sucrose was directly applied onto the running gel. A current of 1.5 ma per gel was applied over a period of thirty minutes. At the end of this time, a bromphenol blue marker, which complexes with albumin, had run approximately 75 percent of the way down the gel. The gels were removed from the glass tubes and cut at the albuminbromphenol blue marker. The section from the origin to the marker was then saved for protein and enzyme location by staining. The length of the stained gel also served as a convenient reference point for relative mobility studies. B u f f e r s a n d R u n n in g G e l Buffers and five percent acrylamide running gel were made as outlined by Smith.4 A tris-borate buffer at a ph of 9.5 was used. All solutions were stored at 4 C until needed. Electrophoresis was carried out at room temperature. L o c a t io n o f A l k a l in e P h o s p h a t a s e A c t iv it y o n t h e G e l The reaction buffer and substrate were made employing the method of Taswell.5 The buffer was a tris-malate at a ph of 9.8. Sodium a-naphthyl phosphate was used as substrate and 4-amino diphenylamine-diazonium sulfate was used as the coupler. Each gel was placed in a tube containing 7 ml of the reagent mixture. The reaction was allowed to proceed for one hour at 4 C. Areas of enzyme activity showed as compact bands of a dark brown color on a yellow background. The yellow background cleared rapidly in a 7 percent acetic acid

3 C H A R A C T E R IZ A T IO N O F S E R U M A L K A L IN E PH O SP H A T A SE 203 c - Bone AP Unheated A - Liver AP Unheated 0 - Bone AP Heated Liver AP Heated F i g u r e la. Liver alkaline phosphatase unheated, (lb) liver alkaline phosphatase heated, ( lc ) bone alkaline phosphatase unheated and (Id) bone alkaline phosphatase heated. Note sharp liver band and broad diffuse bone band. wash. The gels were then photographed and stored at 4 C in small glass tubes con taining distilled water. P h o to gbaph y and to obtain (1 ) relative density of each alka line phosphatase band before and after heating, (2 ) relative mobilities of the band and (3 ) band character. S c a n n in g The stained gels were photographed in 7.5 cm X 1 cm storage tubes filled with dis tilled water. A Polaroid MP-3 Land camera with a 127 mm lens and employing No. 146 L film was used. Four gel tubes were placed side by side on a light source and photographed. The resulting transparency of each stained gel was scanned and band density determined using a Beckman Microzome Densitometer Model R-110. This pro vides a permanent record and allows one Results In figure 1 is shown a characteristic serum alkaline phosphatase pattern from two pa tients, one with biopsy proven hepatitis (figures la and lb ). The other had meta static breast carcinoma to bone with a nega tive liver scan and biopsy (figures lc and Id ). Polaroid transparencies and densi tometer tracings demonstrate that the char acteristic features of liver alkaline phospha tase are (1 ) a narrow sharply defined band

4 204 FORM AN, ZEISS, ETHERIDGE AND GRAYSON F ig u r e 2. Effect o f heat inactivation at 56 C on serum alkaline phosphatase activity in normal subjects, and subjects with hepatic and skeletal disease. MINUTES AT 56 C of enzyme activity, (2) relative heat stability and (3) a mobility of between 54 to 58 mm. Characteristic features of bone alkaline phosphatase (figures lc and Id ) are (1) a diffuse broad band, (2) marked inactivation by heat and ( 3 ) a mobility of between 50 to 54 mm. TABLE I V a l u e s o f R e s i d u a l S e r u m A l k a l i n e P h o s p h a t a s e A c t i v i t y P e r c e n t a g e s O b t a i n e d Number of Patients b y H e a t I n a c t i v a t i o n T e s t Diagnosis Residual Serum Alkaline Phosphatase* Activity (Mean ± S.D.) % 51 Hepatic disease 34.5 ± Skeletal disease 10.6 ± Mixed hepatic and 26.1 ± 8.7 skeletal disease 11 Intestinal disease 52.6 ± 11.9 * Serum alkaline phosphatase remaining after heating at 56 C for 15 minutes. Results of the heat inactivation of bone and hepatic alkaline phosphatase are shown in figure 2. It can be seen that alkaline phosphatase from skeletal origin is rapidly inactivated (56 C) at an exponential rate; half the enzyme disappearing in 5.6 ± 1.5 minutes. In the case of hepatic alkaline phosphatase, heat inactivation occurs at a much slower rate, half the enzyme disappearing in 12 to 15 minutes. Intestinal alkaline phosphatase is quite stable and placental alkaline phosphatase almost completely heat stable. Mixtures of hepatic and bone phosphatases usually gave a normal heat inactivation pattern. Sera in the socalled osteoblastic group showed alkaline phosphatase heat inactivation (56 C, 15 min) to less than 20 percent of original activity and hepatic sera to greater than 34 percent of original activity. Intestinal alkaline phosphatase showed greater than 52 percent of its activity remaining in serum after heat inactivation. The heat inactivation pattern of subjects with both osteoblastic and hepatic diseases

5 205 C H A R A C TE R IZA TIO N O F SE R U M A LK A LIN E PH O SPH A T A SE remained in an intermediate position be tween that of the rapidly inactivated serum of subjects with increased osteoblastic ac tivity' and the slowly inactivated serum of patients with hepatic disease ( table I ). The heat inactivation pattern of alkaline phos phatase in normal serum also lies in an intermediate position between the serum of subjects with increased osteoblastic ac tivity and the serum of patients with he patic disease.4 Normal serum seemed to be have like a mixture containing 50 to 60 per cent skeletal isoenzyme.3 The determina tion of alkaline phosphatase isoenzymes in normal subjects by electrophoretic and C - Bone and Liver AP Mixed d 0 - Bone and Liver AP Mixed Heated F i g u r e 3 c. Bone and liver alkaline phosphatase mixed and ( 3d) bone and liver alkaline phospha tase mixed and heated. Note persistence of liver alkaline phosphatase after heating in combination experiment. TABLE II R e l a t iv e P M o b il it ie s h o sph ata se op S erum A I so en zy m es* l k a l in e Source R f X 100 Hepatitis Serum (liver) Carcinoma Serum (bone) Liver and Bone Mixed Serum Before Heat Inactivation * On 5 percent acrylamide running gel in trisborate buffer at a ph of 9.5, 1.5 milliamps per tube for 30 minutes. thermostability techniques should be studied further before their significance is reported. Only serums with significantly elevated serum alkaline phosphatase levels should be characterized by our procedure. Serum with elevated bone alkaline phos phatase was also mixed with serum contain ing liver alkaline phosphatase. This com bination was subjected to electrophoresis and stained and scanned in the usual man ner. This study was carried out in order to determine if one could characterize serum containing increased bone and liver iso enzymes. The results are shown in Figures 3c and 3d. These patterns demonstrate (1) a band with characteristics between those of liver and bone alkaline phosphatase and (2) bone alkaline phosphatase activity greatly reduced on heating, yielding a nar row band characteristic of liver alkaline phosphatase. Thus, this method is capable of separating liver from bone alkaline phosphatase on the basis of their relative mobilities after heat inactivation. Rf values were calculated by the ratio of the distance from the origin of the run to the band center ( or peak heights on densitometer tracings) in centimeters to gel length in centimeters. In table II is shown the relative mobilities of the alkaline phosphatase bands shown in figures 1 and 3. D iscussion The identification of alkaline phosphatase isoenzymes of different tissue origin in hu

6 206 FORM AN, ZEISS, ETHEBIDGE AND GRAYSON man serum is of important diagnostic significance. The results presented in this study seem to imply that heat inactivation and gel electrophoresis can provide a reliable parameter for measuring the presence of serum alkaline phosphatase activity of skeletal or hepatic origin. Using the method described it has been possible to identify liver or bone alkaline phosphatase in pathological serum. Qualitative identification of the tissue of origin can be accomplished by a visual comparison of the stained isoenzyme in gels of both heated and unheated specimens. When serum contains alkaline phosphatases of both skeletal and hepatic origin, heat inactivation clearly removes the broad bone band leaving a sharp residual liver band. This combined procedure can provide rapid and reliable information concerning the tissue origin of an elevated serum alkaline phosphatase. References 1. F i t z g e r a l d, M. S., F e n n e l l y, J. J., a n d Mc- G e e n e y, K.: The value of differential alkaline phosphatase thermostability in clinical diagnosis. Amer. J. Clin. Path. 51: , M a r s h, W. H., F i n g e r h u t, B., a n d K ir s c h, E.: Adaptation of an alkaline phosphatase method for automatic colorimetric analysis. Clin. Chem. 5: , P o s e n, S., N e a l e, F., a n d C l u b b, J. S.: Heat inactivation in the study of human alkaline phosphatases. Ann. Int. Med. 62: , S m i t h, I., L ig h t s t o n e, P., a n d P e r r y, J.: Separation of human tissue alkaline phosphatases by electrophoresis on acrylamide gels. Clin. Chim. Acta 19: , T a s w e l l, H. F. a n d J e f f e r i e s, D. M.: Separation of bone and liver alkaline phosphatase on starch gel. Amer. J. Clin. Path. 40: , Nothing is more simple than greatness; indeed, to be simple is to be great. Miscellanies: Literary Ethics Emerson