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An investigation of myocardial metabolism by means of isotopes
CLINIC4 CHIMICAACTA
AN INVESTIGATION
OF MYOCARDIAL
333
METABOLISM
BY MEANS
OF ISOTOPES 6*Co-VITAMIN
B,-RETENTION MYO~ARDI~~~, THE EFFECT
Medical Departnae?it
ofthe
CAPACITY
OF NORMAL
AND
INJURED
AND ITS SIGNIFICANCE
OF A HEART
MUSCLE EXTRACT
B JASiNSKI Cantonal
Hospital
Winterthuv,
Zurich
(Switzevland)
(Received September Sth, 1959)
Carbon tetrachloride (Ccl,) IS . principally a liver poison; concomitant damage to other organs, such as the kidneys or heart is hardly mentioned in the literature. The mode of action of CCI, has not yet been clarified. The assumption by GLYNN AND HIMSWORTH~,that the fatty degeneration of liver cells is the result of a lack of oxygen, owing to swelling of the cells and subsequent constriction of vascular capillaries, has not been accepted. Moreover, no explanation has been given for the phenomenon that Ccl, damage to the liver is first seen in the middle and central lobules, no matter by what means, oral or parenteral, the poison is given to the animal. It is probable that the difference in susceptibility of the central and peripheral cells of the liver lobule is due to their difference in function. In this connection, it is interesting that another liver poison, ally1 alcohol, principally affects the periphery of the lobule. It is beyond doubt that an underlying disturbance of the fermentation mechanism is involved in this liver damage. EGER z quite rightly therefore speaks of reversible disturbances of the enzyme system in fatty degeneration in contrast to irreversible disturbances due to cell necrosis. These considerations can be applied to damage to any kind of cell. Thus Ccl, causes damage resulting in severe metabolic disturbances in the organs; an attempt was made to investigate these by various methods, chemical, histochemical etc. Isotopes have brought about a considerable extension of fundamentai investigation. Isotopic and chemical techniques make it possible to follow more accurately the fate of well-defined substances in the entire metabolism, so that important qualitative and quantitative conclusions may be reached about normal and pathological metabolic processes. The isotope technique is particularly important when investigation of the metabolism of an organ is specially difficult; this is undoubtedly the case with the heart. The present in~~estigation is based on the fact, established by means of autoradiography, that the myocardium in comparison with skeletal muscle, retains considerably more radioactive Vitamin B,, (WACO) introduced either parenterally or orallv3’ r 4 It does this to such a degree that with an exposure time of three months and using a heart section 3 mm thick, it produces an obvious darkening on the film negative. This does not happen with a skeletal muscle section+6. Our findings are in complete agreement with results of microbiological determinations of the Vitamin B,, content of the organs concerned. Thus TASTALDI? found in the m~ocardium of an ox 17.6 pug/roe g of fresh tissue, and only 0.25 ,ug,/roog in
NE’l‘H(
1
ml of solution.
)i
1
MYOCARDIAL
METABOLISM
335
cause Ccl, primarily affects the liver parenchyma and damages the cells to the degree of necrosis. Those animals treated with Recosen show, in comparison with the controls, a distinct rise in the radioactivity of the myocardium, whilst the radioactivity of the liver of these animals remains practically unchanged. It now remains to be seen what these results actually mean. It is an established fact that the Vitamin B,,-retaining capacity of the cells is in direct proportion to their functional efficiency. A damaged cell not only loses its existing Vitamin I3l’L,as for example in infectious hepatitis*slO, or in cholemic hepatic necrosis I1, but it also loses its abilitv to retain anyVitamin R,, T;113IX I RESULTS
OF THE
hlEASUREMl3NTS __.
OF RADIOICTIVITY -.__ -___-
Weight of animal -.. ^__.__ iMiCZUZ fil?CZl
.4 vimiil
To.
Guoltp
9s 92 90
“33 238 2.39 2.+0
I06
_..~__ 99
.\verage
I:
Weight hEart ~
rect~ivcd
150 155 150 '99
-.
163.5
GYOU~ 2 : received
tied
G°Co-Titamin
‘4.5
106 92
140 I 1i
Average
100. j
135
422.7
130 125 131
421 364 388
102
155
43s
116 125 I25
358 399 402 394
130.6
-Ior
100
Average
84 93 __~_______ 95.7
138
___----____
B,,
7s 67 53
j.JO O.hj 5.95 8.15 b.612
109 IS8 s i: __ “0.7
8.35 8.85
Go Or
8.80
39
80
6.rg
46
69.5
8.02
rSI.5
io.G
_
___
82 95 j’ 99 98 III
7,55 7.30
80
6.85
8.‘$0
S.Oj 5~90 6.47 6.25 __-.__ 7.08
83 -___ 90 SC
_ -_
Liver comts/g
B,, --+ CCI, + Recosen
94 105 98 137
253
To-Vitamin
of
ZiVPY -~
LIVERS
B,, + Ccl,
s -=
246 247 248 ‘49 250 251 2.52
Weight
counts/g
421 137 6zp 129 106 a-F7 660 I-17 -~----_.~___-._...~ 1’9.7 533.6
I60
GYOU~ 3: received
HEhRTS AXD ---______
_II._...
110
73.5
of
only Tb-Vitamin
399 484 406 342
94
24’ “43 244
OF THE
_~
50 54 49 62 68 63 61
63 58.7
9 --.
..~~~__
supplied to it. This rule does not apply exclusively to liver cells ; it applies to any type of cell in the organism and hence also to the myofibrils. It may therefore be assumed that the reduction in Vitamin B,, uptake is the result of injury to the cell. As long as the details of the mode of action of Ccl, are unknown, it is scarcely possible to be precise about the way in which Ccl, causes damage to heart muscle. Nevertheless, it seems possible from the present knowledge of the role of Vitamin B,, in general metabolism, to derive an opinion as to the character and focus of CCl, action. It is known that Vitamin B,, amongst other substances, plays an important part in the metabolism of cell nuclei. Together with folic acid, it acts as a catalyst for the anabolism of energy-giving substances like uracil, cytosin, thymin, thymidin, pyrimidin, i.e., early stages in the formation of nucleotides and nucleic acids12T13. For this it is CliFZ. Clmli.A&,
j (X900)333-338
the nuclear suhstancrs in particular. At. the sxne time there oc~curs that c-hangs: in mitosis which is characteristic of t’itamin IS,, deficiency, culminating in polypioidosis and polynucleosis of the cells. Other well known consequences of \Itamin R,, deficirq~ are glossitis, atroph). of the gastric muco5a with achylia, funicular myelitis, \kal disturbances, psychological changes and pathological EC& l-1-16. In severr cases of pernicious anaemia, ECG changes are not infrequently seen, in the form of flattened, almost isoelectric T WR\W which arc independent of the degrw of anaemia (Fig. 2). The!- disappear rapidly- under specific ther;lp>-, ix., f’\.en hefow :I rkic in the haemoglobin or the appearanw of a reticulot>%osis, and are thereforc~ direct iildications of the disturbances in metabolism of thr myocardium caused hy Vitamin I<,, dcficienc!,‘7.
337
MYOCARDIAL METABOLISM
In order to examine further the problem of decrease of Vitamin B,, retention in a myocardium damaged by CC&, it is necessary to investigate in which cell components Vitamin B,, exists as a catalyst. By separating the nucleus and mitochondria from the cytoplasm by means of an ultracentrifuge, it has been shown that Vitamin R,, is mainly found in the structural parts of the cell (more than 60%) 18. These are also the sites of the enzyme systems which play an important part in cell metabolism. A disturbance of the enzyme system induced by a cell poison results in a decrease in the Vitamin B,,retention capacity of the cell. This occurs with great regularity in the case Hb
: 7.7 g%
28.3.1957
Hb: 7.7 s% 4+1957
Fig. 2. O.E., born rgo4 C.h. 4721 Ig.jF. Severe pernicious anaemia with pathological ECG. After a total of Go pg Vitamin B,, (4 x 15 ,ug daily) the ECG returned to normal while the Hb remained constant (4+rgj7).
of damage due to Ccl,. If heart muscle extract can increase Vitamin B,,-retention capacity in the myocardium of animals poisoned with CCL,, it may be deduced that Recosen either protects the enzyme systems of the cells from the toxic action of Ccl,, or promotes their regeneration. In both cases definite metabolic processes are concerned at the level of the source of energy i.e. the enzyme system. The results of the investigation are highly significant. Statistic evaluation gave P < 0.01. This means that the heart muscle extract, Recosen, contains active substances which act directly on the metabolic disturbance in the myocardium, apparently via the enzymes. These findings provide a further experimental contribution to the understanding of the favourable therapeutic experiences with this substance. We also believe that these investigations indicate new possibilities for studying the extremely complicated metabolism of the heart and other organs.
The Vitamin B,,-retention capacity of the myocardiunl rapidly diminishes in female rats chronically poisoned with Ccl,. 2. Animals receiving simultaneously the heart muscle extract Recosen, show a significantly higher (P < 0.01) Ti’itamin B,, retention of the m~ocardium. I.
AN INVESTIGATION
OF MYOCARDIAL
333
METABOLISM
BY MEANS
OF ISOTOPES 6*Co-VITAMIN
B,-RETENTION MYO~ARDI~~~, THE EFFECT
Medical Departnae?it
ofthe
CAPACITY
OF NORMAL
AND
INJURED
AND ITS SIGNIFICANCE
OF A HEART
MUSCLE EXTRACT
B JASiNSKI Cantonal
Hospital
Winterthuv,
Zurich
(Switzevland)
(Received September Sth, 1959)
Carbon tetrachloride (Ccl,) IS . principally a liver poison; concomitant damage to other organs, such as the kidneys or heart is hardly mentioned in the literature. The mode of action of CCI, has not yet been clarified. The assumption by GLYNN AND HIMSWORTH~,that the fatty degeneration of liver cells is the result of a lack of oxygen, owing to swelling of the cells and subsequent constriction of vascular capillaries, has not been accepted. Moreover, no explanation has been given for the phenomenon that Ccl, damage to the liver is first seen in the middle and central lobules, no matter by what means, oral or parenteral, the poison is given to the animal. It is probable that the difference in susceptibility of the central and peripheral cells of the liver lobule is due to their difference in function. In this connection, it is interesting that another liver poison, ally1 alcohol, principally affects the periphery of the lobule. It is beyond doubt that an underlying disturbance of the fermentation mechanism is involved in this liver damage. EGER z quite rightly therefore speaks of reversible disturbances of the enzyme system in fatty degeneration in contrast to irreversible disturbances due to cell necrosis. These considerations can be applied to damage to any kind of cell. Thus Ccl, causes damage resulting in severe metabolic disturbances in the organs; an attempt was made to investigate these by various methods, chemical, histochemical etc. Isotopes have brought about a considerable extension of fundamentai investigation. Isotopic and chemical techniques make it possible to follow more accurately the fate of well-defined substances in the entire metabolism, so that important qualitative and quantitative conclusions may be reached about normal and pathological metabolic processes. The isotope technique is particularly important when investigation of the metabolism of an organ is specially difficult; this is undoubtedly the case with the heart. The present in~~estigation is based on the fact, established by means of autoradiography, that the myocardium in comparison with skeletal muscle, retains considerably more radioactive Vitamin B,, (WACO) introduced either parenterally or orallv3’ r 4 It does this to such a degree that with an exposure time of three months and using a heart section 3 mm thick, it produces an obvious darkening on the film negative. This does not happen with a skeletal muscle section+6. Our findings are in complete agreement with results of microbiological determinations of the Vitamin B,, content of the organs concerned. Thus TASTALDI? found in the m~ocardium of an ox 17.6 pug/roe g of fresh tissue, and only 0.25 ,ug,/roog in
NE’l‘H(
1
ml of solution.
)i
1
MYOCARDIAL
METABOLISM
335
cause Ccl, primarily affects the liver parenchyma and damages the cells to the degree of necrosis. Those animals treated with Recosen show, in comparison with the controls, a distinct rise in the radioactivity of the myocardium, whilst the radioactivity of the liver of these animals remains practically unchanged. It now remains to be seen what these results actually mean. It is an established fact that the Vitamin B,,-retaining capacity of the cells is in direct proportion to their functional efficiency. A damaged cell not only loses its existing Vitamin I3l’L,as for example in infectious hepatitis*slO, or in cholemic hepatic necrosis I1, but it also loses its abilitv to retain anyVitamin R,, T;113IX I RESULTS
OF THE
hlEASUREMl3NTS __.
OF RADIOICTIVITY -.__ -___-
Weight of animal -.. ^__.__ iMiCZUZ fil?CZl
.4 vimiil
To.
Guoltp
9s 92 90
“33 238 2.39 2.+0
I06
_..~__ 99
.\verage
I:
Weight hEart ~
rect~ivcd
150 155 150 '99
-.
163.5
GYOU~ 2 : received
tied
G°Co-Titamin
‘4.5
106 92
140 I 1i
Average
100. j
135
422.7
130 125 131
421 364 388
102
155
43s
116 125 I25
358 399 402 394
130.6
-Ior
100
Average
84 93 __~_______ 95.7
138
___----____
B,,
7s 67 53
j.JO O.hj 5.95 8.15 b.612
109 IS8 s i: __ “0.7
8.35 8.85
Go Or
8.80
39
80
6.rg
46
69.5
8.02
rSI.5
io.G
_
___
82 95 j’ 99 98 III
7,55 7.30
80
6.85
8.‘$0
S.Oj 5~90 6.47 6.25 __-.__ 7.08
83 -___ 90 SC
_ -_
Liver comts/g
B,, --+ CCI, + Recosen
94 105 98 137
253
To-Vitamin
of
ZiVPY -~
LIVERS
B,, + Ccl,
s -=
246 247 248 ‘49 250 251 2.52
Weight
counts/g
421 137 6zp 129 106 a-F7 660 I-17 -~----_.~___-._...~ 1’9.7 533.6
I60
GYOU~ 3: received
HEhRTS AXD ---______
_II._...
110
73.5
of
only Tb-Vitamin
399 484 406 342
94
24’ “43 244
OF THE
_~
50 54 49 62 68 63 61
63 58.7
9 --.
..~~~__
supplied to it. This rule does not apply exclusively to liver cells ; it applies to any type of cell in the organism and hence also to the myofibrils. It may therefore be assumed that the reduction in Vitamin B,, uptake is the result of injury to the cell. As long as the details of the mode of action of Ccl, are unknown, it is scarcely possible to be precise about the way in which Ccl, causes damage to heart muscle. Nevertheless, it seems possible from the present knowledge of the role of Vitamin B,, in general metabolism, to derive an opinion as to the character and focus of CCl, action. It is known that Vitamin B,, amongst other substances, plays an important part in the metabolism of cell nuclei. Together with folic acid, it acts as a catalyst for the anabolism of energy-giving substances like uracil, cytosin, thymin, thymidin, pyrimidin, i.e., early stages in the formation of nucleotides and nucleic acids12T13. For this it is CliFZ. Clmli.A&,
j (X900)333-338
the nuclear suhstancrs in particular. At. the sxne time there oc~curs that c-hangs: in mitosis which is characteristic of t’itamin IS,, deficiency, culminating in polypioidosis and polynucleosis of the cells. Other well known consequences of \Itamin R,, deficirq~ are glossitis, atroph). of the gastric muco5a with achylia, funicular myelitis, \kal disturbances, psychological changes and pathological EC& l-1-16. In severr cases of pernicious anaemia, ECG changes are not infrequently seen, in the form of flattened, almost isoelectric T WR\W which arc independent of the degrw of anaemia (Fig. 2). The!- disappear rapidly- under specific ther;lp>-, ix., f’\.en hefow :I rkic in the haemoglobin or the appearanw of a reticulot>%osis, and are thereforc~ direct iildications of the disturbances in metabolism of thr myocardium caused hy Vitamin I<,, dcficienc!,‘7.
337
MYOCARDIAL METABOLISM
In order to examine further the problem of decrease of Vitamin B,, retention in a myocardium damaged by CC&, it is necessary to investigate in which cell components Vitamin B,, exists as a catalyst. By separating the nucleus and mitochondria from the cytoplasm by means of an ultracentrifuge, it has been shown that Vitamin R,, is mainly found in the structural parts of the cell (more than 60%) 18. These are also the sites of the enzyme systems which play an important part in cell metabolism. A disturbance of the enzyme system induced by a cell poison results in a decrease in the Vitamin B,,retention capacity of the cell. This occurs with great regularity in the case Hb
: 7.7 g%
28.3.1957
Hb: 7.7 s% 4+1957
Fig. 2. O.E., born rgo4 C.h. 4721 Ig.jF. Severe pernicious anaemia with pathological ECG. After a total of Go pg Vitamin B,, (4 x 15 ,ug daily) the ECG returned to normal while the Hb remained constant (4+rgj7).
of damage due to Ccl,. If heart muscle extract can increase Vitamin B,,-retention capacity in the myocardium of animals poisoned with CCL,, it may be deduced that Recosen either protects the enzyme systems of the cells from the toxic action of Ccl,, or promotes their regeneration. In both cases definite metabolic processes are concerned at the level of the source of energy i.e. the enzyme system. The results of the investigation are highly significant. Statistic evaluation gave P < 0.01. This means that the heart muscle extract, Recosen, contains active substances which act directly on the metabolic disturbance in the myocardium, apparently via the enzymes. These findings provide a further experimental contribution to the understanding of the favourable therapeutic experiences with this substance. We also believe that these investigations indicate new possibilities for studying the extremely complicated metabolism of the heart and other organs.
The Vitamin B,,-retention capacity of the myocardiunl rapidly diminishes in female rats chronically poisoned with Ccl,. 2. Animals receiving simultaneously the heart muscle extract Recosen, show a significantly higher (P < 0.01) Ti’itamin B,, retention of the m~ocardium. I.