Clinica Chimica Acta, 210 (1992) 63-73 0 1992 Elsevier Science Publishers B.V. All rights reserved. 0009~8981/92/$05.00
63
CCA 05364
Intestinal type alkaline phosphatase hyperphosphatasemia associated with liver cirrhosis Shuichi Saheki”, Masaaki Ochi”, Takahiko Horiuchi b, Yoshikatsu Sakagishi”, Yoko Fujimori-Arai”, Iwao Koyamac, Tsugikazu Komodac and Nozomu Takeuchi” ‘Central Laboratory and Department of Clinical Laboratory Medicine, ‘Department of 1st Internal Medicine. School of Medicine, University of Ehime. Shigenobu-rho, Onsengun, Ehime 791-02 and ‘Department of 1st Biochemistry, Saitama Medical School, Moroyama-machi. Iruma-gun. Saitama 350-04
(Japan) (Received 24 February, 1992; revision received 13 May, 1992; accepted 10 June, 1992)
Key words: Hyperphosphatasemia;
Alkaline phosphatase; Liver cirrhosis; Electrophoresis; Isoelectrofocusing; Sugar moiety
Summary Hyperphosphatasemia due to increased intestinal type serum alkaline phosphatase was noted in a 48-year-old male who had asymptomatic liver cirrhosis. The alkaline phosphatase activity in the serum was 828 U/l (our reference range in adults: 57-194 U/l), 94% of which was of the intestinal type as measured by an immunoprecipitation method. The intestinal component of alkaline phosphatase was separated into two major and some minor components using electrophoresis and isoelectrofocusing. One of the major components had similar mobility to that of a standard intestinal enzyme purified from adult intestine. The components were heat-labile and neuraminidase-resistant. Serial lectin affinity chromatography, however, indicated that sugar chain compositions of the alkaline phosphatase were different from those of the standard tissue intestinal enzyme. These results and further enzymological studies suggest that the patient’s serum alkaline phophatase basically consisted of several intestine-like isoforms.
Correspondence to: Shuichi Saheki, Central Laboratory and Department of Clinical Laboratory Medicine, University of Ehime, Shigenobu-cho, Onsengun, Ehime 791-02, Japan.
Introduction Measurement of alkaline phosphatase (EC 3.1.3.1) activity in serum is commonly used in clinical chemistry. Serum alkaline phosphatase normally consists of three distinctive types of isoenzymes: tissue-unspecific liver/bone/kidney type as a major component and adult intestinal and term placental types as minor components [ 1,2]. Examining these isoenzyme activities in serum has specific applicability in differential diagnosis and analysis of complications. Among them, the intestinal alkaline phosphatase isoenzyme has attracted special interest since its activity is known to be elevated speci~caily in hver cirrhosis [3,4] and Gilbert’s syndrome [S]. The mechanism of the elevation, however, is still unclear. In this report we present a case of a patient with hyperphosphatasemia and liver cirrhosis who had an unusually high serum level of intestinal alkaline phosphatase activity. The isoenzyme was characterized by conventional enzymological studies, as well as by gel electrophoresis using a new buffer system, isoelectrofocusing and serial lectin affinity chromatography. The case exemplified well the intimate correlations between increased intestinal alkaline phosphatase activity in serum and liver cirrhosis. Materials and Methods
The serum from a 4%year-old patient was kept at 4°C or at -20°C before use. For comparison, sera from a hepatitis patient, a 3-year-old male and a full term pregnant woman were selected as serum alkaline phosphatase standards of liver, bone and placenta types, respectively, Commercially available tissue alkaline phosphatase standards (Jookoo Co., Japan) purified from human tissues (liver, adult intestinal mucosa, term placenta and bone tissues from a patient with Paget’s disease) were also used. Enzyme assay Serum alkaline phosphatase activity was measured with an autoanalyzer (TBA 60 S, Toshiba Medical) at 37°C with an assay kit (Sank0 Junyaku Co., Japan) using p-nitrophenyl phosphate as a substrate and N-methylglucamine as a buffer at pH 10.4 [6]. Neuraminidase treatment
The sera were incubated with 0.1 U/ml neuraminidase Koch-Light Ltd., UK) at 37°C for 3 h [7].
(from Vi’hrio cholerae,
Immunoiogicai a~lysis
Mouse monoclonal antibodies against human liver- or adult intestine- or term placenta-alkaline phosphatase isoenzyme were used for immunoprecipitation~
65
Twenty microliters of sample were incubated overnight at 4°C with 2 ~1 of monoclonal antibodies. Twenty microliters of Staphylococcus aureus cells (BDH Lab. Chemicals, UK) were added to the mixture and left to stand for 1 h to adsorb immune complex. After centrifugation at 2000 x g at 4°C for 5 min the alkaline phosphatase activity in the supernatant was measured [8]. Sugar chain analysis
Sugar chains of alkaline phosphatase were analyzed by lectin-affinity chromatography [8]. Concanavalin A (Con A)-Sepharose was obtained from Pharmacia LKB Biotechnology (Bromma, Sweden). Erythroagglutinating phytohemagglutinin (PHA-E)-Pisum sativum agglutinin (PSA)- and wheat germ agglutinin (WGA)-agarose were obtained from E.Y. Lab. (San Mateo, USA). The sample was first loaded on a Con A-Sepharose column. Three fractions were obtained: an unbound fraction (Fraction I), a weakly bound fraction (Fraction II) which was eluted with a buffer containing 10 mmol/l a-methyl-D-mannoside and a strongly bound fraction (Fraction III) which was eluted with a buffer containing 500 mmol/l CYmethyl-D-mannoside. Subsequently, Fraction I was loaded on a PHA-E-agarose column, Fraction II on a PSA-agarose column and Fraction III on a WGA-agarose column. Unbound and bound fractions, designated as A and B were obtained from each column. The yields of the enzyme activity were more than 90% in all the lectin column chromatography. Electrophoresis Agarose gel electrophoresis
Five microliters of each alkaline phosphatase sample was applied on precast gel (Paragon Isopal AP, Beckman Instruments, Inc., USA) and electrophoresis was carried out at 150 V for 25 min in a Tris-Borate buffer. Polyacrylamide slab gel electrophoresis
A thin layer of 3.5% polyacrylamide gel (1 mm thick, 10 x 20 cm) with 10 wells of 5 ~1 capacity was made on a Gel-Bond film (Pharmacia LKB Biotechnology, Sweden). The gel was equilibrated in 25 mmol/l N-methylglucamine buffer containing 10 g/l of Triton X-100 for 1 h before electrophoresis. After 2-4 ~1 of each alkaline phosphatase sample was applied to the each well, electrophoresis was carried out in the same buffer at 2 &cm for 2 h at 4°C. Isoelectrofocusing polyacrylamide gel electrophoresis
A thin layer of 3.5% polyacrylamide gel (2 mm thick, 10 x 20 cm) with 18 wells of 30 ~1 capacity was made on a Gel-Bond film [9]. The gel contained 20 g/l of Ampholine (pH 3.5-9.5: pH 4-6 = 1:1, Phamacia LKB Biotechnology, Sweden) and 10 g/l of Triton X-100. Twenty microliters of each alkaline phosphatase sample was applied to each well and electrofocusing was carried out for about 3 h setting maximum limits to 1.400 in 1.5 mA/cm and 0.6 W/cm.
66
Alkaline phosphatase activity staining
Alkaline phosphatase was detected on the gel by staining the activity after electrophoresis with nitro blue tetrazolium reduction using 5-bromo-4-chloro-3-indolyl phosphate (Sigma Chemical Co., St. Louis, MO, USA) as a substrate [lo]. Heat stability
Samples were incubated at 56°C for 15 min and the remaining alkaline phosphatase activity was measured. Heat stable alkaline phosphatase activity was also analyzed by electrophoresis. Gel permeation chromatography
Alkaline phosphatase was loaded on an HPLC gel permeation column (TSK G3000SW 30 cm + G4OOOSW 30 cm, TOSOH, Japan) and eluted with a buffer containing 20 mmol/l piperazine-n!N’bis(2-ethanesulfonic acid)-Na (pH 6.5), 100 mmol/l KCl, 1 rnmol/l MgS04 and 0.01 mmol/l ZnC12. The elution of alkaline phosphatase activity was monitored and the apparent molecular mass of the alkaline phosphatase isoenzyme was estimated from its elution volume using standard molecular mass markers (Pharmacia LKB Biotechnology, Sweden). Results Clinical data
The case was a 48-year-old male outpatient of blood group B who complained of muscle cramps in the extremities frequently after sudden muscular exertion. He was diagnosed with liver cirrhosis when cholecystectomy was performed at the age of 46. Laboratory examinations showed abnormalities in blood chemistry: albumin = 3.6 (our reference range, RR = 3.9-4.9) g/dl; y-globulin fraction = 29.3 (RR = 8.6-
Fig. 1. Agarose gel electrophoresis. Five microliter/well of alkaline phosphatase sample was analyzed with Paragon Isopal ALP (Beckman Instruments, Inc., USA). X: patient serum; N: normal serum; Lp, Bp, Pp and Ip: human tissue alkaline phosphatase standards purified from liver, bone, placenta and intestinal mucosa respectively; Ls, Bs and Ps: selected sera for alkaline phosphatase standards representing liver, bone and placenta enzymes, respectively.
67
20.0)%; aspartate aminotransferase = 58 (RR 6-23) U/l; alanine aminotransferase = 124 (RR = l-48) U/l; y-glutamyl transpeptidase = 125 (RR = O-31) U/l; alkaline phosphatase = 828 (RR = 57- 194) U/l; total bilirubin = 1.5 (RR = 0.1- 1.1) mg/dl; bile acid = 56 (RR < 10) pmol/l and in serologies: cr-fetoprotein = 617 (RR < 20) q/ml; anti-hepatitis C virus antibody: positive. The moderately high CYfetoprotein level could be explained by the presence of hepatocellular carcinoma as well as liver cirrhosis, but ultrasonography and computed tomography of the abdomen showed only cirrhotic changes on the liver and mild splenomegaly. No other provacative factors nor complications were found. Alkaline phosphatase activity and isoenzymes
The serum alkaline phosphatase activity of the patient was 828 U/l and has been at that high level for the past 3 years. After agarose gel electrophoresis, the alkaline phosphatase activity appeared to consist mainly of two bands which significantly corresponded to intestinal (P-region of serum protein) and minor intestine-like (slightly slower band than that of the bone alkaline phosphatase) enzymes of tissue alkaline phosphatase standards within the limitations of resolution. A third minor broad band was also seen at the position of a liver or bone enzyme (Fig. 1). Immunoprecipitation test for alkaline phosphatase isoenzyme showed that 94% of alkaline phosphatase activity in the serum was of an intestinal enzyme and only 6% was of a tissue unspecific type. To rule out possible genetic predisposition, laboratory examinations were done on consanguinities, but none of the six family members examined had hyperphosphatasemia, abnormal results in alkaline phosphatase or liver function tests. Enzymological study
Some enzymological parameters of alkaline phosphatase in the patient serum were TABLE I Properties of abnormal serum alkaline phosphatase and control alkaline phosphatase isoenzymes Alkaline phosphatase isoenzymes Liver K,,, (mM) Optimal pH Inactivation (%) by 56”C, 10 min Inhibition (“XI)by L-Phe L-Leu L-homoArg Sensitivity to neuraminidase Molecular Mass (kDa)
Bone
Placenta
Intestine
2.52 10.6
2.19 10.5
2.86 10.5
Patient
2.71 10.4
2.88 10.4
74
94
9
22
28
14 17 58
13 22 56
73 26 6
56 8 3
53 10 6
+ 160
+ 160
+ 128
140
138
slab gel electrophoresis. Five microliters/well of alkaline phosphatase sample was Fig. 2. Polyacrylamide gel. It was previously equilibrated and electrophoresed in 25 mmol/l Nappl lied on 3.5% polyacrylamide met1hylglucamine buffer containing 10 g/l of Triton X-100. The patient alkaline phosphatase was c(Impared with the tissue and serum alkaline phosphatase standards.
N
Fig. 3. Polyacrylamide alks dine phosphatase
Ls
Ls
Btr
t
-
ES5
.+
Ps
F’s
X
X
_
3.
_
4
N
slab gel electrophoresis with (+) and without (-) neuraminidase treatment. Set urn was incubated with (+) and without (-) neuraminidase and analyzed by 3 5% polyacrylamide slab gel electrophoresis.
69
examined and they were compared with tissue alkaline phosphatase standards as summarized in Table I. K,,, for the substrate and optimal pH for the patient alkaline phosphatase were similar to those of the standard intestinal isoenzyme. The patient alkaline phosphatase activity was inhibited by the addition of 5 mM of Lphenylalanine, but not by the addition of L-leucine or L-homoarginine, again similar to the standard intestinal and also placental enzyme activities. The heat labile nature of the patient alkaline phosphatase, however, excluded the possibility of a placental type. Gel permeation chromatography indicated that the molecular mass was 138 kDa, close to the value of the intestinal enzyme. All of these results along with those of electrophoretic and immunological examinations indicated that the major form of alkaline phosphatase in the case was of an intestinal type. Minor alkaline phosphatase isoenzyme components
On agarose gel electrophoresis, at least 2 minor components were seen on the anodal side of the major intestinal enzyme in the patient alkaline phosphatase (Fig. 1). According to their mobilities, they could be intestine-like, liver or bone enzymes. To examine these possibilities in minor components, the patient alkaline phosphatase was analyzed using polyacrylamide slab gel electrophoresis and the results were compared with those of tissue and serum alkaline phosphatase standards (Fig. 2). By this method alkaline phosphatase was further separated into multiple bands mainly according to electrical charges. The major components in the
Fig. 4. Isoelectrofocusing electrophoresis. Serum alkaline phosphatase was incubated with (+) and without (-) neuraminidase and analyzed by isoelectrofocusing on a 3.5% polyacrylamide slab gel. *Incubated at 56°C for 15 min before the electrophoresis.
70
serum alkaline phosphatase standards migrated almost to the same positions of the corresponding tissue alkaiine phosphatase on the gel. The major component of the patient alkaline phosphatase had a similar mobility to that of the major intestinal enzyme in the tissue alkaline phosphatase standards. A small amount of liver and bone enzymes were also present in the patient serum. In addition, the patient serum also contained unique intestine-like alkaline phosphatase components on the cathodal side of the major component as indicated at (b) and (c) in Fig. 2. Their mobilities were similar to those of an intestine-like enzyme in tissue alkaline phosphatase standards. Like a major intestinal enzyme these minor components were heat labile and neuraminidase resistant (Fig. 3.) suggesting that an asialo-form of intestine-like or Kasahara isoenzyme was present in the patient serum [5,11,12]. These characteristics were also conlirmed by isoelectrofo~using with and without neuraminidase treatment prior to the isoele~trofo~using (Fig. 4). The patient serum alkaline phosphatase consisted of a major component (p1 = 4.76) and of two minor components (p1 = 4.86, 5.13). The former is considered to be an adult intestinal enzyme and the latter two to be intestine-like isoforms and all three were resistant to neuraminidase digestion. A trace amount of a liver enzyme (p1 = 4) was also re~o~izable in the sample and its pI shifted to 6.3 - 6.8 after neuraminidase treatment. Sugar chain analysis
Neuraminidase resistant property of alkaline phosphatase in the present case is a common characteristic of the intestinal enzyme. It implies that the sugar chains of the enzyme are devoid of accessible sialic acids for neuraminidase. Thus, the sugar chains of the patient enzyme were further analyzed by serial lectin affinity chromatography and they were compared with tissue standard intestinal enzyme (Table II). The results revealed significant differences in sugar chain compositions between the two enzymes. The content of fraction IIIa which has high-mannose type sugar chains f13,14] was higher in the patient’s alkaline phosphatase (49%) than in the standard tissue intestinal enzyme (40/o),while both fraction Ib and IIa contents were lower in the patient’s serum alkaline phosphatase than in the standard intestine
TABLE II Relative amounts of sugar chain fractions obtained by serial lectin affinity chromatography in serum alkaline phosphatase from a patient with hyperphosphatasemia as compared to control duodenum alkaline phosphatase Fractions
Patient (%)
Duodenal (%I)
Ia Ib IIa IIb IIIa IIIb
10 6 12 15 49 8
f2 28 31 12 4 13
71
enzyme. Fraction Ib is a complex type linked to bisecting ~-acetylglucosamine and fraction Ha is a biantennary complex type without fucose linkages. These differences in the sugar chain compositions may reflect differences in the processes of synthesis and maturation of the enzymes in different tissues. Disenssion The secretion of intestinal alkaline phosphatase into the blood stream is very limited under normal conditions, although the serum level of intestinal enzyme is thought to correlate with blood groups 0 and B and secretor phenotypes [14,15]. However, patients with liver diseases and certain nephritis have markedly elevated levels of intestinal alkaline phosphatase in their serum [2,4,5,7,12,16]. In such cases, the ratio of the intestinal enzyme rarely exceeds 50% of the total alkaline phosphatase activity. In the present case, the total serum alkaline phosphatase activity increased to 828 U/l (our reference range: 57-194 U/l) and 94% of the serum alkaline phosphatase consisted of intestinal enzyme. This indicates that the increase of total alkaline phosphatase activity in the patient serum could be solely accounted for by the increase of intestinal type alkaline phosphatase. Although the increase of serum intestinal type alkaline phosphatase activity in liver cirrhosis is definitely related to the pathophysiological status of the disease, the mechanism of the increase is still obscure. When hepatocytes are damaged by posthepatic biliary obstruction induced by intra-hepatic disease, the clearance of plasma alkaline phosphatase by the liver might be disturbed due to the dysfunction of the reti~ulo~ndothelial system [ 171 and intestinal-Orion alkaline phosphatase level might increase in serum 1181.Alternatively, as mentioned earlier by Fishman et al. [4], the increased activity of intestinal alkaline phosphatase in the serum might be related to portal hypertension in liver cirrhosis. In the present case, the patient had mild splenomegaly, a sign of portal hypertension. Under these conditions, this intestinal type alkaline phosphatase could originate from the congested intestine. The conventional electrophoretic analysis of the serum of this patient yielded two alkaline phosphatase bands. The electrophoretic mobility and enzymatic nature of this case of alkaline phosphatase were similar to the tissue standard intestinal alkaline phosphatase and Kasahara isoenzyme from cancerous cells [19]. However, the apparent molecular mass, the isoelectric point and the sugar chain composition of the patient’s alkaline phosphatase were slightly different from the tissue standard intestinal alkaline phosphatase. It has been reported that a hepatoma cell line also produced intestine-like or Kasahara isoenzyme [ 19,201. Moreover, the molecular mass and electrophoretic charge of the intestine-like alkaline phosphatase from the patient serum generally coincided with data obtained by Yamamoto et al. [19]. Given this, it seems that dysfunctional hepatic tissues may produce intestine-like or Kasahara isoenzyme in situ. Regarding sugar moieties, alkaline phosphatase from the patient serum was substantialy rich in high-mannose type sugar chains and hybrid type sugar chains with fucose linkages to the innermost GlcNAc. It implies that the sugar chains of intestinal alkaline phosphatase from the patient’s serum were poorly developed compared to those of the tissue standard intestinal alkaline phosphatase 1131.This may
l‘
be due to extraordinary processing of the sugar chains in alkahne phosphatase molecules, indicating that the expression of glycosidases and glycosyltransferases are distinguishable among respective organs, Taken together, these results indicate that the multiple forms of intestinal alkaline phosphatase from different pathological sources may be influenced by extraordinary shuffling of the sugar chains or ectopic expression of the intestine-like alkaline phosphatase in certain cells. Without doubt this will continue to be an interesting and significant subject for expanded future study. Acknowledgements The authors acknowledge the assistance of Emily Felt in the preparation of the manuscript. This work was supported in part by grants from the ministry of Education, Science and Culture of Japan and from the Ehime Health Foundation. References 1 Stigbrand T. Millin JL, Fishman WH. The genetic basis of alkaline phosphatase expression. In: Alan R Liss, ed. Isozymes. New York: 1982;93-117. 2 Domar U, Danielsson A, Hirano K, Stigbrand T. Alkaline phosphatase isozymes in non-malignant intestinal and hepatic diseases. Stand J Gastroenterol 1988;23:793-800. 3 Sakuma R, Nakayama T, Kitamura M. Molecular heterogeneity of intestinal alkaline phosphatase isozyme in serum from patient with liver cirrhosis (I). Jpn J Clin Chem 1981;10:40-46. 4 Fishman WH, Inglis Ni, Krant MJ. Serum alkaline phosphatase of intestinal origin in patients with cancer and with cirrhosis of the liver. Clin Chim Acta 1965;112:298-313. 5 Lievers AG, Van Essen GG, Beukeveld GJJ, Gazendam J, Dompeling EC, Ten Kate LP, Van Belie SA, Weits J. Familial increased serum intestinal alkaline phosphatase: a new variant associated with Gilbert’s syndrome. J Clin Path01 1990;43:125- 128. 6 Ochi M, Tokunaga K, Shishino K, Murase M, Takeuchi N. Selection of buffer solution for the standardization of serum alkaline phosphatase activity measurement using p-nitrophenyl phosphate as a substrate. Shikoku J Clin Chem 1986;3:37-45 (in Japanese). Miura M, Koyama I, Matsuzaki H, Sakagishi Y, Ikezawa H, Komoda T. Organ specific properties for human urinary alkaline phosphatases. Clin Chim Acta 1988;171:63-74. Komoda T, Sato M, Fttriya K, Sakagishi Y, Sekine T. Allelic and ectopic polymorphism in human placental alkaline phosphatases: sugar chain heterogeneities. Clin Chim Acta 1990;186:203-210. Griffrths J, Black J. Separation and identification of alkaline phosphatase isoenzymes and isoforms in serum of healthy persons by isoelectric focusing. Clin Chem 1987;33:2171-2177. Molecular Cloning, A Laboratory Manual, 2nd edn. Cold Spring Harbor Laboratory Press, 1989. Van Hoof Vo, Lepoutre L, Hoylaerts MF, Chevigne R, De Broe ME. Improved agarose electrophoretic method for separating alkaline phosphatase isoenzymes in serum. Clin Chem 1988;34:1857-1862. 12 Kuwana T, Rosalki SB. Intestinal variant alkaline phosphatase in plasma in diseases. Clin Chem 1990;36:1918-1921. 13 Koyama I, Miura M, Matsuzaki H, Sakagishi Y, Komoda T. Sugar chain heterogeneity of human alkaline phosphatase; differences between normal and tumor associated isozymes. J Chromatogr 1987;413:65-78. 14 Arfors KE, Beckman L, Lundin LD. Genetic variation of human serum phosphatases. Acta Genet 1963;13:89-94. 15 Domar U, Hirano K, Stigbrand T. Serum levels of human alkaline phosphatase isozymes in relation to blood groups. Clin Chim Acta 1991;203:305-314. 16 Moss DW, Parmar CR, Whitaker KB. Comparison of a tumor-derived form of intestinal alkaline phosphatase with foetal and adult intestinal alkaline phosphatases. Clin Chim Acta 1986;158:165- 172.
13 17
Komoda T, Sakagishi Y, Sekine T. Multiple forms of human intestinal alkaline phosphatase: chemical and enzymatic properties and circulating clearances of the fast- and slow-moving enzymes. Clin Chim Acta 1981;117:167-187. 18 Warnes TW, Hine P, Kay G, Smith A. Intestinal alkaline phosphatase in bile: evidence for an enterohepatic circulation. Gut 1981;22:493-498. 19 Yamamoto H, Tanaka M, Nakabayashi H, Sato J, Okochi T, Kishimoto S. Intestinal-type alkaline phosphatase produced by human hepatoblastoma cell line HUH-~ clone 5. Cancer Res 1984;44:339-344 20 Higashino K, Otani R, Kudo S, Yamamura Y. A fetal intestinal-type alkaline phosphatase in hepatocellular carcinoma tissue. Clin Chim Acta 1977;23:1615-1623.
63
CCA 05364
Intestinal type alkaline phosphatase hyperphosphatasemia associated with liver cirrhosis Shuichi Saheki”, Masaaki Ochi”, Takahiko Horiuchi b, Yoshikatsu Sakagishi”, Yoko Fujimori-Arai”, Iwao Koyamac, Tsugikazu Komodac and Nozomu Takeuchi” ‘Central Laboratory and Department of Clinical Laboratory Medicine, ‘Department of 1st Internal Medicine. School of Medicine, University of Ehime. Shigenobu-rho, Onsengun, Ehime 791-02 and ‘Department of 1st Biochemistry, Saitama Medical School, Moroyama-machi. Iruma-gun. Saitama 350-04
(Japan) (Received 24 February, 1992; revision received 13 May, 1992; accepted 10 June, 1992)
Key words: Hyperphosphatasemia;
Alkaline phosphatase; Liver cirrhosis; Electrophoresis; Isoelectrofocusing; Sugar moiety
Summary Hyperphosphatasemia due to increased intestinal type serum alkaline phosphatase was noted in a 48-year-old male who had asymptomatic liver cirrhosis. The alkaline phosphatase activity in the serum was 828 U/l (our reference range in adults: 57-194 U/l), 94% of which was of the intestinal type as measured by an immunoprecipitation method. The intestinal component of alkaline phosphatase was separated into two major and some minor components using electrophoresis and isoelectrofocusing. One of the major components had similar mobility to that of a standard intestinal enzyme purified from adult intestine. The components were heat-labile and neuraminidase-resistant. Serial lectin affinity chromatography, however, indicated that sugar chain compositions of the alkaline phosphatase were different from those of the standard tissue intestinal enzyme. These results and further enzymological studies suggest that the patient’s serum alkaline phophatase basically consisted of several intestine-like isoforms.
Correspondence to: Shuichi Saheki, Central Laboratory and Department of Clinical Laboratory Medicine, University of Ehime, Shigenobu-cho, Onsengun, Ehime 791-02, Japan.
Introduction Measurement of alkaline phosphatase (EC 3.1.3.1) activity in serum is commonly used in clinical chemistry. Serum alkaline phosphatase normally consists of three distinctive types of isoenzymes: tissue-unspecific liver/bone/kidney type as a major component and adult intestinal and term placental types as minor components [ 1,2]. Examining these isoenzyme activities in serum has specific applicability in differential diagnosis and analysis of complications. Among them, the intestinal alkaline phosphatase isoenzyme has attracted special interest since its activity is known to be elevated speci~caily in hver cirrhosis [3,4] and Gilbert’s syndrome [S]. The mechanism of the elevation, however, is still unclear. In this report we present a case of a patient with hyperphosphatasemia and liver cirrhosis who had an unusually high serum level of intestinal alkaline phosphatase activity. The isoenzyme was characterized by conventional enzymological studies, as well as by gel electrophoresis using a new buffer system, isoelectrofocusing and serial lectin affinity chromatography. The case exemplified well the intimate correlations between increased intestinal alkaline phosphatase activity in serum and liver cirrhosis. Materials and Methods
The serum from a 4%year-old patient was kept at 4°C or at -20°C before use. For comparison, sera from a hepatitis patient, a 3-year-old male and a full term pregnant woman were selected as serum alkaline phosphatase standards of liver, bone and placenta types, respectively, Commercially available tissue alkaline phosphatase standards (Jookoo Co., Japan) purified from human tissues (liver, adult intestinal mucosa, term placenta and bone tissues from a patient with Paget’s disease) were also used. Enzyme assay Serum alkaline phosphatase activity was measured with an autoanalyzer (TBA 60 S, Toshiba Medical) at 37°C with an assay kit (Sank0 Junyaku Co., Japan) using p-nitrophenyl phosphate as a substrate and N-methylglucamine as a buffer at pH 10.4 [6]. Neuraminidase treatment
The sera were incubated with 0.1 U/ml neuraminidase Koch-Light Ltd., UK) at 37°C for 3 h [7].
(from Vi’hrio cholerae,
Immunoiogicai a~lysis
Mouse monoclonal antibodies against human liver- or adult intestine- or term placenta-alkaline phosphatase isoenzyme were used for immunoprecipitation~
65
Twenty microliters of sample were incubated overnight at 4°C with 2 ~1 of monoclonal antibodies. Twenty microliters of Staphylococcus aureus cells (BDH Lab. Chemicals, UK) were added to the mixture and left to stand for 1 h to adsorb immune complex. After centrifugation at 2000 x g at 4°C for 5 min the alkaline phosphatase activity in the supernatant was measured [8]. Sugar chain analysis
Sugar chains of alkaline phosphatase were analyzed by lectin-affinity chromatography [8]. Concanavalin A (Con A)-Sepharose was obtained from Pharmacia LKB Biotechnology (Bromma, Sweden). Erythroagglutinating phytohemagglutinin (PHA-E)-Pisum sativum agglutinin (PSA)- and wheat germ agglutinin (WGA)-agarose were obtained from E.Y. Lab. (San Mateo, USA). The sample was first loaded on a Con A-Sepharose column. Three fractions were obtained: an unbound fraction (Fraction I), a weakly bound fraction (Fraction II) which was eluted with a buffer containing 10 mmol/l a-methyl-D-mannoside and a strongly bound fraction (Fraction III) which was eluted with a buffer containing 500 mmol/l CYmethyl-D-mannoside. Subsequently, Fraction I was loaded on a PHA-E-agarose column, Fraction II on a PSA-agarose column and Fraction III on a WGA-agarose column. Unbound and bound fractions, designated as A and B were obtained from each column. The yields of the enzyme activity were more than 90% in all the lectin column chromatography. Electrophoresis Agarose gel electrophoresis
Five microliters of each alkaline phosphatase sample was applied on precast gel (Paragon Isopal AP, Beckman Instruments, Inc., USA) and electrophoresis was carried out at 150 V for 25 min in a Tris-Borate buffer. Polyacrylamide slab gel electrophoresis
A thin layer of 3.5% polyacrylamide gel (1 mm thick, 10 x 20 cm) with 10 wells of 5 ~1 capacity was made on a Gel-Bond film (Pharmacia LKB Biotechnology, Sweden). The gel was equilibrated in 25 mmol/l N-methylglucamine buffer containing 10 g/l of Triton X-100 for 1 h before electrophoresis. After 2-4 ~1 of each alkaline phosphatase sample was applied to the each well, electrophoresis was carried out in the same buffer at 2 &cm for 2 h at 4°C. Isoelectrofocusing polyacrylamide gel electrophoresis
A thin layer of 3.5% polyacrylamide gel (2 mm thick, 10 x 20 cm) with 18 wells of 30 ~1 capacity was made on a Gel-Bond film [9]. The gel contained 20 g/l of Ampholine (pH 3.5-9.5: pH 4-6 = 1:1, Phamacia LKB Biotechnology, Sweden) and 10 g/l of Triton X-100. Twenty microliters of each alkaline phosphatase sample was applied to each well and electrofocusing was carried out for about 3 h setting maximum limits to 1.400 in 1.5 mA/cm and 0.6 W/cm.
66
Alkaline phosphatase activity staining
Alkaline phosphatase was detected on the gel by staining the activity after electrophoresis with nitro blue tetrazolium reduction using 5-bromo-4-chloro-3-indolyl phosphate (Sigma Chemical Co., St. Louis, MO, USA) as a substrate [lo]. Heat stability
Samples were incubated at 56°C for 15 min and the remaining alkaline phosphatase activity was measured. Heat stable alkaline phosphatase activity was also analyzed by electrophoresis. Gel permeation chromatography
Alkaline phosphatase was loaded on an HPLC gel permeation column (TSK G3000SW 30 cm + G4OOOSW 30 cm, TOSOH, Japan) and eluted with a buffer containing 20 mmol/l piperazine-n!N’bis(2-ethanesulfonic acid)-Na (pH 6.5), 100 mmol/l KCl, 1 rnmol/l MgS04 and 0.01 mmol/l ZnC12. The elution of alkaline phosphatase activity was monitored and the apparent molecular mass of the alkaline phosphatase isoenzyme was estimated from its elution volume using standard molecular mass markers (Pharmacia LKB Biotechnology, Sweden). Results Clinical data
The case was a 48-year-old male outpatient of blood group B who complained of muscle cramps in the extremities frequently after sudden muscular exertion. He was diagnosed with liver cirrhosis when cholecystectomy was performed at the age of 46. Laboratory examinations showed abnormalities in blood chemistry: albumin = 3.6 (our reference range, RR = 3.9-4.9) g/dl; y-globulin fraction = 29.3 (RR = 8.6-
Fig. 1. Agarose gel electrophoresis. Five microliter/well of alkaline phosphatase sample was analyzed with Paragon Isopal ALP (Beckman Instruments, Inc., USA). X: patient serum; N: normal serum; Lp, Bp, Pp and Ip: human tissue alkaline phosphatase standards purified from liver, bone, placenta and intestinal mucosa respectively; Ls, Bs and Ps: selected sera for alkaline phosphatase standards representing liver, bone and placenta enzymes, respectively.
67
20.0)%; aspartate aminotransferase = 58 (RR 6-23) U/l; alanine aminotransferase = 124 (RR = l-48) U/l; y-glutamyl transpeptidase = 125 (RR = O-31) U/l; alkaline phosphatase = 828 (RR = 57- 194) U/l; total bilirubin = 1.5 (RR = 0.1- 1.1) mg/dl; bile acid = 56 (RR < 10) pmol/l and in serologies: cr-fetoprotein = 617 (RR < 20) q/ml; anti-hepatitis C virus antibody: positive. The moderately high CYfetoprotein level could be explained by the presence of hepatocellular carcinoma as well as liver cirrhosis, but ultrasonography and computed tomography of the abdomen showed only cirrhotic changes on the liver and mild splenomegaly. No other provacative factors nor complications were found. Alkaline phosphatase activity and isoenzymes
The serum alkaline phosphatase activity of the patient was 828 U/l and has been at that high level for the past 3 years. After agarose gel electrophoresis, the alkaline phosphatase activity appeared to consist mainly of two bands which significantly corresponded to intestinal (P-region of serum protein) and minor intestine-like (slightly slower band than that of the bone alkaline phosphatase) enzymes of tissue alkaline phosphatase standards within the limitations of resolution. A third minor broad band was also seen at the position of a liver or bone enzyme (Fig. 1). Immunoprecipitation test for alkaline phosphatase isoenzyme showed that 94% of alkaline phosphatase activity in the serum was of an intestinal enzyme and only 6% was of a tissue unspecific type. To rule out possible genetic predisposition, laboratory examinations were done on consanguinities, but none of the six family members examined had hyperphosphatasemia, abnormal results in alkaline phosphatase or liver function tests. Enzymological study
Some enzymological parameters of alkaline phosphatase in the patient serum were TABLE I Properties of abnormal serum alkaline phosphatase and control alkaline phosphatase isoenzymes Alkaline phosphatase isoenzymes Liver K,,, (mM) Optimal pH Inactivation (%) by 56”C, 10 min Inhibition (“XI)by L-Phe L-Leu L-homoArg Sensitivity to neuraminidase Molecular Mass (kDa)
Bone
Placenta
Intestine
2.52 10.6
2.19 10.5
2.86 10.5
Patient
2.71 10.4
2.88 10.4
74
94
9
22
28
14 17 58
13 22 56
73 26 6
56 8 3
53 10 6
+ 160
+ 160
+ 128
140
138
slab gel electrophoresis. Five microliters/well of alkaline phosphatase sample was Fig. 2. Polyacrylamide gel. It was previously equilibrated and electrophoresed in 25 mmol/l Nappl lied on 3.5% polyacrylamide met1hylglucamine buffer containing 10 g/l of Triton X-100. The patient alkaline phosphatase was c(Impared with the tissue and serum alkaline phosphatase standards.
N
Fig. 3. Polyacrylamide alks dine phosphatase
Ls
Ls
Btr
t
-
ES5
.+
Ps
F’s
X
X
_
3.
_
4
N
slab gel electrophoresis with (+) and without (-) neuraminidase treatment. Set urn was incubated with (+) and without (-) neuraminidase and analyzed by 3 5% polyacrylamide slab gel electrophoresis.
69
examined and they were compared with tissue alkaline phosphatase standards as summarized in Table I. K,,, for the substrate and optimal pH for the patient alkaline phosphatase were similar to those of the standard intestinal isoenzyme. The patient alkaline phosphatase activity was inhibited by the addition of 5 mM of Lphenylalanine, but not by the addition of L-leucine or L-homoarginine, again similar to the standard intestinal and also placental enzyme activities. The heat labile nature of the patient alkaline phosphatase, however, excluded the possibility of a placental type. Gel permeation chromatography indicated that the molecular mass was 138 kDa, close to the value of the intestinal enzyme. All of these results along with those of electrophoretic and immunological examinations indicated that the major form of alkaline phosphatase in the case was of an intestinal type. Minor alkaline phosphatase isoenzyme components
On agarose gel electrophoresis, at least 2 minor components were seen on the anodal side of the major intestinal enzyme in the patient alkaline phosphatase (Fig. 1). According to their mobilities, they could be intestine-like, liver or bone enzymes. To examine these possibilities in minor components, the patient alkaline phosphatase was analyzed using polyacrylamide slab gel electrophoresis and the results were compared with those of tissue and serum alkaline phosphatase standards (Fig. 2). By this method alkaline phosphatase was further separated into multiple bands mainly according to electrical charges. The major components in the
Fig. 4. Isoelectrofocusing electrophoresis. Serum alkaline phosphatase was incubated with (+) and without (-) neuraminidase and analyzed by isoelectrofocusing on a 3.5% polyacrylamide slab gel. *Incubated at 56°C for 15 min before the electrophoresis.
70
serum alkaline phosphatase standards migrated almost to the same positions of the corresponding tissue alkaiine phosphatase on the gel. The major component of the patient alkaline phosphatase had a similar mobility to that of the major intestinal enzyme in the tissue alkaline phosphatase standards. A small amount of liver and bone enzymes were also present in the patient serum. In addition, the patient serum also contained unique intestine-like alkaline phosphatase components on the cathodal side of the major component as indicated at (b) and (c) in Fig. 2. Their mobilities were similar to those of an intestine-like enzyme in tissue alkaline phosphatase standards. Like a major intestinal enzyme these minor components were heat labile and neuraminidase resistant (Fig. 3.) suggesting that an asialo-form of intestine-like or Kasahara isoenzyme was present in the patient serum [5,11,12]. These characteristics were also conlirmed by isoelectrofo~using with and without neuraminidase treatment prior to the isoele~trofo~using (Fig. 4). The patient serum alkaline phosphatase consisted of a major component (p1 = 4.76) and of two minor components (p1 = 4.86, 5.13). The former is considered to be an adult intestinal enzyme and the latter two to be intestine-like isoforms and all three were resistant to neuraminidase digestion. A trace amount of a liver enzyme (p1 = 4) was also re~o~izable in the sample and its pI shifted to 6.3 - 6.8 after neuraminidase treatment. Sugar chain analysis
Neuraminidase resistant property of alkaline phosphatase in the present case is a common characteristic of the intestinal enzyme. It implies that the sugar chains of the enzyme are devoid of accessible sialic acids for neuraminidase. Thus, the sugar chains of the patient enzyme were further analyzed by serial lectin affinity chromatography and they were compared with tissue standard intestinal enzyme (Table II). The results revealed significant differences in sugar chain compositions between the two enzymes. The content of fraction IIIa which has high-mannose type sugar chains f13,14] was higher in the patient’s alkaline phosphatase (49%) than in the standard tissue intestinal enzyme (40/o),while both fraction Ib and IIa contents were lower in the patient’s serum alkaline phosphatase than in the standard intestine
TABLE II Relative amounts of sugar chain fractions obtained by serial lectin affinity chromatography in serum alkaline phosphatase from a patient with hyperphosphatasemia as compared to control duodenum alkaline phosphatase Fractions
Patient (%)
Duodenal (%I)
Ia Ib IIa IIb IIIa IIIb
10 6 12 15 49 8
f2 28 31 12 4 13
71
enzyme. Fraction Ib is a complex type linked to bisecting ~-acetylglucosamine and fraction Ha is a biantennary complex type without fucose linkages. These differences in the sugar chain compositions may reflect differences in the processes of synthesis and maturation of the enzymes in different tissues. Disenssion The secretion of intestinal alkaline phosphatase into the blood stream is very limited under normal conditions, although the serum level of intestinal enzyme is thought to correlate with blood groups 0 and B and secretor phenotypes [14,15]. However, patients with liver diseases and certain nephritis have markedly elevated levels of intestinal alkaline phosphatase in their serum [2,4,5,7,12,16]. In such cases, the ratio of the intestinal enzyme rarely exceeds 50% of the total alkaline phosphatase activity. In the present case, the total serum alkaline phosphatase activity increased to 828 U/l (our reference range: 57-194 U/l) and 94% of the serum alkaline phosphatase consisted of intestinal enzyme. This indicates that the increase of total alkaline phosphatase activity in the patient serum could be solely accounted for by the increase of intestinal type alkaline phosphatase. Although the increase of serum intestinal type alkaline phosphatase activity in liver cirrhosis is definitely related to the pathophysiological status of the disease, the mechanism of the increase is still obscure. When hepatocytes are damaged by posthepatic biliary obstruction induced by intra-hepatic disease, the clearance of plasma alkaline phosphatase by the liver might be disturbed due to the dysfunction of the reti~ulo~ndothelial system [ 171 and intestinal-Orion alkaline phosphatase level might increase in serum 1181.Alternatively, as mentioned earlier by Fishman et al. [4], the increased activity of intestinal alkaline phosphatase in the serum might be related to portal hypertension in liver cirrhosis. In the present case, the patient had mild splenomegaly, a sign of portal hypertension. Under these conditions, this intestinal type alkaline phosphatase could originate from the congested intestine. The conventional electrophoretic analysis of the serum of this patient yielded two alkaline phosphatase bands. The electrophoretic mobility and enzymatic nature of this case of alkaline phosphatase were similar to the tissue standard intestinal alkaline phosphatase and Kasahara isoenzyme from cancerous cells [19]. However, the apparent molecular mass, the isoelectric point and the sugar chain composition of the patient’s alkaline phosphatase were slightly different from the tissue standard intestinal alkaline phosphatase. It has been reported that a hepatoma cell line also produced intestine-like or Kasahara isoenzyme [ 19,201. Moreover, the molecular mass and electrophoretic charge of the intestine-like alkaline phosphatase from the patient serum generally coincided with data obtained by Yamamoto et al. [19]. Given this, it seems that dysfunctional hepatic tissues may produce intestine-like or Kasahara isoenzyme in situ. Regarding sugar moieties, alkaline phosphatase from the patient serum was substantialy rich in high-mannose type sugar chains and hybrid type sugar chains with fucose linkages to the innermost GlcNAc. It implies that the sugar chains of intestinal alkaline phosphatase from the patient’s serum were poorly developed compared to those of the tissue standard intestinal alkaline phosphatase 1131.This may
l‘
be due to extraordinary processing of the sugar chains in alkahne phosphatase molecules, indicating that the expression of glycosidases and glycosyltransferases are distinguishable among respective organs, Taken together, these results indicate that the multiple forms of intestinal alkaline phosphatase from different pathological sources may be influenced by extraordinary shuffling of the sugar chains or ectopic expression of the intestine-like alkaline phosphatase in certain cells. Without doubt this will continue to be an interesting and significant subject for expanded future study. Acknowledgements The authors acknowledge the assistance of Emily Felt in the preparation of the manuscript. This work was supported in part by grants from the ministry of Education, Science and Culture of Japan and from the Ehime Health Foundation. References 1 Stigbrand T. Millin JL, Fishman WH. The genetic basis of alkaline phosphatase expression. In: Alan R Liss, ed. Isozymes. New York: 1982;93-117. 2 Domar U, Danielsson A, Hirano K, Stigbrand T. Alkaline phosphatase isozymes in non-malignant intestinal and hepatic diseases. Stand J Gastroenterol 1988;23:793-800. 3 Sakuma R, Nakayama T, Kitamura M. Molecular heterogeneity of intestinal alkaline phosphatase isozyme in serum from patient with liver cirrhosis (I). Jpn J Clin Chem 1981;10:40-46. 4 Fishman WH, Inglis Ni, Krant MJ. Serum alkaline phosphatase of intestinal origin in patients with cancer and with cirrhosis of the liver. Clin Chim Acta 1965;112:298-313. 5 Lievers AG, Van Essen GG, Beukeveld GJJ, Gazendam J, Dompeling EC, Ten Kate LP, Van Belie SA, Weits J. Familial increased serum intestinal alkaline phosphatase: a new variant associated with Gilbert’s syndrome. J Clin Path01 1990;43:125- 128. 6 Ochi M, Tokunaga K, Shishino K, Murase M, Takeuchi N. Selection of buffer solution for the standardization of serum alkaline phosphatase activity measurement using p-nitrophenyl phosphate as a substrate. Shikoku J Clin Chem 1986;3:37-45 (in Japanese). Miura M, Koyama I, Matsuzaki H, Sakagishi Y, Ikezawa H, Komoda T. Organ specific properties for human urinary alkaline phosphatases. Clin Chim Acta 1988;171:63-74. Komoda T, Sato M, Fttriya K, Sakagishi Y, Sekine T. Allelic and ectopic polymorphism in human placental alkaline phosphatases: sugar chain heterogeneities. Clin Chim Acta 1990;186:203-210. Griffrths J, Black J. Separation and identification of alkaline phosphatase isoenzymes and isoforms in serum of healthy persons by isoelectric focusing. Clin Chem 1987;33:2171-2177. Molecular Cloning, A Laboratory Manual, 2nd edn. Cold Spring Harbor Laboratory Press, 1989. Van Hoof Vo, Lepoutre L, Hoylaerts MF, Chevigne R, De Broe ME. Improved agarose electrophoretic method for separating alkaline phosphatase isoenzymes in serum. Clin Chem 1988;34:1857-1862. 12 Kuwana T, Rosalki SB. Intestinal variant alkaline phosphatase in plasma in diseases. Clin Chem 1990;36:1918-1921. 13 Koyama I, Miura M, Matsuzaki H, Sakagishi Y, Komoda T. Sugar chain heterogeneity of human alkaline phosphatase; differences between normal and tumor associated isozymes. J Chromatogr 1987;413:65-78. 14 Arfors KE, Beckman L, Lundin LD. Genetic variation of human serum phosphatases. Acta Genet 1963;13:89-94. 15 Domar U, Hirano K, Stigbrand T. Serum levels of human alkaline phosphatase isozymes in relation to blood groups. Clin Chim Acta 1991;203:305-314. 16 Moss DW, Parmar CR, Whitaker KB. Comparison of a tumor-derived form of intestinal alkaline phosphatase with foetal and adult intestinal alkaline phosphatases. Clin Chim Acta 1986;158:165- 172.
13 17
Komoda T, Sakagishi Y, Sekine T. Multiple forms of human intestinal alkaline phosphatase: chemical and enzymatic properties and circulating clearances of the fast- and slow-moving enzymes. Clin Chim Acta 1981;117:167-187. 18 Warnes TW, Hine P, Kay G, Smith A. Intestinal alkaline phosphatase in bile: evidence for an enterohepatic circulation. Gut 1981;22:493-498. 19 Yamamoto H, Tanaka M, Nakabayashi H, Sato J, Okochi T, Kishimoto S. Intestinal-type alkaline phosphatase produced by human hepatoblastoma cell line HUH-~ clone 5. Cancer Res 1984;44:339-344 20 Higashino K, Otani R, Kudo S, Yamamura Y. A fetal intestinal-type alkaline phosphatase in hepatocellular carcinoma tissue. Clin Chim Acta 1977;23:1615-1623.
