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The determination of acetaldehyde in blological samples by head-space gas chromatography
ANALYTICAL
BIOCHEMISTRY
80,
116-124 (1977)
The Determination of Acetaldehyde Samples by Head-Space Chromatography C.J. PETERERIKSSON,HELMUTH Research
Laboratories
in Biological Gas
W. SIPPEL,AND~LOF
of the State Alcohol Monopoly OOIOI Helsinki 10, Finland
(Alko),
A. FORSANDER Box 350,
Received July 21, 1976; accepted January 18, 1977 A method for the determination of acetaldehyde (AcH) in biological samples by head-space gas chromatography is presented. Human venous blood (antecubital), rat blood (heart-punctured) rat liver (freeze-clamped), and rat and mouse brain (freeze-clamped) were used as the biological samples. The method involves two steps, in the first of which the samples are deproteinized with perchloric acid (PCA). Rat blood can, alternatively, be hemolyzed with water. In the second step, the deproteinized supematant or hemolyzed blood is pipetted into a serum bottle, which is sealed with a rubber stopper and brought to 65°C in a sampling turntable. Head-space samples are then automatically taken for GLC analysis. Ethanol causes a nonenzymatic formation of AcH in the PCA supematants of liver and brain, which can be inhibited by the use of thiourea. This reaction is minor in the blood supematants and cannot be inhibited there by thiourea. The method described measures the total AcH content without regard to any binding. Some of the AcH in rat blood was shown to be bound.
Unlike the metabolism of ethanol, the metabolism of its first metabolic product, acetaldehyde (AcH), has, until recently, only been the subject of few investigations. One of the main reasons for this has probably been the difficulties involved in determining AcH in biological samples (1,2). The lack of reliability, specificity, and rapidity in some older procedures, including calorimetric (3), spectrophotometric (4), radiochemical (5), and enzymatic (6) methods, has been emphasized by Duritz and Truitt (l), who introduced the head-space gas chromatographic determination of blood AcH. The determination of AcH in the presence of ethanol in precipitated blood was complicated by a nonenzymatic production of AcH during the incubation prior to the headspace analysis, an effect that appeared to be eliminated by using protein-free filtrates (2,7). Recently, traces of nonenzymatically formed AcH have been found in protein-free acidic supernatants of rat blood containing ethanol (8). Moreover, much larger amounts of AcH were found in liver supernatants under similar conditions (8). These observations showed that the nonenzymatic formation of AcH could not be completely eliminated by 116 Copyright All rights
0 1977 by Academic Press. Inc. of reproductnn in any form reserved.
ISSN ooO3-2697
HEAD-SPACE
CHROMATOGRAPHY
OF ACETALDEHYDE
117
centrifuging away the protein precipitates. An improved method resulted from the discovery that thiourea totally inhibits the nonenzymatic formation of AcH in rat liver supernatants (9). The aim of this investigation was to develop a general method for the head-space determination of AcH in biological samples. Human and rat blood, rat liver and brain tissue, and mouse brain tissue were used in recovery studies. Special attention was given to the use of thiourea in the prevention of the nonenzymatic formation of AcH and to the treatment of the biological samples. MATERIALS
AND METHODS
Biological samples. Male, fed Sprague-Dawley rats were used as the source of rat blood, liver, and brain. Blood was collected by heartpuncture from heparin-treated and pentobarbital-anesthetized animals. Livers and brains from pentobarbital-anesthetized rats were freezeclamped with aluminium tongs precooled in liquid nitrogen. Mouse brains were excised and freeze-clamped from male Swiss albinos. The frozen livers and brains were pulverized in a mortar. Male and female human blood was taken from an antecubital vein and was heparinized. Reagents. AcH (99.5%) and thiourea (p.a.) were obtained from E. Merck Ag, Darmstadt, Germany. The AcH was redistilled before making stock solutions in distilled water. To eliminate any volatile compounds that could interfere with the AcH determinations (9), the thiourea was dried at 90°C for 12 hr before making stock solutions in 0.6~ perchloric acid (PCA). The AcH and thiourea stock solutions were renewed every 2 weeks and month, respectively. Instrument. The gas chromatograph used for the automatic headspace analysis was a Perkin-Elmer F 40 Multifract, equipped with a special liquid circulatory thermostat for the sampling turntable and a new electropneumatic dosing system. Column: 15% polyethylene glycol on Celite 60/100. Temperatures: sample thermostat 65”C, dosing line 17o”C, column oven 75”C, and detector block 140°C. Gas flow rates: hydrogen 35 ml/min, air 300 ml/min, carrier gas (nitrogen) 35 ml/min. Analysis time: 2.5 min/sample. Experimental design. The following basic parameters were varied: (i) The proteins were precipitated from blood, liver, and brain with 0.6 N PCA, and parallel blood samples were hemolyzed with water; (ii) solutions in which samples were diluted to 20, 10 and 6.7% with the 0.6 N PCA solution or water; (iii) the concentration of ethanol was 0 or 10 mM in the 0.6 N PCA or water; (iv) the concentration of thiourea was O-320 mrvt in the 0.6 N PCA or water; (v) AcH was added to the biological samples either before or after precipitation (with PCA) or hemolyzation (with water).
118
ERIKSSON,
SIPPEL
AND
FORSANDER
Sampling techniques. One part blood or frozen tissue powder was transferred to centrifuge tubes at 4”C, and 4, 9, or 14 parts (to give concentrations of 20, 10, and 6.7%) of the ice-cold PCA or water solutions were added. The use of frozen blood was shown to have no effects on the results. For samples to which AcH was added before precipitation or hemolyzation, the time between addition of the AcH and the precipitation or hemolyzation was kept as short as possible (~15 set), except for the experiment in which this time was deliberately varied. The precipitated proteins were centrifuged away at 40008 at 4°C. No AcH was added to the part of the biological sample used to determine the nonenzymatic production of AcH. Head-space methods. Samples, 0.5 ml, of the acidic supernatants of the hemolysates were pipetted into 22-ml serum bottles, sealed with Teflon rubber stoppers, and incubated for 15 min (or longer in the nonenzymatic formation experiments) at 65°C in the sampling turntable; head-space samples were automatically taken by means of the electropneumatic dosing system. AcH levels were determined from the peak heights. Fresh standards containing no biological sample were run daily to fix the 100% recovery. The sensitivity of the method was 0.5 PM in the 0.5-ml sample, which means that we are able to measure AcH concentrations down to 4-5 nmol/ml of blood or g of tissue. Recoveries. In the recovery experiments, the amount of AcH added was 100 nmol/ml or g of biological sample. Each series of experiments was compared with blanks containing saline in place of the biological sample. No endogenous AcH was present in the biological samples, and so, the percentage of recovery was equal to the concentration of AcH found, expressed as nanomoles per milliliter or gram of biological sample. All recoveries reported were determined on the same day that the samples were made. Preliminary trials showed that significant losses of AcH occurred in supernatants or hemolysates stored in the final serum bottles for longer than 24 hr. RESULTS Acetaldehyde
Recoveries
from Blood
Table 1 presents the recovery of AcH from the human and rat blood PCA supernatants and HZ0 hemolysates. Hemolyzed human blood is not suitable for the determination of AcH when ethanol is present because of the strong “spontaneous” formation of AcH (about 200 nmol/ml of blood in 15 min with 10 mM ethanol present). Hemolyzed rat blood, however, seems to be more suitable, but, in this case, the concentration of the blood seems to be crucial. With 6.7% blood concentrations, the AcH recoveries were 3-4% higher, and, with 20% blood
HEAD-SPACE
CHROMATOGRAPHY TABLE
RECOVERY
OF BLOOD
119
OF ACETALDEHYDE I
ACETALDEHYDE’
Recovery [nmol of AcH/ml of blood (%)I Precipitating or hemolyzing solution
Human blood
PCA PCA + 10 mM ethanol Hz0 H,O + 10 mM ethanol
94.7 115.6 131.0 302.4
2 2 ” 2
3.0 4.4 16.2 29.7
Rat blood 94.7 119.3 90.0 105.4
2 t 2 2
6.6 6.1 7.8 15.4
a Samples were treated and analyses were made as described in the Materials and Methods section. AcH, 100 nmol/ml of blood, was added before precipitation (PCA) or hemolyzation (H,O). The final blood concentration was 10% (v/v). Results are given as mean t SD (n = 5).
concentrations, the recoveries were ll- 13% lower than with the 10% concentrations listed in Table 1. An incomplete hemolyzation was noted at blood concentrations of 20%, which could explain the loss of recovery. No such concentration effects were found in the PCA-precipitated blood. However, the recovery of AcH from rat blood was about 10% lower when the AcH was added before the PCA, than when PCA was added first. This effect was not observed in the human and hemolyzed blood experiments. In control experiments without added AcH, the amounts of AcH found in samples with and without ethanol were similar to the corresponding differences shown in Table 1. Acetaldehyde
Recoveries from Liver and Brain
Table 2 shows the recoveries from the rat liver and brain PCA supernatants. With 10 mM ethanol present, a “spontaneous” formation TABLE RECOVERY
OF RAT LIVER
2
AND BRAIN
ACETALDEHYDE~
Recovery [nmol of AcH/g of tissue (%)I Precipitating solution PCA PCA PCA PCA +
+ + + 40
10 mM ethanol 40 mM thiourea 10 mM ethanol mM thiourea
Liver (n = 5)
Brain (n = 4)
101.0 ‘- 6.3 141.7 t 16.5 102.7 2 10.0
100.0 t 2.5 94.9 k 2.7
108.1 k 9.4
a Samples were treated and analyses were made as described in the Materials and Methods section. AcH, 100 nmol/g wet wt of tissue, was added before precipitation with PCA. The final tissue concentration was 10% (w/v). Results are given as mean 2 SD.
120
ERIKSSON, 100
SIPPEL AND FORSANDER
r
TIME BETWEEN PRECIPITATION
ADDITION OF AcH AND OR HEMOLYSATION (min)
FIG. 1. Binding of acetaldehyde in rat blood. Samples were treated and analyses were made as described in the Materials and Methods section, except that blood from decapitated rats was used. PCA precipitations (0) and Hz0 hemolyzations (0) were performed at intervals, after the addition of 100 nmol of AcH/ml of heparinized blood. Final blood concentrations were 20% (v/v), and the temperature was 4°C.
of AcH occurred in the liver supernatants (about 40 nmol/g in 15 min), which, however, could be inhibited by 40 mM thiourea. Similar nonenzymatic formation data from the brain supernatants are reported later in this paper. The tissue concentration was found to have no significant effect on the recoveries, but a 4-8% higher recovery was obtained if the AcH was added after the precipitation instead of (as listed in Table 2) before. Binding of Acetaldehyde
The length of time between the addition of the AcH to rat blood and the PCA precipitation was found to have a marked effect on the AcH recovery. An example of this phenomenon is demonstrated in Fig. I, where the recovery of AcH from a PCA precipitation fell to 55% within 10 min. In contrast, no such effect was found when samples were hemolyzed. These results strongly suggest that there occurred a time-dependent binding of AcH in the blood prior to the hemolyzation or PCA precipitation, so that bound AcH was centrifuged away during the PCA-precipitation procedure. In the hemolyzation method, however, the bound AcH was liberated either during the hemolyzation or during the 15min equilibration at 65°C prior to the head-space analysis. The liberation did not seem to occur during the hemolyzation, because similarly low AcH recoveries were obtained whether or not the blood was hemolyzed before the PCA precipitation.
HEAD-SPACE
The use of Thiourea Formation
CHROMATOGRAPHY
as an Inhibitor
121
OF ACETALDEHYDE
of the Nonenzymatic
Acetaldehyde
The results listed in Tables 1 and 2 show that AcH recoveries are higher when ethanol is present. This was shown to be due to a nonenzymatic formation of AcH in the supernatants during the incubation at 6s”C prior to the head-space analysis (Fig. 2). The reaction was found to proceed linearly for at least the first 45 min of incubation, and the rate was proportional to the ethanol concentrations (5-20 mM> and to the tissue concentrations (6.7-20%). As shown in Fig. 2, the “spontaneous” formation of AcH was lowest in blood, and higher in liver and brain supernatants, with the mouse brain supernatants displaying the highest formation rates. Thiourea was proven to be an efficient inhibitor of nonenzymatic AcH formation in liver and brain supernatants, but no noticeable inhibition was obtained in human and rat blood. Moreover, the use of thiourea in the determination of blood AcH seriously reduced the AcH recovery (Fig. 3). As can be seen in Fig. 3, this effect was only slight in the liver the brain PCA supernatants.
10
20
30 THIOUREA
LO
50
60
70
80
(rnb.7 J
FIG. 2. The effect of thiourea on the ethanol-induced nonenzymatic formation of acetaldehyde in tissue supernatants. PCA supematants containing 10 mM ethanol were made from human blood (O), rat blood (A), rat liver (O), rat brain (A), and mouse brain (m). as described in the Materials and Methods section. The AcH-formation rates were calculated from the linear increase in the AcH concentration during 45 min at 65°C.
122
ERIKSSON,
SIPPEL AND FORSANDER
THIOUREA
(mM)
FIG. 3. The effect of thiourea on the acetaldehyde recovery. PCA supernatants containing 100 pM AcH were made from human blood (0), rat blood (A), rat liver (O), rat brain (A), and mouse brain (a), as described in the Materials and Methods section. Supematants without thiourea were used as base values (100% recovery). (0) Represents the blank PCA solution.
DISCUSSION
Acetaldehyde
Recovery from Blood
Three main factors should be considered in the determination of blood AcH concentrations. The first includes the problems of an artificially increased AcH recovery caused by nonenzymatic AcH formation induced by ethanol; the second is the various possibilities of recovery losses; the third concerns the distribution of AcH in the blood. The first comprehensive study of the nonenzymatic formation of AcH was reported by Truitt (2), who measured AcH in precipitated blood by means of a head-space gas chromatographic method that was almost identical to the one used in this work. Truitt’s main conclusions were that, although the production of AcH required the presence of ethanol, the ethanol itself was not the source of the AcH, and AcH production could be inhibited by using protein-free filtrates. In preliminary experiments, we also found an extremely high production of AcH when the protein precipitations were not centrifuged away, and, subsequently, we used only protein-free acid supernatants. Sippel(9) has shown that a slow production of AcH also occurs in protein-free blood supernatants from rats in the presence of ethanol (8), and he reported that thiourea is a useful inhibitor for this reaction. The occurrence of ethanol-induced nonenzymatic AcH formation in both human and rat blood PCA supernatants was confirmed in the present
HEAD-SPACE
CHROMATOGRAPHY
OF ACETALDEHYDE
123
study. Here, however, thiourea had no inhibitory effect on the formation, which implies that the reaction mechanism must be one other than the ascorbic acid-catalyzed reaction proposed by Sippel (8). Thiourea would, perhaps, be useful with animals containing enough ascorbic acid in their blood to produce the ascorbic acid-catalyzed reaction, but excessive concentrations of thiourea (>20 mM) must be avoided because it reduces the recovery of AcH (Fig. 3). Hemolyzation of the blood seems to be a better way of determining blood AcH in rats, because the ethanol-induced increase of the AcH recovery was less than that when the PCA-supernatants were used (Table 1). Hemolyzation could not be used with human blood, however, because of the large amount of AcH that was formed, as has been reported by Truitt (2). Beside the use of excessive thiourea, another procedure that resulted in a reduced AcH recovery was the addition of AcH before, instead of after, the precipitation. An explanation could be that, before precipitation, AcH is rapidly oxidized in the blood. Even if such a reaction has been reported (l), and proven to exist at 37°C in preliminary experiments, this reaction alone is not likely to explain the 10% loss of recovery that occurred within 15 set at 0°C. The experiments in which the time between AcH addition and precipitation or hemolyzation was varied (Fig. 1) showed that the most probable explanation for the AcH losses was a rapid thermolabile binding of AcH in the blood. This finding suggests that binding may be possible in vivo, resulting in a nonuniform distribution of AcH in the blood in vivo. We are continuing to study the binding of AcH in blood. Acetaldehyde
Recovery
from
Liver and Brain
The determination of liver and brain AcH concentrations has not previously been the subject of any major study. Sippel has demonstrated a strong nonenzymatic AcH formation in liver PCA supernatants in the presence of ethanol (8), which was efficiently inhibited by thiourea (9). This finding was confirmed in the present study, and the presence of thiourea was also found necessary for inhibition of nonenzymatic AcH formation during the determination of brain AcH concentrations. Mouse brain was investigated because several recent papers have reported high brain AcH concentrations during ethanol intoxication (lo12). The amount of AcH found in brain supernatants to which ethanol had been added (Fig. 2) was of such an order that it could easily explain most of the high brain AcH levels reported for both rats and mice (10-15).
124
ERIKSSON,
SIPPEL AND FORSANDER
REFERENCES 1. Duritz, G., and Truitt, E. 2. Truitt, E. B. (1970) Quart. 3. Stotz, E. A. (1943)J. Biol. 4. Burbridge, T. N., Hine,
B. (1964) Quarf. J. Stud. A/c. .I. Stud. Ale. 31, 1- 12. Chem.
25,
498-510.
148, 585-591.
C. N., and Schick, A. F. (1950) .I. Lab. Cfin.
Med.
35,
985-987.
5. Casier, H., and Polet, H. (1958) Arch. Inr. Pharmacodyn. Ther. 113, 439-496. 6. Lundquist, F. (1958) Biochem .f. 68, 172-177. 7. Truitt, E. B. (1971) in Biological Aspects of Alcohol (Roach, M. K., McIsaac, W. M., and Creaven, P. .I., eds.), pp. 212-232, University of Texas Press, Austin, Texas. 8. Sippel, H. W. (1973)Acta Chem. Stand. 27, 541-550. 9. Sippel, H. W. (1972) Acta Chem. Stand. 26, 3398-3400. 10. Littleton, J. M., Griffiths, P. J., and Ortiz, A. (1974) J. Pharm. Pharmacol. 26,81-91. 11. Ortiz, A., Griffiths, P. J., and Littleton, J. M. (1974) J. Pharm. Pharmacol. 26,249-260.
Lin, D. C. (1975) Res. Commun. Chem. Pathol. Pharmacol. 11, 365-371. 13. Kiessling, K. H. (1962) Exp. Cell Res. 27, 367-368. 14. Ridge, J. W. (1%3) Biochem. J. 88, 95-100. 15. Coldwell, B. B., Wiberg, G. S., Trenholm, H. L. (1970) Canad. J. Physiol. 12.
48,254-264.
Pharmncol.
BIOCHEMISTRY
80,
116-124 (1977)
The Determination of Acetaldehyde Samples by Head-Space Chromatography C.J. PETERERIKSSON,HELMUTH Research
Laboratories
in Biological Gas
W. SIPPEL,AND~LOF
of the State Alcohol Monopoly OOIOI Helsinki 10, Finland
(Alko),
A. FORSANDER Box 350,
Received July 21, 1976; accepted January 18, 1977 A method for the determination of acetaldehyde (AcH) in biological samples by head-space gas chromatography is presented. Human venous blood (antecubital), rat blood (heart-punctured) rat liver (freeze-clamped), and rat and mouse brain (freeze-clamped) were used as the biological samples. The method involves two steps, in the first of which the samples are deproteinized with perchloric acid (PCA). Rat blood can, alternatively, be hemolyzed with water. In the second step, the deproteinized supematant or hemolyzed blood is pipetted into a serum bottle, which is sealed with a rubber stopper and brought to 65°C in a sampling turntable. Head-space samples are then automatically taken for GLC analysis. Ethanol causes a nonenzymatic formation of AcH in the PCA supematants of liver and brain, which can be inhibited by the use of thiourea. This reaction is minor in the blood supematants and cannot be inhibited there by thiourea. The method described measures the total AcH content without regard to any binding. Some of the AcH in rat blood was shown to be bound.
Unlike the metabolism of ethanol, the metabolism of its first metabolic product, acetaldehyde (AcH), has, until recently, only been the subject of few investigations. One of the main reasons for this has probably been the difficulties involved in determining AcH in biological samples (1,2). The lack of reliability, specificity, and rapidity in some older procedures, including calorimetric (3), spectrophotometric (4), radiochemical (5), and enzymatic (6) methods, has been emphasized by Duritz and Truitt (l), who introduced the head-space gas chromatographic determination of blood AcH. The determination of AcH in the presence of ethanol in precipitated blood was complicated by a nonenzymatic production of AcH during the incubation prior to the headspace analysis, an effect that appeared to be eliminated by using protein-free filtrates (2,7). Recently, traces of nonenzymatically formed AcH have been found in protein-free acidic supernatants of rat blood containing ethanol (8). Moreover, much larger amounts of AcH were found in liver supernatants under similar conditions (8). These observations showed that the nonenzymatic formation of AcH could not be completely eliminated by 116 Copyright All rights
0 1977 by Academic Press. Inc. of reproductnn in any form reserved.
ISSN ooO3-2697
HEAD-SPACE
CHROMATOGRAPHY
OF ACETALDEHYDE
117
centrifuging away the protein precipitates. An improved method resulted from the discovery that thiourea totally inhibits the nonenzymatic formation of AcH in rat liver supernatants (9). The aim of this investigation was to develop a general method for the head-space determination of AcH in biological samples. Human and rat blood, rat liver and brain tissue, and mouse brain tissue were used in recovery studies. Special attention was given to the use of thiourea in the prevention of the nonenzymatic formation of AcH and to the treatment of the biological samples. MATERIALS
AND METHODS
Biological samples. Male, fed Sprague-Dawley rats were used as the source of rat blood, liver, and brain. Blood was collected by heartpuncture from heparin-treated and pentobarbital-anesthetized animals. Livers and brains from pentobarbital-anesthetized rats were freezeclamped with aluminium tongs precooled in liquid nitrogen. Mouse brains were excised and freeze-clamped from male Swiss albinos. The frozen livers and brains were pulverized in a mortar. Male and female human blood was taken from an antecubital vein and was heparinized. Reagents. AcH (99.5%) and thiourea (p.a.) were obtained from E. Merck Ag, Darmstadt, Germany. The AcH was redistilled before making stock solutions in distilled water. To eliminate any volatile compounds that could interfere with the AcH determinations (9), the thiourea was dried at 90°C for 12 hr before making stock solutions in 0.6~ perchloric acid (PCA). The AcH and thiourea stock solutions were renewed every 2 weeks and month, respectively. Instrument. The gas chromatograph used for the automatic headspace analysis was a Perkin-Elmer F 40 Multifract, equipped with a special liquid circulatory thermostat for the sampling turntable and a new electropneumatic dosing system. Column: 15% polyethylene glycol on Celite 60/100. Temperatures: sample thermostat 65”C, dosing line 17o”C, column oven 75”C, and detector block 140°C. Gas flow rates: hydrogen 35 ml/min, air 300 ml/min, carrier gas (nitrogen) 35 ml/min. Analysis time: 2.5 min/sample. Experimental design. The following basic parameters were varied: (i) The proteins were precipitated from blood, liver, and brain with 0.6 N PCA, and parallel blood samples were hemolyzed with water; (ii) solutions in which samples were diluted to 20, 10 and 6.7% with the 0.6 N PCA solution or water; (iii) the concentration of ethanol was 0 or 10 mM in the 0.6 N PCA or water; (iv) the concentration of thiourea was O-320 mrvt in the 0.6 N PCA or water; (v) AcH was added to the biological samples either before or after precipitation (with PCA) or hemolyzation (with water).
118
ERIKSSON,
SIPPEL
AND
FORSANDER
Sampling techniques. One part blood or frozen tissue powder was transferred to centrifuge tubes at 4”C, and 4, 9, or 14 parts (to give concentrations of 20, 10, and 6.7%) of the ice-cold PCA or water solutions were added. The use of frozen blood was shown to have no effects on the results. For samples to which AcH was added before precipitation or hemolyzation, the time between addition of the AcH and the precipitation or hemolyzation was kept as short as possible (~15 set), except for the experiment in which this time was deliberately varied. The precipitated proteins were centrifuged away at 40008 at 4°C. No AcH was added to the part of the biological sample used to determine the nonenzymatic production of AcH. Head-space methods. Samples, 0.5 ml, of the acidic supernatants of the hemolysates were pipetted into 22-ml serum bottles, sealed with Teflon rubber stoppers, and incubated for 15 min (or longer in the nonenzymatic formation experiments) at 65°C in the sampling turntable; head-space samples were automatically taken by means of the electropneumatic dosing system. AcH levels were determined from the peak heights. Fresh standards containing no biological sample were run daily to fix the 100% recovery. The sensitivity of the method was 0.5 PM in the 0.5-ml sample, which means that we are able to measure AcH concentrations down to 4-5 nmol/ml of blood or g of tissue. Recoveries. In the recovery experiments, the amount of AcH added was 100 nmol/ml or g of biological sample. Each series of experiments was compared with blanks containing saline in place of the biological sample. No endogenous AcH was present in the biological samples, and so, the percentage of recovery was equal to the concentration of AcH found, expressed as nanomoles per milliliter or gram of biological sample. All recoveries reported were determined on the same day that the samples were made. Preliminary trials showed that significant losses of AcH occurred in supernatants or hemolysates stored in the final serum bottles for longer than 24 hr. RESULTS Acetaldehyde
Recoveries
from Blood
Table 1 presents the recovery of AcH from the human and rat blood PCA supernatants and HZ0 hemolysates. Hemolyzed human blood is not suitable for the determination of AcH when ethanol is present because of the strong “spontaneous” formation of AcH (about 200 nmol/ml of blood in 15 min with 10 mM ethanol present). Hemolyzed rat blood, however, seems to be more suitable, but, in this case, the concentration of the blood seems to be crucial. With 6.7% blood concentrations, the AcH recoveries were 3-4% higher, and, with 20% blood
HEAD-SPACE
CHROMATOGRAPHY TABLE
RECOVERY
OF BLOOD
119
OF ACETALDEHYDE I
ACETALDEHYDE’
Recovery [nmol of AcH/ml of blood (%)I Precipitating or hemolyzing solution
Human blood
PCA PCA + 10 mM ethanol Hz0 H,O + 10 mM ethanol
94.7 115.6 131.0 302.4
2 2 ” 2
3.0 4.4 16.2 29.7
Rat blood 94.7 119.3 90.0 105.4
2 t 2 2
6.6 6.1 7.8 15.4
a Samples were treated and analyses were made as described in the Materials and Methods section. AcH, 100 nmol/ml of blood, was added before precipitation (PCA) or hemolyzation (H,O). The final blood concentration was 10% (v/v). Results are given as mean t SD (n = 5).
concentrations, the recoveries were ll- 13% lower than with the 10% concentrations listed in Table 1. An incomplete hemolyzation was noted at blood concentrations of 20%, which could explain the loss of recovery. No such concentration effects were found in the PCA-precipitated blood. However, the recovery of AcH from rat blood was about 10% lower when the AcH was added before the PCA, than when PCA was added first. This effect was not observed in the human and hemolyzed blood experiments. In control experiments without added AcH, the amounts of AcH found in samples with and without ethanol were similar to the corresponding differences shown in Table 1. Acetaldehyde
Recoveries from Liver and Brain
Table 2 shows the recoveries from the rat liver and brain PCA supernatants. With 10 mM ethanol present, a “spontaneous” formation TABLE RECOVERY
OF RAT LIVER
2
AND BRAIN
ACETALDEHYDE~
Recovery [nmol of AcH/g of tissue (%)I Precipitating solution PCA PCA PCA PCA +
+ + + 40
10 mM ethanol 40 mM thiourea 10 mM ethanol mM thiourea
Liver (n = 5)
Brain (n = 4)
101.0 ‘- 6.3 141.7 t 16.5 102.7 2 10.0
100.0 t 2.5 94.9 k 2.7
108.1 k 9.4
a Samples were treated and analyses were made as described in the Materials and Methods section. AcH, 100 nmol/g wet wt of tissue, was added before precipitation with PCA. The final tissue concentration was 10% (w/v). Results are given as mean 2 SD.
120
ERIKSSON, 100
SIPPEL AND FORSANDER
r
TIME BETWEEN PRECIPITATION
ADDITION OF AcH AND OR HEMOLYSATION (min)
FIG. 1. Binding of acetaldehyde in rat blood. Samples were treated and analyses were made as described in the Materials and Methods section, except that blood from decapitated rats was used. PCA precipitations (0) and Hz0 hemolyzations (0) were performed at intervals, after the addition of 100 nmol of AcH/ml of heparinized blood. Final blood concentrations were 20% (v/v), and the temperature was 4°C.
of AcH occurred in the liver supernatants (about 40 nmol/g in 15 min), which, however, could be inhibited by 40 mM thiourea. Similar nonenzymatic formation data from the brain supernatants are reported later in this paper. The tissue concentration was found to have no significant effect on the recoveries, but a 4-8% higher recovery was obtained if the AcH was added after the precipitation instead of (as listed in Table 2) before. Binding of Acetaldehyde
The length of time between the addition of the AcH to rat blood and the PCA precipitation was found to have a marked effect on the AcH recovery. An example of this phenomenon is demonstrated in Fig. I, where the recovery of AcH from a PCA precipitation fell to 55% within 10 min. In contrast, no such effect was found when samples were hemolyzed. These results strongly suggest that there occurred a time-dependent binding of AcH in the blood prior to the hemolyzation or PCA precipitation, so that bound AcH was centrifuged away during the PCA-precipitation procedure. In the hemolyzation method, however, the bound AcH was liberated either during the hemolyzation or during the 15min equilibration at 65°C prior to the head-space analysis. The liberation did not seem to occur during the hemolyzation, because similarly low AcH recoveries were obtained whether or not the blood was hemolyzed before the PCA precipitation.
HEAD-SPACE
The use of Thiourea Formation
CHROMATOGRAPHY
as an Inhibitor
121
OF ACETALDEHYDE
of the Nonenzymatic
Acetaldehyde
The results listed in Tables 1 and 2 show that AcH recoveries are higher when ethanol is present. This was shown to be due to a nonenzymatic formation of AcH in the supernatants during the incubation at 6s”C prior to the head-space analysis (Fig. 2). The reaction was found to proceed linearly for at least the first 45 min of incubation, and the rate was proportional to the ethanol concentrations (5-20 mM> and to the tissue concentrations (6.7-20%). As shown in Fig. 2, the “spontaneous” formation of AcH was lowest in blood, and higher in liver and brain supernatants, with the mouse brain supernatants displaying the highest formation rates. Thiourea was proven to be an efficient inhibitor of nonenzymatic AcH formation in liver and brain supernatants, but no noticeable inhibition was obtained in human and rat blood. Moreover, the use of thiourea in the determination of blood AcH seriously reduced the AcH recovery (Fig. 3). As can be seen in Fig. 3, this effect was only slight in the liver the brain PCA supernatants.
10
20
30 THIOUREA
LO
50
60
70
80
(rnb.7 J
FIG. 2. The effect of thiourea on the ethanol-induced nonenzymatic formation of acetaldehyde in tissue supernatants. PCA supematants containing 10 mM ethanol were made from human blood (O), rat blood (A), rat liver (O), rat brain (A), and mouse brain (m). as described in the Materials and Methods section. The AcH-formation rates were calculated from the linear increase in the AcH concentration during 45 min at 65°C.
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ERIKSSON,
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THIOUREA
(mM)
FIG. 3. The effect of thiourea on the acetaldehyde recovery. PCA supernatants containing 100 pM AcH were made from human blood (0), rat blood (A), rat liver (O), rat brain (A), and mouse brain (a), as described in the Materials and Methods section. Supematants without thiourea were used as base values (100% recovery). (0) Represents the blank PCA solution.
DISCUSSION
Acetaldehyde
Recovery from Blood
Three main factors should be considered in the determination of blood AcH concentrations. The first includes the problems of an artificially increased AcH recovery caused by nonenzymatic AcH formation induced by ethanol; the second is the various possibilities of recovery losses; the third concerns the distribution of AcH in the blood. The first comprehensive study of the nonenzymatic formation of AcH was reported by Truitt (2), who measured AcH in precipitated blood by means of a head-space gas chromatographic method that was almost identical to the one used in this work. Truitt’s main conclusions were that, although the production of AcH required the presence of ethanol, the ethanol itself was not the source of the AcH, and AcH production could be inhibited by using protein-free filtrates. In preliminary experiments, we also found an extremely high production of AcH when the protein precipitations were not centrifuged away, and, subsequently, we used only protein-free acid supernatants. Sippel(9) has shown that a slow production of AcH also occurs in protein-free blood supernatants from rats in the presence of ethanol (8), and he reported that thiourea is a useful inhibitor for this reaction. The occurrence of ethanol-induced nonenzymatic AcH formation in both human and rat blood PCA supernatants was confirmed in the present
HEAD-SPACE
CHROMATOGRAPHY
OF ACETALDEHYDE
123
study. Here, however, thiourea had no inhibitory effect on the formation, which implies that the reaction mechanism must be one other than the ascorbic acid-catalyzed reaction proposed by Sippel (8). Thiourea would, perhaps, be useful with animals containing enough ascorbic acid in their blood to produce the ascorbic acid-catalyzed reaction, but excessive concentrations of thiourea (>20 mM) must be avoided because it reduces the recovery of AcH (Fig. 3). Hemolyzation of the blood seems to be a better way of determining blood AcH in rats, because the ethanol-induced increase of the AcH recovery was less than that when the PCA-supernatants were used (Table 1). Hemolyzation could not be used with human blood, however, because of the large amount of AcH that was formed, as has been reported by Truitt (2). Beside the use of excessive thiourea, another procedure that resulted in a reduced AcH recovery was the addition of AcH before, instead of after, the precipitation. An explanation could be that, before precipitation, AcH is rapidly oxidized in the blood. Even if such a reaction has been reported (l), and proven to exist at 37°C in preliminary experiments, this reaction alone is not likely to explain the 10% loss of recovery that occurred within 15 set at 0°C. The experiments in which the time between AcH addition and precipitation or hemolyzation was varied (Fig. 1) showed that the most probable explanation for the AcH losses was a rapid thermolabile binding of AcH in the blood. This finding suggests that binding may be possible in vivo, resulting in a nonuniform distribution of AcH in the blood in vivo. We are continuing to study the binding of AcH in blood. Acetaldehyde
Recovery
from
Liver and Brain
The determination of liver and brain AcH concentrations has not previously been the subject of any major study. Sippel has demonstrated a strong nonenzymatic AcH formation in liver PCA supernatants in the presence of ethanol (8), which was efficiently inhibited by thiourea (9). This finding was confirmed in the present study, and the presence of thiourea was also found necessary for inhibition of nonenzymatic AcH formation during the determination of brain AcH concentrations. Mouse brain was investigated because several recent papers have reported high brain AcH concentrations during ethanol intoxication (lo12). The amount of AcH found in brain supernatants to which ethanol had been added (Fig. 2) was of such an order that it could easily explain most of the high brain AcH levels reported for both rats and mice (10-15).
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