- Email: [email protected]
The effect of hydrazines and hydroxylamine on the levels of histidine, carnosine, and anserine in rat muscle
Vol . 8 ~ part I, pp . 953-967 1969 . ~e~S~~nc~s ri teat Britain.
Pergamon Press
THE EFFECT OF HYDRAZINES AND HYDROXYLAMINE ON THE LEVELS OF HISTIDINE, CARNOSINE, AND ANSÉRINE IN RAT MUSCLE Finley D . Marshall, Jr . and William C . Yockey Biochemistry Department, School of Medicine The University of South Dakota, Vermillion, South Dakota (Received 5 May 1969 ; in final form 20 June 1969) THE function of the dipeptides carnosine and anserine remains unknown .
The discovery of homocarnosine (1) and carnosine (2) in
brain has stimulated an investigation into the nature of these Recently Marshall and Yockey (3)
dipeptides in excitable tissue .
found that certain drugs will raise the levels of histidine in rat brain and lower the levels of homocarnosine ; while the levels of carnosine did not seem to be significantly altered .
Since some of
the compounds tested were convulsant hydraziäes, the possible alteration in levels of muscle dipeptides was investigated .
The results
of these experiments are reported here . Materials and Methods Albino rats (Holtzman),weighing 350-400 8 . were used in these experiments .
Drugs and chemicals were neutralized if necessary and
injected i .p . in the following amounts :
hydrazine (Hy)
~5 mg/kg ;
(Eastman), 10~ mg/kg ;
],, ],-dimethylhydrazine
X,2-dimethylhydrazine (SDMH)
(UDMH)
(Eastman),
(Aldrich Chemical Co .), 5~~ mg/kg ;
phenylisopropyl hydrazine (PIH) (donated by Lakeside Labs, J . B . 516), 20 m8/kg ; hydroxylamine (HA)
(Eastman), 3U mg/kg .
The amounts
of hydrazines injected were those suggested by Uchida and O'Brien (4) .
The general procedure for the isolation of histidine, carno-
sine and anserine was that reported by Abraham, Pisano, and Udenfriend (2) .
1 ],/2 hr . after injection, the animals were killed by
decapitation and the right gastrocnenious muscle rewoved aad 953
954
DIPEPTiDES IN RAT MUSCLE
Vol . 8 , No.17
immediately frozen until used . The muscle tissue, 2 gm, was homogenized in y ml of cold water for 1,5 minutes in a micro blaring blendor .
The vessel was washed
with 1 ml of water and then 9 ml of absolute ethanol . washings were added to the homogenate .
Both
The combined homogenates
and washings were heated for l~,minutes in a 9~ ° C . water bath, cooled and then were centrifuged at I2,~~0 x (g) for 10 minutes . The supernatant fluid was extracted with an equal volume of a ]. ;1 mixture of isoamyl alcohol-hexane to dissolve lipoidal material .
A volume of water was added which equaled one-fifth
the combined preceding volumes, and the lower aqueous phase was recovered after repeated mixing . The extracted sample was then placed on a Dowex S~-X-y (20~-y~~ mesh) column which had been saturated with Z,ô - lutidine (5),
The column was approximately ]5 cm x ], cm .
The column was
eluted with ~~], M Z,Ô- lutidine and collected in 5-ml fractions . An aliquot of each fraction was assayed by the ninhydrin method or by reaction with Fast Red Salt TRN (donated by General Analine and Film Corp .), or by both methods .
Carnosine and anserine are
usually found together in vertebrate skeletal muscle and are quite difficult to separate into uncontaminated samples by ordinary column chromatography .
Consequently, in order to determine the
amounts of these substances in muscle the recent method of Parker (Ô) was used Yo assay the amount of Carnosine in the presence of anserine and the amount of anserine with Carnosine present . The combined yellow color of the 2,y-dinitrophenyl derivatives of Carnosine and anserine in the purified muscle extract was measured in a Beckman Model DBG-2 spectrophotometer at yap mu .
The
reaction of Carnosine with diazotized p-bromoaniline to give a red
color is used for the quantitative determination of Carnosine .
955
DIPEPTTDES IN RAT MUSCLE
Vol. 8 , No . 17
The optical density of the color was measured at 500 mu in the Beckman Model DBG-2 spectrophotometer .
The carnosine values
determined were then subtracted from the total carnosine-anserine value to obtain the anserine levels . Results The results indicate that anserine and carnosine were significantly lowered in every case except for the non-toxic SDMH .
The
exception of carnosine after treatment with UDMH may be due to the small number of samples .
The effects on histidine are variable,
with UDMH the levels are raised and with HA the levels are lowered . The results of the experiments are shown in Table I .
TABLE I Change in Rat Muscle Constituents after the Intraperitoneal Injection pf Drugs (a) Treatment Control Hy (Hydrazine)
Histidine
(~)± ( )
Carnosine
4
(~~ ±
Anserine
23
(~~ ±
11
78
UDMH
(1,1 - Dimethyl
49
Hydrazine)
SDMH
(1,2 - Dimethyl Hydrazine)
PIH (PhenylisopropylHydrazine)
~~)± 6
+
(~)-
23
9
~~~ ± *
24
+ 2~
amine (a)
42
Results are expressed as umoles/100 g of muscle . Numbers in parentheses are numbers of samples . " P < Q,~ by student's "~" test . ** P < ,UL by student's "t" test .
(~~ ±
110
38 9 _+ S . E . M.
95 6
DIPEPTIDES IN RAT MUSCLE
Vol. 8 , No . 17
Discussion Two possible mechanisms have been proposed for the toxic action of the hydrazines .
One is the interference with vitamin
B6 and enzymatic reactions which require B6, particularly glutamic decarboxylase (],9) .
Another mechanism is the interference with
glucose metabolism and a loss in energy metabolism (10) .
Since
the formation of carnosine and anserine in muscle does not seem to require B6 (11 . 12), another mechanism must be involved in the lowering of these dipegtides in tissues .
Hydrazine could prevent
synthesis by interfering directly in peptide bond formation or indirectly by causing a loss in energy metabolism (13) .
It has
been reported that carnosine and anserine will not leak out of an intact muscle, but there is excessive loss when mechanically damaged (15) .
Roberts et al .
(],6) suggested that some effects of
hydrazine could be explained if it were assumed that the primary action was on a membrane component .
Recently it has also been
reported that hydrazine may damage mitochondrial membranes as part of its toxic action (17) .
These findings along with the
results reported here suggest that in addition to effects on specific enzymes the hydrazines and hydroxylamine may have a more general effect by altering membranes . Acknowledgement The authors would like to acknowledge the support of this work by usrH grant rrB-06137, References J . J . Pisano, J . D . Wilson, L . Cohen, D . Abraham, and S . Udenfriend, J . Biol . Chem . ~, 499 (1961), 2.
D . Abraham, J . J . Pîsano, and S . Udenfriend, Arch . Biochem . Biophys .
~., 210 (1962),
Vol. 8 , No . 17
3,
DIPEPTiDES IN RAT MUSCLE
957
F . D . Marshall, Jr . and W . C . Yockey, Biochem . Pharmacol . ~, 640 (1968),
4,
T . Uchida and R . D . O'Brien, Biochem . Pharmacol . ~, 725 (1964) .
5,
R . I . McManus, J . Biol . Chem . ~, 1398 (1960) .
6,
~ . J . Parker, Jr ., Anal . them . ~, 1359 (1966) .
7,
E . W . Maynert and H . K . Kaji, J . Pharmacol . ex~. Ther . ]~, 114 (1962),
$,
M . A . Medina, H . D . Braymer, and J . L . Reeves, J . Neurochem . 301 (1962),
9,
E . Roberts, "Inhibition in the Nervous System and Gamma-Aminobutyric Acid", Pergamon Press, New York (1960) .
10,
F . P . Underhill, J . Biol . Chem . ],Q, 159 (1966),
11 .
R . E . Winnick and T . E . Winnick, Bull . Soc . Chim . Biol . ~, 1727 (1954),
12,Kalyankar, G . D ., Meister, A ., J . Biol . Chem . ~., 3210 (1959), 13,
S . R . Fortney, D . A . Clark, and E . Stein, J . Pharmac . exp. Ther . 1~, 277 . (1967) ,
14,
A . Pletscher, P . A . Shore, and B . B . Brodie, J . Pharmac .
-p. ex
Ther . ],],~,, $4 (1956) 15 .
R . E . Winnick, S . Moikeha, and T . Winnick, J . Biol . Chem . ~, 3645 (1963),
16,
E . Roberts, D . G . 5imonsen, and E . Roberts, Biochem . Pharmacol . 12. 1445 (1963),
17,
s . H . Lee, Fed . Proc . ~, 423 (1969) .
Pergamon Press
THE EFFECT OF HYDRAZINES AND HYDROXYLAMINE ON THE LEVELS OF HISTIDINE, CARNOSINE, AND ANSÉRINE IN RAT MUSCLE Finley D . Marshall, Jr . and William C . Yockey Biochemistry Department, School of Medicine The University of South Dakota, Vermillion, South Dakota (Received 5 May 1969 ; in final form 20 June 1969) THE function of the dipeptides carnosine and anserine remains unknown .
The discovery of homocarnosine (1) and carnosine (2) in
brain has stimulated an investigation into the nature of these Recently Marshall and Yockey (3)
dipeptides in excitable tissue .
found that certain drugs will raise the levels of histidine in rat brain and lower the levels of homocarnosine ; while the levels of carnosine did not seem to be significantly altered .
Since some of
the compounds tested were convulsant hydraziäes, the possible alteration in levels of muscle dipeptides was investigated .
The results
of these experiments are reported here . Materials and Methods Albino rats (Holtzman),weighing 350-400 8 . were used in these experiments .
Drugs and chemicals were neutralized if necessary and
injected i .p . in the following amounts :
hydrazine (Hy)
~5 mg/kg ;
(Eastman), 10~ mg/kg ;
],, ],-dimethylhydrazine
X,2-dimethylhydrazine (SDMH)
(UDMH)
(Eastman),
(Aldrich Chemical Co .), 5~~ mg/kg ;
phenylisopropyl hydrazine (PIH) (donated by Lakeside Labs, J . B . 516), 20 m8/kg ; hydroxylamine (HA)
(Eastman), 3U mg/kg .
The amounts
of hydrazines injected were those suggested by Uchida and O'Brien (4) .
The general procedure for the isolation of histidine, carno-
sine and anserine was that reported by Abraham, Pisano, and Udenfriend (2) .
1 ],/2 hr . after injection, the animals were killed by
decapitation and the right gastrocnenious muscle rewoved aad 953
954
DIPEPTiDES IN RAT MUSCLE
Vol . 8 , No.17
immediately frozen until used . The muscle tissue, 2 gm, was homogenized in y ml of cold water for 1,5 minutes in a micro blaring blendor .
The vessel was washed
with 1 ml of water and then 9 ml of absolute ethanol . washings were added to the homogenate .
Both
The combined homogenates
and washings were heated for l~,minutes in a 9~ ° C . water bath, cooled and then were centrifuged at I2,~~0 x (g) for 10 minutes . The supernatant fluid was extracted with an equal volume of a ]. ;1 mixture of isoamyl alcohol-hexane to dissolve lipoidal material .
A volume of water was added which equaled one-fifth
the combined preceding volumes, and the lower aqueous phase was recovered after repeated mixing . The extracted sample was then placed on a Dowex S~-X-y (20~-y~~ mesh) column which had been saturated with Z,ô - lutidine (5),
The column was approximately ]5 cm x ], cm .
The column was
eluted with ~~], M Z,Ô- lutidine and collected in 5-ml fractions . An aliquot of each fraction was assayed by the ninhydrin method or by reaction with Fast Red Salt TRN (donated by General Analine and Film Corp .), or by both methods .
Carnosine and anserine are
usually found together in vertebrate skeletal muscle and are quite difficult to separate into uncontaminated samples by ordinary column chromatography .
Consequently, in order to determine the
amounts of these substances in muscle the recent method of Parker (Ô) was used Yo assay the amount of Carnosine in the presence of anserine and the amount of anserine with Carnosine present . The combined yellow color of the 2,y-dinitrophenyl derivatives of Carnosine and anserine in the purified muscle extract was measured in a Beckman Model DBG-2 spectrophotometer at yap mu .
The
reaction of Carnosine with diazotized p-bromoaniline to give a red
color is used for the quantitative determination of Carnosine .
955
DIPEPTTDES IN RAT MUSCLE
Vol. 8 , No . 17
The optical density of the color was measured at 500 mu in the Beckman Model DBG-2 spectrophotometer .
The carnosine values
determined were then subtracted from the total carnosine-anserine value to obtain the anserine levels . Results The results indicate that anserine and carnosine were significantly lowered in every case except for the non-toxic SDMH .
The
exception of carnosine after treatment with UDMH may be due to the small number of samples .
The effects on histidine are variable,
with UDMH the levels are raised and with HA the levels are lowered . The results of the experiments are shown in Table I .
TABLE I Change in Rat Muscle Constituents after the Intraperitoneal Injection pf Drugs (a) Treatment Control Hy (Hydrazine)
Histidine
(~)± ( )
Carnosine
4
(~~ ±
Anserine
23
(~~ ±
11
78
UDMH
(1,1 - Dimethyl
49
Hydrazine)
SDMH
(1,2 - Dimethyl Hydrazine)
PIH (PhenylisopropylHydrazine)
~~)± 6
+
(~)-
23
9
~~~ ± *
24
+ 2~
amine (a)
42
Results are expressed as umoles/100 g of muscle . Numbers in parentheses are numbers of samples . " P < Q,~ by student's "~" test . ** P < ,UL by student's "t" test .
(~~ ±
110
38 9 _+ S . E . M.
95 6
DIPEPTIDES IN RAT MUSCLE
Vol. 8 , No . 17
Discussion Two possible mechanisms have been proposed for the toxic action of the hydrazines .
One is the interference with vitamin
B6 and enzymatic reactions which require B6, particularly glutamic decarboxylase (],9) .
Another mechanism is the interference with
glucose metabolism and a loss in energy metabolism (10) .
Since
the formation of carnosine and anserine in muscle does not seem to require B6 (11 . 12), another mechanism must be involved in the lowering of these dipegtides in tissues .
Hydrazine could prevent
synthesis by interfering directly in peptide bond formation or indirectly by causing a loss in energy metabolism (13) .
It has
been reported that carnosine and anserine will not leak out of an intact muscle, but there is excessive loss when mechanically damaged (15) .
Roberts et al .
(],6) suggested that some effects of
hydrazine could be explained if it were assumed that the primary action was on a membrane component .
Recently it has also been
reported that hydrazine may damage mitochondrial membranes as part of its toxic action (17) .
These findings along with the
results reported here suggest that in addition to effects on specific enzymes the hydrazines and hydroxylamine may have a more general effect by altering membranes . Acknowledgement The authors would like to acknowledge the support of this work by usrH grant rrB-06137, References J . J . Pisano, J . D . Wilson, L . Cohen, D . Abraham, and S . Udenfriend, J . Biol . Chem . ~, 499 (1961), 2.
D . Abraham, J . J . Pîsano, and S . Udenfriend, Arch . Biochem . Biophys .
~., 210 (1962),
Vol. 8 , No . 17
3,
DIPEPTiDES IN RAT MUSCLE
957
F . D . Marshall, Jr . and W . C . Yockey, Biochem . Pharmacol . ~, 640 (1968),
4,
T . Uchida and R . D . O'Brien, Biochem . Pharmacol . ~, 725 (1964) .
5,
R . I . McManus, J . Biol . Chem . ~, 1398 (1960) .
6,
~ . J . Parker, Jr ., Anal . them . ~, 1359 (1966) .
7,
E . W . Maynert and H . K . Kaji, J . Pharmacol . ex~. Ther . ]~, 114 (1962),
$,
M . A . Medina, H . D . Braymer, and J . L . Reeves, J . Neurochem . 301 (1962),
9,
E . Roberts, "Inhibition in the Nervous System and Gamma-Aminobutyric Acid", Pergamon Press, New York (1960) .
10,
F . P . Underhill, J . Biol . Chem . ],Q, 159 (1966),
11 .
R . E . Winnick and T . E . Winnick, Bull . Soc . Chim . Biol . ~, 1727 (1954),
12,Kalyankar, G . D ., Meister, A ., J . Biol . Chem . ~., 3210 (1959), 13,
S . R . Fortney, D . A . Clark, and E . Stein, J . Pharmac . exp. Ther . 1~, 277 . (1967) ,
14,
A . Pletscher, P . A . Shore, and B . B . Brodie, J . Pharmac .
-p. ex
Ther . ],],~,, $4 (1956) 15 .
R . E . Winnick, S . Moikeha, and T . Winnick, J . Biol . Chem . ~, 3645 (1963),
16,
E . Roberts, D . G . 5imonsen, and E . Roberts, Biochem . Pharmacol . 12. 1445 (1963),
17,
s . H . Lee, Fed . Proc . ~, 423 (1969) .