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Some aspects of the chemical and cellular composition of adult rat liver
Experimental
Cell Research
40, 233-251
SOME ASPECTS
OF THE
COMPOSITION P. T. Regional
233
(1965)
IYPE,
P. M.
Research
Laboratory, Medical
CHEMICAL
OF ADULT BHARGAVA
Received
RAT LIVER and
Hyderabad-9, Research,
AND CELLULAR
New
February
India, Delhi,
A.
D. TASKER and Indian
Council
of
India
11, 1965
WE recently described a method [25] for the preparation of rat hepatic cell suspensions which gives an almost quantitative recovery of the hepatic cells in the final suspension; the method takes only 15 min for the preparation of cell suspensions which consist predominantly of single cells and which are virtually free of cell debris and cells other than the hepatic cells present in liver. Further, this method does not involve any enzymatic or drastic physical treatment of the tissue, so that it minimises the loss of intracellular constituents or of cell integrity. Since this method seemed to give a good separation of the hepatic cells from the other cellular and non-cellular material in liver, we felt that it could be used for obtaining useful information on certain aspects of the chemical and cellular composition of liver. In this paper we report the results of estimation of (Q) the number of hepatic (parencells [34] conchpmal) and non-hepatic (i.e., other than the parenchymal) tained in a unit weight of liver or in the whole liver of 3-14 months old rats; (b) the average dry weight of hepatic cells; (c) the percentage of dry weight of liver represented by hepatic cells, non-hepatic cells or non-cellular material; (d) the DNA content of hepatic cells; (e) the percentage of DNA of liver contained in hepatic cells or non-hepatic cells; (f) the extent of occurrence of polynucleated hepatic cells in liver; and (g) the volume of hepatic cells. The variation of the above parameters with increasing dry weight of liver has also been studied; it has been shown that as the weight of liver increases, the number of hepatic cells per unit Lveight of liver falls, whiie the percentage of non-cellular material increases. No definitive information is available on most of the above parameters of liver; the difficulties in the measurement of these values by conventional methods have been pointed out in an earlier, preliminary publication [22]. Experimental
Cell Research
40
234
P. T. Iype, P. M. Bhargava and A. D. Tasker EXPERIMENTAL
Animals.-Adult albino rats of an inbred strain of Nutrition Research Laboratory, Hyderabad, of known sex, age and weight, and fed on an ad libitum diet, were used. Preparation of hepatic cell suspensions.-The cell suspensions were prepared from a known wet weight of perfused liver by the method of Jacob and Bhargava [25], which essentially consists of perfusion of the liver with 30-50 ml of cold (5-10°C) sodium citrate (0.027 M) in calcium-free Locke’s solution [IO], followed by dispersion in 0.25 M sucrose in a specially designed tissue disperser. The dispersate was filtered through a 240-mesh sieve and the residue on the sieve washed with 0.25 M sucrose. Microscopic examination of the residue showed that very few cells or free nuclei, if any, were present; their number was negligible when compared to the number of cells and free nuclei contained in the filtrate. The filtrate was centrifuged at 200 xg and the supernatant saved. The sedimented cells were suspended in 0.9 per cent NaCl and centrifuged as above; this washing with saline was essential to remove adhering sucrose from the hepatic cells which could vitiate the results of dry weight estimations [22]. The supernatant was combined with the earlier supernatant and made up to a known volume; the sedimented hepatic cells were resuspended in 0.9 per cent NaCl. As stated earlier [22], the combined supernatants contained (a) cell debris, including the nuclei, from the small number of hepatic cells broken during the preparation of the cell suspension; (b) material, if any, which leaked out of the hepatic cells during the preparation of the cell suspension; (c) contents of virtually all the cells other than the parenchymal cells present in liver; and (d) almost all the non-cellular material of liver (the material left on the sieve contained the remaining non-cellular material). Since, on an average, only 10 per cent of hepatic cells were broken during the preparation of the cell suspension and since (for reasons discussed later) the material in the second category appeared most unlikely to account for a significant proportion of the wet weight or DNA of the hepatic cells, the combined supernatants obtained as above have been subsequently referred to as the “nonhepatic-cell fraction”. Dry weight determinations.PFor determination of the dry weight of perfused liver, the freshly excised organ was pressed between folds of filter paper, quickly weighed and a known wet weight dried to constant weight at 110°C. The dry weight of hepatic cells in suspension was determined as described earlier [22]. Duplicate estimations were run in each case. They tallied within experimental error; the values given are the average of the two determinations. In some experiments, the approximate dry weight of the non-cellular material and cells other than the hepatic cells in liver was determined experimentally by mixing the non-hepatic-cell fraction obtained as above with the residue left on the sieve during preparation of the cell suspension, and dialysing, first against running water and then against distilled water. The material in the dialysis bag was heated to boiling and the coagulum sedimented in a centrifuge; it was then dried at 110°C to constant weight. The dry weight value thus obtained was found to be close to the value obtained by difference between the dry weight of liver and of hepatic cells contained in the liver, as would be expected since the non-hepatic cell fraction is unlikely to Experimental
Cell Research
40
Chemical
and cellular
composition
of rat
235
liver
contain a significant proportion (on dry weight basis) of dialysable and non-heatcoagulable materials. Hepatic cell count in the cell suspension and the free hepatic cell nuclei count in the non-hepatic-cell fraction.-The concentration of hepatic cells in the cell suspension
and of hepatic cell nuclei in the non-hepatic-cell fraction was determined on a haemocytometer (100 p deep and having a graduated area of 9 mm2divided into 9 major squareseach with an area of 1 mm2 each) under phasecontrast (magnification x SO). Only the hepatic cells were scored in the former case and the hepatic cell nuclei in the latter. The hepatic cell fraction contained very few, if any, hepatic nuclei or cells other than the hepatic cells, and the non-hepatic-cell fraction very few, if any, intact, free hepatic cells. Hepatic cells and their nuclei were distinguishable from other cells and their nuclei by their size. It was assumedthat there was no significant breakage of hepatic cell nuclei during the dispersion of the tissue to single cells becauseof the mild treatment given to the tissue. In some experiments a series of photomicrographs (magnification x128) were taken (35 mm positive contrast film) of the preparation on the haemocytometer so as to cover all its 9 major squares. The hepatic cells in the 9 squareswere counted on a projection screen. Determination of hepatic cell volume.-The films obtained as described above were projected so that a distance of I mm on the haemocytometer was equivalent to 400 mm on the screen; only those photographs in which the hepatic cells were sharply in focus were used. The major and the minor axes of the image of the cells on the screen were measured accurately; the mean divided by 400 was taken as the average diameter and the volume then calculated as for a sphere. Estimation of multinucleated cells.-The hepatic cell suspension,diluted to an appropriate cell concentration, was stained with 2 per cent aqueous, chloroform-washed methyl green by mixing a drop of this stain with about 10 ml of the cell suspension. The proportion of hepatic cells containing 1, 2, 3, or 4 nuclei was then determined by scoring randomly selected 300-800 cells under a microscope. Determination of DNA.-DNA was estimated in the hepatic cells and in the nonhepatic-cell fraction as described earlier [22]; the material remaining on the sieve during the preparation of the cell suspensionwas added to the non-hepatic-cell fraction before the estimation. In some experiments, the total DNA content of whole liver was experimentally determined as above; this value tallied, within experimental error, with the value for the DNA content of liver obtained by the addition of the DNA content of the hepatic cell and the non-hepatic-cell fractions. Calculations.-The following values were experimentally determined as described above: (a) the wet and the dry weights (and thus the moisture content) of liver; (b) the number of hepatic cell nuclei in the non-hepatic-cell fraction per gram dry weight of liver; (c) the number of hepatic cells recovered in the cell suspensionper gram dry weight of liver; (d) the dry weight of a known number of hepatic cells; (e) the DNA content of a known number of hepatic cells; (f) the DNA content of non-hepatic-cell fraction derived from a known wet weight of liver; (g) the percentage of mono-, bi-, tri-, and tetra-nucleated hepatic cells in the hepatic cell suspension;and (h) the percentage of hepatic cells falling within different, specified volume ranges. Other values given were calculated from the above data as indicated in the notes to the tables. Experimenlal
Cell
Research
40
236
P. T. Iype, P. M. Bhargava
and A. D. Tasker
RESULTS
Tables I-IV give the results of the various estimations for nineteen, 3-14month-old rats. The following observations made for these animals are considered significant: I (parts l-4). The recovery of hepatic cells in cell suspensionsprepared from rat liver, the number, dry weight and DNA of rat hepatic and non-hepatic cells, and the dry weight of non-cellular material in rat liver.a TABLE
Part 1 Cell suspensions
Data used
on animal
Moisture in liver
No. of free hepatic cell nuclei
No. of hepatic cells corresponding to the free nuclei
Per g dry wt. of liver (millions)
Per g dry wt. of liver (millions)
No. of intact hepatic cells Per g dry wt. of liver (millions)
Per cent of total no. of hepatic cells
(J)
wt.
Age
Sex
&I
(months)
(g)
(per cent of wet wt. of liver)
(A)
(‘3
((2
CD)
(El
w
(G)
W)
(1)
6 12 11 10 7 8 17 20 16 22 13 24 5 1 4 9 3 2 18
* r” 6 6 9 ? 0 9 d 9 9 0” CT * i 6 3 s 6
100 120 150 180 135 135 150 175 160 185 195 230 295 290 290 295 310 310 270
3
0.74 1.14 1.17 1.19 1.37 1.47 1.47 1.50 1.76 1.76 1.78 2.14 2.28 2.45 2.70 2.85 3.02 3.14 3.18
so.4 71.0 75.8 71.5 73.2 71.3 73.3 73.2 74.3 73.5 79.3 76.9 79.9 76.0 77.8 76.7 74.1 75.0 72.4
76.0 41.0 63.2 32.6 44.0 28.3 46.8 71.5 36.1 33.6 37.8 19.9 52.6
67 36 56 29 39 25 41 58 32 26 32 16 40
24.9
21
352 430 320 440 330 245 260 332 247 170 206 207 240 234 17x 275 323 338 198
71.0-80.4 75.0 0.7
19.9-76.0 42.8 4.3
16-67 36 4
170-440 280 18
Animal no.
Range Mean Standard Experimentnt
3 3 3 3 3 3 9 3 13 6 9 12 12 14 9 12 12 7
deviation Cell Research
40
Dry wt. of liver
34.0
28 -
84.0 92.2 85.1 93.8 89.4 90.7 86.3 85.1 88.2 86.4 86.5 92.8 85.1
90.9
90.4 84.0-93.8 88.5 0.8
Chemical
and cellular
composition
237
of rat liver
and Bhargava’s method gives a near-quantitative (range 84-93 per cent; mean 89 k 0.8 per cent) yield of hepatic cells in the final cell suspension (Table I). (1)
Jacob
(2)
The
liver
contains
196-469
x lo6
(mean
320 k 6 X 106)
hepatic
cells
per g dry weight of liver, or 52-135 x lo6 (mean 81 & 28 x 106) hepatic cells per g wet weight of liver, or 310-863 X lo6 (mean 514 + 117 X 106) hepatic cells per liver (Table I). The number of other (non-hepatic) cells in liver a The values in the columns H, J to M, 0 to Q, S to Z, and AA to KK, have been
&[based
Y=
arrived at as given below; the values in the other columns were determined experimentally as described in the text: H-
Gxa(a=1.13,1.18,1.23ori.3l,for3, 6, 9 and 13-month-old vely; see text and Table
J=
I ~ H+I
K=
H+I.
L-
ExK. L
M =
~ L+z
O-
KxN.
P-
13x0.
Qz
rats, II).
respecti-
6.7 ,ug (stromal Z==
E x Y.
AA=
,>
x 100. BB
j: 100.
KxR.
7’-
EXS.
1: =
T -----x T + w
V =
\I’=
sz
CC-
of
of DNA per lo6 diploid cells cells of liver) of rat [15]].
x 100.
ExBB.
100.
b -(H x R) (b = DNA in the mixture of the non-hepatic-cell fraction and the material left on the sieve during the preparation of the liver cell suspension, derived from liver tissue corresponding to 1 g dry wt.; this quantity was determined experimentally as described in the text but is not given in the Table).
EE = 1000 ~ (0 + BB). FF-
E xEE.
GG=
FF ~ x 100. E x 1000
HH=S+V.
ExV.
II=
ExHH.
w
JJ=
K+Y.
~ T+W
content
average diameter of the “non-hepatic” cells is 10 p, that the cells are spherical, and that their dry wt. per unit volume is the same as that of the hepatic cells; the average dry wt. and the diameter of a hepatic cell (which has been assumed to be spherical in suspension) have been taken to be 1.22 pg and 2.65 ,a, respectively; the values for the hepatic cells are based on the measurements for the various animals, made as described in the text].
E x 1000 S=
known
= Y x 0.0655 [the factor of 0.0655 is based on the assumptions that the
x 100.
2
d
on the
x 100.
KK=L+Z. Experimental
Cell Research
40
5 $
g B 3
‘2
1.17 1.19 1.37 1.47 1.47 1.50 1.76 1.76 1.78 2.14 2.28
11 10 7 8 17 20 16 22 13
deviation
320
Standard
Mean
6
2.7
40.1
514
117
28.8-55.0
48.1
38.1 29.0 55.0 43.2 28.8 30.5 40.9
45.0
42.4
-
CM)
Per cenl of toLa1 no. of cells in live1
310-863
697
219
196-469
863
303 -
369 270 301 390 279 196 238 223 280
397 443 585 491 344 424 478 639
310 532 440 558 505
419 466
376 469
CL)
Per liver (millions)
Number
cells
W)
Per g dry wt. of liver (millions)
Hrputic
Range
2.45 2.70 2.85 3.02 3.14 3.18
0.74 1.14
6 12
24 5 1 4 9 3 2 18
(W
(g)
vfr;“-;.
(A)
Animal no.
2
on unimol
Datu used
I (continued).
Part
TABLE
224
0.12
1.30
392 43
619 50
331-944
-889
0.75-2.42
944
331 820 491 448 398 740 789
658 425 533 524 587 663
(P)
liver (mg)
935
331
Per
weight
1.18 1.34
1.27 2.42 1.10 1.31
224 346 346
225 547 279 255
0.75 1.40 1.00 1.30 0.94 1.55 1.24
889 373 455 441 428 451
(0)
Per g dry wt. of liver Cm&
2.12 0.80 1.21 0.94 1.16 1.67
(NJ
Per million cells Cm&
Dry
12.1-19.7 14.7 0.2
39.2 4.3
13.8
-
15.2 15.8 -
12.8 19.7
12.1 15.9 12.2 16.9
12.3
-
CR)
Per million cells cw)
22.4-88.9
29.4
33.1
27.9 25.5 22.4 34.6 34.6
88.9 37.3 45.5 44.1 42.8 45.1 22.5 54.7
(Q)
Per cent of total dry wt. of liver
6600 4240 290
3530-
-
4180
-
6600 3570 3860 3620 3530
4290 3680
4470
4610 -
(3
Per g dry wt. of liver t&9
DNA
CT)
liver (I+?)
7,220 659
.5,39011,900
6,120 6,310 5,510 9,890 6,280 6,780 G,440 7,550
5,390 -
Per
42.775.5 58.6 2.8
59.7 59.4 42.7 75.5 59.2 54.3 49.8 62.0
57.4 -
W)
Per cent of total DNA in liver
h $ i.,o 3s z 2 z
on animal
Ihta
used
1.17 1.37 1.47 1.47 1.50 1.76 1.76 1.78 2.14 2.85
11 7 8 17 20 16 22 13 24 9
deviation
(E)
(A)
Range Mean Standard
69
Animal no.
Dry wt. of liver
3
Non-hepatic
2140-4950 3020 275
3410 3020 2940 4950 2140 2460 3250 3640 2160 2180
(V
Per g dry of liver (PP)
I (continued).
Part
TABLE
wt.
4000 4130 4320 7270 3210 4330 5710 6480 4630 6220
m
Per liver u-4
DNA
3210-7270 5030 413
cells
24.5-57.3 41.4 2.8
42.6 40.3 40.6 57.3 24.5 40.8 45.7 50.2 38.0 34.3
F)
7 Per cent of total DNA in liver
319-738 450 41
510 450 438 738 319 367 484 544 323 326
w
7 Per g dry wt. of liver (millions)
478-1080 751 62
596 617 644 1080 478 646 853 968 691 929
w
Per liver (millions)
Number
45.0-71.2 59.9 2.7
57.6 55.0 61.9 71.0 45.0 56.8 71.2 69.5 59.1 51.9
(AA)
Per cent of total no. of cells in liver
21-48 29 3
33 29 29 48 21 24 32 36 21 21
@W
Per g dry wt. of liver (w)
weight
31-71 49 4
39 40 42 71 31 42 56 63 45 61
(CC)
Per liver (w)
Dry
2.1-4.8 2.9 0.3
3.3 2.9 2.9 4.8 2.1 2.4 3.2 3.6 2.1 2.1
(DD)
Per cent of total dry wt. of liver
240
P. T. Iype, P. M. Bhargava
and A. D. Tasker
I (continued).
TABLE Part
4
Data used
an animal
Non-cellular
material Dry
weight
ll0.
(9)
Per g dry wt. of liver (w)
(A)
(J-3
WE)
(FF)
Animal
11
Dry wt. of liver
Per
liver (mg)
Total Per cent of total dry wt. of liver
Per g dry wt. of liver
DNA
Per
Total
liver
0%)
(.&a
(GG)
WW
(11)
Per g dry wt. of liver (millions)
(JJ)
no.
of cells
Per liver (millions)
WK)
1.17
511
,598
51.1
8020
9,390
7
1.37
542
743
54.2
7480
10,200
859
1120
8
1.47
520
765
52.0
7230
10,600
708
1040
17
1.47
727
72.7
8620
12,700
20
1.50
432
43.2
8730
13,100
709
1060
16
1.76
697
1230
69.7
6030
10,600
646
1140
22
1.76
714
1260
71.4
7100
12,500
680
1200
13
1.78
741
1320
74.1
7260
12,900
782
1390
24
2.14
633
1350
63.3
5690
12,200
546
1170
9
2.85
647
1850
64.7
6360
18,100
929
1790
432-741
59%
43.2-74.1
5690-8730
9,390-
546-1040
1030-
Range
1070 648
1850 Mean Standard
616 deviation
1080
34
125
1030
1040
18,100 61.6 3.4
7250 324
12,200 770
1520
1790 778
1250
149
247
is 319-738 X lo6 (mean 450 k 41 X 10”) per g dry weight of liver, of i.?197 X lo6 (mean 114 +36 x 106) per g wet weight of liver, or 478-1080 x 106 (mean 751 &62 x 106) per liver. The number of non-hepatic cells in liver exceeds that of hepatic cells, the latter representing 29-55 per cent (mean 40.1 1.2.7 per cent) and the former 45-71 per cent (mean 59.9 & 2.7 per cent) of the total number of cells in liver. Earlier experiments in which cells were counted in tissue sections also indicated that the number of non-hepatic cells exceeds that of hepatic cells in liver [l]. Our observations are, however, at slight variance with the conclusions of Daoust [9] that at least 60 per cent of liver cell population is represented by parenchymal cells. (3) Dry weight of hepatic cells (average of the cell population from one animal) varies from 0.75-2.42 x 1OP mg (mean 1.30 i 0.12 X 1O-6 mg) per cell. The dry weight of hepatic cells does not appear to have been estimated earlier, but an approximate value can be arrived at from the data of De, Chatterjee and Bose [ll]. These authors, by quantitative phase contrast Experimental
Ceil Research
40
241
Chemical and cellular composition of rat liver
microscopy, found that in 5 ,u thick sections, the dry weights of cytoplasm, heterochromatin and nucleolus of hepatic cells were 0.486, 0.702 and 1.226 ~~g/~2. If it is assumed that the cytoplasm of hepatic cells represents the least dense and the nucleolus the most dense part of the cell and that the II. Relative occurrence of cells with
TABLE
Data
4ge (months) 3 7 9
on animal
1, 2, 3 or 4 nuclei in rat liuer.
used
Total in the exam -
Cells containing
Ses
Wt. (fz)
% Total Wet no. of wt. of cells liver examined (9)
3 3” e
160 270 195
6.9 11.5 7.7
897 793 451
7 1 nucleus -
2 nuclei -
3 nuclei -
4 nuclei -
No.
Per cent
NO.
Per cent
No.
Per cent
783 662 353
87.3 83.5 78.3
108 124 95
12.0 15.6 21.1
6 6 2
0.7 0.8 0.4
No. 1 1
Per cent
KO.
0.1 0.2
3017 932 553
average volume of a hepatic. cell is 10,600 cp (Table III), it can be calculated from the data of the above workers that the dry weight of a hepatic cell would lie between 1.03 and 2.58 x 10m6 mg; the dry weight value would be likely to be closer to 1.03 x 1O-6 mg (cf. the average value of 1.30 X 1O-6 mg observed by us) than to 2.58 x lo-” mg since the cytoplasm accounts for a major proportion of the volume of the hepatic cells. (4) The livers of 3-g-month-old animals contain 12-21 per cent bi-nucleated cells and less than 1 per cent tri- and tetra-nucleated cells (Table II). The presence of tri- and tetra-nucleated cells in liver does not appear to have been described earlier. The presence of bi-nucleated cells in liver is well known [12, 19, 401. (5) For every animal examined, the volume of 81-100 per cent of the hepatic cells was found to be between 5100 and 20,100 C,U (Table III). Usually, a small number of cells smaller than 5100 ct~ (up to 3420 cp) and larger than 20,100 C,U (up to 36,800 cp) were found. The average volume of a hepatic cell, however, varied from rat to rat within much narrower limits (8630-13,800 c,u; mean 10,600 k 1480 c,u). The precise volume of hepatic cells does not appear to have been determined earlier. (6) The total number of cells (of all kinds) in liver is ,5461040 x lo6 (mean 778 + 149 x 106) per g dry weight of liver, or 1266277 X lo6 (mean 197 k42 x 106) per g wet weight of liver, or 1030-1790 x lo6 (mean 1250 k Esperimenkd
Cell Research 40
8 ;: * %
?
G z
-
Sex
8 3 6 9 ? Q ? 8 6 d 5 CT CT o^ 6
6 11 10 7 17 20 13 14 24 15 9 3 2 18 19
Data
III.
Expt. no.
TABLE
3 3 3 3 3 9 6 12 9 12 Q 12 12 7 7
Age (months)
on animal
Variation
100 150 180 135 147 175 195 245 230 265 295 312 310 270 300
Wt. (g)
used
with
0.74 1.17 1.19 1.37 1.47 1.50 1.78 1.88 2.14 2.19 2.85 3.0’2 3.14 3.18 3.61
139 169 161 128 125 121 116 160 131 135 161 173 110 91 149
Total \ no. Dry wt. of cells of liver examined 69
liver weight
0.7 7.7 1.9 2.3 0.8 1.7 0 2.5 0 1.5 0.6 0 0 0 0.7
7.2 20.7 32.9 18.0 13.6 19.0 12.1 25.6 8.4 11.1 9.9 1.7 3.6 4.4 5.4
Percentage 3420 5100 to to 5100 7050 (c,u) (c/J)
in the relative
33.8 38.4 34.7 44.5 34.4 32.2 42.2 30.6 23.7 29.6 32.9 12.7 17.3 26.4 20.2
7050 to 9450 (CP)
of cells
38.1 18.4 19.9 25.0 25.6 28.2 27.6 20.6 40.1 34.0 31.7 41.0 26.4 30.7 32.9
17.3 10.6 9.3 9.4 18.4 13.2 17.2 13.7 19.3 16.4 19.9 34.2 18.1 25.3 25.5
12,400 to 16,000 (ccc)
falling
2.2 4.2 0.6 0.8 4.8 4.9 0.9 6.3 5.4 5.9 5.0 6.4 24.6 11.0 8.7
0 0 0.6 0 1.6 0 0 0.6 3.1 0.7 0 2.9 9.1 1.1 6.0
0.7 0 0 0 0.8 0.8 0 0 0 0.7 0 0.6 0.9 1.1 0
24,700 to 30,600 (w)
volume
20,100 to 24,700 (CP)
various
0 0 0 0 0 0 0 0 0 0 0 0.6 0 0 0.7
(
10,400 8,960 8,630 9,010 10,500 9,900 9,780 9,600 11,200 10,600 10,500 12,600 13,800 11,900 12,300
Average volume of a cell (cl4
volumes.
30,600 to 36,800 (cl4
ranges
cells of different
16,000 to 20,100 (w)
within
of rat hepatic
9450 to 12,400 (cl4
examined
proportion
Chemical
and cellular
composition
of rat liver
243
247 x 106) per liver (Table I). This number has been estimated earlier from the DNA content of liver tissue or of an average liver nucleus, both values being determined chemically [ 12, 16, 17, 431. Fakuda and Sibatani [16, 171 give the number of cells in liver as 1790 x lo6 for a rat whose liver weights 12 g, 1060 x lo6 for a rat whose liver weighs 5.7 g, and 668 X lo6 for a rat whose liver weighs 2.6 g. Williams [43] has reported values up to 4000 X lo6 cells per liver in adult rats. Thompson, Heagy, Hutchison and Davidson [41] reported that there is, on an average, 9.27 X lo-l2 g DNA per nucleus in rat liver; the total amount of DNA in livers of the same rats varied from 217-286 mg per 100 g liver. From these data it can be calculated that the number of nuclei in the liver of an adult animal, whose liver weighs 10 g would be 2340-3070 X 106; the number of cells in liver would be lower in view of the presence of bi-nucleated hepatic cells. In most of the animals (liver wet weight 3.8-11.5 g) examined by us, the total number of cells in liver was found to be less than 1500 x lo6 per liver. Thus, while some of the values for the total number of cells in adult rat liver observed by other workers fall within the range observed by us, other values are abnormally high; this may be partly due to no correction for polyploidy of hepatic cells having been applied in the earlier investigations, In any event, it is difficult to assess the validity of the values for total number of cells in liver observed by earlier workers for reasons which have already been stated by us [22]. (7) On an average, the hepatic cells appear to account for less than half (range 22.4-88.9 per cent; mean 39.2 i4.3 per cent) and the non-cellular material for more than half (range 43.2-74.1 per cent; mean 61.6 + 3.4 per cent) of the dry weight of liver; the non-hepatic cells in liver probably contribute less than 5 per cent (range 2.1-4.8 per cent; mean 2.9 k 0.3 per cent) towards the dry weight of liver. It must be emphasised that in the estimations reported here the amount of material which may have leaked out of the hepatic cells during the dispersion of the tissue to a suspension of single cells has not been taken into account. Therefore, the true values for the contribution of hepatic cells towards the dry weight of liver may be slightly higher than those stated above. ,4 large percentage of the constituents of the hepatic cells, say more than 20 per cent, is unlikely to have leaked out during the dispersion of the tissue since (i) the cells possess most of the normal metabolic functions of liver; thus, they can respire [23], synthesise protein [5] and RNA [26], can recognise their own kind both in vitro and in vivo [6], and contain glycogen [24]; (ii) the dry weight of hepatic cells obtained by us is close to the value calculated from the data of De, Chatterjee and Bose [l l] on the density of cytoplasm and nuclei of hepatic cells in tissue sections. However, Experimental
Cell
Research
40
244
P. T. Igpe, P. Ail. Bhargava TABLE
Correlation
IV. Significance of’ various correlations. No. of animalsa
between
Dry
wt. of liver
Average
Dry
wt. of liver
No. of hepatic
Dry
wt. of liver
Dry
Dry
wt. of liver
DNA
Dry
wt. of liver
Dry wt. of non-cellular liver
Dry
wt. of liver
Total
Dry
wt. of liver
‘Total no. of hepatic hcpatic cellsjliver
No. of hepatic
cells/liver cells/liver
and il. D. Tasker
volume
of hepatic
cells
cells/liver
wt. of hepatic of hepatic
cells/liver
cells/liver material/
DNAjliver and non-
Correlation coefficientb
Significance level (P value)
15
0.81
< 0.001
15
0.72
< 0.01
15
0.65
< 0.01
10
0.75
< 0.02
10
0.92
< 0.001
10
0.85
< 0.01
10
0.74
c 0.02 < 0.01
DNA
of hepatic
cells/liver
10
0.86
DNA
of hepatic
cells/liver
10
0.86
< 0.01
of non-
10
0.45
> 0.1
wt. of
15
- 0.67
i 0.01
dry
15
-- 0.53
< 0.03
Dry
wt. of hepatic
Dry
wt. of liver
DNA (or no. or dry hepatic cellsjliver
Dry
wt. of liver
No. of hepatic liver
Dry
wt. of liver
Dry
Dry
wt. of liver
wt.)
cells/g
wt. of hepatic of liver
dry
cells/g
wt.
10
- 0.65
< 0.05
10
pO.il
< 0.05
DNA (or no. or dry wt.j of nonhepatic cellsjg dry wt. of liver
10
- 0.05
> 0.1
DNA (or no. or dry wt.) of nonhepatic cells/g dry wt. of liver
10
- 0.46
> 0.1
cells/liver
DNA (or no. or dry hepatic cells/liver
10
0.08
> 0.1
Dry
wt. of IO6 hepatic
cells
19
0.11
> 0.1
No. of hepatic
cells/liver
Dry
wt. of 10’ hepatic
cells
15
- 0.32
10.1
No. of hepatic of liver
cells/g
dry
wt.
Dry
wt. of IO6 hepatic
cells
15
-.- 0.13
> 0.1
ISo. of hepalic of liver
cells/g
dry wt.
Dry
15
0.59
< 0.05
Total
DNA/g
dry
wt. of liver
No. of hepatic of liver
cells/g
dry wt.
Dry wt. of non-cellular dry wt. of liver
No. of hepatic of liver
cells/g
dry
Dry
wt. of liver
No. of hepatic Dry
wt.
wt.
of liver
Dry wt. of hepatic wt. of liver No. of hepatie of liver
cells/g
cells/g
Ezperimenfal
wt. of hepatic of liver
material/g
wt.)
of non-
cells/g
dry
wt.
dry
DNA of hepatic of liver
cells/g
dry
wt.
10
0.84
< 0.01
dry wt.
DNA of hepatic of liver
cells/g
dry wt.
10
0.72
< 0.02
Cell Research
40
Chemical
and cellular
composition
of rat liver
245
even if it is assumed that 25 per cent of the material of the hepatic cells leaks out into the non-hepatic-cell fraction during dispersion of the tissue, it can be calculated that the non-cellular material in liver will represent, on an average, nearly 50 per cent of the dry weight of the tissue. So far, it has not been recognised that in liver the amount of extracellular material may equal or exceed that of intracellular material on dry weight basis. (8) Liver contains 0.57-0.87 per cent (mean 0.73 iO.03 per cent) DNA per g dry weight, or 0.13-0.23 per cent (mean 0.18 ? 0.04 per cent) DNA per g wet weight, or 9-18 mg (mean 12.2 5 0.8 mg) DNA per liver. These values are within the range reported recently by Horvath [21]. The higher values (20.8-28.6 mg per liver) obtained by several earlier workers [16, 17, 38, 411 for adult rats were based on phosphorus estimations and may be in error due to the presence of non-DNA phosphorus in the DNA fraction obtained by the classical Schmidt and Thannhauser fractionation procedure. (9) Hepatic cells contain a little more than half (43-76 per cent; mean 58.6 F. 2.8 per cent), and the other cells a little less than half (25-57 per cent; mean 41.4-2.8 per cent) of the total DNA of liver. (10) The DNA content of the average hepatic (parenchymal) cell varies between 12.1 and 19.7 x lo-l2 g; the average values for 3-, 6-, 9- and 13month-old animals were found to be 13.1, 15.2, 15.5 and 19.7 x 10-12 g per cell. Since 3-, 6- and g-month-old animals contained 13, 18 and 23 per cent more hepatic nuclei than hepatic cells (Table II), and if it is assumed that 13-month-old animals contain 30 per cent more hepatic nuclei than cells [19]. the average DSA content per hepatic nucleus for 3-, 6-, 9- and 13-month-old animals can be calculated to be 11.6, 12.8, 13.1 and 15.2 x lo-l2 g. Earlier workers, using chemical estimation of DNA in isolated liver cell nuclei [2, 7, 8, 13, 14, 16-18, 31, 32, 411, or estimation of DNA in whole liver tissue and division of the value obtained by the number of nuclei deria Where the number of animals is given as 10, the data for animals no. 7, 8, 9, 11, 13, 16, 17, 20, 22 and 24 (Table I) was used for arriving at the correlation coefficient; when the number is given as 15, the data for animals no. 5, 6, 7, 8, 9, 10, 11, 12, 13, 16, 17, 18, 20, 22 and 24 (Table I) was used; and when the number is given as 19, the data for all the animals given in Table I was used. b Product moment correlation coefficient,
,,i-;” Y=
(x - 5) (y - ij) > sx * sy
where n is the number of animals; Sx and Sy are the standard deviations of the two variables and y, respectively; and Z and g are the means of the various values of x and y, respectively. ’ Correlation coefficients with P > 0.05 are considered not significant. Experimental
Cell Research
x
40
246
P. T. Iype, P. M. Bhargava
and A. D. Tasker
ved from the tissue [35, 361, have given values ranging from 5.9 to 15.4 x lo-l2 g DNA per nucleus. The lower values (in comparison to ours) obtained by several earlier workers may be due to their preparation of nuclei having contained nuclei from cells other than hepatic (parenchymal) cells, in contrast to the present investigation in which DNA was estimated in a preparation which consisted almost entirely of hepatic cells. The number of nuclei from the non-hepatic cells in a preparation of total nuclei derived from liver is likely to be about the same as the number of nuclei from hepatic cells. The nuclei from non-hepatic cells of liver would contain only the normal complement of DNA corresponding to diploid cells, in contrast to nuclei from hepatic cells which are known to be polyploid; as such the presence of nuclei from non-hepatic cells in appreciable numbers in a preparation of nuclei derived from whole liver would give a lower average DNA content per nucleus than would be obtained with a preparation consisting entirely of hepatic cells. In fact, the extent of polyploidy observed in the liver of adult rats [3, 4, 12, 29, 30, 33, 371 is so high as to make the values of DNA per average hepatic nucleus obtained in the present investigation more likely than those obtained in the earlier investigations, which probably gave the DNA content of an average nucleus derived from the various cells present in liver, rather than of an average hepatic (parenchymal) cell nucleus. DISCUSSION
The data presented in Tables I-III were statistically analysed primarily with a view to determine the pattern, if any, in the variation with the dry weight of liver, in (i) the number, dry weight and DNA of hepatic and nonhepatic cells; (ii) the dry weight of non-cellular material, and (iii) the volume of hepatic cells. The correlation coefficients which appeared to be of interest are given in Table IV. This analysis allows the following conclusions to be drawn: (a) The percentage recovery of hepatic cells obtained in the cell suspensions prepared by the method of Jacob and Bhargava [25] is independent of the age or the dry weight of liver (Table I). (b) The percentage of binucleated hepatic cells in liver rises with the age of the animal (Table II). (c) The percentage of hepatic cells with larger volume, and the average volume of cells, increase as the dry weight of liver increases (Tables III and IV). Seise and Stegmann [39] and Helweg-Larsen [20] reported that average nuclear and cellular volume increase in direct proportion to the logarithm of liver weight in mouse. This is also to be expected in view of the observed increase of the number of binucleated cells in liver with ageExperimental
Cell
Research
40
Chemical
and cellular
composition
247
of rat liver
(d) As the dry weight of liver increases, the following parameters also increase (Tables I and IV): (i) the number, and as would therefore be expected, the dry weight and the DNA of hepatic cells in the whole liver; (ii) the dry weight of non-cellular material in the whole liver; (iii) the total DNA of liver; and (iv) the total number of cells (hepatic plus non-hepatic) in liver. Again, as may be expected on the basis of the correlations under (i) above, there was a significant, positive correlation between the DNA of hepatic cells per liver, and either the number or the dry weight of hepatic cells per liver. However, no correlation was found between the dry weight of liver and the DNA (or number or dry weight) of non-hepatic cells per liver; the latter parameters seemed to vary little during liver growth from 1.2 to 2.9 g dry weight. These observations show that during growth of rats beyond the age of 3 months, there is a rise in hepatic and total cell number, the non-cellular material, and the hepatic cell and total tissue DNA, while there is no statistically significant rise in the number of non-hepatic cells. (e) As the liver weight increases, the number of hepatic cells (or the weight of these cells) per g of liver decreases Tables I and IV). This is also indicated by a significant negative correlation between the dry weight of liver and the total DNA per g dry weight of liver; in view of the observed lack of significant variation in the number of non-hepatic cells with increase in liver weight, the above negative correlation could be explained only if, with an increase in the dry weight of liver, there is a concurrent decrease in the number of hepatic cells, which decrease more than compensates for the increase in DNA of the average hepatic cell due to polyploidy. The decrease in the dry weight of hepatic cells per g liver observed with increasing dry weight of liver, would be expected to be accompanied either by a concurrent increase in the percentage of non-cellular material in liver, or/and by an increase in the number and weight of non-hepatic cells. The former possibility is strongly supported by a high significant negative correlation between the number of hepatic cells per g dry weight of liver and the dry weight of non-cellular material per g dry weight of liver, and no significant negative correlation between (i) the number of hepatic cells per g dry weight of liver and the number (or DNA or dry weight) of non-hepatic cells per g dry weight of liver; (ii) dry weight of liver and the number (or DNA or dry weight) of non-hepatic cells per g dry weight of liver; and (iii) the number of hepatic cells per liver and the number (or DNA or dry weight) of non-hepatic cells per liver. Thus, it would appear that as the already adult (3-month-old) animals get older, the percentage of non-cellular material in liver increases, and the percentage of the material derived from the parenchymal cells of Experimental
Cell
Research
40
248
P. T. Iype, P. M. Bhargava
and A. D. Tasker
liver decreases. Our observations in this respect are supported by the earlier report of Johnson and Albert [27] who found that in mouse, the number of nuclei progressively decreased from 281 + 10 x lo6 to 211 -t 11 x lo6 per g wet weight of liver, as the liver weight increased from 1.04 + 0.03 to 1.29 + 0.03 g. It would, therefore, appear that the cause of the reduction of various enzymes (e.g., those involved in the esterillcation of phosphorus or oxidation of butyrate; [42]) in liver with age (the enzyme activity being expressed as activity per unit weight of liver or per mg protein), observed by earlier investigators, may be due to a reduction in the number of hepatic cells per unit weight of liver and a corresponding increase in the non-cellular material which is unlikely to contain these enzymes. Our observations, therefore, lead us to endorse the view of Jones, Bitensky, Chayen and Cunningham [28] (who concluded that during chemical carcinogenesis of liver, the number of hepatic cells per unit weight of liver falls) that biochemical comparisons involving animals of different ages must take into consideration the “tissue dilution artifact” which may arise due to a reduction in the number of one kind of cell in the tissue and a concomitant increase in the number of other kinds of cells or of non-cellular material. (f) There is no correlation between the dry weight of hepatic cells (which varies from 0.75 to 2.42 x 1O-6 mg per cell) and either the dry weight of liver or the number of hepatic cells per liver or the number of hepatic cells per g dry weight of liver (Tables I and IV). In the case of 14 out of 19 animals, the dry weight varied between narrow limits (0.94-1.40 x 1OP mg per hepatic cell) and this variation appeared uncorrelated to any of the parameters studied. The average dry weight of adult hepatic cells in an animal would, therefore appear to vary randomly and little; this view is supported by a significant positive correlation between the number of hepatic cells per g dry weight of liver and the dry weight of hepatic cells per g dry weight of liver. This conclusion, together with the positive correlation between dry weight of liver and hepatic cell volume, would suggest a lowering of the average density of the hepatic cells with increase in liver weight. (g) As the dry weight of hepatic cells per g dry weight of liver (or the number of hepatic cells per g dry weight of liver, with which value the dry weight of hepatic cells per g dry weight of liver was directly correlated) increases, the DNA of hepatic cells per g dry weight of liver also increases (Tables I and IV), suggesting that larger cell size may be associated with larger DNA content.
Experimental
Cell
Research
40
Chemical
and cellular
composition
249
of rat liver
SUMMARY 1. Jacob and Bhargava’s method of preparing rat hepatic cell suspensions gives a recovery of 84-93 per cent (mean 89 k 0.8 per cent) of the hepatic parenchymal cells present initially in adult rat liver. 2. The liver of 3%14-month-old rats contains 196-469 X lo6 (mean 320 + 6 x 106) hepatic cells per g dry weight of liver, or 52-135 X lo6 (mean 81 + 28 x 10”) hepatic cells per g wet weight of liver, while the number of non-hepatic cells is 319-738 X lo6 (mean 450 k 41 X 106) per g dry weight of liver, or 75-197 x lo6 (mean 114 2 36 X 106) per g wet weight of liver. The hepatic cells represent 29-55 per cent (mean 40.1 k 2.7 per cent) and the non-hepatic cells 45-71 per cent (59.9 k 2.7 per cent) of the total cell population of liver. The number of hepatic cells per g dry weight of liver decreases with increase in dry weight of liver; no significant correlation was found between the dry weight of liver and the number of non-hepatic cells per g dry weight of liver. 3. The number of hepatic and non-hepatic cells in the whole liver is 310863 x lo6 (mean 514 + 117 X 106) and 478-1080 X 106 (mean 751 k 62 X 106), respectively. k. The total number of cells in liver is 546-1040 x lo6 (mean 778 1149 x 106) per g dry weight of liver, or 126-277 X lo6 (mean 197 k42 X 106) per g wet weight of liver, or 1030-1790 X 106 (mean 1250 k 247 X 106) per liver. 5. The dry weight of hepatic cells varies from 0.75-2.42 mg (mean 1.30 * 0.12 mg) per lo6 cells. 6’. On an average, the hepatic cells appear to be responsible for a little less than half, the non-hepatic cells for less than 5 per cent, and the noncellular material for about half the dry weight of liver. The percentage of non-cellular material in liver (on dry weight basis) increases with an increase in the dry Lveight of liver and a decrease in the number of hepatic cells per unit weight of liver. 7. The livers of 3-g-month-old animals contain 12-21 per cent binucleated hepatic cells and less than 1 per cent tri- and tetra-nucleated hepatic cells. The number of hinucleated cells increases with age. S. The volume of a hepatic cell usually varies between 5100 and 20,100 CPU;only a small fraction of the hepatic cells of liver have a volume which is outside this range. The average volume of hepatic cells from any animal varies between X630-12,300 cp (mean 10,600 k1480 cp) and increases with increase in the dry weight of liver. Y. Liver contains 0.57-0.87 per cent (mean 0.73 + 0.03 per cent) DNA 17 - G31818
Experimental
Cell Research
40
250
P. T. Iype, P. M. Bhargava
and A. Il. Tasker
per g dry weight of liver, or 0.13-0.23 per cent (mean 0.18 k 0.04 per cent) DNA per g wet weight of liver, or 9-18 mg (mean 12.2 10.8 mg) DNA per liver. 20. The hepatic cells in liver contain a little more than half (43-56 per cent; mean 58.6 2 2.8 per cent), and the other cells a little less than half (25-57 per cent; mean 41.4 1-2.8 per cent) of the total DNA of liver. 11. The DNA content of average hepatic cell varies between 12.1 and 19.7 X lo-l2 g, and of an average hepatic cell nucleus betlveen 11.6 and 15.2 x lo-l2 g. 12. The results are discussed vis-a-vis earlier observations in respect of the above-mentioned parameters of liver. This work was done during the tenure of a Senior Research Fellowship of the Council of Scientific and Industrial Research, New Delhi, awarded to one of us (P. T. I.), which is gratefully acknowledged. We are also grateful to Dr S. Husain Zaheer, Director-General, Council of Scientific and Industrial Research, New Delhi, and to Dr G. S. Sidhu, Director, Regional Research Laboratory, Hyderabad, for encouragement. We should also like to express our gratitude to Mrs Shanta Madhavan of Nutrition Research Laboratory, Hyderabad, for help with the statistical evaluation of data. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
ABERCROMBIE, M. and HARKNESS, R. D., Proc. Roy. Sot. B 138, 544 (1951). ALBERT, S., JOHNSON, R. M. and WAGSHAL, R. R., Science 117, 551 (1953). ALFERT, M. and GESCHWIND, I. I., Exptl Cell Res. 15, 230 (1958). BEATTY, R. A., Parthenogenesis and Polyploidy in Mammalian Development. Cambridge University Press, Cambridge, 1957. BHARGAVA, K. and BHARGAVA, P. M., Life Sciences p. 477 (1962). ~ Unpublished. CAMPBELL, R. M. and KOSTERLITZ, H. W., Science 115, 84 (1952). CUNNINGHAM, L., GRIFFIN, A. C. and LUCK, J. M., J. Gen. Pkysiol. 34, 59 (1950). DAOUST, R., in R. W. BRAUER (ed.), Liver Function, p. 3. American Institute of Biological Sciences, 1958. DAWSON. R. M. C.. ELLIOTT. D. C.. ELLIOTT. W. H. and JONES, K. M., Data for Biochemical Research, p.‘209. Oxfdrd University Press, London, 1959. DE, P., CHATTERJEE, R. and BOSE, A., Ezyptl Cell Res. 27, 168 (1962). DOLJANSKI, F., Int. Rev. Cytol. 10, 217 (1960). DOUNCE. A. L.. TISHKOFF. G. H.. BARNETT, S. R. and FREER, R. M., J. Gen. Pkysiol. 33,
626 (1950j. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.
ELY, J. 0. and Ross, M. H., Science 114, 70 (1951). FALZONE, J. A., BARROWS, C. H. and YIENGST, M. J., Expfl FUKUDA, M. and SIBATANI, A., Experientia 9, 27 (1953).
__
(Tokyo) 40, 95 (1953). Nature 168, 248 (1951). ~ ibid. 171. 611 (1953). HELWEG-LARSEN, G. F., ‘Acfa Pafkol. Microbial. HORVATH, G., Nature 200, 261 (1963). IYPE, P. T. and BHARGAVA, P. M., Life Sciences, ~ Biockem. J. 94, 284 (1965).
Res. 26, 552 (1962).
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HARRISON,
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M. F.,
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92, 1 (1952).
Chemical 24.
~
27. 29.
30. 31.
JOHXSON, R. JONES, G. R. KOULISH, S. LAQUERRIERE, LEUCHTENBERGER,
Res. 3,
32. 33. 34. 35. 36. 37. 38. 39. 40. 41.
42. 43.
composition
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Unpublished.
25. JACOB, S. T. 26. __ Biochem. 28.
and cellular
P. M., Exptl Cell Res. 27, 453 (1962). (1965). M. and ALBERT, S., Proc. Sot. ExptZ Biol. Med. 102, 373 (1959). N., BITENSKY, L., CHAYEN, J. and CUNNINGHAN, G. J., Nature 191,1203 (1961). and LESSLER, M. A., Cancer Res. 22, 1188 (1962). R., Nature 180, 1199 (1957). C., LEUCHTENBERGER, R., VENDRELY, C. and VENDRELY, R., ExptI Cett 240 (1952). C., VENDRELY, R. and VENDRELY, C., Proc. Natl Acad. Sci. Wash. 37, and
BHARGAVA,
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LEUCHTESBERGER, 33 (1951). NAORA, H., J. Biophys. Biochem. Cytol. 3, 949 (1957). POI’PER, H. and SCHAFFNER, F., Liver: Structure and Function, p. 7. McGraw-Hill, New York, 1957. PRICE, J. M. and LAIRD, A. K., Cancer Res. 10, 650 (1950). PRICE, J. M., MILLER, E. C., MILLER, J. A. and WEBER, G. M., Cancer Res. 10, 18 (1950). RIGLER, R., Exptl CeZZ Res. 28, 260 (1962). SCHMIDT, G. and THANNHAUSER, S. J., J. Biot. Chem. 161, 83 (1945). SIESS, U. and STEGMANN, H., Virchows Arch. Pathot. Anat. Physiot. 318, 534 (1950). ST. AUBIS, P. M. G. and BUCHER, N. L. R., Anat. Rec. 112, 797 (1952). THO~XSOX-, R. Y., HEAGY, F. C., HUTCHISON, W. C. and DAVIDSOX, J. N., Biochem. J. 53, 460 (1953). WEISBACH, E. C. and GARBUS, J., Nafure 178, 1225 (1956). \\‘ILLIAMS, .J. N., J. Nutrition 73, 199 (1961).
Experimental
Cell Research
40
Cell Research
40, 233-251
SOME ASPECTS
OF THE
COMPOSITION P. T. Regional
233
(1965)
IYPE,
P. M.
Research
Laboratory, Medical
CHEMICAL
OF ADULT BHARGAVA
Received
RAT LIVER and
Hyderabad-9, Research,
AND CELLULAR
New
February
India, Delhi,
A.
D. TASKER and Indian
Council
of
India
11, 1965
WE recently described a method [25] for the preparation of rat hepatic cell suspensions which gives an almost quantitative recovery of the hepatic cells in the final suspension; the method takes only 15 min for the preparation of cell suspensions which consist predominantly of single cells and which are virtually free of cell debris and cells other than the hepatic cells present in liver. Further, this method does not involve any enzymatic or drastic physical treatment of the tissue, so that it minimises the loss of intracellular constituents or of cell integrity. Since this method seemed to give a good separation of the hepatic cells from the other cellular and non-cellular material in liver, we felt that it could be used for obtaining useful information on certain aspects of the chemical and cellular composition of liver. In this paper we report the results of estimation of (Q) the number of hepatic (parencells [34] conchpmal) and non-hepatic (i.e., other than the parenchymal) tained in a unit weight of liver or in the whole liver of 3-14 months old rats; (b) the average dry weight of hepatic cells; (c) the percentage of dry weight of liver represented by hepatic cells, non-hepatic cells or non-cellular material; (d) the DNA content of hepatic cells; (e) the percentage of DNA of liver contained in hepatic cells or non-hepatic cells; (f) the extent of occurrence of polynucleated hepatic cells in liver; and (g) the volume of hepatic cells. The variation of the above parameters with increasing dry weight of liver has also been studied; it has been shown that as the weight of liver increases, the number of hepatic cells per unit Lveight of liver falls, whiie the percentage of non-cellular material increases. No definitive information is available on most of the above parameters of liver; the difficulties in the measurement of these values by conventional methods have been pointed out in an earlier, preliminary publication [22]. Experimental
Cell Research
40
234
P. T. Iype, P. M. Bhargava and A. D. Tasker EXPERIMENTAL
Animals.-Adult albino rats of an inbred strain of Nutrition Research Laboratory, Hyderabad, of known sex, age and weight, and fed on an ad libitum diet, were used. Preparation of hepatic cell suspensions.-The cell suspensions were prepared from a known wet weight of perfused liver by the method of Jacob and Bhargava [25], which essentially consists of perfusion of the liver with 30-50 ml of cold (5-10°C) sodium citrate (0.027 M) in calcium-free Locke’s solution [IO], followed by dispersion in 0.25 M sucrose in a specially designed tissue disperser. The dispersate was filtered through a 240-mesh sieve and the residue on the sieve washed with 0.25 M sucrose. Microscopic examination of the residue showed that very few cells or free nuclei, if any, were present; their number was negligible when compared to the number of cells and free nuclei contained in the filtrate. The filtrate was centrifuged at 200 xg and the supernatant saved. The sedimented cells were suspended in 0.9 per cent NaCl and centrifuged as above; this washing with saline was essential to remove adhering sucrose from the hepatic cells which could vitiate the results of dry weight estimations [22]. The supernatant was combined with the earlier supernatant and made up to a known volume; the sedimented hepatic cells were resuspended in 0.9 per cent NaCl. As stated earlier [22], the combined supernatants contained (a) cell debris, including the nuclei, from the small number of hepatic cells broken during the preparation of the cell suspension; (b) material, if any, which leaked out of the hepatic cells during the preparation of the cell suspension; (c) contents of virtually all the cells other than the parenchymal cells present in liver; and (d) almost all the non-cellular material of liver (the material left on the sieve contained the remaining non-cellular material). Since, on an average, only 10 per cent of hepatic cells were broken during the preparation of the cell suspension and since (for reasons discussed later) the material in the second category appeared most unlikely to account for a significant proportion of the wet weight or DNA of the hepatic cells, the combined supernatants obtained as above have been subsequently referred to as the “nonhepatic-cell fraction”. Dry weight determinations.PFor determination of the dry weight of perfused liver, the freshly excised organ was pressed between folds of filter paper, quickly weighed and a known wet weight dried to constant weight at 110°C. The dry weight of hepatic cells in suspension was determined as described earlier [22]. Duplicate estimations were run in each case. They tallied within experimental error; the values given are the average of the two determinations. In some experiments, the approximate dry weight of the non-cellular material and cells other than the hepatic cells in liver was determined experimentally by mixing the non-hepatic-cell fraction obtained as above with the residue left on the sieve during preparation of the cell suspension, and dialysing, first against running water and then against distilled water. The material in the dialysis bag was heated to boiling and the coagulum sedimented in a centrifuge; it was then dried at 110°C to constant weight. The dry weight value thus obtained was found to be close to the value obtained by difference between the dry weight of liver and of hepatic cells contained in the liver, as would be expected since the non-hepatic cell fraction is unlikely to Experimental
Cell Research
40
Chemical
and cellular
composition
of rat
235
liver
contain a significant proportion (on dry weight basis) of dialysable and non-heatcoagulable materials. Hepatic cell count in the cell suspension and the free hepatic cell nuclei count in the non-hepatic-cell fraction.-The concentration of hepatic cells in the cell suspension
and of hepatic cell nuclei in the non-hepatic-cell fraction was determined on a haemocytometer (100 p deep and having a graduated area of 9 mm2divided into 9 major squareseach with an area of 1 mm2 each) under phasecontrast (magnification x SO). Only the hepatic cells were scored in the former case and the hepatic cell nuclei in the latter. The hepatic cell fraction contained very few, if any, hepatic nuclei or cells other than the hepatic cells, and the non-hepatic-cell fraction very few, if any, intact, free hepatic cells. Hepatic cells and their nuclei were distinguishable from other cells and their nuclei by their size. It was assumedthat there was no significant breakage of hepatic cell nuclei during the dispersion of the tissue to single cells becauseof the mild treatment given to the tissue. In some experiments a series of photomicrographs (magnification x128) were taken (35 mm positive contrast film) of the preparation on the haemocytometer so as to cover all its 9 major squares. The hepatic cells in the 9 squareswere counted on a projection screen. Determination of hepatic cell volume.-The films obtained as described above were projected so that a distance of I mm on the haemocytometer was equivalent to 400 mm on the screen; only those photographs in which the hepatic cells were sharply in focus were used. The major and the minor axes of the image of the cells on the screen were measured accurately; the mean divided by 400 was taken as the average diameter and the volume then calculated as for a sphere. Estimation of multinucleated cells.-The hepatic cell suspension,diluted to an appropriate cell concentration, was stained with 2 per cent aqueous, chloroform-washed methyl green by mixing a drop of this stain with about 10 ml of the cell suspension. The proportion of hepatic cells containing 1, 2, 3, or 4 nuclei was then determined by scoring randomly selected 300-800 cells under a microscope. Determination of DNA.-DNA was estimated in the hepatic cells and in the nonhepatic-cell fraction as described earlier [22]; the material remaining on the sieve during the preparation of the cell suspensionwas added to the non-hepatic-cell fraction before the estimation. In some experiments, the total DNA content of whole liver was experimentally determined as above; this value tallied, within experimental error, with the value for the DNA content of liver obtained by the addition of the DNA content of the hepatic cell and the non-hepatic-cell fractions. Calculations.-The following values were experimentally determined as described above: (a) the wet and the dry weights (and thus the moisture content) of liver; (b) the number of hepatic cell nuclei in the non-hepatic-cell fraction per gram dry weight of liver; (c) the number of hepatic cells recovered in the cell suspensionper gram dry weight of liver; (d) the dry weight of a known number of hepatic cells; (e) the DNA content of a known number of hepatic cells; (f) the DNA content of non-hepatic-cell fraction derived from a known wet weight of liver; (g) the percentage of mono-, bi-, tri-, and tetra-nucleated hepatic cells in the hepatic cell suspension;and (h) the percentage of hepatic cells falling within different, specified volume ranges. Other values given were calculated from the above data as indicated in the notes to the tables. Experimenlal
Cell
Research
40
236
P. T. Iype, P. M. Bhargava
and A. D. Tasker
RESULTS
Tables I-IV give the results of the various estimations for nineteen, 3-14month-old rats. The following observations made for these animals are considered significant: I (parts l-4). The recovery of hepatic cells in cell suspensionsprepared from rat liver, the number, dry weight and DNA of rat hepatic and non-hepatic cells, and the dry weight of non-cellular material in rat liver.a TABLE
Part 1 Cell suspensions
Data used
on animal
Moisture in liver
No. of free hepatic cell nuclei
No. of hepatic cells corresponding to the free nuclei
Per g dry wt. of liver (millions)
Per g dry wt. of liver (millions)
No. of intact hepatic cells Per g dry wt. of liver (millions)
Per cent of total no. of hepatic cells
(J)
wt.
Age
Sex
&I
(months)
(g)
(per cent of wet wt. of liver)
(A)
(‘3
((2
CD)
(El
w
(G)
W)
(1)
6 12 11 10 7 8 17 20 16 22 13 24 5 1 4 9 3 2 18
* r” 6 6 9 ? 0 9 d 9 9 0” CT * i 6 3 s 6
100 120 150 180 135 135 150 175 160 185 195 230 295 290 290 295 310 310 270
3
0.74 1.14 1.17 1.19 1.37 1.47 1.47 1.50 1.76 1.76 1.78 2.14 2.28 2.45 2.70 2.85 3.02 3.14 3.18
so.4 71.0 75.8 71.5 73.2 71.3 73.3 73.2 74.3 73.5 79.3 76.9 79.9 76.0 77.8 76.7 74.1 75.0 72.4
76.0 41.0 63.2 32.6 44.0 28.3 46.8 71.5 36.1 33.6 37.8 19.9 52.6
67 36 56 29 39 25 41 58 32 26 32 16 40
24.9
21
352 430 320 440 330 245 260 332 247 170 206 207 240 234 17x 275 323 338 198
71.0-80.4 75.0 0.7
19.9-76.0 42.8 4.3
16-67 36 4
170-440 280 18
Animal no.
Range Mean Standard Experimentnt
3 3 3 3 3 3 9 3 13 6 9 12 12 14 9 12 12 7
deviation Cell Research
40
Dry wt. of liver
34.0
28 -
84.0 92.2 85.1 93.8 89.4 90.7 86.3 85.1 88.2 86.4 86.5 92.8 85.1
90.9
90.4 84.0-93.8 88.5 0.8
Chemical
and cellular
composition
237
of rat liver
and Bhargava’s method gives a near-quantitative (range 84-93 per cent; mean 89 k 0.8 per cent) yield of hepatic cells in the final cell suspension (Table I). (1)
Jacob
(2)
The
liver
contains
196-469
x lo6
(mean
320 k 6 X 106)
hepatic
cells
per g dry weight of liver, or 52-135 x lo6 (mean 81 & 28 x 106) hepatic cells per g wet weight of liver, or 310-863 X lo6 (mean 514 + 117 X 106) hepatic cells per liver (Table I). The number of other (non-hepatic) cells in liver a The values in the columns H, J to M, 0 to Q, S to Z, and AA to KK, have been
&[based
Y=
arrived at as given below; the values in the other columns were determined experimentally as described in the text: H-
Gxa(a=1.13,1.18,1.23ori.3l,for3, 6, 9 and 13-month-old vely; see text and Table
J=
I ~ H+I
K=
H+I.
L-
ExK. L
M =
~ L+z
O-
KxN.
P-
13x0.
Qz
rats, II).
respecti-
6.7 ,ug (stromal Z==
E x Y.
AA=
,>
x 100. BB
j: 100.
KxR.
7’-
EXS.
1: =
T -----x T + w
V =
\I’=
sz
CC-
of
of DNA per lo6 diploid cells cells of liver) of rat [15]].
x 100.
ExBB.
100.
b -(H x R) (b = DNA in the mixture of the non-hepatic-cell fraction and the material left on the sieve during the preparation of the liver cell suspension, derived from liver tissue corresponding to 1 g dry wt.; this quantity was determined experimentally as described in the text but is not given in the Table).
EE = 1000 ~ (0 + BB). FF-
E xEE.
GG=
FF ~ x 100. E x 1000
HH=S+V.
ExV.
II=
ExHH.
w
JJ=
K+Y.
~ T+W
content
average diameter of the “non-hepatic” cells is 10 p, that the cells are spherical, and that their dry wt. per unit volume is the same as that of the hepatic cells; the average dry wt. and the diameter of a hepatic cell (which has been assumed to be spherical in suspension) have been taken to be 1.22 pg and 2.65 ,a, respectively; the values for the hepatic cells are based on the measurements for the various animals, made as described in the text].
E x 1000 S=
known
= Y x 0.0655 [the factor of 0.0655 is based on the assumptions that the
x 100.
2
d
on the
x 100.
KK=L+Z. Experimental
Cell Research
40
5 $
g B 3
‘2
1.17 1.19 1.37 1.47 1.47 1.50 1.76 1.76 1.78 2.14 2.28
11 10 7 8 17 20 16 22 13
deviation
320
Standard
Mean
6
2.7
40.1
514
117
28.8-55.0
48.1
38.1 29.0 55.0 43.2 28.8 30.5 40.9
45.0
42.4
-
CM)
Per cenl of toLa1 no. of cells in live1
310-863
697
219
196-469
863
303 -
369 270 301 390 279 196 238 223 280
397 443 585 491 344 424 478 639
310 532 440 558 505
419 466
376 469
CL)
Per liver (millions)
Number
cells
W)
Per g dry wt. of liver (millions)
Hrputic
Range
2.45 2.70 2.85 3.02 3.14 3.18
0.74 1.14
6 12
24 5 1 4 9 3 2 18
(W
(g)
vfr;“-;.
(A)
Animal no.
2
on unimol
Datu used
I (continued).
Part
TABLE
224
0.12
1.30
392 43
619 50
331-944
-889
0.75-2.42
944
331 820 491 448 398 740 789
658 425 533 524 587 663
(P)
liver (mg)
935
331
Per
weight
1.18 1.34
1.27 2.42 1.10 1.31
224 346 346
225 547 279 255
0.75 1.40 1.00 1.30 0.94 1.55 1.24
889 373 455 441 428 451
(0)
Per g dry wt. of liver Cm&
2.12 0.80 1.21 0.94 1.16 1.67
(NJ
Per million cells Cm&
Dry
12.1-19.7 14.7 0.2
39.2 4.3
13.8
-
15.2 15.8 -
12.8 19.7
12.1 15.9 12.2 16.9
12.3
-
CR)
Per million cells cw)
22.4-88.9
29.4
33.1
27.9 25.5 22.4 34.6 34.6
88.9 37.3 45.5 44.1 42.8 45.1 22.5 54.7
(Q)
Per cent of total dry wt. of liver
6600 4240 290
3530-
-
4180
-
6600 3570 3860 3620 3530
4290 3680
4470
4610 -
(3
Per g dry wt. of liver t&9
DNA
CT)
liver (I+?)
7,220 659
.5,39011,900
6,120 6,310 5,510 9,890 6,280 6,780 G,440 7,550
5,390 -
Per
42.775.5 58.6 2.8
59.7 59.4 42.7 75.5 59.2 54.3 49.8 62.0
57.4 -
W)
Per cent of total DNA in liver
h $ i.,o 3s z 2 z
on animal
Ihta
used
1.17 1.37 1.47 1.47 1.50 1.76 1.76 1.78 2.14 2.85
11 7 8 17 20 16 22 13 24 9
deviation
(E)
(A)
Range Mean Standard
69
Animal no.
Dry wt. of liver
3
Non-hepatic
2140-4950 3020 275
3410 3020 2940 4950 2140 2460 3250 3640 2160 2180
(V
Per g dry of liver (PP)
I (continued).
Part
TABLE
wt.
4000 4130 4320 7270 3210 4330 5710 6480 4630 6220
m
Per liver u-4
DNA
3210-7270 5030 413
cells
24.5-57.3 41.4 2.8
42.6 40.3 40.6 57.3 24.5 40.8 45.7 50.2 38.0 34.3
F)
7 Per cent of total DNA in liver
319-738 450 41
510 450 438 738 319 367 484 544 323 326
w
7 Per g dry wt. of liver (millions)
478-1080 751 62
596 617 644 1080 478 646 853 968 691 929
w
Per liver (millions)
Number
45.0-71.2 59.9 2.7
57.6 55.0 61.9 71.0 45.0 56.8 71.2 69.5 59.1 51.9
(AA)
Per cent of total no. of cells in liver
21-48 29 3
33 29 29 48 21 24 32 36 21 21
@W
Per g dry wt. of liver (w)
weight
31-71 49 4
39 40 42 71 31 42 56 63 45 61
(CC)
Per liver (w)
Dry
2.1-4.8 2.9 0.3
3.3 2.9 2.9 4.8 2.1 2.4 3.2 3.6 2.1 2.1
(DD)
Per cent of total dry wt. of liver
240
P. T. Iype, P. M. Bhargava
and A. D. Tasker
I (continued).
TABLE Part
4
Data used
an animal
Non-cellular
material Dry
weight
ll0.
(9)
Per g dry wt. of liver (w)
(A)
(J-3
WE)
(FF)
Animal
11
Dry wt. of liver
Per
liver (mg)
Total Per cent of total dry wt. of liver
Per g dry wt. of liver
DNA
Per
Total
liver
0%)
(.&a
(GG)
WW
(11)
Per g dry wt. of liver (millions)
(JJ)
no.
of cells
Per liver (millions)
WK)
1.17
511
,598
51.1
8020
9,390
7
1.37
542
743
54.2
7480
10,200
859
1120
8
1.47
520
765
52.0
7230
10,600
708
1040
17
1.47
727
72.7
8620
12,700
20
1.50
432
43.2
8730
13,100
709
1060
16
1.76
697
1230
69.7
6030
10,600
646
1140
22
1.76
714
1260
71.4
7100
12,500
680
1200
13
1.78
741
1320
74.1
7260
12,900
782
1390
24
2.14
633
1350
63.3
5690
12,200
546
1170
9
2.85
647
1850
64.7
6360
18,100
929
1790
432-741
59%
43.2-74.1
5690-8730
9,390-
546-1040
1030-
Range
1070 648
1850 Mean Standard
616 deviation
1080
34
125
1030
1040
18,100 61.6 3.4
7250 324
12,200 770
1520
1790 778
1250
149
247
is 319-738 X lo6 (mean 450 k 41 X 10”) per g dry weight of liver, of i.?197 X lo6 (mean 114 +36 x 106) per g wet weight of liver, or 478-1080 x 106 (mean 751 &62 x 106) per liver. The number of non-hepatic cells in liver exceeds that of hepatic cells, the latter representing 29-55 per cent (mean 40.1 1.2.7 per cent) and the former 45-71 per cent (mean 59.9 & 2.7 per cent) of the total number of cells in liver. Earlier experiments in which cells were counted in tissue sections also indicated that the number of non-hepatic cells exceeds that of hepatic cells in liver [l]. Our observations are, however, at slight variance with the conclusions of Daoust [9] that at least 60 per cent of liver cell population is represented by parenchymal cells. (3) Dry weight of hepatic cells (average of the cell population from one animal) varies from 0.75-2.42 x 1OP mg (mean 1.30 i 0.12 X 1O-6 mg) per cell. The dry weight of hepatic cells does not appear to have been estimated earlier, but an approximate value can be arrived at from the data of De, Chatterjee and Bose [ll]. These authors, by quantitative phase contrast Experimental
Ceil Research
40
241
Chemical and cellular composition of rat liver
microscopy, found that in 5 ,u thick sections, the dry weights of cytoplasm, heterochromatin and nucleolus of hepatic cells were 0.486, 0.702 and 1.226 ~~g/~2. If it is assumed that the cytoplasm of hepatic cells represents the least dense and the nucleolus the most dense part of the cell and that the II. Relative occurrence of cells with
TABLE
Data
4ge (months) 3 7 9
on animal
1, 2, 3 or 4 nuclei in rat liuer.
used
Total in the exam -
Cells containing
Ses
Wt. (fz)
% Total Wet no. of wt. of cells liver examined (9)
3 3” e
160 270 195
6.9 11.5 7.7
897 793 451
7 1 nucleus -
2 nuclei -
3 nuclei -
4 nuclei -
No.
Per cent
NO.
Per cent
No.
Per cent
783 662 353
87.3 83.5 78.3
108 124 95
12.0 15.6 21.1
6 6 2
0.7 0.8 0.4
No. 1 1
Per cent
KO.
0.1 0.2
3017 932 553
average volume of a hepatic. cell is 10,600 cp (Table III), it can be calculated from the data of the above workers that the dry weight of a hepatic cell would lie between 1.03 and 2.58 x 10m6 mg; the dry weight value would be likely to be closer to 1.03 x 1O-6 mg (cf. the average value of 1.30 X 1O-6 mg observed by us) than to 2.58 x lo-” mg since the cytoplasm accounts for a major proportion of the volume of the hepatic cells. (4) The livers of 3-g-month-old animals contain 12-21 per cent bi-nucleated cells and less than 1 per cent tri- and tetra-nucleated cells (Table II). The presence of tri- and tetra-nucleated cells in liver does not appear to have been described earlier. The presence of bi-nucleated cells in liver is well known [12, 19, 401. (5) For every animal examined, the volume of 81-100 per cent of the hepatic cells was found to be between 5100 and 20,100 C,U (Table III). Usually, a small number of cells smaller than 5100 ct~ (up to 3420 cp) and larger than 20,100 C,U (up to 36,800 cp) were found. The average volume of a hepatic cell, however, varied from rat to rat within much narrower limits (8630-13,800 c,u; mean 10,600 k 1480 c,u). The precise volume of hepatic cells does not appear to have been determined earlier. (6) The total number of cells (of all kinds) in liver is ,5461040 x lo6 (mean 778 + 149 x 106) per g dry weight of liver, or 1266277 X lo6 (mean 197 k42 x 106) per g wet weight of liver, or 1030-1790 x lo6 (mean 1250 k Esperimenkd
Cell Research 40
8 ;: * %
?
G z
-
Sex
8 3 6 9 ? Q ? 8 6 d 5 CT CT o^ 6
6 11 10 7 17 20 13 14 24 15 9 3 2 18 19
Data
III.
Expt. no.
TABLE
3 3 3 3 3 9 6 12 9 12 Q 12 12 7 7
Age (months)
on animal
Variation
100 150 180 135 147 175 195 245 230 265 295 312 310 270 300
Wt. (g)
used
with
0.74 1.17 1.19 1.37 1.47 1.50 1.78 1.88 2.14 2.19 2.85 3.0’2 3.14 3.18 3.61
139 169 161 128 125 121 116 160 131 135 161 173 110 91 149
Total \ no. Dry wt. of cells of liver examined 69
liver weight
0.7 7.7 1.9 2.3 0.8 1.7 0 2.5 0 1.5 0.6 0 0 0 0.7
7.2 20.7 32.9 18.0 13.6 19.0 12.1 25.6 8.4 11.1 9.9 1.7 3.6 4.4 5.4
Percentage 3420 5100 to to 5100 7050 (c,u) (c/J)
in the relative
33.8 38.4 34.7 44.5 34.4 32.2 42.2 30.6 23.7 29.6 32.9 12.7 17.3 26.4 20.2
7050 to 9450 (CP)
of cells
38.1 18.4 19.9 25.0 25.6 28.2 27.6 20.6 40.1 34.0 31.7 41.0 26.4 30.7 32.9
17.3 10.6 9.3 9.4 18.4 13.2 17.2 13.7 19.3 16.4 19.9 34.2 18.1 25.3 25.5
12,400 to 16,000 (ccc)
falling
2.2 4.2 0.6 0.8 4.8 4.9 0.9 6.3 5.4 5.9 5.0 6.4 24.6 11.0 8.7
0 0 0.6 0 1.6 0 0 0.6 3.1 0.7 0 2.9 9.1 1.1 6.0
0.7 0 0 0 0.8 0.8 0 0 0 0.7 0 0.6 0.9 1.1 0
24,700 to 30,600 (w)
volume
20,100 to 24,700 (CP)
various
0 0 0 0 0 0 0 0 0 0 0 0.6 0 0 0.7
(
10,400 8,960 8,630 9,010 10,500 9,900 9,780 9,600 11,200 10,600 10,500 12,600 13,800 11,900 12,300
Average volume of a cell (cl4
volumes.
30,600 to 36,800 (cl4
ranges
cells of different
16,000 to 20,100 (w)
within
of rat hepatic
9450 to 12,400 (cl4
examined
proportion
Chemical
and cellular
composition
of rat liver
243
247 x 106) per liver (Table I). This number has been estimated earlier from the DNA content of liver tissue or of an average liver nucleus, both values being determined chemically [ 12, 16, 17, 431. Fakuda and Sibatani [16, 171 give the number of cells in liver as 1790 x lo6 for a rat whose liver weights 12 g, 1060 x lo6 for a rat whose liver weighs 5.7 g, and 668 X lo6 for a rat whose liver weighs 2.6 g. Williams [43] has reported values up to 4000 X lo6 cells per liver in adult rats. Thompson, Heagy, Hutchison and Davidson [41] reported that there is, on an average, 9.27 X lo-l2 g DNA per nucleus in rat liver; the total amount of DNA in livers of the same rats varied from 217-286 mg per 100 g liver. From these data it can be calculated that the number of nuclei in the liver of an adult animal, whose liver weighs 10 g would be 2340-3070 X 106; the number of cells in liver would be lower in view of the presence of bi-nucleated hepatic cells. In most of the animals (liver wet weight 3.8-11.5 g) examined by us, the total number of cells in liver was found to be less than 1500 x lo6 per liver. Thus, while some of the values for the total number of cells in adult rat liver observed by other workers fall within the range observed by us, other values are abnormally high; this may be partly due to no correction for polyploidy of hepatic cells having been applied in the earlier investigations, In any event, it is difficult to assess the validity of the values for total number of cells in liver observed by earlier workers for reasons which have already been stated by us [22]. (7) On an average, the hepatic cells appear to account for less than half (range 22.4-88.9 per cent; mean 39.2 i4.3 per cent) and the non-cellular material for more than half (range 43.2-74.1 per cent; mean 61.6 + 3.4 per cent) of the dry weight of liver; the non-hepatic cells in liver probably contribute less than 5 per cent (range 2.1-4.8 per cent; mean 2.9 k 0.3 per cent) towards the dry weight of liver. It must be emphasised that in the estimations reported here the amount of material which may have leaked out of the hepatic cells during the dispersion of the tissue to a suspension of single cells has not been taken into account. Therefore, the true values for the contribution of hepatic cells towards the dry weight of liver may be slightly higher than those stated above. ,4 large percentage of the constituents of the hepatic cells, say more than 20 per cent, is unlikely to have leaked out during the dispersion of the tissue since (i) the cells possess most of the normal metabolic functions of liver; thus, they can respire [23], synthesise protein [5] and RNA [26], can recognise their own kind both in vitro and in vivo [6], and contain glycogen [24]; (ii) the dry weight of hepatic cells obtained by us is close to the value calculated from the data of De, Chatterjee and Bose [l l] on the density of cytoplasm and nuclei of hepatic cells in tissue sections. However, Experimental
Cell
Research
40
244
P. T. Igpe, P. Ail. Bhargava TABLE
Correlation
IV. Significance of’ various correlations. No. of animalsa
between
Dry
wt. of liver
Average
Dry
wt. of liver
No. of hepatic
Dry
wt. of liver
Dry
Dry
wt. of liver
DNA
Dry
wt. of liver
Dry wt. of non-cellular liver
Dry
wt. of liver
Total
Dry
wt. of liver
‘Total no. of hepatic hcpatic cellsjliver
No. of hepatic
cells/liver cells/liver
and il. D. Tasker
volume
of hepatic
cells
cells/liver
wt. of hepatic of hepatic
cells/liver
cells/liver material/
DNAjliver and non-
Correlation coefficientb
Significance level (P value)
15
0.81
< 0.001
15
0.72
< 0.01
15
0.65
< 0.01
10
0.75
< 0.02
10
0.92
< 0.001
10
0.85
< 0.01
10
0.74
c 0.02 < 0.01
DNA
of hepatic
cells/liver
10
0.86
DNA
of hepatic
cells/liver
10
0.86
< 0.01
of non-
10
0.45
> 0.1
wt. of
15
- 0.67
i 0.01
dry
15
-- 0.53
< 0.03
Dry
wt. of hepatic
Dry
wt. of liver
DNA (or no. or dry hepatic cellsjliver
Dry
wt. of liver
No. of hepatic liver
Dry
wt. of liver
Dry
Dry
wt. of liver
wt.)
cells/g
wt. of hepatic of liver
dry
cells/g
wt.
10
- 0.65
< 0.05
10
pO.il
< 0.05
DNA (or no. or dry wt.j of nonhepatic cellsjg dry wt. of liver
10
- 0.05
> 0.1
DNA (or no. or dry wt.) of nonhepatic cells/g dry wt. of liver
10
- 0.46
> 0.1
cells/liver
DNA (or no. or dry hepatic cells/liver
10
0.08
> 0.1
Dry
wt. of IO6 hepatic
cells
19
0.11
> 0.1
No. of hepatic
cells/liver
Dry
wt. of 10’ hepatic
cells
15
- 0.32
10.1
No. of hepatic of liver
cells/g
dry
wt.
Dry
wt. of IO6 hepatic
cells
15
-.- 0.13
> 0.1
ISo. of hepalic of liver
cells/g
dry wt.
Dry
15
0.59
< 0.05
Total
DNA/g
dry
wt. of liver
No. of hepatic of liver
cells/g
dry wt.
Dry wt. of non-cellular dry wt. of liver
No. of hepatic of liver
cells/g
dry
Dry
wt. of liver
No. of hepatic Dry
wt.
wt.
of liver
Dry wt. of hepatic wt. of liver No. of hepatie of liver
cells/g
cells/g
Ezperimenfal
wt. of hepatic of liver
material/g
wt.)
of non-
cells/g
dry
wt.
dry
DNA of hepatic of liver
cells/g
dry
wt.
10
0.84
< 0.01
dry wt.
DNA of hepatic of liver
cells/g
dry wt.
10
0.72
< 0.02
Cell Research
40
Chemical
and cellular
composition
of rat liver
245
even if it is assumed that 25 per cent of the material of the hepatic cells leaks out into the non-hepatic-cell fraction during dispersion of the tissue, it can be calculated that the non-cellular material in liver will represent, on an average, nearly 50 per cent of the dry weight of the tissue. So far, it has not been recognised that in liver the amount of extracellular material may equal or exceed that of intracellular material on dry weight basis. (8) Liver contains 0.57-0.87 per cent (mean 0.73 iO.03 per cent) DNA per g dry weight, or 0.13-0.23 per cent (mean 0.18 ? 0.04 per cent) DNA per g wet weight, or 9-18 mg (mean 12.2 5 0.8 mg) DNA per liver. These values are within the range reported recently by Horvath [21]. The higher values (20.8-28.6 mg per liver) obtained by several earlier workers [16, 17, 38, 411 for adult rats were based on phosphorus estimations and may be in error due to the presence of non-DNA phosphorus in the DNA fraction obtained by the classical Schmidt and Thannhauser fractionation procedure. (9) Hepatic cells contain a little more than half (43-76 per cent; mean 58.6 F. 2.8 per cent), and the other cells a little less than half (25-57 per cent; mean 41.4-2.8 per cent) of the total DNA of liver. (10) The DNA content of the average hepatic (parenchymal) cell varies between 12.1 and 19.7 x lo-l2 g; the average values for 3-, 6-, 9- and 13month-old animals were found to be 13.1, 15.2, 15.5 and 19.7 x 10-12 g per cell. Since 3-, 6- and g-month-old animals contained 13, 18 and 23 per cent more hepatic nuclei than hepatic cells (Table II), and if it is assumed that 13-month-old animals contain 30 per cent more hepatic nuclei than cells [19]. the average DSA content per hepatic nucleus for 3-, 6-, 9- and 13-month-old animals can be calculated to be 11.6, 12.8, 13.1 and 15.2 x lo-l2 g. Earlier workers, using chemical estimation of DNA in isolated liver cell nuclei [2, 7, 8, 13, 14, 16-18, 31, 32, 411, or estimation of DNA in whole liver tissue and division of the value obtained by the number of nuclei deria Where the number of animals is given as 10, the data for animals no. 7, 8, 9, 11, 13, 16, 17, 20, 22 and 24 (Table I) was used for arriving at the correlation coefficient; when the number is given as 15, the data for animals no. 5, 6, 7, 8, 9, 10, 11, 12, 13, 16, 17, 18, 20, 22 and 24 (Table I) was used; and when the number is given as 19, the data for all the animals given in Table I was used. b Product moment correlation coefficient,
,,i-;” Y=
(x - 5) (y - ij) > sx * sy
where n is the number of animals; Sx and Sy are the standard deviations of the two variables and y, respectively; and Z and g are the means of the various values of x and y, respectively. ’ Correlation coefficients with P > 0.05 are considered not significant. Experimental
Cell Research
x
40
246
P. T. Iype, P. M. Bhargava
and A. D. Tasker
ved from the tissue [35, 361, have given values ranging from 5.9 to 15.4 x lo-l2 g DNA per nucleus. The lower values (in comparison to ours) obtained by several earlier workers may be due to their preparation of nuclei having contained nuclei from cells other than hepatic (parenchymal) cells, in contrast to the present investigation in which DNA was estimated in a preparation which consisted almost entirely of hepatic cells. The number of nuclei from the non-hepatic cells in a preparation of total nuclei derived from liver is likely to be about the same as the number of nuclei from hepatic cells. The nuclei from non-hepatic cells of liver would contain only the normal complement of DNA corresponding to diploid cells, in contrast to nuclei from hepatic cells which are known to be polyploid; as such the presence of nuclei from non-hepatic cells in appreciable numbers in a preparation of nuclei derived from whole liver would give a lower average DNA content per nucleus than would be obtained with a preparation consisting entirely of hepatic cells. In fact, the extent of polyploidy observed in the liver of adult rats [3, 4, 12, 29, 30, 33, 371 is so high as to make the values of DNA per average hepatic nucleus obtained in the present investigation more likely than those obtained in the earlier investigations, which probably gave the DNA content of an average nucleus derived from the various cells present in liver, rather than of an average hepatic (parenchymal) cell nucleus. DISCUSSION
The data presented in Tables I-III were statistically analysed primarily with a view to determine the pattern, if any, in the variation with the dry weight of liver, in (i) the number, dry weight and DNA of hepatic and nonhepatic cells; (ii) the dry weight of non-cellular material, and (iii) the volume of hepatic cells. The correlation coefficients which appeared to be of interest are given in Table IV. This analysis allows the following conclusions to be drawn: (a) The percentage recovery of hepatic cells obtained in the cell suspensions prepared by the method of Jacob and Bhargava [25] is independent of the age or the dry weight of liver (Table I). (b) The percentage of binucleated hepatic cells in liver rises with the age of the animal (Table II). (c) The percentage of hepatic cells with larger volume, and the average volume of cells, increase as the dry weight of liver increases (Tables III and IV). Seise and Stegmann [39] and Helweg-Larsen [20] reported that average nuclear and cellular volume increase in direct proportion to the logarithm of liver weight in mouse. This is also to be expected in view of the observed increase of the number of binucleated cells in liver with ageExperimental
Cell
Research
40
Chemical
and cellular
composition
247
of rat liver
(d) As the dry weight of liver increases, the following parameters also increase (Tables I and IV): (i) the number, and as would therefore be expected, the dry weight and the DNA of hepatic cells in the whole liver; (ii) the dry weight of non-cellular material in the whole liver; (iii) the total DNA of liver; and (iv) the total number of cells (hepatic plus non-hepatic) in liver. Again, as may be expected on the basis of the correlations under (i) above, there was a significant, positive correlation between the DNA of hepatic cells per liver, and either the number or the dry weight of hepatic cells per liver. However, no correlation was found between the dry weight of liver and the DNA (or number or dry weight) of non-hepatic cells per liver; the latter parameters seemed to vary little during liver growth from 1.2 to 2.9 g dry weight. These observations show that during growth of rats beyond the age of 3 months, there is a rise in hepatic and total cell number, the non-cellular material, and the hepatic cell and total tissue DNA, while there is no statistically significant rise in the number of non-hepatic cells. (e) As the liver weight increases, the number of hepatic cells (or the weight of these cells) per g of liver decreases Tables I and IV). This is also indicated by a significant negative correlation between the dry weight of liver and the total DNA per g dry weight of liver; in view of the observed lack of significant variation in the number of non-hepatic cells with increase in liver weight, the above negative correlation could be explained only if, with an increase in the dry weight of liver, there is a concurrent decrease in the number of hepatic cells, which decrease more than compensates for the increase in DNA of the average hepatic cell due to polyploidy. The decrease in the dry weight of hepatic cells per g liver observed with increasing dry weight of liver, would be expected to be accompanied either by a concurrent increase in the percentage of non-cellular material in liver, or/and by an increase in the number and weight of non-hepatic cells. The former possibility is strongly supported by a high significant negative correlation between the number of hepatic cells per g dry weight of liver and the dry weight of non-cellular material per g dry weight of liver, and no significant negative correlation between (i) the number of hepatic cells per g dry weight of liver and the number (or DNA or dry weight) of non-hepatic cells per g dry weight of liver; (ii) dry weight of liver and the number (or DNA or dry weight) of non-hepatic cells per g dry weight of liver; and (iii) the number of hepatic cells per liver and the number (or DNA or dry weight) of non-hepatic cells per liver. Thus, it would appear that as the already adult (3-month-old) animals get older, the percentage of non-cellular material in liver increases, and the percentage of the material derived from the parenchymal cells of Experimental
Cell
Research
40
248
P. T. Iype, P. M. Bhargava
and A. D. Tasker
liver decreases. Our observations in this respect are supported by the earlier report of Johnson and Albert [27] who found that in mouse, the number of nuclei progressively decreased from 281 + 10 x lo6 to 211 -t 11 x lo6 per g wet weight of liver, as the liver weight increased from 1.04 + 0.03 to 1.29 + 0.03 g. It would, therefore, appear that the cause of the reduction of various enzymes (e.g., those involved in the esterillcation of phosphorus or oxidation of butyrate; [42]) in liver with age (the enzyme activity being expressed as activity per unit weight of liver or per mg protein), observed by earlier investigators, may be due to a reduction in the number of hepatic cells per unit weight of liver and a corresponding increase in the non-cellular material which is unlikely to contain these enzymes. Our observations, therefore, lead us to endorse the view of Jones, Bitensky, Chayen and Cunningham [28] (who concluded that during chemical carcinogenesis of liver, the number of hepatic cells per unit weight of liver falls) that biochemical comparisons involving animals of different ages must take into consideration the “tissue dilution artifact” which may arise due to a reduction in the number of one kind of cell in the tissue and a concomitant increase in the number of other kinds of cells or of non-cellular material. (f) There is no correlation between the dry weight of hepatic cells (which varies from 0.75 to 2.42 x 1O-6 mg per cell) and either the dry weight of liver or the number of hepatic cells per liver or the number of hepatic cells per g dry weight of liver (Tables I and IV). In the case of 14 out of 19 animals, the dry weight varied between narrow limits (0.94-1.40 x 1OP mg per hepatic cell) and this variation appeared uncorrelated to any of the parameters studied. The average dry weight of adult hepatic cells in an animal would, therefore appear to vary randomly and little; this view is supported by a significant positive correlation between the number of hepatic cells per g dry weight of liver and the dry weight of hepatic cells per g dry weight of liver. This conclusion, together with the positive correlation between dry weight of liver and hepatic cell volume, would suggest a lowering of the average density of the hepatic cells with increase in liver weight. (g) As the dry weight of hepatic cells per g dry weight of liver (or the number of hepatic cells per g dry weight of liver, with which value the dry weight of hepatic cells per g dry weight of liver was directly correlated) increases, the DNA of hepatic cells per g dry weight of liver also increases (Tables I and IV), suggesting that larger cell size may be associated with larger DNA content.
Experimental
Cell
Research
40
Chemical
and cellular
composition
249
of rat liver
SUMMARY 1. Jacob and Bhargava’s method of preparing rat hepatic cell suspensions gives a recovery of 84-93 per cent (mean 89 k 0.8 per cent) of the hepatic parenchymal cells present initially in adult rat liver. 2. The liver of 3%14-month-old rats contains 196-469 X lo6 (mean 320 + 6 x 106) hepatic cells per g dry weight of liver, or 52-135 X lo6 (mean 81 + 28 x 10”) hepatic cells per g wet weight of liver, while the number of non-hepatic cells is 319-738 X lo6 (mean 450 k 41 X 106) per g dry weight of liver, or 75-197 x lo6 (mean 114 2 36 X 106) per g wet weight of liver. The hepatic cells represent 29-55 per cent (mean 40.1 k 2.7 per cent) and the non-hepatic cells 45-71 per cent (59.9 k 2.7 per cent) of the total cell population of liver. The number of hepatic cells per g dry weight of liver decreases with increase in dry weight of liver; no significant correlation was found between the dry weight of liver and the number of non-hepatic cells per g dry weight of liver. 3. The number of hepatic and non-hepatic cells in the whole liver is 310863 x lo6 (mean 514 + 117 X 106) and 478-1080 X 106 (mean 751 k 62 X 106), respectively. k. The total number of cells in liver is 546-1040 x lo6 (mean 778 1149 x 106) per g dry weight of liver, or 126-277 X lo6 (mean 197 k42 X 106) per g wet weight of liver, or 1030-1790 X 106 (mean 1250 k 247 X 106) per liver. 5. The dry weight of hepatic cells varies from 0.75-2.42 mg (mean 1.30 * 0.12 mg) per lo6 cells. 6’. On an average, the hepatic cells appear to be responsible for a little less than half, the non-hepatic cells for less than 5 per cent, and the noncellular material for about half the dry weight of liver. The percentage of non-cellular material in liver (on dry weight basis) increases with an increase in the dry Lveight of liver and a decrease in the number of hepatic cells per unit weight of liver. 7. The livers of 3-g-month-old animals contain 12-21 per cent binucleated hepatic cells and less than 1 per cent tri- and tetra-nucleated hepatic cells. The number of hinucleated cells increases with age. S. The volume of a hepatic cell usually varies between 5100 and 20,100 CPU;only a small fraction of the hepatic cells of liver have a volume which is outside this range. The average volume of hepatic cells from any animal varies between X630-12,300 cp (mean 10,600 k1480 cp) and increases with increase in the dry weight of liver. Y. Liver contains 0.57-0.87 per cent (mean 0.73 + 0.03 per cent) DNA 17 - G31818
Experimental
Cell Research
40
250
P. T. Iype, P. M. Bhargava
and A. Il. Tasker
per g dry weight of liver, or 0.13-0.23 per cent (mean 0.18 k 0.04 per cent) DNA per g wet weight of liver, or 9-18 mg (mean 12.2 10.8 mg) DNA per liver. 20. The hepatic cells in liver contain a little more than half (43-56 per cent; mean 58.6 2 2.8 per cent), and the other cells a little less than half (25-57 per cent; mean 41.4 1-2.8 per cent) of the total DNA of liver. 11. The DNA content of average hepatic cell varies between 12.1 and 19.7 X lo-l2 g, and of an average hepatic cell nucleus betlveen 11.6 and 15.2 x lo-l2 g. 12. The results are discussed vis-a-vis earlier observations in respect of the above-mentioned parameters of liver. This work was done during the tenure of a Senior Research Fellowship of the Council of Scientific and Industrial Research, New Delhi, awarded to one of us (P. T. I.), which is gratefully acknowledged. We are also grateful to Dr S. Husain Zaheer, Director-General, Council of Scientific and Industrial Research, New Delhi, and to Dr G. S. Sidhu, Director, Regional Research Laboratory, Hyderabad, for encouragement. We should also like to express our gratitude to Mrs Shanta Madhavan of Nutrition Research Laboratory, Hyderabad, for help with the statistical evaluation of data. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
ABERCROMBIE, M. and HARKNESS, R. D., Proc. Roy. Sot. B 138, 544 (1951). ALBERT, S., JOHNSON, R. M. and WAGSHAL, R. R., Science 117, 551 (1953). ALFERT, M. and GESCHWIND, I. I., Exptl Cell Res. 15, 230 (1958). BEATTY, R. A., Parthenogenesis and Polyploidy in Mammalian Development. Cambridge University Press, Cambridge, 1957. BHARGAVA, K. and BHARGAVA, P. M., Life Sciences p. 477 (1962). ~ Unpublished. CAMPBELL, R. M. and KOSTERLITZ, H. W., Science 115, 84 (1952). CUNNINGHAM, L., GRIFFIN, A. C. and LUCK, J. M., J. Gen. Pkysiol. 34, 59 (1950). DAOUST, R., in R. W. BRAUER (ed.), Liver Function, p. 3. American Institute of Biological Sciences, 1958. DAWSON. R. M. C.. ELLIOTT. D. C.. ELLIOTT. W. H. and JONES, K. M., Data for Biochemical Research, p.‘209. Oxfdrd University Press, London, 1959. DE, P., CHATTERJEE, R. and BOSE, A., Ezyptl Cell Res. 27, 168 (1962). DOLJANSKI, F., Int. Rev. Cytol. 10, 217 (1960). DOUNCE. A. L.. TISHKOFF. G. H.. BARNETT, S. R. and FREER, R. M., J. Gen. Pkysiol. 33,
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__
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32. 33. 34. 35. 36. 37. 38. 39. 40. 41.
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LEUCHTESBERGER, 33 (1951). NAORA, H., J. Biophys. Biochem. Cytol. 3, 949 (1957). POI’PER, H. and SCHAFFNER, F., Liver: Structure and Function, p. 7. McGraw-Hill, New York, 1957. PRICE, J. M. and LAIRD, A. K., Cancer Res. 10, 650 (1950). PRICE, J. M., MILLER, E. C., MILLER, J. A. and WEBER, G. M., Cancer Res. 10, 18 (1950). RIGLER, R., Exptl CeZZ Res. 28, 260 (1962). SCHMIDT, G. and THANNHAUSER, S. J., J. Biot. Chem. 161, 83 (1945). SIESS, U. and STEGMANN, H., Virchows Arch. Pathot. Anat. Physiot. 318, 534 (1950). ST. AUBIS, P. M. G. and BUCHER, N. L. R., Anat. Rec. 112, 797 (1952). THO~XSOX-, R. Y., HEAGY, F. C., HUTCHISON, W. C. and DAVIDSOX, J. N., Biochem. J. 53, 460 (1953). WEISBACH, E. C. and GARBUS, J., Nafure 178, 1225 (1956). \\‘ILLIAMS, .J. N., J. Nutrition 73, 199 (1961).
Experimental
Cell Research
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