Alkaline Phosphatase Isoenzymes in Feline Serum using Agarose Gel Alkaline Phosphatase Kit Method
an
Barbara S. Horney, Andrea J. Farmer, Allan MacKensie, David J. Honor and Susan Buczkowski
ABSTRACT Total serum alkaline phosphatase (ALP) activity is the product of the combined activity of isoenzymes from a number of tissue sources. In this study, a commercialy available kit for electrophoretic separation of ALP isoenzymes in an agarose gel was used to separate ALP isoenzymes in feline tissue extracts and serum. Five separate bands of ALP activity were identified. These bands were numbered 1 to 5 with band 1 having the most anodal migration. The tissue of origin corresponding to the migration position of the isoenzymes are as follows: Band 3 was the liver isoenzyme, band 4 was the bone isoenzyme and ALP isoenzymes of both intestine and kidney migrate in the position labelled band 5. Band 1 appears to be related to albumin and does not represent true ALP activity. The tissue source of band 2 (a and b) was not identified. Serum ALP activity of mature, healthy cats is primarily of liver origin. Immature cats ( < 1 year of age) have a greater proportion of the bone isoenzyme in the serum.
RESUME L'activite totale de la phosphatase alcaline (ALP) serique est le resultat de l'activite combinee des isoenzymes de differentes sources tissulaires. Dans la presente etude, une trousse commerciale pour la separation des isoenzymes de I'ALP sur agarose a ete utilisee, a partir d'extraits tissulaires et de serum de chat. Cinq bandes d'activite d'ALP
ont ete identifiees et numerotees de 1 * 5, la bande 1 pr&sentant Ia plus grande migration anodale. L'origine tissulaire correspondant a la position migratoire des isoenzymes est comme suit: la bande 3 etait l'isoenzyme h6patique, la bande 4 etait l'isoenzyme osseux et la bande 5 representait les isoenzymes de l'intestin et du rein. La bande 1 semble reliee a l'albumine et ne represente pas une activitO vraie de l'ALP. La source tissulaire de Ia bande 2 (a et b) n'a pas e identifiee. L'activite serique de l'ALP des chats sains et adultes est principalement d'origine h6patique. Les chats immatures (moins d'un an) ont une plus grande proportion d'isoenzyme osseux dans leur serum. (Traduit par Dr Michel Fontaine)
Serum alkaline phosphatase (ALP) is a measurement of the combined activity of individual isoenzymes derived from different tissue sources. Identification of the various isoenzymes can provide useful information regarding the organ source of increased serum ALP activity. Increases in serum ALP activity in cats are usually not as dramatic as in other species, however, serum ALP is a useful enzyme for detection of biliary disease in the cat (1,2,3) as well as certain nonhepatic disease states (4). The various isoenzymes of feline ALP have been separated and identified using polyacrylamide (PAGE) (5) and cellulose acetate (6) gel electrophoresis. The current study was undertaken to investigate the electrophoretic mobility of feline ALP isoenzymes present in serum and various tissue
extracts using a commercially available agarose gel electrophoresis kit designed for high resolution separation of human liver and bone ALP isoenzymes. Extracts of feline liver, intestine, kidney and bone were prepared as follows. One part of tissue (100 g) was mixed with four parts (400 mL) of saline in a blender for five minutes. One part of n-butanol (100 mL) was added and the mixture was stirred with a magnetic mixer for 30 min and then centrifuged at 2500 g for 15 min. The bottom aqueous layer was removed to be used for isoenzyme separation. These extracts were frozen at - 20°C until used. Prior to electrophoresis, the extract was diluted with pooled, heat inactivated (3 h at 56°C) feline serum to yield ALP activity of 100-400 U/L (DACOS Chemistry Analyzer, Dart Reagent Systems, Coulter Electronics,
Hialeah, Florida). Serum samples from 14 cats which had no abnormalities on physical examination, hematology or serum chemistry were selected from material submitted to the Atlantic Veterinary College Diagnostic Laboratory. These samples were used fresh or held at -20°C until processed. The separation of feline ALP isoenzymes was achieved by electrophoresis through agarose gel (Titan Gel, Helena Laboratories, Beaumont, Texas). Five microliters of pretreated serum or tissue extract were electrophoresed through a horizontal agarose gel in barbital-sodium barbital buffer (supplied) for 28 min at room temperature at 250 volts using the Titan gel chamber and the Gelman power supply (0-500 v Gelman Instrument Co., Ann
Department of Pathology and Microbiology, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, Prince Edward Island CIA 4P3 (Horney, Farmer, MacKensie, Honor) and Department of Veterinary Pathology, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, Saskatchewan S7N OWO (Buczkowski). Support for Andrea Farmer was provided through a DVM Student Summer Research Award from the Canadian Veterinary Research Trust Fund. Submitted January 6, 1992.
Can J Vet Res 1992; 56: 373-375
373
TABLE I. Relative migration values (Rm) for
tissue extract electrophoresis
.r
alkaline phosphatase
..,.
*:
.,-
j f:.,
_
.,g
..S i.
,m
;
;
f
-a
Tissue Liverc Liverd Boned Intestined
Kidneyd
na
4 5 3 3 4
Mean 0.221 0.49 0.33 0.165 0.20
Range 0.114-0.31 0.48 -0.0 0.27 -0.37 0.15 -0.17 0.17 -0.27
SDb 0.081 0.008 0.053 0.013 0.045
aNumber of repeated runs bStandard deviation c Undiluted dDiluted with inactive serum
making identification of separate iso-
enzyme bands difficult. The hepatic isoenzyme demonstrated
Fig. 1. Example of ALP electrophoretic gels and densitometer scans of extracts of feline liver, bone, kidney and intestine. ( ) indicates sample application point. Each sample was run in duplicate. "Control serum" is of human origin and contains bone and liver isoenzymes.
Arbor, Michigan). The gels were stained for ALP activity with the titan gel alkaline phosphatase (HR) reagent using 5-bromo-4-chloro-3-indolyl phosphate-p-toluidine salt (BCIP) as the color indicator and nitro blue tetrazolium as a stabilizer. Incubation of the gels was carried out at 45°C for 30 min using the Helena Laboratories incubation oven dryer (IOD). The gels were dried at 60°C for 10 min in the Helena Laboratories IOD. Gels were scanned within 15 min (to avoid interference with increasing background staining) using a densitometer (Helena Laboratories, Beaumont, Texas). The scan initiation point was marked by drawing a line 25 mm from the cathodal edge of the gel (3.5 mm on the cathodal side of the application point). This point was chosen as the initiation point in order to include any bands which migrated slightly cathodally. The migration distances were measured from scan initiation to peak staining as indicated on the densitometer scan. This calculation was chosen to avoid zero and negative numbers for bands that had little or reverse migration respectively. Relative migration distances (Rm) of the various isoenzymes were calculated by dividing the migration distance of the isoenzyme from the scan initiation point by the migration distance of band 1. Absolute activity of ALP in U/L was calculated by multiplying the measured 374
relative area below the curve (density) for each peak (excluding the albumin peak) by the total measured ALP activity. The baseline was raised to eliminate variable background staining in a small number of the scans. Tissue extracts showed different electrophoretic mobility of ALP activity when comparison was made between undiluted or saline diluted samples and those diluted with heat inactivated serum. The results reported here are those of serum diluted extracts in order to allow comparison with the migration of isoenzymes found in serum (Fig. 1). Statistical comparison of tissue extract Rm distances was done by Analysis of Variance (ANOVA) followed by a Scheffe's test using the SAS computer package (Statistical Analysis Systems version 6ed, SAS Institute Inc., Cary, North Carolina, 1987). Similar to our findings, the migration distance of the liver isoenzyme was reported to be altered toward the anode when tissue extracts were diluted with inactivated serum using the PAGE gel technique (5). The agarose gel electrophoresis kit method is very sensitive, separate bands were obtained at activity levels as low as 28 U/L of serum ALP. Neuraminidase treatment of the sample has been thought to have little value in ALP isoenzyme identification in cats (5). We found that omitting the pretreatment step resulted in the widening of the electrophoretic bands
the greatest mobility of the tissue ALP extracts followed by bone, kidney and intestine respectively (Fig. 1). The mean relative electrophoretic mobilities (Rm values) are listed in Table I. The Rm value of the isoenzymes from bone and liver were significantly different from each other (p < 0.05). The bands of intestine and kidney origin overlapped in their migration. In the normal mature cat, up to five bands of ALP activity are present in the serum (Fig. 2). The bands were identified as band 1 through 5 with band 1 having the most anodal migration. Based on comparison of Rm (Table II) distances between these bands and the tissue extracts, the tissue sources of these isoenzymes were identified as follows: 2 (a,b) - source unknown, 3 - liver, 4 - bone and 5 (present variably) kidney/intestine. The most anodally migrating band, labelled as band 1, was present in all sera, including heat inactivated sera with a measured ALP activity of zero. This band corresponded to the position occupied by albumin when the gel was stained with amido black to demonstrate protein. Absolute activities of ALP were calculated from the densitometer scan and the measured total ALP activity without inclusion of the albumin band which did not appear to be true ALP activity (Table II). In mature cats, the liver isoenzyme exhibited the greatest activity (range = 11.6-24.2 U/L). In immature (
an
Barbara S. Horney, Andrea J. Farmer, Allan MacKensie, David J. Honor and Susan Buczkowski
ABSTRACT Total serum alkaline phosphatase (ALP) activity is the product of the combined activity of isoenzymes from a number of tissue sources. In this study, a commercialy available kit for electrophoretic separation of ALP isoenzymes in an agarose gel was used to separate ALP isoenzymes in feline tissue extracts and serum. Five separate bands of ALP activity were identified. These bands were numbered 1 to 5 with band 1 having the most anodal migration. The tissue of origin corresponding to the migration position of the isoenzymes are as follows: Band 3 was the liver isoenzyme, band 4 was the bone isoenzyme and ALP isoenzymes of both intestine and kidney migrate in the position labelled band 5. Band 1 appears to be related to albumin and does not represent true ALP activity. The tissue source of band 2 (a and b) was not identified. Serum ALP activity of mature, healthy cats is primarily of liver origin. Immature cats ( < 1 year of age) have a greater proportion of the bone isoenzyme in the serum.
RESUME L'activite totale de la phosphatase alcaline (ALP) serique est le resultat de l'activite combinee des isoenzymes de differentes sources tissulaires. Dans la presente etude, une trousse commerciale pour la separation des isoenzymes de I'ALP sur agarose a ete utilisee, a partir d'extraits tissulaires et de serum de chat. Cinq bandes d'activite d'ALP
ont ete identifiees et numerotees de 1 * 5, la bande 1 pr&sentant Ia plus grande migration anodale. L'origine tissulaire correspondant a la position migratoire des isoenzymes est comme suit: la bande 3 etait l'isoenzyme h6patique, la bande 4 etait l'isoenzyme osseux et la bande 5 representait les isoenzymes de l'intestin et du rein. La bande 1 semble reliee a l'albumine et ne represente pas une activitO vraie de l'ALP. La source tissulaire de Ia bande 2 (a et b) n'a pas e identifiee. L'activite serique de l'ALP des chats sains et adultes est principalement d'origine h6patique. Les chats immatures (moins d'un an) ont une plus grande proportion d'isoenzyme osseux dans leur serum. (Traduit par Dr Michel Fontaine)
Serum alkaline phosphatase (ALP) is a measurement of the combined activity of individual isoenzymes derived from different tissue sources. Identification of the various isoenzymes can provide useful information regarding the organ source of increased serum ALP activity. Increases in serum ALP activity in cats are usually not as dramatic as in other species, however, serum ALP is a useful enzyme for detection of biliary disease in the cat (1,2,3) as well as certain nonhepatic disease states (4). The various isoenzymes of feline ALP have been separated and identified using polyacrylamide (PAGE) (5) and cellulose acetate (6) gel electrophoresis. The current study was undertaken to investigate the electrophoretic mobility of feline ALP isoenzymes present in serum and various tissue
extracts using a commercially available agarose gel electrophoresis kit designed for high resolution separation of human liver and bone ALP isoenzymes. Extracts of feline liver, intestine, kidney and bone were prepared as follows. One part of tissue (100 g) was mixed with four parts (400 mL) of saline in a blender for five minutes. One part of n-butanol (100 mL) was added and the mixture was stirred with a magnetic mixer for 30 min and then centrifuged at 2500 g for 15 min. The bottom aqueous layer was removed to be used for isoenzyme separation. These extracts were frozen at - 20°C until used. Prior to electrophoresis, the extract was diluted with pooled, heat inactivated (3 h at 56°C) feline serum to yield ALP activity of 100-400 U/L (DACOS Chemistry Analyzer, Dart Reagent Systems, Coulter Electronics,
Hialeah, Florida). Serum samples from 14 cats which had no abnormalities on physical examination, hematology or serum chemistry were selected from material submitted to the Atlantic Veterinary College Diagnostic Laboratory. These samples were used fresh or held at -20°C until processed. The separation of feline ALP isoenzymes was achieved by electrophoresis through agarose gel (Titan Gel, Helena Laboratories, Beaumont, Texas). Five microliters of pretreated serum or tissue extract were electrophoresed through a horizontal agarose gel in barbital-sodium barbital buffer (supplied) for 28 min at room temperature at 250 volts using the Titan gel chamber and the Gelman power supply (0-500 v Gelman Instrument Co., Ann
Department of Pathology and Microbiology, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, Prince Edward Island CIA 4P3 (Horney, Farmer, MacKensie, Honor) and Department of Veterinary Pathology, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, Saskatchewan S7N OWO (Buczkowski). Support for Andrea Farmer was provided through a DVM Student Summer Research Award from the Canadian Veterinary Research Trust Fund. Submitted January 6, 1992.
Can J Vet Res 1992; 56: 373-375
373
TABLE I. Relative migration values (Rm) for
tissue extract electrophoresis
.r
alkaline phosphatase
..,.
*:
.,-
j f:.,
_
.,g
..S i.
,m
;
;
f
-a
Tissue Liverc Liverd Boned Intestined
Kidneyd
na
4 5 3 3 4
Mean 0.221 0.49 0.33 0.165 0.20
Range 0.114-0.31 0.48 -0.0 0.27 -0.37 0.15 -0.17 0.17 -0.27
SDb 0.081 0.008 0.053 0.013 0.045
aNumber of repeated runs bStandard deviation c Undiluted dDiluted with inactive serum
making identification of separate iso-
enzyme bands difficult. The hepatic isoenzyme demonstrated
Fig. 1. Example of ALP electrophoretic gels and densitometer scans of extracts of feline liver, bone, kidney and intestine. ( ) indicates sample application point. Each sample was run in duplicate. "Control serum" is of human origin and contains bone and liver isoenzymes.
Arbor, Michigan). The gels were stained for ALP activity with the titan gel alkaline phosphatase (HR) reagent using 5-bromo-4-chloro-3-indolyl phosphate-p-toluidine salt (BCIP) as the color indicator and nitro blue tetrazolium as a stabilizer. Incubation of the gels was carried out at 45°C for 30 min using the Helena Laboratories incubation oven dryer (IOD). The gels were dried at 60°C for 10 min in the Helena Laboratories IOD. Gels were scanned within 15 min (to avoid interference with increasing background staining) using a densitometer (Helena Laboratories, Beaumont, Texas). The scan initiation point was marked by drawing a line 25 mm from the cathodal edge of the gel (3.5 mm on the cathodal side of the application point). This point was chosen as the initiation point in order to include any bands which migrated slightly cathodally. The migration distances were measured from scan initiation to peak staining as indicated on the densitometer scan. This calculation was chosen to avoid zero and negative numbers for bands that had little or reverse migration respectively. Relative migration distances (Rm) of the various isoenzymes were calculated by dividing the migration distance of the isoenzyme from the scan initiation point by the migration distance of band 1. Absolute activity of ALP in U/L was calculated by multiplying the measured 374
relative area below the curve (density) for each peak (excluding the albumin peak) by the total measured ALP activity. The baseline was raised to eliminate variable background staining in a small number of the scans. Tissue extracts showed different electrophoretic mobility of ALP activity when comparison was made between undiluted or saline diluted samples and those diluted with heat inactivated serum. The results reported here are those of serum diluted extracts in order to allow comparison with the migration of isoenzymes found in serum (Fig. 1). Statistical comparison of tissue extract Rm distances was done by Analysis of Variance (ANOVA) followed by a Scheffe's test using the SAS computer package (Statistical Analysis Systems version 6ed, SAS Institute Inc., Cary, North Carolina, 1987). Similar to our findings, the migration distance of the liver isoenzyme was reported to be altered toward the anode when tissue extracts were diluted with inactivated serum using the PAGE gel technique (5). The agarose gel electrophoresis kit method is very sensitive, separate bands were obtained at activity levels as low as 28 U/L of serum ALP. Neuraminidase treatment of the sample has been thought to have little value in ALP isoenzyme identification in cats (5). We found that omitting the pretreatment step resulted in the widening of the electrophoretic bands
the greatest mobility of the tissue ALP extracts followed by bone, kidney and intestine respectively (Fig. 1). The mean relative electrophoretic mobilities (Rm values) are listed in Table I. The Rm value of the isoenzymes from bone and liver were significantly different from each other (p < 0.05). The bands of intestine and kidney origin overlapped in their migration. In the normal mature cat, up to five bands of ALP activity are present in the serum (Fig. 2). The bands were identified as band 1 through 5 with band 1 having the most anodal migration. Based on comparison of Rm (Table II) distances between these bands and the tissue extracts, the tissue sources of these isoenzymes were identified as follows: 2 (a,b) - source unknown, 3 - liver, 4 - bone and 5 (present variably) kidney/intestine. The most anodally migrating band, labelled as band 1, was present in all sera, including heat inactivated sera with a measured ALP activity of zero. This band corresponded to the position occupied by albumin when the gel was stained with amido black to demonstrate protein. Absolute activities of ALP were calculated from the densitometer scan and the measured total ALP activity without inclusion of the albumin band which did not appear to be true ALP activity (Table II). In mature cats, the liver isoenzyme exhibited the greatest activity (range = 11.6-24.2 U/L). In immature (
