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Quantitative estimation of free and bound serum aldehyde
13
CLINICA CHIMICA ACTA
QUANTITATIVE
GERARD
QUASH
ESTIMATION OF FREE AND BOUND SERUM ALDEHYDE
AND KRISHNA
Department of Biochemistry,
MAHARAJ
University of the West Indies, Mona, Kingston 7
(Jamaica)
(Received April znd, 1970)
SUMMARY
I. On heating human serum with As,O, in the presence of methanol, the level of aldehyde is increased. The increase, expressed as a percentage of the total aldehyde content, is referred to as the bound aldehyde. 2. In the sera of patients with early malignancy, the value of bound aldehyde is found to be significantly lower than that of normal sera, but as metastases occur, it rises above the normal value. 3. In the sera of patients with malignancy, but who responded to treatment, the value for bound aldehyde is similar to that found in the sera of patients with non-malignant disease.
The literature contains many references underlying the importance of polyamines to the growth of micro-organism+, plants2 and animal cells undergoing regeneration3. An increase in polyamine levels has also been shown to take place in the sera of patients with cancer and chronic infection 4. Polyamines could affect growth by acting either directly on the DNA dependent RNA polymerase reactions, or indirectly on the amino acid pool and hence making available to the cell more t-RNA amino acid complexes which are the immediate regulators of RNA synthesis“. Another mechanism which explains the involvement of the polyamines in growth regulation is the inhibitory action of their metabolic breakdown products. Bachrach et aL7 have shown that spermine dialdehyde: CHO(CH,),NH(CH,),NH(cH,),CHO, acts as a potent inhibitor of growth, the molecule itself forming cross links across the two strands of DNA. Other aldehydes which have been shown to exert a growth inhibitory effect are malondialdehyde CHO-CH,-CHOs, methylglyoxal CH,-CO-CHO (ref. g) and “retine” a derivative of methyl glyoxalr” found to occur naturally in clamsll, animal cells12 and human urine13. Unequivocal evidence for the presence of “retine” in tissue homogenates can be obtained only if it is released from its bound carrier by pre-treatment of the homogenate with As,O,, whereas it occurs in an unbound form in human urine13. We therefore undertook a quantitative estimation of the aldehydes released C&z. Chim. Acta,
zg
(1970)
17-16
I4
QUASH,MAHARAJ
by treating human serum with As,O,. To assess specifically this release of bound aldehyde, the free aldehydes present in each serum were evaluated by measuring the aldehyde content of the serum without the As,O, treatment. MATERIALS
AND
METHODS
Source of seYa
All sera were obtained from patients admitted to the Hospital of the University of the West Indies and the Hope Cancer Institute. (I)
Liberation
of aldehydes
The use of As,O, for the liberation of aldehydes was based essentially on the observations of Egyiid et al.14 and modified for serum as follows : to IO mg As,O, were added 0.2 ml serum and 0.9 ml of 50% methanol. The control consisted of 0.2 ml serum+o.g ml of methanol. The contents of both tubes were thoroughly mixed on a Vortex mixer, heated at 70~ for 30 min and then cooled to room temperature for aldehyde estimation. Bound aldehyde expressed as a percentage of total aldehyde = (Aldehyde content +As,O,) - (Aldehyde content -As,O,)
x IOO
(aldehyde content+As,O,) (2)
Estimation
of aldehyde
The method was essentially that described by Bachrach et al.16 for the estimation of spermine aldehyde. To each tube was added 0.5 ml of 0.4% N methyl benzolone thiazolone hydrazone hydrochloride. The tubes were incubated at room temperature for 30 min then 2.5 ml of 0.2% FeCl, was added to each tube, followed by a further incubation at room temperature for 15 min. The tubes were then centrifuged at 3000 rev./min for 15 min in a refrigerated centrifuge, and the supernatant read at 660 nm in a Zeiss spectrophotometer Model PMQ II. All estimations were done in duplicate. RESULTS
AND
DISCUSSION
Before the technique for liberating aldehydes was applied to unknown sera, the different steps of the procedure, viz. temperature, time of heating and amount of As,O,, were investigated on samples of normal sera. The method was as described under MATERIALS AND METHODS except for the variable shown. The values for bound aldehyde expressed as a percentage are shown in Table I. The results show that maximum release of aldehydes occurs on heating the serum at 70’ for 30 min in the presence of IO mg of As,O,. With these parameters established, the unknown sera were investigated. The sera were divided into 3 main groups: (A) from patients with histologically proven malignant disease; (B) from patients with histologically proven malignant disease who had responded to treatment; (C) from patients with non-malignant disease. The mean values for bound aldehydes in these 3 groups are shown in Table II. To see whether there was in fact any significant difference between these 3 groups, 2 types of statistical analyses were performed. Clin. Chim. Acta,
30 (1970) 13-16
FREEANDBOUNDSERUMALDEHYDE
TABLE
I5
I
VALUES
OF BOUND
ALDEHYDE
IN
NORMAL
SERA
Temp. of heating
Bound aldehyde %
Time of heating min
BOW&d aldehyde %
60” 7o”
39.4 46.9
‘5 30
30.2 41.1
IO
39.4 38.0
75”
42.6
60
37.9
12
38.2
80”
31.2
TABLE
mg A g208 added 8
Bound aldehyde %
II
VALUES
OF BOUND
Group *
No.
A
23
B
II
C * See text.
30
ALDEHYDE
in group
IN SERA
IN VARIOUS
DISEASE
STATES
Bound aldehyde percentage mean and S.E. + 2.26 51.5 zt 3.27 45.7 f 1.98 31.5
Analysis of variance By this method of analysis, a variance ratio of 16.63 was obtained, which means that there are significant differences between the groups even at the I?; point. (I)
(2)
A “t” test between the diflerent
groups
Groups
t value
P
A compared with B A compared with C B compared with C
5.02 4.70 I .42
< .OOI < ,001 < .2
From these analyses it can therefore be seen that of the 3 groups of sera analysed, the values for bound aldehydes in the malignant group are significantly lower than those of either the non-malignant or malignant treated groups, whereas there is no significant difference between the latter 2 groups. Not included in the malignant group are 3 sera from patients with metastases, who had values for bound aldehyde of 74.6, 84.00 and 83.00. The sample size was too small for any meaningful statistical analysis to be made. In the non-malignant group there was one patient with homozygous sickle cell disease, where the aldehyde released on As,O, treatment was also high, viz. 72.82. While we have no explanation for this latter value, the high values for bound aldehyde in the sera of patients with metastases must stand out as being in contradistinction to the low value found in early malignancy. These results cannot be interpreted at present, since the quantitative estimations done here do not permit us to say whether the high values during metastases are due to the same aldehyde(s) measured in early malignancy. Nevertheless, the findings (a) that there is a difference in the bound aldehyde content between the sera of patients with malignant and nonmalignant disease and (b) that this difference no longer exists when the patients with malignant disease respond to treatment, strongly suggest that there is some relationClin. Chim. Acta, 30 (1970)
13-16
QUASH, MAHARA J
16
ship between bound serum aldehydes and growth. An investigation into the nature of this relationship will be reported in the following paper. ACKNOWLEDGEMENT
We are grateful to our colleagues at University College Hospital of the West Indies and the Hope Cancer Institute for the samples of serum. REFERENCES I H. KIHARA AND R. SNELL, Proc. Natl. Acad. Sci. U.S., 43 (1957) 867. 2 S. Cocuccr AND N. BAGNI, Life Sci., 7 (1968) 113. 3 A. RAINA, J. JKNNE AND M. SIIMES,Biochim.Biophys. Acta, 123 (1966) 197. 4 G. QUASH AND M. WILSON, West Indian Med. J., XVI (1967) 81. 5 K. ABRAHAM, Europ. J. Biochem., 5 (1968) 143. 6 D. EZEKIEL AND H. BROCKMAN, J. Mol.Biol., 31 (1968) 541. 7 U. BACHRACH, S. ABZUG AND A. BEKIERKUNST, Biochim.Biophys. Acta, 134(1967) 8 B. BROOKS AND 0. KLAMERTH, Europ. J. Biochem., 5 (1968) 178. 9 H. OTSUKA AND L. EGY~D, Cancer Res., 27 (1967) 1498. 10 L. EGY~~D. Proc. Natl. Acad. Sci. U.S., 54 (1965) zoo. 11 M. SCHMEER, Science, 144 (1964) 413. 12 A. SZENT-GYC~RGYI, A. HEGYELI AND J.MCLAUGHLIN, Science,140(1963) 1391. 13 A. HEGYELI, J.MCLAUGHLIN AND A. SZENT-GYGRGYI, Science,142 (1963) 391. 14 L. EGY~_?D,J.MCLAUGHLIN AND A. SZENT-GYGRGYI, Proc. Natl. Acad. Sci. U.S., 1422. 15 U. BACHRACH AND B. RECHES, Anal. Biochem., 17 (1966) 38.
Clin. Chim. Acta,
30 (1970) 13-16
174.
57 (1967)
CLINICA CHIMICA ACTA
QUANTITATIVE
GERARD
QUASH
ESTIMATION OF FREE AND BOUND SERUM ALDEHYDE
AND KRISHNA
Department of Biochemistry,
MAHARAJ
University of the West Indies, Mona, Kingston 7
(Jamaica)
(Received April znd, 1970)
SUMMARY
I. On heating human serum with As,O, in the presence of methanol, the level of aldehyde is increased. The increase, expressed as a percentage of the total aldehyde content, is referred to as the bound aldehyde. 2. In the sera of patients with early malignancy, the value of bound aldehyde is found to be significantly lower than that of normal sera, but as metastases occur, it rises above the normal value. 3. In the sera of patients with malignancy, but who responded to treatment, the value for bound aldehyde is similar to that found in the sera of patients with non-malignant disease.
The literature contains many references underlying the importance of polyamines to the growth of micro-organism+, plants2 and animal cells undergoing regeneration3. An increase in polyamine levels has also been shown to take place in the sera of patients with cancer and chronic infection 4. Polyamines could affect growth by acting either directly on the DNA dependent RNA polymerase reactions, or indirectly on the amino acid pool and hence making available to the cell more t-RNA amino acid complexes which are the immediate regulators of RNA synthesis“. Another mechanism which explains the involvement of the polyamines in growth regulation is the inhibitory action of their metabolic breakdown products. Bachrach et aL7 have shown that spermine dialdehyde: CHO(CH,),NH(CH,),NH(cH,),CHO, acts as a potent inhibitor of growth, the molecule itself forming cross links across the two strands of DNA. Other aldehydes which have been shown to exert a growth inhibitory effect are malondialdehyde CHO-CH,-CHOs, methylglyoxal CH,-CO-CHO (ref. g) and “retine” a derivative of methyl glyoxalr” found to occur naturally in clamsll, animal cells12 and human urine13. Unequivocal evidence for the presence of “retine” in tissue homogenates can be obtained only if it is released from its bound carrier by pre-treatment of the homogenate with As,O,, whereas it occurs in an unbound form in human urine13. We therefore undertook a quantitative estimation of the aldehydes released C&z. Chim. Acta,
zg
(1970)
17-16
I4
QUASH,MAHARAJ
by treating human serum with As,O,. To assess specifically this release of bound aldehyde, the free aldehydes present in each serum were evaluated by measuring the aldehyde content of the serum without the As,O, treatment. MATERIALS
AND
METHODS
Source of seYa
All sera were obtained from patients admitted to the Hospital of the University of the West Indies and the Hope Cancer Institute. (I)
Liberation
of aldehydes
The use of As,O, for the liberation of aldehydes was based essentially on the observations of Egyiid et al.14 and modified for serum as follows : to IO mg As,O, were added 0.2 ml serum and 0.9 ml of 50% methanol. The control consisted of 0.2 ml serum+o.g ml of methanol. The contents of both tubes were thoroughly mixed on a Vortex mixer, heated at 70~ for 30 min and then cooled to room temperature for aldehyde estimation. Bound aldehyde expressed as a percentage of total aldehyde = (Aldehyde content +As,O,) - (Aldehyde content -As,O,)
x IOO
(aldehyde content+As,O,) (2)
Estimation
of aldehyde
The method was essentially that described by Bachrach et al.16 for the estimation of spermine aldehyde. To each tube was added 0.5 ml of 0.4% N methyl benzolone thiazolone hydrazone hydrochloride. The tubes were incubated at room temperature for 30 min then 2.5 ml of 0.2% FeCl, was added to each tube, followed by a further incubation at room temperature for 15 min. The tubes were then centrifuged at 3000 rev./min for 15 min in a refrigerated centrifuge, and the supernatant read at 660 nm in a Zeiss spectrophotometer Model PMQ II. All estimations were done in duplicate. RESULTS
AND
DISCUSSION
Before the technique for liberating aldehydes was applied to unknown sera, the different steps of the procedure, viz. temperature, time of heating and amount of As,O,, were investigated on samples of normal sera. The method was as described under MATERIALS AND METHODS except for the variable shown. The values for bound aldehyde expressed as a percentage are shown in Table I. The results show that maximum release of aldehydes occurs on heating the serum at 70’ for 30 min in the presence of IO mg of As,O,. With these parameters established, the unknown sera were investigated. The sera were divided into 3 main groups: (A) from patients with histologically proven malignant disease; (B) from patients with histologically proven malignant disease who had responded to treatment; (C) from patients with non-malignant disease. The mean values for bound aldehydes in these 3 groups are shown in Table II. To see whether there was in fact any significant difference between these 3 groups, 2 types of statistical analyses were performed. Clin. Chim. Acta,
30 (1970) 13-16
FREEANDBOUNDSERUMALDEHYDE
TABLE
I5
I
VALUES
OF BOUND
ALDEHYDE
IN
NORMAL
SERA
Temp. of heating
Bound aldehyde %
Time of heating min
BOW&d aldehyde %
60” 7o”
39.4 46.9
‘5 30
30.2 41.1
IO
39.4 38.0
75”
42.6
60
37.9
12
38.2
80”
31.2
TABLE
mg A g208 added 8
Bound aldehyde %
II
VALUES
OF BOUND
Group *
No.
A
23
B
II
C * See text.
30
ALDEHYDE
in group
IN SERA
IN VARIOUS
DISEASE
STATES
Bound aldehyde percentage mean and S.E. + 2.26 51.5 zt 3.27 45.7 f 1.98 31.5
Analysis of variance By this method of analysis, a variance ratio of 16.63 was obtained, which means that there are significant differences between the groups even at the I?; point. (I)
(2)
A “t” test between the diflerent
groups
Groups
t value
P
A compared with B A compared with C B compared with C
5.02 4.70 I .42
< .OOI < ,001 < .2
From these analyses it can therefore be seen that of the 3 groups of sera analysed, the values for bound aldehydes in the malignant group are significantly lower than those of either the non-malignant or malignant treated groups, whereas there is no significant difference between the latter 2 groups. Not included in the malignant group are 3 sera from patients with metastases, who had values for bound aldehyde of 74.6, 84.00 and 83.00. The sample size was too small for any meaningful statistical analysis to be made. In the non-malignant group there was one patient with homozygous sickle cell disease, where the aldehyde released on As,O, treatment was also high, viz. 72.82. While we have no explanation for this latter value, the high values for bound aldehyde in the sera of patients with metastases must stand out as being in contradistinction to the low value found in early malignancy. These results cannot be interpreted at present, since the quantitative estimations done here do not permit us to say whether the high values during metastases are due to the same aldehyde(s) measured in early malignancy. Nevertheless, the findings (a) that there is a difference in the bound aldehyde content between the sera of patients with malignant and nonmalignant disease and (b) that this difference no longer exists when the patients with malignant disease respond to treatment, strongly suggest that there is some relationClin. Chim. Acta, 30 (1970)
13-16
QUASH, MAHARA J
16
ship between bound serum aldehydes and growth. An investigation into the nature of this relationship will be reported in the following paper. ACKNOWLEDGEMENT
We are grateful to our colleagues at University College Hospital of the West Indies and the Hope Cancer Institute for the samples of serum. REFERENCES I H. KIHARA AND R. SNELL, Proc. Natl. Acad. Sci. U.S., 43 (1957) 867. 2 S. Cocuccr AND N. BAGNI, Life Sci., 7 (1968) 113. 3 A. RAINA, J. JKNNE AND M. SIIMES,Biochim.Biophys. Acta, 123 (1966) 197. 4 G. QUASH AND M. WILSON, West Indian Med. J., XVI (1967) 81. 5 K. ABRAHAM, Europ. J. Biochem., 5 (1968) 143. 6 D. EZEKIEL AND H. BROCKMAN, J. Mol.Biol., 31 (1968) 541. 7 U. BACHRACH, S. ABZUG AND A. BEKIERKUNST, Biochim.Biophys. Acta, 134(1967) 8 B. BROOKS AND 0. KLAMERTH, Europ. J. Biochem., 5 (1968) 178. 9 H. OTSUKA AND L. EGY~D, Cancer Res., 27 (1967) 1498. 10 L. EGY~~D. Proc. Natl. Acad. Sci. U.S., 54 (1965) zoo. 11 M. SCHMEER, Science, 144 (1964) 413. 12 A. SZENT-GYC~RGYI, A. HEGYELI AND J.MCLAUGHLIN, Science,140(1963) 1391. 13 A. HEGYELI, J.MCLAUGHLIN AND A. SZENT-GYGRGYI, Science,142 (1963) 391. 14 L. EGY~_?D,J.MCLAUGHLIN AND A. SZENT-GYGRGYI, Proc. Natl. Acad. Sci. U.S., 1422. 15 U. BACHRACH AND B. RECHES, Anal. Biochem., 17 (1966) 38.
Clin. Chim. Acta,
30 (1970) 13-16
174.
57 (1967)