A granulocyte test for detection of cattle heterozygous for mannosidosis

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Clinica Chimica Acta, 88 (1978) 429-435 @ ElsevierlNorth-Holland Biomedical Press

CCA 9473

A GRANULOCYTE TEST FOR DETECTION OF CATTLE HETEROZYGOUS FOR MANNOSIDOSIS

P.J. HEALY

*, P.J. NICHOLLS

** and PETRUSIA

BUTREJ

New South Wales Department of Agriculture, Veterinary Research Station, Roy Watts Road, Glenfield, New South Wales, 2167 (Australia) (Received

January

31st, 1978)

ar-Mannosidase and @-N-acetylglucosaminidase activities were found to differ between lymphocytes and granulocytes isolated from bovine blood. Activities of both enzymes in granulocyte preparations were found to be related to the eosinophil content of the preparations. A procedure is described that allows definition of the mannosidosis genotype of adult cattle by giving consideration to the influence of eosinophil content of granulocjrte preparations upon the activity of ar-mannosidase relative to that of ~-~-acetylglucos~inid~e.

Introduction Estimation of the activity of acidic cw-mannosidase, EC 3.2.1.52 (aM) in plasma is the primary test used for detection of cattle heterozygous for the lysosomal storage disease, mannosidosis [1,2]. As age, sex and other unknown factors also influence plasma oM activity [3], there is a requirement for a secondary test to allow definition of the genotype of animals that give inconclusive results with the plasma test, or are to be tested as individu~s. Leucocyte tests are used to confirm diagnoses of a number of lysosomal storage diseases of man [4]. Beutler et al. ]5] demonstrated that lysosomal enzyme activities vary between different classes of human leucocytes. A lymphocyte test has been proposed as a secondary test for detection of cattle heterozygous for mannosidosis, but this test is not reliable as some overlap occurs between samples from heterozygotes and from normal homozygotes [ 61. In this communication we describe investigations of a granulocyte test for detection of heterozygotes for bovine mannosidosis. In selection of procedures * To whom correspondence should be addressed. ** Present address: Biometrical Branch, Department Wales, Australia, 2000.

of Agriculture, Pbillip Street, Sydney, New South

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we considered the fact that many animals at risk will be in areas remote from laboratories. Therefore, the test had to be applicable to samples transported considerable distances. It is well known that EDTA is superior to heparin as an anticoagulant in preserving leucocyte morphology, hence we elected to investigate a test based upon examination of EDTA-treated blood. As crM is Zn’+ dependent [7], we have investigated the effect of addition of Zn” to media used to extract enzymes from leucocytes, in order to reverse the inhibitory effect caused by Zn*’ chelation by EDTA. The majority of leucocyte tests used in investigation of lysosomal storage diseases in man are based upon estimation of enzyme activities in relation to protein content of leucocyte extracts. We considered that it would be more appropriate to relate cwMactivity in granulocyte extracts to that of acidic P-N-acetylglucosaminidase (/3-2-acetamido2-deoxy-D-glucoside acetamidodeoxyglucohydrolase, EC 3.2.1.30 (NAG)), as activity of this lysosomal enzyme is not affected by heterozygosity [S] and the enzyme assay could be expected to be more precise than current methods of protein assay. Materials and methods Animals

The animals used in this investigation were: 1. Ten adult Australian Illawarra Shorthorn cows and 10 3-month-old calves of the same breed, The animals were from a dairy herd at the Veterinary Research Station, Glenfield, that contained no amimals known to be heterozygous for mannosidosis. 2. 252 adult Australian Illawarra Shorthorn, Friesian, Angus and Murray Grey cows that were considered to be homozygous-normal for mannosidosis, based upon results of estimation of plasma (YMactivity. 3.17 adult Angus and Murray Grey cows considered to be heterozygous for mannosidosis. All animals had plasma cwMactivities less than 50% of the mean activity of groups of animals they were grazing with. Samples

Blood samples were collected from the external jugular vein into lOO-ml vessels containing 150 mg disodium EDTA, or from the coccygeal vessels into evacuated tubes containing 15 mg EDTA. Unless otherwise stated, the samples were held for 24 h at 4°C before commencing leucocyte isolations. Isolation of leucocy tes

All procedures

were carried out at room temperature:

1. Lymphocytes

After 10 ml of blood was centrifuged at 1500 X g for 15 min, the buffy coat was harvested into 2 ml of 0.154 M NaCl (saline). The packed cells were resuspended in 2 ml of saline and centrifuged as before, the resultant buffy coat was pooled with the previous buffy coat and carefully layered over 3 ml of FicolPaque (Pharmacia, Uppsala, Sweden) and centrifuged at 400 X g for 40 min. The interface was harvested into 2 ml saline and centrifuged at 100 X g for

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5 min. The resultant pellet was resuspended in saline and centrifuged again at 100 X g for 5 min. Erythrocytes in the preparation were lysed by suspension in 0.1 M NH&l and the lymphocytes deposited by centrifugation at 400 X g for 10 min. A smear of the cell preparations was made, the cells were then resuspended in saline and centrifuged at 1500 X g for 5 min. The pellet was resuspended in 4 ml of saline and the number of cells determined by counting with an electronic particle counter (Coulter model F). After a further centrifugation at 400 X g for 10 min the pellet was drained, by inversion of the tube, and the cells resuspended in 1 ml of 0.2% aqueous Triton X-100, containing 1 mM ZnClz. The preparation was then frozen until required for analysis. 2. Granulocy tes Packed cells, remaining after harvesting the buffy coats, were washed again with saline, then decanted into a 50-ml tube. The cells were mixed with 20 ml distilled water followed 30 set later by 5 ml of 0.77 M NaCl. The mixture was centrifuged at 1500 X g for 10 min and the resultant pellet washed with saline. The few remaining erythrocytes were lysed with NH&l, as described above, and the granulocytes were washed with saline. Smears were made of the preparations, cell numbers determined and then the cells resuspended in triton and frozen, as described above. Analytical procedures Enzyme activities aM activity in extracts of the leucocytes was determined using the procedure described by Healy and Cole [2]. NAG activity in 0.025 ml of extract was estimated using 0.25 ml of 2.5 mM p-nitrophenyl-iVacetyl-fl-D-glucosaminide (Koch Light Colnbrook, U.K.) in 0.1 M citrate phosphate buffer, pH 4.3. Amount of p-nitrophenol released was determined by spectrophotometry at 405 nm, as in the estimation of crM activity. Protein concentration in leucocyte extracts was determined using the procedure of Bradford [ 91. Preliminary estimations demonstrated close agreement between results obtained by this procedure and the Lowry method [lo] in estimation of the protein content of leucocyte extracts, r = 0.978, n = 10. Examination of leucocyte smears Smears of the leucocyte preparations were stained with “Diff Quik” (Harleco, Gibbestown, New Jersey, U.S.A.). Estimates of granulocyte content of lymphocyte preparations were made by counting 400 cells along the periphery of the smears. Composition of the granulocyte preparations was determined by counting 200 cells in a series of traverses across the breadth of the smears. Statistical methods Significance of differences between means were estimated using t-tests. For the group of 252 adult normal and 17 adult heterozygote animals the logarithmic transformation of both the reciprocal of the enzyme ratio (cuM/ NAG) and per cent eosinophils in granulocyte preparations were analysed by least-squares regression equations.

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The technique of linear discriminant functions [ 111 was used to provide a method of classifying animals into either group, based on logarithms of the reciprocal of the enzyme ratio and per cent eosinophils in granulocyte preparations. Results Lymphocyte and granulocyte preparations were made at 2, 24 and 48 h after collection of blood from the 10 Australian Illawarra Shorthorn cows and calves. Lymphocyte preparations from all animals were relatively pure, granulocyte content in all cases being estimated to be less than 1 per cent. Granulocyte preparations from the calves consisted mainly of neutrophils, with eosinophil content varying from 0.5% to 4%, mean 1%. On the other hand, granulocyte preparations from the adult animals were found to be mixtures of neutrophils and eosinophils, mean eosinophil content being 36%, range 6-62%. Lymphocyte content of the granulocyte preparations was less than 2% in all cases. In the preparations made at 48 h up to 10% of cells were unidentifiable. Table I presents results of analyses of these preparations. Within each type of preparation there were no significant differences in protein content in extracts prepared at 2 and 24 h. Activity of aM was higher in lymphocyte preparations made at 24 h than in those made at 2 h (P < 0.01). There were no significant differences in NAG activity in lymphocyte preparations made at 2 and 24 h or between aM and NAG activities in granulocytes prepared at 2 and 24 h. In some preparations made at 48 h, protein content and enzyme activities were lower than those in preparations made at 2 and 24 h. There were no significant differences in protein content or enzyme activities in extracts prepared from TABLE I MEAN PROTEIN CONTENT AND ENZYME ACTIVITIES IN EXTRACTS OF LEUCOCYTES PREPARATIONS MADE AT VARIOUS TIMES AFTER COLLECTION OF BLOOD FROM CATTLE CONSIDERED TO BE NORMAL FOR MANNOSIDOSIS n = 10, S.E.M. in parentheses. HOlUS

Lymphocytes

after collection

cows

Protein (pg/106 cells)

2 24 48

1.78 1.25 1.09

(0.27) (0.09) (0.07)

1.55 1.44 1.33

(0.11) (0.11) (0.18)

NAG (m I.U./pg protein)

2 24 48

0.225 0.262 0.199

(0.034) (0.022) (0.025)

0.189 0.232 0.175

(0.022) (0.032) (0.020)

0.360 0.382 0.334

(0.060) (0.071) (0.061)

0.127 0.107 0.121

(0.014) (0.017) (0.021)

NAG (m I.U./106

2 24 48

0.324 0.321 0.212

(0.031) (0.028) (0.028)

0.288 0.313 0.222

(0.035) (0.031) (0.028)

7.07 7.14 5.77

(1.02) (0.99) (0.73)

1.82 1.47 1.31

(0.066) (0.088) (0.082)

ffM (m I. U. /pg protein)

2 24 48

0.013 0.021 0.014

(0.003) (0.003) (0.002)

0.009 0.017 0.011

(0.002) (0.002) (0.001)

0.101 0.113 0.096

(0.020) (0.024) (0.020)

0.012 0.010 0.012

(0.001) (0.001) (0.002)

cuM (m I.U./106

2 24 48

0.018 0.025 0.015

(0.003) (0.004) (0.002)

0.014 0.024 0.015

(0.003) (0.004) (0.002)

1.97 (0.35) 2.11 (0.38) 1.46 (0.30)

cells)

cells)

Granulocytes Calves

Calves

cows 20.6 20.5 18.9

(1.07) (2.08) (1.74)

15.8 16.2 13.2

(1.61) (1.92) (1.72)

0.18 (0.028) 0.15 (0.017) 0.14 (0.016)

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lymphocytes from cows and calves. On the other hand, protein content and enzyme activities were higher in extracts of granulocytes from cows than in those from calves. Protein content and enzyme activities of granulocyte extracts were greater than in extracts of lymphocytes. This was most evident in samples from cows. The mean (YM activity per unit of NAG activity (enzyme ratio) in extracts of lymphocytes isolated at 24 h was not significantly different between cows and calves, ratios being 0.073 (S.E.M. 0.008) and 0.077 (S.E.M. 0.012) respectively. Mean enzyme ratio was higher in granulocytes from cows, 0.297 (S.E.M. 0.017) than calves, 0.106 (S.E.M. 0.014). In the granulocyte preparations from cows, made at 24 h, the ratio of enzyme activities was related to the number of eosinophils in the preparation. The relation can be described by the equation. (Log enzyme ratio))l = 0.868 - 0.205 X log (per cent eosinophils). Lymphocyte and granulocyte preparations from 10 adult Australian Illawarra Shorthorn cows were divided into 2 aliquots prior to resuspension in triton. One aliquot was resuspended in triton containing 1 mM ZnCl,, the other in triton without added ZnCl,. There were no significant differences in NAG activity between extracts of the aliquots, but cwM activity was significantly lower in extracts without ZnClz (P < 0.001). The effect of inclusion of ZnClz in triton is demonstrated by differences in enzyme ratios. For lymphocytes 0.117 (S.E.M. 0.012) with ZnCl, and 0.064 (S.E.M. 0.011) without ZnClz, and for granulocytes 0.23 (S.E.M. 0.011) and 0.189 (S.E.M. 0.011) respectively. Inclusion of 1 mM ZnCl, in the triton used to resuspend cell preparations results in a concentration of 0.091 mM ZnCl, in the reaction mixtures used for estimation of enzyme activities. When extracts prepared in triton without added ZnCl, were analysed using substrate preparations with and without ZnClz (concentration 0.091 mM), there were no significant differences in either NAG of cuM activities. Granulocytes preparations were made from duplicate blood samples taken from the same 10 cows. Precision of the method for estimation of enzyme activity ratios was found to be 1% of the mean value. Precision was calculated using the formula dm, where d is the difference between results of duplicate estimations. Granulocyte preparations were made from samples collected from 252 normal and 17 heterozygote animals. Fig. 1 illustrates the relationships between enzyme ratios and eosinophil content of the preparations. The preparations from the normal and heterozygote animals contained between 0.5%-71% and 9.5%-43% eosinophils, respectively. Regression equations relating enzyme ratios to per cent eosinophils were: Normals: (log enzyme ratio))l = 1.217 (+S.E.M. 0.028) - 0.380 (kO.023) X log (per cent eosinophils), r = -0.728. Heterozygotes: (log enzyme ratio)-l = 1.768 (kO.206) - 0.409 (kO.150) X log (per cent eosinophils), r = -0.576 The regression equations were compared assuming that the linear relation for heterozygotes would be consistent for preparations over the range of per cent eosinophils observed in the normal group. Both slope coefficients were significantly negative (P < 0.001 and P < 0.05, respectively, but were not significantly different however, the intercepts were significantly different (P <

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1.6 -

-0.4

0.0

0.4 log

08 I%EOSINOPHILSl

1.2

1.6

Fig. 1. Plot of log transformed values for per cent eosinophils and reciprocal of the ratio of or-mannosidase and P-N-acetylglucosaminidase activities in granulocyte preparations from 252 normal (0) and 17 heterozygote (a) cattle, A discriminant line is shown.

0.001). The regression lines can be regarded as parallel, with a common slope of -0.381 (20.022). The linear combination of the 2 measures which best discriminated between the 2 groups was (log enzyme ratio)-’ + 0.431 log (per cent eosinophils), which had means of 1.277 (+S.E.M. 0.115) and 1.799 (kO.114) for the two groups. This discriminant function was highly significant (Fz 267= 163.7, P < 0.001). A discriminant line, (log enzyme ratio)-l = 1.538 - 0.431 X log (per cent eosinophils), based upon a point midway between the means of the 2 groups, is shown graphically in Fig. 1. The estimated probability of misclassification of either normal or heterozygote animals was found to be 1.2%, i.e. 3 normal animals out of the 252 could be misclassified. From Fig. 1, it can be seen that 2 normal animals were misclassified. Discussion The results presented in this paper show that granulocyte test based upon consideration of the relative activities of (YMand NAG in relation to eosinophil content of the preparations is a suitable method for determination of the mannosidosis genotype of adult cattle. It is difficult to obtain an accurate estimate of the eosinophil content of granulocyte preparations containing less than 2-3s eosinophils. As young calves are not likely to have a significant number of eosinophils in their circulation this test may not be applicable when it is desired to determine their genotype. Results presented in Table I show that aM activity in granulocyte preparations may be 100 times that in lymphocyte preparations. A minor contamination of lymphocyte preparations with eosinophils could result in classification of a heterozygote animal as a homozygote-normal if aM activity is considered in relation to protein content of the extracts [ 61.

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Snaith [ 71 reported that IBMis activated by pre-incubation of EDTA-treated but not fully by the presence of Zn*’ in the reaction mixenzyme with Zn*‘, ture used to determine its activity, Our observations confirm this finding. We observed that the degree of activation stimulated by ZnCL varied from preparation to preparation. It is possible that homozygote-normal animals could be classified as heterozygotes if the inhibitory effect, caused by chelation of Zn2+ by EDTA is not reversed by inclusion of Zn2’ in media used to extract the enzyme from leucocyte preparations [6]. Mixed leucocyte prep~ations are frequently used in investigation of lysozomal storage diseases in man [ 12-161. We appreciate that eosinophil content of granulocyte preparations from bovine blood may be much higher than that likely to be found in similar preparations from other species. Also, that there are probably significant differences in substrate specificities for the lysosomal enzymes between different species. Nevertheless the results of this study vindicate the concern expressed by Beutler et al. [ 51 and Thompson et al. [6] that careful attention must be given to the composition of leucocyte preparations when it is desired to discriminate between heterozygous and homozygotenormal subjects. References 1 Jolly, R.D.. Digby. J.G. and RammeII, C.G. (1976)

N.Z. Vet. J. 22, 218-222 2 Healy, P.J. and Cole, A.E. (1976) Aust. Vet. J. 52,385-386 3 Jolly, R.D.. Thompson, K.G.. Tse, C.A., Mumford, R.E. and Merrill. M. (1974) N.Z. Vet. J. 22,155160 4 Stanbury, J.B.. Wynagaarden, J.B. and Fredrickson, D.F. (1972) The metabolic basis of inherited diseases, McGraw I-Ii& New York 5 BeutIer, E., K&I, W., Matsumoto, F. and PaugaIis. G. (1976) J. EXP. Med. 143.975-980 6 Thompson, K.G., Jolly, R.D. and Winchester, B.G. (1976) Biochem. Med. 15.233-240 7 Snaith, S.M. (1977) Bioehem. J. 163.557-564 8 Hocking, J.D., JoBy, R.D. and Bat& R.D. (1972) Biochem. J. 128,69-78 9 Bradford, M.M. (1976) Anal. Biochem. 72.248-254 10 Hartee. E.F. (1972) AnaI. Biochem. 48.422427 11 Snedecor, G.W. and Cochran. W.G. (1967) Statistical Methods, 6th edn.. Iowa State University Press, Ames, Iowa 12 Matsuda. I.. Arashima, S., Anakura. M. and Oka, Y. (1973) Clin. Chim. Acta 48,9-13 13 Peters. S.P., Lee, R.E. and Glow, R.H. (1975) Clin. Chim. Acta 60, 391-396 14 Liem, K.O. and Hooghinkel, G.J.M. (1975) Clin. Chim. Acta 60,259-262 15 Troost, J., van der Heijden. M.C. and StaaI, G.E.J. (1976) Clin. Chim. Acta 73,321-327 16 Suzuki, Y.. Fukuoka, K.. Wey, J.J. and Handa, S. (1977) Clin. Chim. Acta 75.91-97