ANALYTICAL
BIOCHEMISTRY
84,
Determination Automated
384-392 (1978)
of Acetaldehyde in Blood Using Distillation and Fluorometry
ALLAN R. STOWELL,KATHRYN E. CROW,ROBERT AND RICHARD D. BATT' Department
of Chemistry, Biochemistry Palmerston North,
and Biophysics, New Zealand
M. GREENWAY, Massey
University,
Received March 3 1, 1977; accepted August 24, 1977 A sensitive enzymic method for the determination of acetaldehyde in human blood has been developed. The method may be operated in a semiautomated or fully automated mode and involves continuous-flow distillation of samples with fluorometry. Levels of acetaldehyde between 0.5 and 20 kmol/liter in blood may be determined, using either yeast or sheep liver aldehyde dehydrogenases.
As part of a continuing study on the metabolism of ethanol in humans, a sensitive and reliable method for the determination of acetaldehyde in blood was required. The method commonly used involves gas chromatography (1,2) and this technique has been used by the authors for the simultaneous determination of acetaldehyde and ethanol in human blood samples. Although sensitive and specific, gas chromatography has not been completely reliable, primarily because of the irreversible binding of acetaldehyde to column packings. It was therefore decided to develop an alternative analytical procedure. Although it had been suggested by Lundquist in 1958 (3) that distillation of biological samples (such as deproteinized blood supematants) followed by fluorometric determination of NADH produced enzymatically ‘may provide a sensitive method for acetaldehyde assay, no account of such a method has been reported. Distillation had been suggested as a means of removing interfering substances and fluorometry to provide the required sensitivity. The continuous-flow manifold for the distillation of volatile aldehydes and ketones in a stream of nitrogen, as described by Duncombe and Shaw (4), was found to operate satisfactorily with samples containing very low concentrations of acetaldehyde, and this was combined with enzymatic NAD-linked oxidation and fluorometric measurement of NADH to give a simple and reliable assay which could be used routinely. 1 To whom reprint requests should be addressed. 0003-2697/78/0842-0384$02.00/O Copyri.ght All rights
0 1978 by Academic F’ress. Inc. of reproduction in any form reserved.
384
AUTOMATED
ACETALDEHYDE
MATERIALS
ASSAY
385
AND METHODS
Reagents
Sheep liver cytoplasmic aldehyde dehydrogenase (EC 1.2.1.3) was prepared by the methods described by Crow et al (5). Solutions containing 500-3000 enzyme units/ml2 were stored at 4°C for up to 2 months in phosphate buffer, pH 7.3, and 0.1% (v/v) mercaptoethanol. Yeast aldehyde dehydrogenase (EC 1.2.1.5), grade II, was obtained from Sigma Chemical Co., St. Louis, Missouri. Acetaldehyde and NAD+ were obtained from BDH Chemicals Ltd. (Poole, United Kingdom). Acetaldehyde was redistilled before use and stored at 4°C for up to 6 months. Dilute solutions were freshly prepared when required. Blood used for recovery experiments was either fresh heparinized human venous blood or outdated blood bank blood, no difference being found between the two in the recovery of added acetaldehyde. Apparatus
A Turner (Model 430) spectrofluorometer was used for all fluorescence determinations. An automated chemistry adapter (No. 430-035)3 was used for automated fluorescence determinations together with an RDK potentiometric chart recorder. For manual determinations, a single-position sample compartment (No. 430-011)4 was used with round, borosilicate glass cuvettes (12 X 75 mm). An Ismatek mp-25 peristaltic pump (Ismatek sa. Zurich, Switzerland) was fitted with tygon tubing. Glass coils, cactus fittings, and otherjunctions which were used with the Autoanalyser Sampler II were standard items of Technicon equipment. The heating bath employed was a standard laboratory thermostated water bath capable of maintaining stable temperatures up to 95 + 1°C. Semiautomated
Assay
Manifold. The manifold illustrated in Fig. 1 was used for the semiautomated assay of acetaldehyde, with manual collection of acetaldehyde samples in buffer into fluorometer cuvettes from the second gas/liquid separator (g). The transit time for a sample was estimated by timing a bubble from the sampler to the coil (c) and from the separator (g) to the sample outlet, 2 set being added to allow for the passage time in the nitrogen stream. Sample collection was phased so that collection extended * One unit = 0.001 absorbance units/min nmol of NADWmin. 3 G. K. Turner Associates catalog number. ’ G. K. Turner Associates catalog number.
and
is equivalent
to the formation
of 0.483
386
STOWELL
ET AL. P-WASTE
L
FIG. 1. Flow diagram of the acetaldehyde distillation manifold. (a) Single waterjacketed coil; (b) DO cactus; (c) single mixing coil; (d) Dl cactus; (e) Double mixing coil; (f) (g) gas/liquid separators, described in Ref. (4).
from 5 set before the estimated time of appearance of acetaldehyde in the outlet tube to 5 set after its estimated disappearance at the outlet. The nitrogen flow rate could be varied between 100 and 1200 ml/min and the heating bath temperature could be kept constant at any temperature between 30 and 95°C. Procedure. The buffer used for all assays was 15 mM pyrophosphate, pH 9.3. Sampling began after the temperature of the heating bath and the N2 flow rate had both been adjusted to desired values and the buffer had been pumping through the system for at least 5 min. Sample volume was 1.5-2.0 ml and the sampling rate was 20/hr with a 1:2 sample:wash ratio. Sample collection was carried out as described above using stopwatch timing and, after collection, each sample was covered with Parafilm (Gallenkamp, United Kingdom) to prevent loss of acetaldehyde. Following the completion of sample collection, 0.5 ml of a freshly prepared solution containing NAD+ (1.5 mM) and sheep liver cytoplasmic aldehyde dehydrogenase (84 units/ml) was added to each sample and the two solutions were mixed by inversion. Complete conversion of acetaldehyde in the sample to acetate was obtained within 15-20 min at room temperature and the fluorescence due to
AUTOMATED
ACETALDEHYDE
ASSAY
387
reduced NAD+ was then determined using an excitation wavelength of 350 nm and an emission wavelength of 460 nm. A standard curve was prepared for each assay using aqueous acetaldehyde standards. Although the yeast enzyme was used mainly for the fully automated procedure, it was found to be suitable for use in the semiautomated procedure, if 100 mM pyrophosphate buffer, pH 8.0, was used and K+ (700 mM) and mercaptoethanol (O.l%, v/v) were added to the enzyme/ NAD+ reagent. Fully Automated Assay Manifold. The same distillation manifold used for the semiautomated assay method was used in conjunction with an analytical manifold (Fig. 2). To avoid wastage of enzyme from continuous pumping, an extra sampling probe was attached to the existing moving arm of the sampler by an extension arm to ensure that enzyme solution was aspirated only when the sample was being pumped. A separate wash reservoir was constructed for the enzyme sampler. The transmission
>d
FIG. 2. Analytical manifold for the fully automated assay system. (a) Single mixing coil; (b) A0 junction; (c) H3 cactus; (d) 40-ft delay coil (1.6-mm i.d.) thermostated at 37°C.
388
STOWELL
ET AL.
tubing carrying the enzyme solution was adjusted in length so that the entry of enzyme into the H3 cactus coincided with the entry of acetaldehyde from the sample. Procedure. Since satisfactory results could be achieved only by using yeast aldehyde dehydrogenase (discussed in Results) with the fully automated system, some changes in reagents were necessary. The buffer used was 100 mM pyrophosphate, pH 8.0, and because the yeast enzyme is K+ activated, KC1 (700 mM) was included in the NAD+ solution (1.5 mM). The enzyme solution was prepared by dissolving crystalline enzyme in 100 mM pyrophosphate buffer, pH 8.0, containing 0.1% (v/v) mercaptoethanol; an enzyme concentration found to give satisfactory standard curves was 0.4 units/ml.5 The lag time from initiation of sampling to recorder response was approximately 15 min. The enzyme solution was kept ice-cold throughout the operation period of the manifold. Treatment of blood samples prior to assay. Blood samples were deproteinized with an equal volume of ice-cold 1 M perchloric acid and protein-free supematants obtained by centrifugation were assayed without further treatment. Supematants not assayed immediately were stored in sealed autoanalyzer cups for up to 24 hr at 4°C. For recovery experiments, concentrated acetaldehyde solutions were added to blood samples so that the sample volume was increased by no more than 1%. Deproteinization was carried out after thorough mixing of the acetaldehyde and blood. RESULTS
Characteristics
of the Distillation
Manifold
The results presented in this section have all been obtained with the semiautomated assay method using the sheep liver enzyme. Effect of NzfEow rate on acetaldehyde transferfrom samples. At a distillation temperature of 90°C the efficiency of transfer of acetaldehyde from sample to buffer was found to increase with decreasing nitrogen flow rate between 1200 and 100 ml/min. There was a 100% difference between the acetaldehyde transfer efficiencies at these maximum and minimum flow rates. In order to obtain maximum sensitivity, the nitrogen flow rate was routinely set and maintained at a level between 100 and 400 ml/min. Normally, the rate was 130 mYmin. Determination of optimum distillation temperature. Optimum distillation temperature was determined by taking duplicate lo-PM acetaldehyde 5 One enzyme unit (as defined by the Sigma Chemical Co.) will oxidize 1.0 pmol of acetaldehyde to acetic acid per minute at pH 8.0 and 25°C in the presence of NAD+, K+, and P-mercaptoethanol.
AUTOMATED
ACETALDEHYDE
ASSAY
389
standards and blanks through the normal assay procedure at different heating-bath temperatures using a nitrogen flow rate of 130 mYmin for all samples. A heating-bath temperature of 90°C was found to give optimum distillation efficiency. Condenser temperature. Refrigeration of the water entering the condenser coil jacket to 2°C increased acetaldehyde transfer from sample to buffer by only 6% compared with the use of tap water flowing through the jacket at a temperature of approximately 17°C. Refrigeration was therefore considered unnecessary. E’jkiency of distillation. Using established conditions of temperature and nitrogen flow rate (90°C and 130 mYmin), the efficiency with which acetaldehyde was transferred from sample to buffer was determined. Results obtained by distillation of acetaldehyde standards using the semiautomated procedure were compared with values obtained by direct addition of acetaldehyde standards to buffer before the addition of NAD+ and the enzyme. The efficiency obtained was 46 2 3%. Standard curves. Initial attempts to produce satisfactory standard curves using a 2: 1 sample:wash ratio were unsuccessful. Reproducibility was poor, especially when standards were run in a random order. Use of a 1:2 sample:wash ratio was found to improve reproducibility markedly. The standard curve shown in Fig. 3 was produced by randomly sampling standards and blanks giving maximum errors, associated with single measurements of acetaldehyde in the range of O-20 FM in blood, of about to.5 PM. The significance of levels below 0.5 PM could be determined by assaying such samples several times. The assay system as described will also measure acetaldehyde concentrations up to 100 PM (corresponding to 200 FM in blood) with some deviation from linearity. Accuracy. Table 1 shows that the measurement of acetaldehyde added to blood is close to 100% compared with aqueous standards. Good recoveries of acetaldehyde from blood could be achieved only if the blood was chilled (O-PC) before acetaldehyde addition. It is well known that acetaldehyde added to blood at higher temperatures rapidly disappears and that cooling of blood samples inhibits this disappearance (6,7). No correction was made for the solid content of blood when calculating recoveries, and since identical recoveries were obtained from plasma samples and controls it would seem that no correction is necessary. The high recovery further suggests that the efficiency of acetaldehyde distillation from perchloric supematants is the same as that from aqueous solutions. Acetaldehyde in concentrations of up to 200 PM was found to be stable in deproteinized blood or plasma supematants for at least 24 hr if supematants were kept in sealed containers at 4°C. Specificity. Since both sheep liver and yeast aldehyde dehydrogenases
390
STOWELL
0
2.5
ET AL.
5.0 ACETALDEHYDE
7.5
IO.0
pM
FIG. 3. Standard curve obtained using the semiautomated assay procedure. The mean and range of five determinations are represented for each concentration. The figures by each point represent one standard deviation.
react with a number of aldehydes (3,5), the methods described are specific only for volatile aldehydes that will react with these enzymes. It is unlikely that human blood would contain any volatile aldehydes other than acetaldehyde, which is usually found in measurable quantities only during the metabolism of ethanol. Using the sheep liver enzyme, aqueous solutions of propionaldehyde could be assayed with the same efficiency as acetaldehyde, and butyraldehyde at an 81% efficiency compared with acetaldehyde. Formaldehyde was found to react very slowly with the enzyme when samples containing O-10 FM concentrations were carried through the normal assay procedure; stable fluorescence readings could not be obtained. No reaction was obtained with acetone at concentrations of up to 130 PM, and ethanol at high concentrations (8%) had no effect on the assay of acetaldehyde. Ethanol interference only occurred when blood supernatants prepared from blood containing ethanol were assayed. This was due to the production of acetaldehyde while the blood sample was being deproteinized and not to the assay procedure. When assaying blood samples containing ethanol it was necessary to
AUTOMATED
ACETALDEHYDE TABLE
MEASUREMENT
OF ACETALDEHYDE
Acetaldehyde added to whole blood (pmoYliter)
1 ADDED
TO WHOLE
BLOOD
Acetaldehyde measured (PmoYliter) Mean”
0.0
4.0 12.0 20.0
391
ASSAY
0"
4.0 12.0 19.8
Range -
11.8-12.3 19.0-20.4
a N = 4 for each acetaldehyde concentration. b No significant levels of acetaldehyde were found in the blank blood samples used for this experiment. However levels of about 1.0 pM were commonly found in other blank samples.
construct a correction curve by adding varying amounts of ethanol to ethanol-free blood samples, deproteinizing, and assaying for acetaldehyde in the normal way. Acetaldehyde formed during the deproteinization step was proportional to blood ethanol concentration. If the blood ethanol concentrations of actual samples were known, then an appropriate correction could be made, a procedure employed by other workers (1). The Fully Automated Assay
Initial attempts to use the sheep liver enzyme in the fully automated system were unsuccessful. Sensitivity was found to drop markedly with time over a 2- to 3-hr running period, and this may have been due to a loss of enzyme activity in a dilute solution. Use of the yeast enzyme overcame this problem and satisfactory sensitivity and reproducibility were achieved (Fig. 4). The effect of ethanol on the assay, using the yeast enzyme, was found to be the same as when the sheep enzyme was used. DISCUSSION
The sensitivity of these methods (minimum detectable level CO.25 PM) is a considerable improvement on previously published enzymatic methods for acetaldehyde determinations (3,8,9), the most sensitive published method being that of Lundquist (3) which permitted detection of acetaldehyde at a minimum level of 0.1 &ml (2.3 PM) in plasma. The increased sensitivity makes the methods eminently suitable for the measurement of blood acetaldehyde in subjects after consuming ethanol (up to 1 g/kg body weight) when the acetaldehyde level has been found to range from cl-20 PM (unpublished results). The distillation step employed in this assay makes it responsive to volatiles only and so more specific than methods involving direct addition
392
STOWELL
ET AL.
ACETALDEHYDE
>
PM
FIG. 4. Standard curve obtained using the fully automated assay system. The mean and range of five determinations are represented for each acetaldehyde concentration. The figures by each point represent one standard deviation.
of biological extracts to reaction mixtures (3,8,9). Such methods (3,8,9) also require that the protein precipitant be neutralized prior to mixing of the sample with the enzyme, and this step has been eliminated. The semiautomated method has been used routinely for the assay of blood acetaldehyde concentrations in the range O-20 PM, and is convenient to use if samples number no more than about 20. For larger sample numbers, it is more efficient to use the fully automated procedure. ACKNOWLEDGMENT This research project was supported, in part, by a grant from the Medical Research Council of New Zealand.
REFERENCES 1. Korsten, M. A., Matsuzaki, S., Feinman, L., and Lieber, C. S. (1975) N. Engl. J. Med. 292, 386-389. 2. Eriksson, C. J. P. (1975) in The Role of Acetaldehyde in the Actions of Ethanol (Lindros, K. O., and Eriksson, C. J. P., eds.), pp. 9-18, Kauppakijapaino, Helsinki. 3. Lundquist, F. (1958) Biochemistry 68, 172-177. 4. Duncombe, R. E., and Shaw, W. H. C. (1966) in Automation in Analytical Chemistry (Kawaran, E., Jerome, H., and Stamm, O., eds.), Vol. II, pp. 15-18, Mediad Inc., White Plains, New York. 5. Crow, K. E., Kitson, T. M., MacGibbon, A. K. H., and Batt, R. D. (1974) Biochim. Biophys. Acta 350, 121-128. 6. Stotz, E. (1943)J. Biol. Chem. 148, 585-591. 7. Duritz, G., and Truitt, E. B. (1964) Quart. J. Stud. Alcohol 25, 498-510. 8. Tottmar, O., and Marchner, H. (1976) Acta Pharmacol. Toxicol. 38, 366-375. 9. Bemt, E., and Bergmeyer, H. U. (1974) in Methods of Enzymatic Analysis (Bergmeyer, H. U., ed.), Vol. 3, pp. 1506-1509, Academic Press, New York and London.
BIOCHEMISTRY
84,
Determination Automated
384-392 (1978)
of Acetaldehyde in Blood Using Distillation and Fluorometry
ALLAN R. STOWELL,KATHRYN E. CROW,ROBERT AND RICHARD D. BATT' Department
of Chemistry, Biochemistry Palmerston North,
and Biophysics, New Zealand
M. GREENWAY, Massey
University,
Received March 3 1, 1977; accepted August 24, 1977 A sensitive enzymic method for the determination of acetaldehyde in human blood has been developed. The method may be operated in a semiautomated or fully automated mode and involves continuous-flow distillation of samples with fluorometry. Levels of acetaldehyde between 0.5 and 20 kmol/liter in blood may be determined, using either yeast or sheep liver aldehyde dehydrogenases.
As part of a continuing study on the metabolism of ethanol in humans, a sensitive and reliable method for the determination of acetaldehyde in blood was required. The method commonly used involves gas chromatography (1,2) and this technique has been used by the authors for the simultaneous determination of acetaldehyde and ethanol in human blood samples. Although sensitive and specific, gas chromatography has not been completely reliable, primarily because of the irreversible binding of acetaldehyde to column packings. It was therefore decided to develop an alternative analytical procedure. Although it had been suggested by Lundquist in 1958 (3) that distillation of biological samples (such as deproteinized blood supematants) followed by fluorometric determination of NADH produced enzymatically ‘may provide a sensitive method for acetaldehyde assay, no account of such a method has been reported. Distillation had been suggested as a means of removing interfering substances and fluorometry to provide the required sensitivity. The continuous-flow manifold for the distillation of volatile aldehydes and ketones in a stream of nitrogen, as described by Duncombe and Shaw (4), was found to operate satisfactorily with samples containing very low concentrations of acetaldehyde, and this was combined with enzymatic NAD-linked oxidation and fluorometric measurement of NADH to give a simple and reliable assay which could be used routinely. 1 To whom reprint requests should be addressed. 0003-2697/78/0842-0384$02.00/O Copyri.ght All rights
0 1978 by Academic F’ress. Inc. of reproduction in any form reserved.
384
AUTOMATED
ACETALDEHYDE
MATERIALS
ASSAY
385
AND METHODS
Reagents
Sheep liver cytoplasmic aldehyde dehydrogenase (EC 1.2.1.3) was prepared by the methods described by Crow et al (5). Solutions containing 500-3000 enzyme units/ml2 were stored at 4°C for up to 2 months in phosphate buffer, pH 7.3, and 0.1% (v/v) mercaptoethanol. Yeast aldehyde dehydrogenase (EC 1.2.1.5), grade II, was obtained from Sigma Chemical Co., St. Louis, Missouri. Acetaldehyde and NAD+ were obtained from BDH Chemicals Ltd. (Poole, United Kingdom). Acetaldehyde was redistilled before use and stored at 4°C for up to 6 months. Dilute solutions were freshly prepared when required. Blood used for recovery experiments was either fresh heparinized human venous blood or outdated blood bank blood, no difference being found between the two in the recovery of added acetaldehyde. Apparatus
A Turner (Model 430) spectrofluorometer was used for all fluorescence determinations. An automated chemistry adapter (No. 430-035)3 was used for automated fluorescence determinations together with an RDK potentiometric chart recorder. For manual determinations, a single-position sample compartment (No. 430-011)4 was used with round, borosilicate glass cuvettes (12 X 75 mm). An Ismatek mp-25 peristaltic pump (Ismatek sa. Zurich, Switzerland) was fitted with tygon tubing. Glass coils, cactus fittings, and otherjunctions which were used with the Autoanalyser Sampler II were standard items of Technicon equipment. The heating bath employed was a standard laboratory thermostated water bath capable of maintaining stable temperatures up to 95 + 1°C. Semiautomated
Assay
Manifold. The manifold illustrated in Fig. 1 was used for the semiautomated assay of acetaldehyde, with manual collection of acetaldehyde samples in buffer into fluorometer cuvettes from the second gas/liquid separator (g). The transit time for a sample was estimated by timing a bubble from the sampler to the coil (c) and from the separator (g) to the sample outlet, 2 set being added to allow for the passage time in the nitrogen stream. Sample collection was phased so that collection extended * One unit = 0.001 absorbance units/min nmol of NADWmin. 3 G. K. Turner Associates catalog number. ’ G. K. Turner Associates catalog number.
and
is equivalent
to the formation
of 0.483
386
STOWELL
ET AL. P-WASTE
L
FIG. 1. Flow diagram of the acetaldehyde distillation manifold. (a) Single waterjacketed coil; (b) DO cactus; (c) single mixing coil; (d) Dl cactus; (e) Double mixing coil; (f) (g) gas/liquid separators, described in Ref. (4).
from 5 set before the estimated time of appearance of acetaldehyde in the outlet tube to 5 set after its estimated disappearance at the outlet. The nitrogen flow rate could be varied between 100 and 1200 ml/min and the heating bath temperature could be kept constant at any temperature between 30 and 95°C. Procedure. The buffer used for all assays was 15 mM pyrophosphate, pH 9.3. Sampling began after the temperature of the heating bath and the N2 flow rate had both been adjusted to desired values and the buffer had been pumping through the system for at least 5 min. Sample volume was 1.5-2.0 ml and the sampling rate was 20/hr with a 1:2 sample:wash ratio. Sample collection was carried out as described above using stopwatch timing and, after collection, each sample was covered with Parafilm (Gallenkamp, United Kingdom) to prevent loss of acetaldehyde. Following the completion of sample collection, 0.5 ml of a freshly prepared solution containing NAD+ (1.5 mM) and sheep liver cytoplasmic aldehyde dehydrogenase (84 units/ml) was added to each sample and the two solutions were mixed by inversion. Complete conversion of acetaldehyde in the sample to acetate was obtained within 15-20 min at room temperature and the fluorescence due to
AUTOMATED
ACETALDEHYDE
ASSAY
387
reduced NAD+ was then determined using an excitation wavelength of 350 nm and an emission wavelength of 460 nm. A standard curve was prepared for each assay using aqueous acetaldehyde standards. Although the yeast enzyme was used mainly for the fully automated procedure, it was found to be suitable for use in the semiautomated procedure, if 100 mM pyrophosphate buffer, pH 8.0, was used and K+ (700 mM) and mercaptoethanol (O.l%, v/v) were added to the enzyme/ NAD+ reagent. Fully Automated Assay Manifold. The same distillation manifold used for the semiautomated assay method was used in conjunction with an analytical manifold (Fig. 2). To avoid wastage of enzyme from continuous pumping, an extra sampling probe was attached to the existing moving arm of the sampler by an extension arm to ensure that enzyme solution was aspirated only when the sample was being pumped. A separate wash reservoir was constructed for the enzyme sampler. The transmission
>d
FIG. 2. Analytical manifold for the fully automated assay system. (a) Single mixing coil; (b) A0 junction; (c) H3 cactus; (d) 40-ft delay coil (1.6-mm i.d.) thermostated at 37°C.
388
STOWELL
ET AL.
tubing carrying the enzyme solution was adjusted in length so that the entry of enzyme into the H3 cactus coincided with the entry of acetaldehyde from the sample. Procedure. Since satisfactory results could be achieved only by using yeast aldehyde dehydrogenase (discussed in Results) with the fully automated system, some changes in reagents were necessary. The buffer used was 100 mM pyrophosphate, pH 8.0, and because the yeast enzyme is K+ activated, KC1 (700 mM) was included in the NAD+ solution (1.5 mM). The enzyme solution was prepared by dissolving crystalline enzyme in 100 mM pyrophosphate buffer, pH 8.0, containing 0.1% (v/v) mercaptoethanol; an enzyme concentration found to give satisfactory standard curves was 0.4 units/ml.5 The lag time from initiation of sampling to recorder response was approximately 15 min. The enzyme solution was kept ice-cold throughout the operation period of the manifold. Treatment of blood samples prior to assay. Blood samples were deproteinized with an equal volume of ice-cold 1 M perchloric acid and protein-free supematants obtained by centrifugation were assayed without further treatment. Supematants not assayed immediately were stored in sealed autoanalyzer cups for up to 24 hr at 4°C. For recovery experiments, concentrated acetaldehyde solutions were added to blood samples so that the sample volume was increased by no more than 1%. Deproteinization was carried out after thorough mixing of the acetaldehyde and blood. RESULTS
Characteristics
of the Distillation
Manifold
The results presented in this section have all been obtained with the semiautomated assay method using the sheep liver enzyme. Effect of NzfEow rate on acetaldehyde transferfrom samples. At a distillation temperature of 90°C the efficiency of transfer of acetaldehyde from sample to buffer was found to increase with decreasing nitrogen flow rate between 1200 and 100 ml/min. There was a 100% difference between the acetaldehyde transfer efficiencies at these maximum and minimum flow rates. In order to obtain maximum sensitivity, the nitrogen flow rate was routinely set and maintained at a level between 100 and 400 ml/min. Normally, the rate was 130 mYmin. Determination of optimum distillation temperature. Optimum distillation temperature was determined by taking duplicate lo-PM acetaldehyde 5 One enzyme unit (as defined by the Sigma Chemical Co.) will oxidize 1.0 pmol of acetaldehyde to acetic acid per minute at pH 8.0 and 25°C in the presence of NAD+, K+, and P-mercaptoethanol.
AUTOMATED
ACETALDEHYDE
ASSAY
389
standards and blanks through the normal assay procedure at different heating-bath temperatures using a nitrogen flow rate of 130 mYmin for all samples. A heating-bath temperature of 90°C was found to give optimum distillation efficiency. Condenser temperature. Refrigeration of the water entering the condenser coil jacket to 2°C increased acetaldehyde transfer from sample to buffer by only 6% compared with the use of tap water flowing through the jacket at a temperature of approximately 17°C. Refrigeration was therefore considered unnecessary. E’jkiency of distillation. Using established conditions of temperature and nitrogen flow rate (90°C and 130 mYmin), the efficiency with which acetaldehyde was transferred from sample to buffer was determined. Results obtained by distillation of acetaldehyde standards using the semiautomated procedure were compared with values obtained by direct addition of acetaldehyde standards to buffer before the addition of NAD+ and the enzyme. The efficiency obtained was 46 2 3%. Standard curves. Initial attempts to produce satisfactory standard curves using a 2: 1 sample:wash ratio were unsuccessful. Reproducibility was poor, especially when standards were run in a random order. Use of a 1:2 sample:wash ratio was found to improve reproducibility markedly. The standard curve shown in Fig. 3 was produced by randomly sampling standards and blanks giving maximum errors, associated with single measurements of acetaldehyde in the range of O-20 FM in blood, of about to.5 PM. The significance of levels below 0.5 PM could be determined by assaying such samples several times. The assay system as described will also measure acetaldehyde concentrations up to 100 PM (corresponding to 200 FM in blood) with some deviation from linearity. Accuracy. Table 1 shows that the measurement of acetaldehyde added to blood is close to 100% compared with aqueous standards. Good recoveries of acetaldehyde from blood could be achieved only if the blood was chilled (O-PC) before acetaldehyde addition. It is well known that acetaldehyde added to blood at higher temperatures rapidly disappears and that cooling of blood samples inhibits this disappearance (6,7). No correction was made for the solid content of blood when calculating recoveries, and since identical recoveries were obtained from plasma samples and controls it would seem that no correction is necessary. The high recovery further suggests that the efficiency of acetaldehyde distillation from perchloric supematants is the same as that from aqueous solutions. Acetaldehyde in concentrations of up to 200 PM was found to be stable in deproteinized blood or plasma supematants for at least 24 hr if supematants were kept in sealed containers at 4°C. Specificity. Since both sheep liver and yeast aldehyde dehydrogenases
390
STOWELL
0
2.5
ET AL.
5.0 ACETALDEHYDE
7.5
IO.0
pM
FIG. 3. Standard curve obtained using the semiautomated assay procedure. The mean and range of five determinations are represented for each concentration. The figures by each point represent one standard deviation.
react with a number of aldehydes (3,5), the methods described are specific only for volatile aldehydes that will react with these enzymes. It is unlikely that human blood would contain any volatile aldehydes other than acetaldehyde, which is usually found in measurable quantities only during the metabolism of ethanol. Using the sheep liver enzyme, aqueous solutions of propionaldehyde could be assayed with the same efficiency as acetaldehyde, and butyraldehyde at an 81% efficiency compared with acetaldehyde. Formaldehyde was found to react very slowly with the enzyme when samples containing O-10 FM concentrations were carried through the normal assay procedure; stable fluorescence readings could not be obtained. No reaction was obtained with acetone at concentrations of up to 130 PM, and ethanol at high concentrations (8%) had no effect on the assay of acetaldehyde. Ethanol interference only occurred when blood supernatants prepared from blood containing ethanol were assayed. This was due to the production of acetaldehyde while the blood sample was being deproteinized and not to the assay procedure. When assaying blood samples containing ethanol it was necessary to
AUTOMATED
ACETALDEHYDE TABLE
MEASUREMENT
OF ACETALDEHYDE
Acetaldehyde added to whole blood (pmoYliter)
1 ADDED
TO WHOLE
BLOOD
Acetaldehyde measured (PmoYliter) Mean”
0.0
4.0 12.0 20.0
391
ASSAY
0"
4.0 12.0 19.8
Range -
11.8-12.3 19.0-20.4
a N = 4 for each acetaldehyde concentration. b No significant levels of acetaldehyde were found in the blank blood samples used for this experiment. However levels of about 1.0 pM were commonly found in other blank samples.
construct a correction curve by adding varying amounts of ethanol to ethanol-free blood samples, deproteinizing, and assaying for acetaldehyde in the normal way. Acetaldehyde formed during the deproteinization step was proportional to blood ethanol concentration. If the blood ethanol concentrations of actual samples were known, then an appropriate correction could be made, a procedure employed by other workers (1). The Fully Automated Assay
Initial attempts to use the sheep liver enzyme in the fully automated system were unsuccessful. Sensitivity was found to drop markedly with time over a 2- to 3-hr running period, and this may have been due to a loss of enzyme activity in a dilute solution. Use of the yeast enzyme overcame this problem and satisfactory sensitivity and reproducibility were achieved (Fig. 4). The effect of ethanol on the assay, using the yeast enzyme, was found to be the same as when the sheep enzyme was used. DISCUSSION
The sensitivity of these methods (minimum detectable level CO.25 PM) is a considerable improvement on previously published enzymatic methods for acetaldehyde determinations (3,8,9), the most sensitive published method being that of Lundquist (3) which permitted detection of acetaldehyde at a minimum level of 0.1 &ml (2.3 PM) in plasma. The increased sensitivity makes the methods eminently suitable for the measurement of blood acetaldehyde in subjects after consuming ethanol (up to 1 g/kg body weight) when the acetaldehyde level has been found to range from cl-20 PM (unpublished results). The distillation step employed in this assay makes it responsive to volatiles only and so more specific than methods involving direct addition
392
STOWELL
ET AL.
ACETALDEHYDE
>
PM
FIG. 4. Standard curve obtained using the fully automated assay system. The mean and range of five determinations are represented for each acetaldehyde concentration. The figures by each point represent one standard deviation.
of biological extracts to reaction mixtures (3,8,9). Such methods (3,8,9) also require that the protein precipitant be neutralized prior to mixing of the sample with the enzyme, and this step has been eliminated. The semiautomated method has been used routinely for the assay of blood acetaldehyde concentrations in the range O-20 PM, and is convenient to use if samples number no more than about 20. For larger sample numbers, it is more efficient to use the fully automated procedure. ACKNOWLEDGMENT This research project was supported, in part, by a grant from the Medical Research Council of New Zealand.
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