equine samples analyzed at the Onta- rio Veterinary College over an 18 .... Chemical Co., St. Louis, Missouri. 3Fischer Scientific Co., Fair Lawn, New Jersey.
Examination of the Origin of Increased Equine Serum Alkaline Phosphatase Concentrations K.F. TRUEMAN, J.H. LUMSDEN AND B.J. McSHERRY
Department of Pathology, Ontario Veterinary College, University of Guelph, Guelph, Ontario NI G 2 WI
SUMMARY
Serum alkaline phosphatase activity was found to be increased in 32.6% of equine samples analyzed at the Ontario Veterinary College over an 18 month period. An attempt was made using sensitivity to L-phenylalanine and heat to identify the origin of increased serum alkaline phosphatase isoenzymes present in 44 clinical cases. No difference in sensitivity to either procedure was observed for serum alkaline phosphatase from groups of foals and horses representing different clinical problems. Alkaline phosphatase of osseous tissue origin appeared to be the major source of activity for each group of animals reported. R ASU M t
Determination de l'origine de 1'elevation de la concentration de la phosphatase alcaline, dans le serum de chevaux Cette etude s'etalait sur une periode de 18 mois et elle a permis de constater une augmentation de l'activite de la phosphatase alcaline serique, dans 32,6% des echantillons de serum equin analyses 'a cette fin, au College veterinaire de l'Ontario. On tenta de determiner l'origine de l'augmentation des isoenzymes de la phosphatase alcaline serique qu'on detecta dans 44 cas cliniques, en utilisant l'epreuve de sensibilite 'a la L-phenylalanine et 'a la chaleur. On ne decela pas de difference dans la sensibilite 'a l'une ou l'autre epreuve, de la part de groupes de poulains ou de chevaux atteints de diverses conditions cliniques. La phosphatase alcaline qui originait du tissu osseux sembla representer la principale source d'activite, pour chacun des groupes precites.
I NTRODUCTION nates from hepatobiliary, skeletal or Serum alkaline phosphatase (AP) intestinal sources. In this report an concentrations have been used by cli- attempt is made using L-phenylalanine nicians as an indication of altered cel- sensitivity and thermostability to idenlular function. In the horse as in other tify the origin of the isoenzyme(s) in animals, the enzyme originates in cells some horses with elevated serum AP from several organs (5). The diagnos- activity. Serum AP concentrations tic value of increased serum alkaline observed during an 18 month period phosphatase is therefore related to the are tabulated. ability of the clinician to correlate the observation with clinical findings or MATERIALS AND METHODS the laboratory capability of detecting During an 18 month period 1 619 the tissue or organ of origin. Several equine sera were tested for total AP techniques have been described by activity by the automated American which isoenzymes of AP can be char- Monitor KDA method' using Pacterized in tissue or organ extracts nitrophenylphosphate as substrate, pH of 10.2 at 37°C. These sera origi(5). The sensitivity of intestinal AP to nated from hospital and field clinical L-phenylalanine has been widely cases. Some animals were tested more reported (5,9) and differences in ther- than once. The serum AP concentramostability between bone, liver and tions were compared with the laboraintestinal AP described ( 11,14). L- tory reference values of 220-600 phenylalanine sensitivity has been I. U./ L previously determined for clinused to measure intestinal AP in serum ically healthy horses. samples from normal horses and those Samples of jejunum, liver and rib with various disease problems (2). It were obtained within one hour of was concluded that an increase in death from a six year old horse which serum concentration of intestinal AP had no evidence of intestinal, hepatic was associated with evidence of intesor skeletal disease. Tissue extracts tinal damage, but suggested that inter- were prepared in normal buffered pretation may be difficult when total saline as recommended (2), stored at serum AP is increased. It has been 4°C, and examined within 24 hours of found that the increased AP in perito- preparation. neal fluid from horses with colic was of The extracts were suitably diluted granulocytic rather than of intestinal and tested for total AP using a manual origin greatly decreasing the diagnos- method.2 P-nitrophenylphosphate tic specificity for intestinal damage (6). was used as substrate and an incubaSerum AP is one of the enzymes tion period of 30 minutes at 37°C with included in the Ontario Veterinary a glycine buffer at pH 10.5. The reacCollege equine biochemistry examina- tion was terminated with 0.02 N tion of clinical cases. Increased con- sodium hydroxide and the optical dencentrations are frequently observed sity at 410 nm determined. but often unexplained. It is considered Sensitivity to L-phenylalanine3 was that the serum AP most likely origi- examined by adding varying quanti-
Present address of Dr. Trueman: 18 Mabb Street, Kenmore, Brisbane, Queensland, Australia. 'American Monitor KDA, Indianapolis, Indiana. 2Sigma Chemical Co., St. Louis, Missouri. 3Fischer Scientific Co., Fair Lawn, New Jersey.
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Can Vet J 1983; 24: 108-111.
ties of L-phenylalanine to the glycine squamous cell carcinoma of the eye. bone and intestinal extracts to varying buffer and retesting the extracts. The Group 2B (n=5) included horses with concentrations of L-phenylalanine final concentration in the reaction various diagnoses such as rabies, pleu- (manual technique) is presented in mixture varied from 0.95 mmol/ L to ritis, peritonitis and a ruptured eso- Figure 1. With 7.7 mmol/ L L30.8 mmol/ L L-phenylalanine. A con- phagus. Four horses with intestinal phenylalanine in the reaction mixture centration of L-phenylalanine was symptoms were included in Group 2C, 70% of intestinal AP, 16% of liver AP selected which would give the greatest three were postsurgical colic cases with and 29% of bone AP activity was inhipercentage differentiation of intestinal confirmed salmonellosis in one case bited. This concentration appeared to isoenzymes from those of liver and and suspected in another. The fourth provide optimal differentiation of bone origin. case had intestinal colic which intestinal from liver and bone AP. Heat sensitivity was determined by responded to medical treatment. Heating at 56°C for ten minutes incubating 0.5 mL of each extract at Group 2D (n=2) contained a horse resulted in virtually complete inactiva560 C for exactly ten minutes, retesting with navicular disease and another tion of bone AP while 61.5% and 4.3% and comparing with the total serum with an apical fracture of the sesamoid of activity was lost for liver and intesAP concentration. bone. tinal AP respectively. Forty-nine sera with increased AP Serum AP concentrations were Storage of sera at -20°C for up to concentrations were collected over a examined by group for sensitivity to nine weeks resulted in a mean loss of two month period from 44 clinical L-phenylalanine and thermostability. 12% of the initial AP activity hospital cases. Forty-five of the serum The influence of storage at -20°C was (range + 9.3% to -24%). samples were stored at -20° C and examined as determined by the autoSerum AP activity determined by within nine weeks total AP retested a) mated method. The total serum AP the automated method (l.U./L) was by the automated method b) by the activity in stored sera was compared found to compare to those determined Sigma manual method before and for the automated and manual by the manual method (Sigma after the addition of c) L-phenylalanine methods. units/ mL) times 1 2.4 with a standard and d) exposure to heat as previously deviation of 7.2 (Table 11). described. Four sera held at 4°C were RESULTS L-phenylalanine sensitivity and tested in a similar manner within 48 The distribution of serum AP activity thermostability of serum AP for the hours of collection. The sera were for 1 619 equine samples collected dur- samples from the different clinical thawed and tested in four batches ing an 18 month period is summarized groups are presented in Table 1I. There along with a control pool of ten equine in Table 1. Sensitivity of AP in liver, was no apparent difference in AP sensera with normal AP concentration. sitivity to L-phenylalanine or thermoTABLE I The 44 cases were arbitrarily divided stability between the groups. EQUINE SERUM ALKALINE PHOSPHATASE into two groups according to age, ACTIVITIES OBSERVED OVER AN D IS C U S S I 0 N Group 1 less than and Group 2 greater 18 MONTH PERIOD (n=1619) than 12 months of age (Table 1I). Of the 1 619 foal and adult horse serum These groups were further subdivided AP determinations, 32.6% were Number of according to clinical and laboratory Range (I.U./ L) Samples % increased in relationship to reference data. Group I A (n= 14) contained foals 0-599 (normal) intervals previously established for 1088 67.2 with no evidence of organic disease. 600-999 336 20.7 adult horses. Foals have higher serum 1000-1999 165 10.1 Seven were preanesthesia samples AP levels than adult horses presuma2000-6000 30 1.8 obtained prior to either hernia or casbly related to bone growth (1). The tration operations. The other animals in this group had minor injuries such 100 as contracted tendons or lameness not associated with bone lesions. Group ; ^AA liver I B (n=6) consisted of foals with evi8O0 \ X bone X dence of systemic disease such as 0 Intestine * endocarditis, purpura, severe lacerations with infection and one rabies 60O suspect. Foals in Group IC (n=3) had C diarrhea of at least three or four days duration. Group I D (n=2) included 40F one animal with a fractured radius and E ulna and another with nonhealing 201 0 fractured splint bone. CL The mature animals were subdivided in a similar manner. Group 2A (n=8) included cases with minor condi09 19 38 7.7 15 4 3008 tions such as cryptorchids, lameness mmole/L L-phenylalanine not associated with bone changes, rec- FIGURE 1. Alkaline phosphatase activity in tissue extracts when treated with varying concentrations tal prolapse, pneumovagina and of L-phenalylanine. Z
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TABLE 11 MEANS AND STANDARD DEVIATIONS OF TOTAL ALKALINE PHOSPHATASE DETERMINED BY AN AUTOMATED METHOD ON FRESH SERA, AND MANUAL METHOD ON FROZEN SERA FROM GROUPS OF FOALS (1) AND HORSES (2) WITH DIFFERENT CLINICAL CONDITIONS. SENSITIVIT'Y TO 7.7 MMOL L L-PHENYLALANINE AND HEA-1- Al' 56°C FOR TEN MINUTES Is ALSO SHOWN No. in Group Group 14 IA 6 IB 3 IC 2 ID 2A 8 2B 5 4 2C 2 2D
Automated method fresh sera
(I.U./L) 1067 ± 459 1274± 535 968 ± 140 957± 15 752 ± 125 767 ± 203 1258 ± 431 664± 28
Manual method frozen sera (Sigma Unit/mL) 8.3 ± 3.2 10.4± 3.8 6.8 ± 0.7 6.7± 1.5 6.1 ± 1.2 6.5 ± 2.5 9.4 ± 2.0 5.2±0.2
serum AP from the 14 foals in group 1 A was increased likely for this physiological reason rather than any apparent clinical disease. It is interesting to note the variety of conditions associated with increased AP levels in the other groups. In no case was there direct evidence of hepatobiliary disease which is frequently associated with increased serum AP in other species (3,13). It has been reported that 15 mmol/ L of L-phenylalanine gave approximately 80% inhibition of intestinal extract AP, while liver and bone extracts were inhibited by 15% and 20% respectively (2). This is in contrast to the present observations where 7.7 mmol/ L L-phenylalanine gave the greatest percentage differentiation between intestinal AP, and that of liver or bone. For the lower concentration of L-phenylalanine similar degrees of inhibition have been reported for other species (9,12), except the 29% inhibition of bone AP in the present study is greater than
expected. The resistance of intestinal AP to heat was similar to previous reports (5,14). However, the complete inhibition of bone AP and 61.5% inhibition of liver AP is greater than described for other species (11,14). Temperature and time are important factors which affect the heat inactivation rates (10,14). At 560 C for 15 minutes almost complete inactivation of equine liver AP has been described [5). Handling and storage of serum samples is one of several factors that influence AP determinations (15). A 25% decrease in activity after six months at -10°C has been reported (15). Other workers (8) found freezing 110
L-Phenylalanine (% inhibited) 26.7 3.0 25.7 3.2 27.9 2.8 28.5±5.0 28.2 ± 2.6 25.9 ± 3.5 27.3 ± 4.8 25.7±0.3
Thermostability (% inhibited) 90.9 4.2 88.8 1.4 89.1 +2.0 89.5±0.3 88.6 2.3 86.1 ± 2.2 85.7 ± 2.7 91.4± 1.1
for four days increased AP activity 1.5% (range 4% to + 5%). In the present work a mean 12% decrease in AP activity in sera stored at -20°C was found. There appears to be no information available with regard to the influence of freezing (-20°C) and storing tissue AP isoenzymes. Human extracts stored at -80°C were found to be stable over a period of several months (7). However, if there is variable stability of tissue isoenzymes when stored at -20° C, storage of the 45 serum samples may have influenced our attempts to detect organ related isoenzymes. The similar sensitivity of sera from all groups to L-phenylalanine and to heat, would indicate that in no group was a significant amount of intestinal AP present. Small increases in intestinal AP have been found in the serum of horses with diarrhea or intestinal parasitism. This was not detected in the seven animals with history of intestinal disease in this study. As three of the four adult horses were postsurgical cases, this may have attributed to the increased serum AP detected. In a recent report of 50 horses with colic (6), serum AP values were found to be within the normal range. Thermostability of serum AP has been found useful in separating hepatobiliary from skeletal disease in human medicine (4,11). No difference for the clinical groups reported here was observed, although hepatobiliary disease was not suspected in any case. The marked susceptibility of equine sera and bone extract to heat reported in this and other studies (5) would suggest that a large proportion of equine serum AP is of osseous origin. In human sera a thermosensitivitv result-
ing in less than 25% remaining activity is considered to be indicative of increased osteoblastic activity (4). The "normal" foals of group I A had increased serum AP in relation to adult horses but the similar thermosensitivity of other groups would suggest the increased AP to be of osseous origin. In normal children increased serum AP is derived mainly from bone due to increased osteoblastic activity (I 1) and the same interpretation appears to apply for foals. Serum AP isoenzyme classification using L-phenylalanine inhibition or heat sensitivity provided limited direction in identifying the organ or tissue of origin for the clinical cases reported. Further development is required before these techniques will be of significant assistance to the diagnostician. REFERENCES 1. BLACKMORE DJ, ELTON D. Enzyme activity in the serum of thoroughbred horses in the United Kingdom. Equine Vet J 1975; 7: 34-39. 2. BLACKMORE DJ, PALMER A. Phenylalanine inhibited P-nitrophenyl phosphate activity in the serum as an indicator of intestinal cellular disruption in the horse. Res Vet Sci 1977; 23: 146-152. 3. EVERETT RM, DUNCAN JR, PRASSE KW. Alka-
line phosphatase, leucine aminopeptidase and alanine aminotransferase activities with obstructive and toxic hepatic disease in cats. Am J Vet Res 1977; 38: 963-966. 4. FITZGERALD MXM, FENNELLY JJ, McGEENEY K.
The value of differential alkaline phosphatase thermostability in clinical diagnoses. Am J Clin Path 1969; 51: 194-201. 5. FROSCHER BG, NAGODE LA. lsoenzymes of equine alkaline phosphatase. Am J Vet Res 1979; 40: 1514-1517. 6. FROSCHER BG, NAGODE LA. Origin and importance of increased alkaline phosphatase activity in peritoneal fluids of horses with colic. Am J Vet Res 1981; 42: 888-891. 7. GERHARDT W, LYKKEGAARD NIELSEN M, VAGN NIELSEN 0, OLSEN JS, STATLAND BE.
Routine measurements of liver and bone alkaline phosphatase in human serum: differential inhibition by L-phenylalanine and carbamide (urea) on the LKB 8600 reaction rate analyzer. Clin Chim Acta 1974; 53: 281290. 8. MASSION CG, FRANKENFELD JK. Alkaline
phosphatase: liability in fresh and frozen human serum and in lyophilized control material. Clin Chem 1972; 18: 366-373. 9. NAGODE LA. KOESTNER A. STERNMEYER CL.
Organ identifying properties of alkaline phosphatases from canine tissues. Clin
Chim Acta 1969; 26: 45-54. 10. POSEN S. NEALE FC, CLUBB JS. Heat inactivation in the study of human alkaline phosphatases. Ann Intern Med 1965; 62: 1234-1243. 11. RATLIFF CR, HALL FF, CULP TWr GEVEDON RE, WESTFALL CL. The differentiation of hepatic and skeletal alkaline phosphatase by ther-
mofractionation. Gastroenterology 1972; 58: 22-29. 12. REYNOSO G, ELIAS EG. MITTELMAN A. The
contribution of the intestinal mucosa to the total serum alkaline phosphatase activity. Am J Clin Path 1971; 56: 707-712. 13. ROGERS WA. Source of serum alkaline phosphatase in clinically normal and diseased
dogs: a clinical study. J Am Vet Med Assoc 1976; 168: 934-937. 14. SAINI PK. SAINI SK. Origin of serum alkaline phosphatase in the dog. Am J Vet Res 1978; 39: 1510-1513. 15. YOUNG DS, PESTANER IlC, GIBBERMAN V. Effects of drugs on clinical laboratory tests. Clin Chem 1975; 21: 246D-248D.
LETTER TO THE EDITOR
An Open Letter to the World Veterinary Congress Against Nuclear War
superpowers cannot in any sense be considered to be weapons of war. They are instruments of global suicide as likely to be set off by accident as DEAR SIR: by intention (6). As such, they should Enclosed please find an open letter be of concern to every citizen of the addressed to the World Veterinary world. Congress. In hopes that the issues Many national and international raised therein can be widely discussed medical and scientific associations by veterinarians before the Congress, I have taken a public stand in favor of a have submitted the letter to the Cana- nuclear freeze and nuclear disarmadian Veterinary Journal, the Austral- ment. They have done so not only as ian Veterinary Journal, the Journal of sane and responsible world citizens, the American Veterinary Medical but as professional groups with speAssociation and The Veterinary cific areas of expertise. While the surRecord. vival of the human race may not, While many scientific and profes- strictly speaking, be a veterinary consional bodies have addressed this sub- cern, the well-being of animal life on ject, we as veterinarians have acted to this planet definitely lies within our date as if the redirection of funds from domain of professional interest and veterinary and human health care and expertise. research to bomb building - and The World Veterinary Congress indeed as if the annihilation of animal provides an unprecedented opportunlife on this planet - have nothing to ity for veterinarians to make a nonpardo with us. I think it high time this tisan, global, stand against the stealing situation be redressed. Since I will not of money from veterinary and health have funding to attend the Congress, I concerns to feed the nuclear beast. am also hoping with this letter to stir This, surely, is an issue beyond the interested people who are able to scope of political bickering, an ethical attend to carry the issue through to issue on which even humane societies some kind of resolution. and intensive veal farmers, electron microscopists and epidemiologists, To the World Veterinary Congress right wingers, left wingers and centre In 1962, the New England Journal fielders can agree. of Medicine published the results of a I would like to suggest that this issue landmark study on the medical conse- be brought before the full congress and quences of thermonuclear war (1-5). that a statement, however brief and to Since that time, evidence has accumu- the point, be made and distributed to lated that the nuclear arsenals of the the appropriate powers that be. It is a Can Vet J 1983; 24: 111.
naive worldview indeed, and one not shared by the animals we care for, that sees the choices as "our side", "their side", and suicide. If we cannot bring ourselves to speak, undividedly, on behalf of the human race, let's at least make some noise on behalf of the animals. It may do more to promote animal and human well-being than all the vaccines in the world. DAVID WALTNER-TOEWS, D.V.M. 126-252 Stone Road West, Guelph, Ontario N1 G 2 V7
References I. GARLAND J. The medical consequences of thermonuclear war: editor's note. N Engl J Med 1962; 266: 1126-1127. 2. ERVIN FR, GLAZIER JB. ARONOW S, NATHAN D, COLEMAN R, AVERY N, SHOHET S. LEEMAN C.
3.
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The medical consequences of thermonuclear war. l. Human and ecologic effects in Massachusetts of an assumed thermonuclear attack on the United States. N Engl J Med 1962; 266: 1127-1137. SIDEL VW. GEIGER HJ. LOWN B. The medical consequences of thermonuclear war. 11. The physicians role in the postattack period. N Engl J Med 1962; 266: 1137-1145. ARONOW s. The medical consequences of thermonuclear war. 111. A glossary of radiation terminology. N Engl J Med 1962; 266: 1145-1149. LEIDERMAN PH. MENDELSON JH. The medical consequences of thermonuclear war. IV Some psychiatric and social aspects of the defense shelter program. N Engl J Med 1962; 266: 1149-1155. MULLER JE. On accidental nuclear war. Newsweek 1982; March 1, p.9.
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