PROTEIN CHARACTERISTICS OF SERUM AND BILE ALKALINE PHOSPHATASE

Transcription

1 GASTROENTEROLOGY Copyright 1966 by The Williams & Wilkins Co. Vol. 50, No.5 Printed in U.S.A.. PROTEN CHARACTERSTCS OF SERUM AND BLE ALKALNE PHOSPHATASE CHARLES E. POPE,, M.D., AND SDNEY R. COOPERBAND, M.D. Evans Memorial Department of Clinical Research, University Hospitals; the Vand V Medical Services, Boston City Hospital; and the Department of Medicine, Boston University School of Medicine, Boston. Massach7lsetts Although considerable attention has been directed to the source of the enzyme alkaline phosphatase found in serum, there has been little investigation to determine the origin of the alkaline phosphatase found in bile. f, as is usually assumed,! the alkaline phosphatase activity of bile is simply derived from serum, both serum and bile alkaline phosphatase might be expected to have similar protein characteristics. We have applied two methods of protein characterization, starch gel electrophoresis and ultracentrifugation in sucrose density gradients,to determine whether the enzymatic alkaline phosphatase proteins found in serum and in bile are identical. Our studies indicate that the alkaline phosphatase proteins found in serum and in bile are similar in molecular size, but show different electrophoretic distribution patterns in starch gel. These observations support the suggestion that bile alkaline phosphatase is not directly or solely derived from the serum enzyme, hence the biliary system does not represent a primary path of excretion for this protein. Received November 29, Accepted December 31, Address requests for reprints to: Dr. Sidney R. Cooperband, 15 Stoughton Street, Boston, Massachusetts This investigation was supported in part by Public H ealth Service Grant AM , from the National nstitute of Arthritis and Metabolic Diseases, and by Public Health Service Direct Traineeship AT-571 (for C. E. P.). We would like to thank Drs. B. E. Kodsi and. A. D. Bouchier and the Surgical Services of the Boston University Medical Center for help in obtaining the bile specimens. We appreciate Dr. Franz J. ngelfinger's critical assistance in these studies and his review of this manuscript. 631 Materials and Methods Normal bile was obtained from the gall bladders of 15 patients undergoing laparotomy for diseases unrelated to the hepatobiliary tree. Abnormal bile was obtained from 31 patients with cholelithiasis or inflammatory disease of the hepatobiliary system. Specimens were obtained by direct needle puncture of the gall bladder shortly after opening the abdomen and before manipulation of the biliary tree. Specimens were analyzed either immediately or after storage at -20 C. Storage at this temperature for 6 months, and repeated freezing and thawing of the specimens, did not change the isozyme pattern, although the total enzyme activity tended to decrease with the age of the specimen. Only fresh specimens were utilized for ultracentrifugal analysis. Sera from 60 normal patients and 6 patients with surgically proven obstructive jaundice were obtained and stored at -20 C before analysis. The isozyme pattern of each specimen was determined by horizontal starch gel electrophoresis in M tris(hydroxymethyl)aminomethane (Tris) citrate buffer at ph After electrophoresis the gel was divided horizontally and one-half was stained for alkaline phosphatase activity according to Boyer: The other half of each gel was stained for protein with Amido black lob stain." Serum specimens from normal donors or the patients with obstructive jaundice were run simultaneously with each set of bile specimens analyzed. The location of alkaline phosphatase activity in bile was correlated with the location of the alkaline phosphatase activity of the serum, and also with the protein pattern of the serum as determined by the Amido black lob stain. Alkaline phosphatase was measured chemically in the bile and sera by the method of King and Wootton.' The sedimentation characteristics of alkaline phophatase from eight biles and four sera were examined by preparative ultracentrifugation (Spinco model L) in sucrose density gradients. All four sera analyzed contained high

2 632 POPE AND COOPERBAND Vol. 50, No.5 SERUM PROTEN DSTRBUTKlN Normal serum SOZYMES OF ALKALNE PHOSPHATASE FOUND N HUMAN BLE AND SERUM oriqin ~ -0 0 DDODOD DO 00 DOD ". 1'- ~ ' HoploOlobins 4 fjfo.t PO'- Alb. lip. a-2 olb. alb. w V> ~ Obstructi\le :: jooodice serum "- V> 0 :: "- w Normal bile z :J "" --' "" Abnormal bile,, SOZYME NUMBER p ~ - FG. 1. Electrophoretic distribution of alkaline phosphatase isozymes from normal serum and serum from patients with obstructive jaundice; from normal bile and bile from patients with abnormalities of the hepatobiliary system. These are correlated with electrophoretic mobility of the serum proteins. -The different isozymes are numbered by their relative anodal mobility. levels of alkaline phosphatase due to biliary obstruction. A slight modification of the method of Edelman et a1. 5 was used. A linear gradient ranging from 10% to 40% sucrose in 0.01 M phosphate-buffered saline (0.14 M NaC), ph 7.4, was constructed in the Spinco SW -39 swinging bucket head, and 0.5 ml of bile or serum was layered on top. The tubes were then spun for 24 hr at 39,000 rpm. These tubes were fractionated by drop collecting from the bottom. Ten drop fractions were generally obtained. Alkaline phosphatase activity and optical density at 280 m,u were determined for each fraction. Alkaline phosphatase was determined by using an identical aliquot of each fraction for a colorimetric blank. One serum and two biles were generally run simultaneously for each experiment. Results Figure 1 shows the general isozyme patterns for alkaline phosphatase found in normal and abnormal bile, and in serum from normal subjects and patients with obstructive jaundice. The enzyme distribution was related to the electrophoretic distribution of the serum proteins. There were five isozymes demonstrable in our studies with mobilities that ranged between that of the serum y- and,b-globulins. We have numbered the isozymes in a manner analogous to the numbering used for lactic + dehydrogenase isozymes. 6 The isozyme which migrated most rapidly toward the anode was given the number 1. The isozyme which barely moved toward the anode was no. 5. sozymes 3, 4, and 5 are seen as accentuations in a diffuse staining reaction in the region between the serum y-globulin and slow Y2-globulin. sozyme 5, which is very near the origin, showed some evidence of splitting in approximately one-third of the biles studied. n 60 normal sera, alkaline phosphatase showed only one isozyme band, no. 1. Sera from patients with obstructive jaundice showed additional bands with mobilities similar to isoenzymes 3, 4, and 5 of bile alkaline phosphatase. These serum patterns are similar to those described by Chiandussi et aj.7 Bile demonstrated some variation in isozyme patterns. Figure 2 is a compilation of the isozyme patterns seen in all the biles studied. The predominant isozyme found in normal and pathological bile is no. 5. n one-third of the normal biles, isozymes 2, 3, and 4 were also present. All possible combinations of these types were seen. sozyme 1 was demonstrated in only 1 of 15 normal biles. Abnormal bile, obtained from actively inflamed gall bladders, differed only in having a higher incidence of isozyme 1. We have studied three patients from whom both gall bladder and subsequent T-tube bile was available. We could detect no change in the bile isozyme pattern of these patients following cholecystectomy, All sera demonstrated the usual protein pattern on starch gel. n contrast, normal bile did not show a staining reaction for protein. n 13 abnormal biles, particularly in bile removed from acutely inflamed gallbladders, a faint protein band with the same mobility as serum albumin was seen. The demonstrated multiplicity in isozyme pattern in bile might be due to chemical alteration of the serum enzymes by constituents in bile. To determine whether this could occur, normal serum and serum with elevated alkaline phosphatase levels were mixed with varying quantities of normal bile and then incubated for 4 to 24 hr at 4 C and 37 C. The normal biles did not

3 May 1966 SERUM AND BLE ALKALNE PHOSPHATASE 633 NORMAL BLE D ABNORMAL BLE SOENZYME NUMBER FG. 2. Summary of the various isozymes seen in 15 normal biles and 31 biles from patients with abnormalities of the hepatobiliary system. 2 contain any isozyme 1. The mixtures were then subjected to electrophoresis. n all cases the serum isozyme (no. 1) retained its original mobility in the,b-globulin range, and did not appear to lose any enzyme activity. n no case was the serum enzyme pattern made to resemble any of the bile alkaline phosphatase patterns by this treatment. The electrophoretic differences between bile and serum alkaline phosphatase could be due to partial destruction or alteration of the serum enzyme in passage through the liver ces. Cleavage or hydrolysis of the serum enzyme would alter the size and ultracentrifugation characteristics of this protein. Preparative density gradient ultracentrifugation studies were therefore undertaken to determine whether the bile enzymes sedimented differently than serum enzymes. Figure 3 is a representative experiment. Both normal bile and icteric serum were fractionated in a sucrose density gradient, and the distribution of enzyme was determined. The enzyme positions were com""' pared with the general serum protein pattern distribution as determined by the optical density determinations at 280 mp.. The bile specimens were also read at 280 mp', but in these samples the light absorption was primarily due to bilirubin pigments. No major difference in the sedimentation characteristics of the enzymes was observed among four normal biles, four abnormal biles, and four sera. Those enzymes present in bile and serum all sedimented in the range of 4 to 10 S. Representative tubes from the fastest, median, and slowest sedimenting region of the alkaline phosphatase peak were subjected to electrophoresis in starch gel. All of these fractions showed isozyme patterns identical with those of the original sample. Discussion Although hepatobiliary excretion has been assumed to be the major pathway for the removal of serum alkaline phosphatase, results of experiments designed to test this hypothesis have not agreed. Dalgaard,8 using biliary fistula dogs, found that serum and bile alkaline phosphatase were inversely correlated, and concluded that the bile alkaline phosphatase was derived from the serum. Le Veen et aj.9 felt that they could quantitatively recover intravenously injected alkaline phosphatase activity from

4 634 POPE AND COOPERBAND Vol. 50, No.5 2.' SERUM --OPTCAL DENSTY , ALKAUNE PHOSPHATASE BLE 1.5 4S d',,,",,, \,'",,0..., ',,, ~ ' \' } ~ 4 a -- 34' BOTTOM OF TOP OF BOTTOM or TOP OF GRAO[NT GtAOfENT GRADENT GRADEN'r FRACTON NUMBER FG. 3. Ultracentrifuge distribution of alkaline phosphatase in the serum from a patient with obstructive jaundice (left) and from normal bile (right). The sedimentation and approximate S values of the serum protein as determined by optical density at 280 m", are given for comparison. "," the bile of dogs. Sterling and Winsten/o however, studying human subjects with T tube drainage of the bile duct, found no correlation between serum and bile levels of alkaline phosphatase. Polin et alp drew the same conclusion in experimental animals. n addition, Frankl and Merritt,2 using dogs, found no evidence for serumbile transfer of other enzymes such as glutamic oxaloacetic transaminase, glutamic pyruvic transaminase, and lactic dehydrogenase. The alkaline phosphatase found in bile could be entirely derived from the cells of the liver. Alkaline phospha.tase activity has been demonstrated in granules, both in the parenchymal cells of the liver and to a lesser extent in the biliary ductular epithelium Although this activity could represent alkaline phosphatase derived from the serum, recent work by Sebesta et aj.5 has shown that the isolated perfused cat liver is capable of synthesizing the alkaline phosphatase in the bile recovered from his preparations. Proof of transfer of the serum alkaline phosphatase into the bile requires that the serum alkaline phosphatase have some unique recognizable characteristic. Determination of a unique isozyme pattern is one such characteristic. sozymes are subfractions of a measurable enzyme which have the s a substrate m ~ specificity but, as defined by the nature of their separation, different protein characteristics. 6 The electrophoretic mobility and relative distribution of isoenzyme activity had been demonstrated to be characteristic for the tissue from which these originated. 6 Our results show a difference between the isozyme patterns of bile and those of serum. n only one of the normal biles did we demonstrate a faint band corresponding to the normal serum pattern (isozyme 1). Conversely, the bands found in bile are not found in normal serum. The presence of the no. 1 isozyme ("serum" isozyme) in some of the abnormal biles might be explained on the basis of increased permeability of the inflamed gall bladder wall to serum proteins and enzymes. This suggestion is strengthened by

5 May 1966 SERUM AND BLE ALKALNE PHOSPHATASE 635 the finding that protein with mobility of serum albumin was always present in abnormal biles containing isoenzyme 1. Earlier investigations comparing the alkaline phosphatase isozymes in serum and bile have been conflicting. Rosenberg,17 using a starch block technique, did not demonstrate a marked electrophoretic difference between the enzymes present in bile and in serum. His technique of detecting isozymes, however, required elution from the starch block; his recoveries from bile were only 20% of the activity added, which might have been responsible for his failure to detect a difference. Haije and DeJong,18 analyzing one bile sample on agar gel, also reported no difference between bile and serum. Estborn,19 comparing one serum with one bile sample, felt there was a small but distinct difference between the mobilities of the alkaline phosphatase samples. However, Chiandussi et al.,7 examining three bile specimens by horizontal starch gel electrophoresis, found the main activity in bile to be at the origin, thus demonstrating a difference between serum and bile. Keiding,20 using starch block analysis, also was able to demonstrate a difference in the mobility of bile and serum alkaline phosphatase. Recently, Gordon 21 has studied alkaline phosphatase in a number of body fluids and tissue extracts, using vertical starch gel electrophoresis. He also has demonstrated a difference between the bile and serum enzyme distribution. Our data demonstrate a clear difference in the electrophoretic distributions of the serum and bile enzymes. t would seem either that the alkaline phosphatase activity found in bile is not derived from serum, or that the serum enzyme is altered during the process of excretion. We could not demonstrate any evidence for physical or biological alteration in the serum enzyme by exposure to bile constituents. Serum and bile can be incubated together for as long as 24 hours without a change in isozyme patterns. The ultracentrifuge analyses indicate that serum and bile alkaline phosphatase are approximately the same molecular size. Thus, the isozymes found in bile probably do not represent fragments of the normal serum alkaline phosphatase with different electrophoretic mobilities. These experiments, however, do not conclusively rule out hepatic alteration. t is still possible that intramolecular reorganization of the tertiary structure could occur, resulting in difference in electrophoretic mobility, but without denaturation and with maintenance of enzyme activity. Such a transformation, however, would be unusual. Our results are consistent with the interpretation that most of the alkaline phosphatase activity in bile is not an excretory product derived from the serum. Summary 1. The isozymes of alkaline phosphatase found in the bile do not resemble those found in the serum. 2. ncubation of bile with serum does not convert the serum alkaline phosphatase isozymes to a pattern resembling those found in bile. 3. Ultracentrifugation of serum and bile shows that serum and bile alkaline phosphatase have approximately similar molecular sizes. 4. These findings are consistent with the hypothesis that bile alkaline phosphatase does not represent an excretion product of serum alkaline phosphatase. REFERENCES 1. Gutman, A. B Serum alkaline phosphatase activity in diseases of the skeletal and hepatobiliary systems. Amer. J. Med. 27: l. 2. Boyer, S. H Alkaline phosphatase in human sera and placentae. Science 134: Smithies, O Zone electrophoresis in starch gels ; group variations in serum proteins of normal human adults. Biochem. J. 61: King, E. J., and 1. D. P. Wootton Micro-analysis in medical biochemistry, Ed. 3, p. 85. J. and A. Churchill, Ltd., L ondon. 5. Edelman, G. M., H. G. Kunkel, and E. C. Franklin nteraction of the rheumatoid factor with antigen-antibody complexes and aggregated 'Y-globulin. J. Exp. Med. 108: Vesell, E. S., K. C. Osterland, A. G. Bearn,

6 636 POPE AND COOPERBAND Vol. 50, No.5 and H. G. Kunkel soenzymes of lactic dehydrogenase; their alterations in arthritic synovial fluid and sera. J. Clin. nvest. 41: Chiandussi, L., S. F. Greene, and S. Sherlock Serum alkaline phosphatase fractions in hepato-biliary and bone diseases. Clin. Sci. 1313: Dalgaard, J. B Serum and bile phosphatase in biliary fistula dogs. Acta Physiol. Scand. 16: LeVeen, H. H., L. J. Talbot, M. Restuccia, and J. R. Barberio Metabolism and excretion of alkaline phosphatase; relation to liver function and determination of maximal secretory rates of liver. J. Lab. Clin. Med. 36: Sterling, J., and S. Winsten Enzymes in bile. J. Einstein Med. Cent. 7: Polin, S. G., M. A. Spellberg, L. Teitelman, and M. Okumura Origin of elevation of serum alkaline phosphatase in hepatic disease. Gastroenterology 413: Frankl, H. D., and J. H. Merritt Enzyme activity in the serum and common duct bile of dogs. Arne'. J. Gastroent. 31: Sherlock, S., and V. Walshe Hepatic alkaline phosphatase; histological and microchemical studies on liver tissue in normal subjects and in liver and bone disease. J. Path. Bact. 59: Wachstein, M., and F. G. Zak Distribution of alkaline phosphatase in the human liver; a study of postmortem material. Arch. Path. 42: Sebesta, D. G., F. J. Bradshaw, and D. J. Prockop Source of elevated serum alkaline phosphatase activity in biliary obstruction; studies using isolated liver perfusion. Gastroenterology 47: Markert, C. L., and F. Moller Multiple forms of enzymes: tissue, ontogenetic, and species specific patterns. Proc. Nat. Acad. Sci. U. S. A.. i5: Rosenberg, 1. N Zone electrophoretic studies of serum alkaline phosphatase. J. Clin. nvest. 38: Haije, W. G., and M. DeJong soenzyme patterns of serum alkaline phosphatase in agar-gel electrophoresis and their clinical significance. Clin. Chim. Acta 8: Estborn, B Visualization of acid and alkaline phosphatase after starch-gel electrophoresis of seminal plasma, serum, and bile. Nature (London) 184: Keiding, N. R The alkaline phosphatase fractions of human lymph. Clin. Sci. 26: Gordon, S Alkaline phosphatase isoenzymes in serum and tissues. S. Afr. Med. J. 39: