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A quantitative cytochemical study of acid phosphatases in hepatocytes of different ploidy classes from aging rats
Experimental Gerontology, Vol. 17, pp. 267-272, 1982 Printed in the USA. All rights reserved.
0531-5565/82/040267-06503.00/0 Copyright ©1982Pergamon Press Ltd
A QUANTITATIVE CYTOCHEMICAL STUDY OF ACID PHOSPHATASES IN HEPATOCYTES OF DIFFERENT PLOIDY CLASSES FROM AGING RATS
J . MIDDLETON a n d P . B . GAHAN Biology Department, Queen Elizabeth College, Campden Hill Road, London W8 7AH, England
(Received 24 August 1981) A b s t r a c t - A quantitative cytochemical study of isolated hepatocytes from young, middle-aged
and old adult rats has shown a doubling of acid naphthol AS-BI phosphatase activity as the hepatocytes shift from the diploid to the tetraploid state, and an overall increase in activity in cells from old animals. There was a complete inhibition of all acid phosphatase activity with 10-2M sodium molybdate, but only a 9007o inhibition in hepatocytes from livers of rats of 12-25 months-old animals with 10-2M sodium fluoride, the resistant activity possibly representing adenyl cyclase activity. In contrast, 10-2M ouabain inhibited 70-80070 of the activity in hepatocytes from 12-15months-old animals, this level of inhibition decreasing with increasing age, and implying a decrease in transport ATPase activity and an increased lysosomal phosphatase activity with increasing age of the rats.
INTRODUCTION PREVIOUS QUANTITATIVE cytochemical studies of acid naphthol AS-BI phosphatase activities in isolated hepatocytes from young and middle-aged adult rats have shown (a) an increased activity of almost 100070 when the euploid value of the liver cell doubles, and (b) that 70-80070 of this activity is ouabain sensitive regardless of euploidy value (Middleton and Gahan, 1979). The present work concerns an extension of these studies on isolated hepatocytes from rats of ages up to 32 months, together with the effects of various inhibitors on the acid phosphatases investigated.
MATERIALS AND METHODS Materials Adult female BN/BiRij rats aged from 12-32 months were obtained from the specific pathogen free colony maintained at the Institute for Experimental Gerontology, TNO, Rijswijk (Z.H.) in the Netherlands. These animals were maintained in small groups under constant temperature (21°C) and relative humidity conditions (6007o). This strain of rat has a mean life span of 30.5 months and the advantage that neoplastic nodules and other lesions originating from hepatocytes are almost totally absent from old female BN/BiRij rats (Hollander, 1976). Suspensions of isolated hepatocytes were dissociated from rat livers by use of collagenase (Sigma Type I) and hyaluronidase (Sigma Type I) (Van Bezooijen et al., 1974; 1977). The isolated hepatocytes were maintained in 267
268
J MIDDI,IETON AND P.B. G A H A N
Dulbecco's medium plus 10% foetal calf serum. Over 90% of the cells used in the current experiments were viable as indicated by the trypan blue dye exclusion test (Grankvist et aL, 1977). The cells were prepared on microscope slides as previously described (Middleton and Gahan, 1979). Quantification of acid phosphatase activity and DNA content of individual hepatocytes
Hepatocytes were incubated for acid phosphatase in a medium containing naphthol AS-BI phosphate as substrate and fast red violet LB as the diazonium salt at pH 5.4 for 75 min at 37°C. The enzyme reaction endproduct for each cell was measured by its absorbance at 535 nm using a Vickers M86 integrating microdensitometer (Middleton and Gahan, 1979). Subsequently, the enzyme reaction end-product was removed from the cells by extraction in 40°7on-heptanol in 1,1,2,2-tetrachloroethane (Maggi and Chng, 1966) after which the DNA was stained by means of the Feulgen reaction. The DNA values of the hepatocyte nuclei were measured by integrating microdensitometry at a wavelength of 540 nm. Rat sperm and white blood cell nuclei similarly treated served as standard material to establish haploid and diploid DNA values respectively. RESULTS A c i d p h o s p h a t a s e activities
Mean values for hepatocytes o f different ploidy classes f r o m rats o f different ages are given in Table 1 alongside previously reported data for y o u n g adult rats (Middleton and G a h a n , 1979) for comparison. The a p p r o x i m a t e doubling o f activity previously seen on shifting f r o m diploid cells (1 × 2c) to tetraploid cells (2 × 2c; 1 x 4c) is repeated in cells f r o m 25 m o n t h s and 32 m o n t h s old animals. However, this is not so for the shift f r o m tetraploidy to octoploidy when the increase in enzyme activity is only about 50°7o (Table 1). C o m p a r i s o n o f the activities f r o m each age against 121A m o n t h s as 100070 for each ploidy class shows a similar activity for 15m but a continuing increase in activity at 25 and 32 m o n t h s , the activity doubling in the 1 x 2c and 1 x 4c values at 32 m o n t h s (Table 1). lnhibitors
S o d i u m m o l y b d a t e (10-2M) completely inhibited all activity in all hepatocytes f r o m each age. H o w e v e r , sodium fluoride (10-2M) only inhibited 90070 o f the activity in hepatocytes f r o m rats o f 12½-25 m o n t h s . At 30 m o n t h s , 99-100070 o f activity was inhibited by the fluoride ions. The data for o u a b a i n inhibition (Table 2) show that 70-80°7o inhibition occurs at 12V2 and 15 m o n t h s , declining slightly to a b o u t 6507o at 25 m o n t h s , and d r o p p i n g to 38070 or less by 32 months. DISCUSSION C o m p a r i s o n o f the m e a n values o f acid p h o s p h a t a s e activities in the isolated hepatocytes show a doubling in activity on shifting f r o m diploid to tetraploid values in the 2 5 - m o n t h animal and almost the same in the 3 2 - m o n t h animals. This is as previously reported for y o u n g e r adults (Middleton and G a h a n , 1979). The main difference lies in the failure to double the activity f r o m 4c to 8c in the older animals which m a y be interpreted as either an inability to double the activity, or the lack o f need to double the activity in late age. Equally, such a doubling o f activity need not be expected since a c o m p a r i s o n o f the results at 12-15 m o n t h s and those o f 25 and 32 m o n t h s shows a trend towards a doubling in the total a m o u n t o f activity for the 2c and 4c cells, and a 60-70070 increase for the higher ploidy values (Table 1). Since the cell volumes appear to remain relatively constant within
121Yz* 1s 25 32 32
15f 25 32 32
‘70Activity compared with lx2c ceils = 100%
% Activity compared with 12lYzm = 100%
13 (10) 12 (3) 26 (16) 39(24) 37 (15)
97 177 203 208
100 100 100 100 100
61 f 59 f 108 zt 124 * 127 zt
1 x 2c
**NOD
= Absolute Integrated Optical Density.
87 160 128 158
239 215 217 151 182
37 (38) 34 (20) 47 (23) 66 (5) 63 (14)
2 x 2c 146 * 127 + 234 + 187 zt 231 f
‘Figure for 12% month animal is from one cell only.
*From Middleton and Gahan (1979).
12’S* 151 25 32 32
Mean reaction product (AIOD)** + SD
Age (months)
1 x4c
98 171 170 199
207 210 200 173 198
49 (27) 42 (30) 97 (31) 61 (28) 86 (60)
91 154 133 158
410 385 357 268 310
f I f zt *
2X4c 250 227 386 354 394
f
266 237 377 299 377
_ -
89 142 112 142
318 444
527 366 403
733 -
569 + 104 (12) 454 zt 89 (25) 512 f 109(19)
447 (1) -
1 x 16~
436 402 349 242 297
f 50 (3) CIE53 (3) * 80 (80) f 56 (103) * 122 (57)
1 x 8c
2 x 8c
S.D. FOR HEPATOCYTES OF VARIOUS CLASSES
MEASURED IS IN PARENTHESES.
Ploidy class
OF CELLS
END-PRODUCT PER CELL
THE NUMBER
126 CIE29 (214) 124 zt 25 (182) 216 f 37 (107) 214 + 48 (69) 251 f 65 (107)
OLD.
PHOSPHATASE REACTION
FROM RATS 12-32 MONTHS
TABLE 1. MEAN ACID NAPHTHOL AS-BI
z
5 9 2 r:
z
12½ 15 25 32 32
% inhibition v test (Table 1)
13 17 38 78 88
+ ± ± + + 79 71 65 37 31
5 7 l0 30 24
(7) (8) (11) (9) (13)
1 × 2c
* A I O D = A b s o l u t e I n t e g r a t e d O p t i c a l Density.
12½ 15 25 32 32
Mean reaction product (AIOD)* ±S.D.
Age (months) 27 37 84 162 144 81 71 64 23 38
-4- 5 (20) ± 13 (21) ± 12 (15) 4- 6 2 ( 2 ) -4- 41 (8)
2 x 2c 25 37 72 160 166
± + ± ± 480 70 67 25 34
6 (94) 12 (118) 15 (62) 35(12) 56 (98)
1 x 4c 50 70 136 254 258
P l o i d y classes
TABLE 2. EFFECT OF OUABAIN INHIBITION.
10 (18) 22 (29) 32 (17) 52(17) 59 (42)
80 69 65 28 34
-4± ± ± ±
2 x 4c
80 138 244 251
78 66 63 18 33
58 (1) ± 37 (4) ± 27 (53) ± 112(11) ± 90 (34)
1 x 8c
--67 7 21
187 ± 48 (8) 423 ± 1 2 0 ( 4 ) 405 ± 143 (15)
---
2 x 8c
:> z >
o > z 7z z
~-
O
ACID PHOSPHATASES IN HEPATOCYTES
271
each ploidy class regardless of age, then this may be interpreted as a real increase in acid phosphatase activity of the cells from the older animals. These data are in agreement with previously published biochemical data from hepatocytes of the same strain of rats (Sleyster and Knook, 1980). Naphthol AS-BI phosphate is a broad-spectrum substrate (Barka, 1961; Gahan et al., 1978; Meijer et al., 1980) and will demonstrate at least three liver cell acid phosphatases. Total inhibition in the presence of molybdate ions has confirmed the activity of acid phosphatase. Furthermore, 90o70 inhibition was achieved with sodium fluoride in cells from 12-25-months animals and total inhibition at 32 months also indicating the presence of acid phosphatase. However, a small fluoride-resistant fraction which is absent from 32 months material has been noted in a variety of tissues (Maggi et al., 1966; Maggi and Carbonell, 1969; Gahan et al., 1978) and may be indicative of adenyl cyclase activity in the hepatocytes. Some 70-80070 inhibition is achieved with ouabain in hepatocytes from 12 V2-15-month rats, though with increasing age of the rat there is a decrease in the amount of activity inhibited (Table 2). The degree of inhibition is similar for all ploidy classes of a given age group. These results enable a distinction to be made between the classical lysosomal acid phosphatase which is ouabain resistant and sensitive to fluoride and molybdate, and the ouabain-sensitive fraction which may represent an ATPase fraction. The former fraction increases in activity in total amount in a small way until 25 months and then sharply by 32 months. The latter responds in the converse fashion, namely, decreases slowly to 25 months and drops sharply by 32 months. The ouabain-sensitive acid phosphatase is not restricted to the cell membrane but is seen also to be associated with cytoplasmic granules. Electron microscope cytochemical studies of acid naphthol AS-BI phosphatases in mouse kidney and seminal vesicle and hamster cardiac muscle have shown them to be associated with lysosomes, golgi stacks and endoplasmic reticulum (Maggi, 1973). Hence if this ouabainsensitive fraction represents a transport phosphatase, at least part of this may be associated with the lysosomal system. The enzyme substrate employed in these studies may be considered as demonstrating an increased lysosomal phosphatase activity with increased age, and a transport phosphatase which is diminished in activity in old age. These findings also confirm the need to study genuinely old adults in addition to those from late middle age in order to ascertain real changes related to aging. Acknowledgements-We wish to thank Dr. D.L. Knook for supplying the liver material and for facilities in his laboratory for part of this work. We are indebted for financial assistance to the Science Research Council, U.K. (to P.B.G.) and to the E.E.C. Special Concerted Action Group on Cell Ageing and Organ Function.
REFERENCES BARKA, T. (1961) J. Histochem. Cytochem. 9, 542. GAHAN, P.B., DAWSON, A.L. and FmLDINO, J. (1978) Ann. Bot. 42, 1413. GI~NKVIST, K., LERNMARK, A. and TALIEDAL, I. (1977) Biochem. J. 162, 19. HOLLANDER, C.F. (1976) Lab. Ann. Sci. 26, 320. MAGGI, V. (1973) Lysosomes. In: CellBiology in Medicine. (Edited by E.E. BITTAR), 215, John Wiley and Sons, New York. MAGGI, V. and CARBONELL, A.W. (1969) Histochem. J. 1, 383.
272
J. MIDDLETONAND P.B. GAHAN
MAGGI, V. and CHNO, W.L. (1966) Histochemie 7, 267. MAGGI, V., FRANKS, L.M. and CARBONELL, A.W. (1966) Histochemie 6, 305. MEIJER, A.E.F.H., VAN DER LOOS, C.M. and SCHUURHUIZEN, P.W. (1980) Histochemistry 67, 23. MIDDLETON, J. and GAHAN, P.B. (1979) Histochem J. 11, 649. SLEYSTER, E.C. and KNOOK, D.L. (1980) Mech. Aging Develop. 14, 443. VAN BEZOOIJEN, C.F.A., GRELL, T. and KNOOK, D.L. 0977) Mech. Aging Develop. 6, 244. VAN BEZOOIJEN, C.F.A., VAN NOORD, M.J. and I(NOOK, D.L. (1974) Mech. Aging Develop. 3, 107.
0531-5565/82/040267-06503.00/0 Copyright ©1982Pergamon Press Ltd
A QUANTITATIVE CYTOCHEMICAL STUDY OF ACID PHOSPHATASES IN HEPATOCYTES OF DIFFERENT PLOIDY CLASSES FROM AGING RATS
J . MIDDLETON a n d P . B . GAHAN Biology Department, Queen Elizabeth College, Campden Hill Road, London W8 7AH, England
(Received 24 August 1981) A b s t r a c t - A quantitative cytochemical study of isolated hepatocytes from young, middle-aged
and old adult rats has shown a doubling of acid naphthol AS-BI phosphatase activity as the hepatocytes shift from the diploid to the tetraploid state, and an overall increase in activity in cells from old animals. There was a complete inhibition of all acid phosphatase activity with 10-2M sodium molybdate, but only a 9007o inhibition in hepatocytes from livers of rats of 12-25 months-old animals with 10-2M sodium fluoride, the resistant activity possibly representing adenyl cyclase activity. In contrast, 10-2M ouabain inhibited 70-80070 of the activity in hepatocytes from 12-15months-old animals, this level of inhibition decreasing with increasing age, and implying a decrease in transport ATPase activity and an increased lysosomal phosphatase activity with increasing age of the rats.
INTRODUCTION PREVIOUS QUANTITATIVE cytochemical studies of acid naphthol AS-BI phosphatase activities in isolated hepatocytes from young and middle-aged adult rats have shown (a) an increased activity of almost 100070 when the euploid value of the liver cell doubles, and (b) that 70-80070 of this activity is ouabain sensitive regardless of euploidy value (Middleton and Gahan, 1979). The present work concerns an extension of these studies on isolated hepatocytes from rats of ages up to 32 months, together with the effects of various inhibitors on the acid phosphatases investigated.
MATERIALS AND METHODS Materials Adult female BN/BiRij rats aged from 12-32 months were obtained from the specific pathogen free colony maintained at the Institute for Experimental Gerontology, TNO, Rijswijk (Z.H.) in the Netherlands. These animals were maintained in small groups under constant temperature (21°C) and relative humidity conditions (6007o). This strain of rat has a mean life span of 30.5 months and the advantage that neoplastic nodules and other lesions originating from hepatocytes are almost totally absent from old female BN/BiRij rats (Hollander, 1976). Suspensions of isolated hepatocytes were dissociated from rat livers by use of collagenase (Sigma Type I) and hyaluronidase (Sigma Type I) (Van Bezooijen et al., 1974; 1977). The isolated hepatocytes were maintained in 267
268
J MIDDI,IETON AND P.B. G A H A N
Dulbecco's medium plus 10% foetal calf serum. Over 90% of the cells used in the current experiments were viable as indicated by the trypan blue dye exclusion test (Grankvist et aL, 1977). The cells were prepared on microscope slides as previously described (Middleton and Gahan, 1979). Quantification of acid phosphatase activity and DNA content of individual hepatocytes
Hepatocytes were incubated for acid phosphatase in a medium containing naphthol AS-BI phosphate as substrate and fast red violet LB as the diazonium salt at pH 5.4 for 75 min at 37°C. The enzyme reaction endproduct for each cell was measured by its absorbance at 535 nm using a Vickers M86 integrating microdensitometer (Middleton and Gahan, 1979). Subsequently, the enzyme reaction end-product was removed from the cells by extraction in 40°7on-heptanol in 1,1,2,2-tetrachloroethane (Maggi and Chng, 1966) after which the DNA was stained by means of the Feulgen reaction. The DNA values of the hepatocyte nuclei were measured by integrating microdensitometry at a wavelength of 540 nm. Rat sperm and white blood cell nuclei similarly treated served as standard material to establish haploid and diploid DNA values respectively. RESULTS A c i d p h o s p h a t a s e activities
Mean values for hepatocytes o f different ploidy classes f r o m rats o f different ages are given in Table 1 alongside previously reported data for y o u n g adult rats (Middleton and G a h a n , 1979) for comparison. The a p p r o x i m a t e doubling o f activity previously seen on shifting f r o m diploid cells (1 × 2c) to tetraploid cells (2 × 2c; 1 x 4c) is repeated in cells f r o m 25 m o n t h s and 32 m o n t h s old animals. However, this is not so for the shift f r o m tetraploidy to octoploidy when the increase in enzyme activity is only about 50°7o (Table 1). C o m p a r i s o n o f the activities f r o m each age against 121A m o n t h s as 100070 for each ploidy class shows a similar activity for 15m but a continuing increase in activity at 25 and 32 m o n t h s , the activity doubling in the 1 x 2c and 1 x 4c values at 32 m o n t h s (Table 1). lnhibitors
S o d i u m m o l y b d a t e (10-2M) completely inhibited all activity in all hepatocytes f r o m each age. H o w e v e r , sodium fluoride (10-2M) only inhibited 90070 o f the activity in hepatocytes f r o m rats o f 12½-25 m o n t h s . At 30 m o n t h s , 99-100070 o f activity was inhibited by the fluoride ions. The data for o u a b a i n inhibition (Table 2) show that 70-80°7o inhibition occurs at 12V2 and 15 m o n t h s , declining slightly to a b o u t 6507o at 25 m o n t h s , and d r o p p i n g to 38070 or less by 32 months. DISCUSSION C o m p a r i s o n o f the m e a n values o f acid p h o s p h a t a s e activities in the isolated hepatocytes show a doubling in activity on shifting f r o m diploid to tetraploid values in the 2 5 - m o n t h animal and almost the same in the 3 2 - m o n t h animals. This is as previously reported for y o u n g e r adults (Middleton and G a h a n , 1979). The main difference lies in the failure to double the activity f r o m 4c to 8c in the older animals which m a y be interpreted as either an inability to double the activity, or the lack o f need to double the activity in late age. Equally, such a doubling o f activity need not be expected since a c o m p a r i s o n o f the results at 12-15 m o n t h s and those o f 25 and 32 m o n t h s shows a trend towards a doubling in the total a m o u n t o f activity for the 2c and 4c cells, and a 60-70070 increase for the higher ploidy values (Table 1). Since the cell volumes appear to remain relatively constant within
121Yz* 1s 25 32 32
15f 25 32 32
‘70Activity compared with lx2c ceils = 100%
% Activity compared with 12lYzm = 100%
13 (10) 12 (3) 26 (16) 39(24) 37 (15)
97 177 203 208
100 100 100 100 100
61 f 59 f 108 zt 124 * 127 zt
1 x 2c
**NOD
= Absolute Integrated Optical Density.
87 160 128 158
239 215 217 151 182
37 (38) 34 (20) 47 (23) 66 (5) 63 (14)
2 x 2c 146 * 127 + 234 + 187 zt 231 f
‘Figure for 12% month animal is from one cell only.
*From Middleton and Gahan (1979).
12’S* 151 25 32 32
Mean reaction product (AIOD)** + SD
Age (months)
1 x4c
98 171 170 199
207 210 200 173 198
49 (27) 42 (30) 97 (31) 61 (28) 86 (60)
91 154 133 158
410 385 357 268 310
f I f zt *
2X4c 250 227 386 354 394
f
266 237 377 299 377
_ -
89 142 112 142
318 444
527 366 403
733 -
569 + 104 (12) 454 zt 89 (25) 512 f 109(19)
447 (1) -
1 x 16~
436 402 349 242 297
f 50 (3) CIE53 (3) * 80 (80) f 56 (103) * 122 (57)
1 x 8c
2 x 8c
S.D. FOR HEPATOCYTES OF VARIOUS CLASSES
MEASURED IS IN PARENTHESES.
Ploidy class
OF CELLS
END-PRODUCT PER CELL
THE NUMBER
126 CIE29 (214) 124 zt 25 (182) 216 f 37 (107) 214 + 48 (69) 251 f 65 (107)
OLD.
PHOSPHATASE REACTION
FROM RATS 12-32 MONTHS
TABLE 1. MEAN ACID NAPHTHOL AS-BI
z
5 9 2 r:
z
12½ 15 25 32 32
% inhibition v test (Table 1)
13 17 38 78 88
+ ± ± + + 79 71 65 37 31
5 7 l0 30 24
(7) (8) (11) (9) (13)
1 × 2c
* A I O D = A b s o l u t e I n t e g r a t e d O p t i c a l Density.
12½ 15 25 32 32
Mean reaction product (AIOD)* ±S.D.
Age (months) 27 37 84 162 144 81 71 64 23 38
-4- 5 (20) ± 13 (21) ± 12 (15) 4- 6 2 ( 2 ) -4- 41 (8)
2 x 2c 25 37 72 160 166
± + ± ± 480 70 67 25 34
6 (94) 12 (118) 15 (62) 35(12) 56 (98)
1 x 4c 50 70 136 254 258
P l o i d y classes
TABLE 2. EFFECT OF OUABAIN INHIBITION.
10 (18) 22 (29) 32 (17) 52(17) 59 (42)
80 69 65 28 34
-4± ± ± ±
2 x 4c
80 138 244 251
78 66 63 18 33
58 (1) ± 37 (4) ± 27 (53) ± 112(11) ± 90 (34)
1 x 8c
--67 7 21
187 ± 48 (8) 423 ± 1 2 0 ( 4 ) 405 ± 143 (15)
---
2 x 8c
:> z >
o > z 7z z
~-
O
ACID PHOSPHATASES IN HEPATOCYTES
271
each ploidy class regardless of age, then this may be interpreted as a real increase in acid phosphatase activity of the cells from the older animals. These data are in agreement with previously published biochemical data from hepatocytes of the same strain of rats (Sleyster and Knook, 1980). Naphthol AS-BI phosphate is a broad-spectrum substrate (Barka, 1961; Gahan et al., 1978; Meijer et al., 1980) and will demonstrate at least three liver cell acid phosphatases. Total inhibition in the presence of molybdate ions has confirmed the activity of acid phosphatase. Furthermore, 90o70 inhibition was achieved with sodium fluoride in cells from 12-25-months animals and total inhibition at 32 months also indicating the presence of acid phosphatase. However, a small fluoride-resistant fraction which is absent from 32 months material has been noted in a variety of tissues (Maggi et al., 1966; Maggi and Carbonell, 1969; Gahan et al., 1978) and may be indicative of adenyl cyclase activity in the hepatocytes. Some 70-80070 inhibition is achieved with ouabain in hepatocytes from 12 V2-15-month rats, though with increasing age of the rat there is a decrease in the amount of activity inhibited (Table 2). The degree of inhibition is similar for all ploidy classes of a given age group. These results enable a distinction to be made between the classical lysosomal acid phosphatase which is ouabain resistant and sensitive to fluoride and molybdate, and the ouabain-sensitive fraction which may represent an ATPase fraction. The former fraction increases in activity in total amount in a small way until 25 months and then sharply by 32 months. The latter responds in the converse fashion, namely, decreases slowly to 25 months and drops sharply by 32 months. The ouabain-sensitive acid phosphatase is not restricted to the cell membrane but is seen also to be associated with cytoplasmic granules. Electron microscope cytochemical studies of acid naphthol AS-BI phosphatases in mouse kidney and seminal vesicle and hamster cardiac muscle have shown them to be associated with lysosomes, golgi stacks and endoplasmic reticulum (Maggi, 1973). Hence if this ouabainsensitive fraction represents a transport phosphatase, at least part of this may be associated with the lysosomal system. The enzyme substrate employed in these studies may be considered as demonstrating an increased lysosomal phosphatase activity with increased age, and a transport phosphatase which is diminished in activity in old age. These findings also confirm the need to study genuinely old adults in addition to those from late middle age in order to ascertain real changes related to aging. Acknowledgements-We wish to thank Dr. D.L. Knook for supplying the liver material and for facilities in his laboratory for part of this work. We are indebted for financial assistance to the Science Research Council, U.K. (to P.B.G.) and to the E.E.C. Special Concerted Action Group on Cell Ageing and Organ Function.
REFERENCES BARKA, T. (1961) J. Histochem. Cytochem. 9, 542. GAHAN, P.B., DAWSON, A.L. and FmLDINO, J. (1978) Ann. Bot. 42, 1413. GI~NKVIST, K., LERNMARK, A. and TALIEDAL, I. (1977) Biochem. J. 162, 19. HOLLANDER, C.F. (1976) Lab. Ann. Sci. 26, 320. MAGGI, V. (1973) Lysosomes. In: CellBiology in Medicine. (Edited by E.E. BITTAR), 215, John Wiley and Sons, New York. MAGGI, V. and CARBONELL, A.W. (1969) Histochem. J. 1, 383.
272
J. MIDDLETONAND P.B. GAHAN
MAGGI, V. and CHNO, W.L. (1966) Histochemie 7, 267. MAGGI, V., FRANKS, L.M. and CARBONELL, A.W. (1966) Histochemie 6, 305. MEIJER, A.E.F.H., VAN DER LOOS, C.M. and SCHUURHUIZEN, P.W. (1980) Histochemistry 67, 23. MIDDLETON, J. and GAHAN, P.B. (1979) Histochem J. 11, 649. SLEYSTER, E.C. and KNOOK, D.L. (1980) Mech. Aging Develop. 14, 443. VAN BEZOOIJEN, C.F.A., GRELL, T. and KNOOK, D.L. 0977) Mech. Aging Develop. 6, 244. VAN BEZOOIJEN, C.F.A., VAN NOORD, M.J. and I(NOOK, D.L. (1974) Mech. Aging Develop. 3, 107.