Burvenich, Christian

Roets, E

Vandeputte-Van Messom, G

English

Burvenich, Christian Paul G.

Ghent University Academic Bibliography

Changes in N-Acetyl-13-D-Glucosaminidase Activities in Relation to Other Milk Components Throughout Normal Lactation in the Guinea Pig E. ROETS, C. BURVENICH, end G. VANDEPU'n'E-VAN MESSOM Department of Veterinary Physiology Veterinary Faculty of the University of Ghent Casinoplein 24, B-9000 Gent, Belgium ABSTRACT Changes in milk production, NAGase activities, Na, C1, K, lactose, total N, and fat were followed throughout normal ac- tation in 26 healthy guinea pigs. Appro- priate microassays were used and all de- terminations were performed on the same small skim milk sample (microhematocrit tube). A very sensitive and rapid spec- trofluorimetric determination f NAGase in 10-~tl skim milk samples was evaluat- ed. Four days after parturition, daily milk production reached a maximum of about 20 ml. Thereafter milk yield decreased progressively to about 7 ml on d 15. During the entire course of lactation, a progressive increase in NAGas¢, total N, fat, Na, and CI was observed. However, K and lactose decreased. Highly signifi- cant positive correlations between NAG- ase and Na, C1, fat, and total N were evident, whereas correlations were nega- tive between NAGase and K, lactose, and milk production. These changes can be explained on basis of alterations that oc- cur at the level of the blood-milk barrier. The short lactation period of the guinea pig, the easy handling and milking prac- tices, together with the available sensitive micromethods can lead to the establish- ment of a convenient guinea pig mastitis model. INTRODUCTION The guinea pig has been chosen by several authors (11, 14) as a suitable model for study- ing the physiology of normal milk secretion Rcc.giveA February 13, 1988. Ac.c¢ptcd June 16, 1989. during an entire lactation. A more extensive study with an excellent analysis of milk pro- duction, macroingredients, and macrominerals has been performed recently (1, 2, 3, 16). In these studies during lactation, milk K and lac- tose decreased, whereas Na and C1 increased. These changes, normally occurring after about d 6 of lactation, are attributed to an increase of the paracellular movement of ions and small molecules between extracellular fluid and milk (13). This is in agreement with the changes observed in milk during late lactation in the cow. The guinea pig has also been used to study leukocyte migration in the milk during experimentally induced endotoxin mastitis (9, 10). Concomitant changes in the concentrations of some milk components are typical for the diagnosis of bovine mastitis, i.e., a decrease of lactose and K and an increase of Na, CI, and serum albumin concentration. An influx of polymorphonuclear blood cells into milk occurs as well (18). It appears that of the different enzymes ecreted into the milk, the lysosomal enzyme NAGase is a good parameter for the diagnosis of mastiris. Significant correlations between NAGase activity and SCC (6, 7), CI, lactose, and lactoferrin (12) have been demon- strated in cow's milk. Moreover, NAGase ac- tivity was indicative of the severity of inflam- marion of the udder. The establishment of a guinea pig mastitis model, in which determination f milk produc- tion and composition by daily milking is per- formed, could be very useful for preliminary studies of the inflammation of the mammary gland. Experimentally induced mastiffs experi- ments on lactating cows are very expensive and time-consuming. The objective of the present work was to study the changes in NAGase activities in rela- tion to other components in milk throughout normal actation in guinea pigs. All determina- tions were performed on the same milk sample 1989 J DahT Sci 72:3197-3203 3197 3198 ROErS ET AL. collected uring the entire lactation period (15 d). Furthermore, the possibility to use a very sensitive (10 Ixl skim milk) and rapid spec- trofluorimetric determination of NAGase as a marker of inflammation was evaluated. MATERIALS AND METHODS Animals Healthy female common guinea pigs were mated (one male per group of 6 to 10 females). To avoid individual variability, experiments were performed during second lactation. Twenty-six guinea pigs at about 6 wk of age were used. The guinea pigs were ear-tagged and kept under controlled temperature (19 ± I'C) in a box with straw on the floor. They were fed at regular times (0800 and 1600 h) with commercial guinea pig pellets, carrots, cabbage, and grass. This diet was regularly supplemented with ascorbic acid in the drinking water, which was provided for ad libitum in- take. The weight of the mothers on the day of the second parturition was 944 + 52 g (mean ± SE). Collection of Milk In order to ensure the presence of sufficient milk in the mammary glands, the mothers were separated from the litter for 6 h before milking. Milking occurred daily during the lactation period. Although the use of oxytocin during milking has been recommended in literature (8), we did not use it because: 1) the injections induce different degrees of stress, 2) oxytoein may alter milk electrolyte composition, and 3) only minor differences in the milk volume ob- tained with or without oxytocin, were observed in preliminary experiments. Milk was obtained at 1400 h by means of machine designed by McKenzie and Anderson (8). The guinea pig was placed on its back in the operator's hand and ann. By gentle massage of the glands and under a slight negative pressure (20 + cm Hg), milk was collected in preweighed plastic vials. Milk from each of the two mammary glands of an animal was sampled separately. After being weighed both milk samples were mixed for further analysis. Because the rate of milk secre- tion is constant, daily milk production (MP) (g/ 24 h) was calculated by multiplying the amount of milk produced in 6 h (between 0800 and 1400 h) by 4. Chemical Determinations Because guinea pig milk contains high amounts of fat (>6%), and fat sometimes influ- ences some chemical determinations, we de- cided to determine the different milk constitu- ents in fat-free milk. Fresh milk (about 70 ktl) was collected directly into capillary tubes of 75-ram long, sealed, and cenlrifugated at 4"C for 15 rain at 12,000 x g in a microhematocrit centrifuge (Hawksley Ltd., Lancing, Sussex). After centrifugation, the length of the column of fat, which was clearly separated from the skim milk, was measured and calculated as a percentage of the total volume of milk in the tube. To collect he skim milk, the upper part of the capillary tube just below the boundary of the packed fat was cut off, and appropriate volumes (10 ~tl for NAGase, K and Na; 20 p.l for lactose and CI; 100 ktl for total N) were obtained with a 50-~tl microsyringe and stored at -20"C until analysis. Sodium and K were estimated by means of flame photometry; C1 was measured with a chloride meter. Lactose was assayed without deproteinization according to Yamashita and Watanabe (19), adapted for guinea pig milk by Mepham and Beck (11). Total N (TN) was determined by Kjeldahl. Oeterminatlon of N-Acetyl-I~.D-Glucosamin idase and Milk Components The lysosomal enzyme NAGase was mea- sured in skim milk with a fluorimetric proce- dure using 4-methylumbelliferyl-N-acetyl-~-D- glucosaminide as substrate. Skim milk was used, because in cow's milk, 91% of the total activity is present in the skim milk fraction (7). Moreover, when using whole milk, turbidity problems were often encountered and spec- trofluorimetric readings were inaccurate. These problems, which are probably due to the high amounts of fat in guinea pig milk, could not be overcome by using a small volume (10 lal) of fresh milk or by improving fat dispersion by incorporating a detergent (.1% Triton X-100) in the buffer solution. The enzymatic reaction was started by add- ing lOlal of skim milk to a tube containing 200 Journal of Dairy Science Vol. 72, No. 12, 1989 N-ACETYL-[3-D-GLUCOSAMINIDASE AND MILK COMPONENTS 3199 TABLE 1. Concentrations and relative changes of Na, K. CI, lactose, fat, NAGase, and total N in guinea pig milk during sectmd lactation. Parameter Mean SEM Range CMrve course Na, mM 20.8 1.6 13.8-33.8 Increase K, mM 23.4 1.3 12.0--28.0 Decrease CI, mM 45.5 4.5 24.5-79.0 Increase Lactose, g/100 g 4.3 .2 2.9-5.1 Decrease Fat, % 11.0 .6 8.0-15.8 Increase NAGase, riM/rain per ml 20.6 1.9 11.2-38.7 Increase Total N, mg/100 mg 767 40 631-1074 Increase gl of 2 mM 4-methylumbelliferyl-N-acetyl-~- D-glucosaminide in .25 M citrate buffer (pH 4.4), and incubated for 10 rain at 37"C. The reaction was terminated by the addition of 5.5 ml of .1 M carbonate buffer (pH 10), and the released 4-methylumbelliferone was measured at an excitation wavelength of 365 nm and an emission wavelength of 450 nm, using a spec- trofluodphotometer. Enzyme activity was ex- pressed as nanomoles of liberated 4-methylum- belliferone per minute in 1 ml of skim milk. RESULTS In Table 1, data of the different milk compo- nents during the 15 successive d of lactation were averaged for all animals (n -- 26). With the exception of K and lactose, all milk compo- nents analyzed increased in concentration pro- gressively during lactation, differing by about a factor two to three times between start and finish. The mean daily average of the twenty-six animals for MP and NAGase are shown in Figure 1. Daily MP varied among animals and ranged from 5.9 to 18.9 g (mean of 11.8 ± 1.2 g]24 h). The MP increased rapidly until d 4 and then declined slowly until d 10. During the last part of the lactation, MP was low and amounted to about 7 g. The NAGase activities decreased from 23.5 ± 1.5 nM/min per ml on d 1 to 11.2 + .9 nM/min per ml on d 4 and then increased steadily to 38.7 ± 2.6 nM/min per ml on d 15. Mean changes in the concentrations of fat and TN and also shown in Figure 1. The changes in Na, K, Cl, and lactose are in Figure 2. At the onset of lactation, average lactose and fat concentrations were 4.8 ± .2 and 10.9 ± .8 g/100 g, respectively. At the end of lactation (d 15) these parameters averaged 2.9 + .5 and 15.8 + 1.6 g/100 g. Total N decreased from 823 + 62 mg/100 ml on d 1 to 624 + 52 mg% on d 6 and then increased steadily to 1031 ± 79 mg/100 ml on d 15; Na and CI showed a similar pattern, At the onset of lactation, Na and CI concentra- tions were 14.7 ± 1.0 and 24.5 + 1.3 raM, respectively, whereas by d 15 they were 33.8 ± 5.0 and 79.0 + 5.4 raM, Potassium fell continu- ously during lactation from 28.0 + .1 to 12.0 ± 5.0 mM (d 15). In order to investigate whether secretion of the different milk components are subjected to a common mechanism of control, correlation coefficients between the different pairs of pa- rameters were calculated (Table 2). Highly pos- itive or negative correlation coefficients (P<.001) observed between all parameters dem- onstrate a linear relationship of their concentra- tions to each other. DI,~,U,~IO N Changes in MP, NAGase activities, and el, Na, K, lactose, fat, and TN concentrations were investigated throughout the second lactation in 26 healthy guinea pigs. Using micromethods and fat-free milk, all these analyses could easily be performed on about 250 gl of milk. Apart from the Kjeldahl N determination, the differ- ent analyses were done on microamounts of skim milk (10 to 20 gl), As no deproteinization of the samples is required and stable reagents were used, these analyses were quick and rather inexpensive. Significant changes in MP, CI, Na, K, lactose, and fat concentrations during lacta- tion were observed and the data are in agree- merit with those from literature (1, 2, 3). In these lactation studies, during established lacta- tion (d 1 to about d 7), milk composition remains nearly constant, indicating the presence Jourllal of Dairy Science Vol. 72, No. 12, 1989 3200 m E t- = u  E =E =-  ¢= 0 < Z 40 35 30 25 20 15 10 5 ROETS ET AL. (A) / .0- NAGase I , /~ /~ t ~  I / l ~  .0-Milk Production l / / / I  I I I I I I I I I I I I : : 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Days 21 18 15 12 3 e-  ,q, ¢q UI =- O = m  0 -i 0 L_  1100 /~ 16 E (B 0 1000 0 14  - -  E 800 4,,, al Nitrogen 0 I-- 8 60O 500 : : : : : : : : : I : I : : 6 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Days Figure 1. Mean and SEM for milk production and concentrations of NAGas¢, (A), total N, and fat (B) throughout lactation in 26 guinea pig sows. Journal of Dairy Science Vol. 72, No. 12, 1989 30 :~ 22 " i  20 o is 10 N-ACETYL-~D-GLUCOSAMINIDASE AND MILK COMPONENTS ./t o~+io--o- -o.~,}. ._ .+~ (A) , f 1++o+, 1\1 ' ' ' ' ' ' ' ' ' ' I I I I • , • • • • • • • • 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Days 35 30 25 2O 15 10 3201 +; 80 (B) .)t s s H.  - ,o ¢ - -+- ' - -N  +o,  0 - - - -  0 = 60 I \ !~!  I+ I¢  , s  E ¢= ~.O- Chloride (1~ • ~ _ _  U 0 • '~ 40 3.5 ,~ m tO 30 3.0 ,:,--° | 2o • . . . . .  : . . : . , , , , 2 .s  1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Days Figure 2. Mean data and SEM for conccntratiotm of g and Na (A), (21 and l~tose (B) throughout lactation m 26guinea pig sows. of  a "tight" blood-milk barrier in the mammary gland of these animals. In goats (4) and cows (17) hematocrit read- ings of  fat content in milk were overestimated by about 20% as compared with classical meth- ods. Due to the small amount of milk collected in our experiments, the Gerber method (sample volume: 11 ml milk) could not be compared Journal of Dairy Science Vol. 72, No. 12, 1989 3202 ROETS El" AL. TABLE 2. Correlation coefficient for pairs of parameters collected from 26 guinea pigs during 15 consecutive d. All values were highly significant (P<.001). Milk Parameter Na C1 Lactose Fat Total N NAGase yield K -.98 -.96 .97 -.90 -.89 -.91 .86 Na .98 -.97 .94 .86 .90 -.88 CI -.98 .90 .79 .87 -.88 Lactose -.91 -.79 -.90 .93 Fat .82 .92 -.87 Total N .86 -.82 NAGase -.87 with results obtained by hematocrit centrifuga- fion. It was found, however, that milk fat val- ues measured with both methods were highly correlated (4, 17). The NAGase activity of guinea pig's milk has not been determined by others. In compari- son with all other parameters, the change in concentration from the start to the end of lacta- tion was most striking with NAGase (fourfold increase). As correlations with the other milk components were highly significant O'able 2), determination of the enzyme seems to be a good and suitable parameter for estimating changes in secretion of milk of guinea pigs. These correlations were also found in milk of cows, some of  which were affected by mastitis (6, 7, 12), With the exception of d 1, normal NAGase activities (d 2 to 6) amounted to about 13 nM/min per ml and were twice as high as the activities measured in normal quarter milk of the cow [2.9 (15); 5.1 (7); and 5.3 nM/min per ml (5)]. The high NAGase activity (23 riM/ min per ml) noted on d 1, together with the elevated concentrations of TN and fat in milk, are possibly due to the secretion of colostrum. Because correlations were good between SCC and NAGase activities in cows, the deter- mination of the enzyme in milk reflects the number of ceils in milk. At the end of lactation, a disintegration of the blood-milk barrier oc- curs, and an increase of the paraceUular secre- tion therefore results in an increase of Na and CI and a decrease of K and lactose in milk (13). During that phenomenon, SCC increases, prob- ably from the influx of typical blood proteins into the mammary gland. It is therefore not surprising that correlations between NAGase activities and concentrations of electrolytes in milk were observed. Due to the very short lactation period of the guinea pig and the easy handling and milking Journal of Dairy Sciertee Vol. 72, No. 12, 1989 practices of the animals, several experiments can be done at once. These advantages and the availability of sensitive micro-methods to eval- uate changes in milk composition can lead to the establishment of a convenient guinea pig mastitis model. ACKNOWLEDGMENTS This study was upported by the Belgian NFWO (Grant Number 3.0091.89). We thank the Upjohn Company, Belgium, for supplying the lactating guinea pigs. REFERENCES 1 Anderson, R.R., M. S. Saiah, L. G. Sheffield, and W. N. McKenzie, Jr. 1984. Milk production and lactation curve of guinea pigs. J. Dairy Sci. 67:185. 2 Anderson, R. R., and D. D. Chavis. 1986. Changes in macroingredients of guinea pig milk through lactation. J. Dairy Sci. 69:2268. 3 Anderson, R. R., and L. G. Sheffield. 1988. Macrominer- ais in guinea pig milk during 21 days of lactation. J. Dairy Sci. 71:337. 4 Fleet, I. R., and J. L. Linzell. 1964. A rapid method f estimating fat in very small quantities of milk. J. Physiol. (Lood.) 175:15P. 5 Kitchen, B. J., and G. Middleton. 1976. Enzymatic methods for the estimation of the somatic cell count in bovine milk. J Dairy Res. 43:491. 6 Kitchen, B. J., G. Middleton, and M. Salmon. 1978. Bovine turk N-acetyl-~-ghicosaminidase activity and its significance in the detection of almormal udders secre- tions. J. Dairy Res. 45:15. 7 Kunkel, J. R., R. B. Bushnell, J. Cullor, and J. Aleong. 1987. Studies on induced Klebsiella mastitis with rela- tionships among N-acetyl-l~-D-glucosaminidase, bacte- riai and somatic cell counts. Cornell Vet. 77:225. 8 McKenzie, W. N., Jr., and R. R. Anderson. 1979. A modified device for collecting milk from guinea pigs. J. Dairy Sci. 62:1469. 9 McKenzie, W. N., Jr., and R. R. Anderson. 1981. Endotoxin induced migration of leukocytes from blood to milk. J. Dairy Sci. 64:227. 10 M_cKenzie, W.N., Jr., and R. R. Anderson. 1981. Endo- N-ACErYL-[3-D-GLUCOSAMINIDASE AND MILK COMPONENTS 3203 toxin dose-related leukocytosis in milk of guinea piss. J. Dairy Sci. 64:2234. 11 Mepham, T. B., and N.F.G. Beck. 1973. Variation in the yield and composition ofmilk throughout lactation in the guinea pig (Carla porcellus). Comp. Biocbem. Physiol. 45:273. 12 Obara, Y., and M. Komatsu. 1984. Relationship between N-acctyl-~D-glucosammidase activity and cell count, lactose, chloride, or lactoferrin i  cow milk. J. Dairy Res. 67:1043. 13 Pe.aker, M., and J. C. Taylor. 1975. Milk secretion i  the rabbit:changes during lactation and the mechanism ofion transport. J. Physiol. (Lond.) 253:527. 14 Peaker, M., C.D.R. Jones, J. A. Good¢, and J. L. Linzell. 1975. Changes in milk composition during lactation in the guinea-pig, and the ffect of prolactin. J. Endocfinol. 67:307. 15 Py0tala, S. 1987. Effect of some vehicles used in intramammary mastiffs preparations on inflammation indicators in milk. J. Vet. Pharmacol. Ther. 10:248. 16 Sheffield, L. G., and R. R. Anderson. 1985. Relationship between milk production and mammary gland indices of guinea pigs. J. Dairy Sci. 68:638. 17 Vandeputte-Van Messom, G., and C. Burvenich. 1989. Comparison of fat and cream content in normal and mastitic milk of cows. Vet. Q. 11:61. 18 Vandeputte-Van Messom, G., C. Burvenich, E. Roets, and L. A. Dcvriez¢. 1988. Effect of bovine somatot~'opin on milk yield and composition during Escherichia coli induced mastiffs in lactating cows: some preliminary results. Vlaam~ Diergeneeskd. Tijdschr. 57:53. 19 Yamashlta, M., and F. Watanab¢. 1973. Determination of sexum glucose with o-toluidine without glacial acetic acid. Clin. China. Acta 47:211. Journal of Dairy Science Vol. 72, No. 12, 1989 

Changes in N-acetyl-beta-D-glucosaminidase activities in relation to other milk components throughout normal lactation in the guinea-pig.

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Changes in N-acetyl-beta-D-glucosaminidase activities in relation to other milk components throughout normal lactation in the guinea-pig.

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