BIOCHEMICAI
MEDICINE
18. 308-314 ( 1977)
A Method for Estimation and Concentration
of Acetone Radioactivity in Blood and Urine’
A. C. HAFF AND G. A. REICHARD. Lankenau
Hospital,
Division
of Research,
Philadelphia,
JR. Pennsylvania
19151
Received March 28, 1977
Chemical methods to determine ketone-body concentrations in biological fluids are generally unable to distinguish between acetone and acetoacetate (AcAc)~ because of spontaneous decarboxylation of AcAc to form acetone under conditions of the assay systems employed (1). A method is described for the estimation of acetone radioactivity and concentration in blood and urine. AcAc is enzymatically converted to /3-hydroxybutyrate (fl-OHB), following which acetone is isolated by distillation. Radioactivity in acetone is measured by precipitation of the acetone in the distillate as the Denigks salt which is counted. Acetone concentration is determined in the same distillate by an automated colorimetric procedure. Isolation of acetone as the Denigks salt has frequently been used to assay radioactivity of the ketone bodies (2, 3), however, a problem remains with regard to the determination of acetone concentrations. Many methods have been reported, but the spectrophotometric ones lacked the sensitivity necessary for the accurate estimation of microgram quantities. It was decided to modify the method of Procos (4) to an automated system in which temperature, mixing time, and stability of reagent background could be more easily controlled and monitored. MATERIALS
AND METHODS
Technicon AutoAnalyzer components and manifold construction used are illustrated in Fig. 1. The all-glass distillation apparatus is similar to that described by Weichselbaum and Somogyi (9, but with some modification (Fig. 2). ‘This work was supported by NIH Research Grant AM-13527. *Abbreviations used in this paper: AcAc, acetoacetate: P-OHB, P-hydroxybutyrate.
308 Copyright @ 1977 by Academic Press. Inc. All rights of reproduction in any form reserved
ISSN 0062944
ACETONE
CONCENTRATION
309
IN BLOOD AND URINE sampler
I/
30/hr 1 2 sample wash
double
480 15mm
to
ratlo
nm F/C
FIG. I.
Flow diagram for automated assay of acetone.
Reagents (i) Phosphate buffer, pH 7.0 (100 pmole/ml): (a) 13.6 g of KH,POJliter, (b) 17.4 g of K,HPOJliter. Combine 390 ml of (a) with 610 ml of (b). Adjust pH to 7.0 with 5 N HCl. (ii) NADH (45 pmolelml): 600 mg of NADH (Boehringer Mannheim) in 20 ml of 1% NaHCO,. (iii) p-OHB dehydrogenase: 15 pmole/ml (Boehringer Mannheim). (iv) HgSO,: 7.3 g of red mercuric oxide in 100 ml of 4 N H,SO,. (v) 50% H,SO, (v/v). (vi) Toluene-phosphor scintillation fluid: 5 g of 2.5-diphenyloxazole and 0.3 g of 2.2~p-phenylene bis-5phenyloxazole in 1 liter of toluene. (vii) Bray’s scintillation fluid: 120 g of naphthalene, 8 g of PPO (Packard), 0.4 g of POPOP (Packard), 40 ml of ethylene glycol, 200 ml of methanol. Bring to total volume of 2000 ml with dioxane. (viii) Color reagent: Place 400 ml of 4.8 N (exact) KOH in a beaker. While mixing on a magnetic stirrer, slowly add 5 ml of salicylaldehyde.
310
HAFF AND REICHARLI
FIG. 2. Distillation apparatus consisting of: (A) heating mantle, controlled by rheostat: (B) 250 ml-boiling flask with microseparatory funnel attached to side arm; (C) distillation trap; (D) West condenser, “no hold-up”; (E) angled distillation spout (special order from Lab Glass, N. J.): (F) 25-ml volumetric flask packed in ice.
Allow to stand at room temperature for 1 hr, then filter through No. 12 folded filter paper. This reagent should be prepared fresh daily. (ix) Stock acetone standard (1 @mole/ml): Working acetone standards to cover a range of 0.02-I pmole/ml. All glassware and water used in the preparation of standards must be chilled. Standards should be prepared immediately before use. Conversion of AcAc to POHB Blood samples are collected in heparinized tubes and centrifuged at 4°C. A plasma or urine aliquot (containing not more than 10 pmole of AcAc) is added to a two-necked 250-ml boiling flask containing 50 ml of deionized water, 2 ml of phosphate buffer, 1 ml of NADH, and 0.2 ml of P-OHB dehydrogenase. The microseparatory funnel attached to the side neck contains 2 ml of water. The boiling flask is immediately connected to the distillation apparatus and all joints are water sealed. The tip of the distillation spout is immersed in 5 ml of deionized water contained in a 2%ml volumetric flask packed in ice. Conversion of AcAc to /3-OHB takes place at room temperature over a 2thr incubation period. At the end of the incubation period, 2 ml of H,SO, are layered under the water in the separatory funnel. The stopcock is opened to allow the addition of the sulfuric acid, but the water head is maintained in the funnel. This system has been found to stoichiometri-
ACETONE
CONCENTRATION
IN
BLOOD
AND
URINE
311
tally convert 10 pmole of AcAc to fi-OHB in 29 hr. Following conversion, the mixture is distilled for 20 min at a rate of about 0.8 ml/min. The distillate is brought to a final volume of 25 ml with deionized water. Estimation of Radioactivity in Acetone Twenty milliliters of distillate are placed in a tared, 50-m& screwcapped centrifuge tube and 30 pmole of carrier AcAc are added. Acetone is precipitated as the Deniges salt when 6 ml of HgSOd and 3 ml of 50% H&SO., are added and the sealed tube is boiled for 45 min. The precipitate is washed twice with iced deionized water and then with acetone, and finally dried in vacua over silica gel. We have consistently observed complete recovery of acetone as the Deniges salt with a ratio of 1.16 mg of salt/pmole of AcAc or acetone, a value almost exactly that originally reported by Van Slyke (6). A known amount of Denigbs salt is transferred to a counting vial and suspended in 10 ml of toluene-phosphor scintillation fluid and 0.5 g of thixcin (Baker Castor Oil Co., Bayonne, N. J.). The sealed vials are shaken on a mechanical shaker for 2 hr before counting. Automated
Assay for Acetone
The remaining distillate is analyzed for acetone concentration in accordance with the flow diagram outlined in Fig. 2. Acetone concentrations of the distillates should fall within the optimal range of the color determination of 0.02-l pmole/ml. A freshly prepared set of working standards covering this range is used in each run. Samples are aspirated at the rate of 30/hr, using a cam with a 1:2 sample-to-wash ratio (Technicon Corp.). They are mixed with color reagent and passed into the 40-ft time-delay coil of a heating bath thermostatically controlled at 60°C. Intensity of color is measured photometrically at 480 nm. RESULTS
The validity of the enzymatic conversion of AcAc to P-OHB was substantiated as follows. [3-14C]AcAc was added to acetone-free plasma. Varying amounts of AcAc were added to aliquots of this plasma to give AcAc concentrations of 1,2,5,7, and 10 pmole/ml. The plasma standards were then enzymatically converted under the conditions described. Two milliliters of distillate were counted in Bray’s solution and 20 ml of the distillate were used to prepare a Deniges salt. The procedure was repeated on numerous occasions. Consistently, the absence of radioactivity in the Deniges salt and the distillate indicated that conversion of up to 10 pmole of AcAc was complete after 2) hr of incubation at room temperature. A similar set of standards, containing in addition [2-14Cl acetone and carrier acetone to give a concentration of 2 pmole/ml, was treated in the
312
HAFF
AND
REICHARD
same manner. Recovery of acetone counts from the distillate counted both in Bray’s solution and in the Deniges salt was 96-1040/o. When this experiment was repeated using aqueous standards, essentially the same percentage of counts was recovered. Distillates from the same standards were assayed for acetone concentration using the automated method previously described. The mean recovery for both aqueous and plasma standards was 95%. Color development agrees with Beer’s law over a range of acetone concentration from 0.02 to 1 @mole (Fig. 3): there is little experimental deviation. Noise levels are consistently low, and reproducibility of standard curves on a day-to-day basis has proved to be good. The suspension of Deniges salt in a toluene-PPO-POPOP-thixcin scintillation fluid results in a decrease in counting efficiency associated with counting radioactivity in heterogenous systems (7, 8). Correction for this loss of efficiency can be made arithmetically. An aqueous standard of
7- r I FIG.
3.
Recorder
tracing
of standard
curve
for acetone
determination
ACETONE
CONCENTRATlON
IN BLOOD AND URINE
313
TABLE 1 CORRECTION FOR DECREASE IN COUNTING EFFICIENCY IN HETEROGENOUS SYSTEM dpm [Wlacetone added 6,397 8,739 61,360 32,147 8,607 8,631
dpm recovered from Denigks salt
dpm recovered in distillate 6,419 8,679 65,413 3 1,475 8,588 8,506
4,095 7,149 54,166 23,682 5.970 6,602
Correction factor 1.30 1.21 1.21 1.33 1.44 1.29
[2-*4C] acetone and 30 pmoles of carrier AcAc is distilled and an aliquot of the distillate is counted in Bray’s solution. The acetone in a second aliquot is precipitated as the Denigis salt and counted in the scintillation gel described above. A factor which will correct for the loss in counting efficiency is arrived at by comparison of the two sets of counts. The consistency of this number has been repeatedly demonstrated (Table l), but to eliminate possible error this procedure is carried out with each group of experimental samples. In fed or overnight-fasted humans, plasma acetone concentrations were found to be less than 0.1 mM. Values found in other groups studied are outlined in Table 2. Using a chromatographic technique, Sulway and Malins (9) report similar findings in diabetic ketoacidotic patients. DISCUSSION
In the automated procedure described, the normality of the KOH solution, used in the preparation of the color reagent, is critical. When solutions of KOH with normalities of 4.0,4.4,4.6,4.8, and 5.0 were used in its preparation, color reaction increased markedly with normality. The reagent prepared with 5 N KOH, however, crystallized out at room temperature. To maintain uniformity of standards from day to day, it is TABLE 2 PLASMA ACETONE (@mole/ml)
Nonobese, 3-day fasted Obese, 3-day fasted Obese, 21-day fasted Ketotic diabetic Ketoacidotic diabetic
N
Mean
Range
6 6 3 10 12
0.8 0.3 1.4 0.4 5.0
(0.5-1.4) (0.2-0.5) (1.0-1.7) (0.1-0.9) (1.7-12.4)
314
HAFF AND REICHARD
mandatory that the normality of the KOH solution be exact. In order to ensure constant baseline values, the color reagent should be allowed to stand for I hr at room temperature before use. When used immediately following its preparation. an upward drift in baseline is occasionally observed. SUMMARY
A method is described for the measurement of acetone radioactivity blood and urine. An automated procedure allows for the determination acetone concentration in an aliquot of the same sample.
in of
REFERENCES 1. Peden, V. H.. J. Lab. Clin. Med. 63, 332 (1964). 2. Bates, M. W., Krebs, H. A., and Williamson, D. H., Biochem J. 110, 655 (1968). 3. McGarry, J. D., Guest, M. J., and Foster, D. W., J. Biol. Chem. 245, 4382 (1970). 4. Procos, J., Cfin. Chem. 7, 97 (1961). 5. Weichselbaum, T. E., and Somogyi. M., J. Biol. Chem. 140, 5 (1941). 6. Van Slyke, D. D., J. Biol. Chem. 32, 455 (1917). 7. White, C. G., and Helf, S., Nucleonics 14, 46 (1956). 8. Nathan, D. G., Davidson, J. D., Waggoner, J. C., and Berlin, N. I., J. Lab. Clin. Med. 52, 915 (1958). 9.
Sulway, M. J., and Malins, J. M.. Lancer 2, 736 (1970).
MEDICINE
18. 308-314 ( 1977)
A Method for Estimation and Concentration
of Acetone Radioactivity in Blood and Urine’
A. C. HAFF AND G. A. REICHARD. Lankenau
Hospital,
Division
of Research,
Philadelphia,
JR. Pennsylvania
19151
Received March 28, 1977
Chemical methods to determine ketone-body concentrations in biological fluids are generally unable to distinguish between acetone and acetoacetate (AcAc)~ because of spontaneous decarboxylation of AcAc to form acetone under conditions of the assay systems employed (1). A method is described for the estimation of acetone radioactivity and concentration in blood and urine. AcAc is enzymatically converted to /3-hydroxybutyrate (fl-OHB), following which acetone is isolated by distillation. Radioactivity in acetone is measured by precipitation of the acetone in the distillate as the Denigks salt which is counted. Acetone concentration is determined in the same distillate by an automated colorimetric procedure. Isolation of acetone as the Denigks salt has frequently been used to assay radioactivity of the ketone bodies (2, 3), however, a problem remains with regard to the determination of acetone concentrations. Many methods have been reported, but the spectrophotometric ones lacked the sensitivity necessary for the accurate estimation of microgram quantities. It was decided to modify the method of Procos (4) to an automated system in which temperature, mixing time, and stability of reagent background could be more easily controlled and monitored. MATERIALS
AND METHODS
Technicon AutoAnalyzer components and manifold construction used are illustrated in Fig. 1. The all-glass distillation apparatus is similar to that described by Weichselbaum and Somogyi (9, but with some modification (Fig. 2). ‘This work was supported by NIH Research Grant AM-13527. *Abbreviations used in this paper: AcAc, acetoacetate: P-OHB, P-hydroxybutyrate.
308 Copyright @ 1977 by Academic Press. Inc. All rights of reproduction in any form reserved
ISSN 0062944
ACETONE
CONCENTRATION
309
IN BLOOD AND URINE sampler
I/
30/hr 1 2 sample wash
double
480 15mm
to
ratlo
nm F/C
FIG. I.
Flow diagram for automated assay of acetone.
Reagents (i) Phosphate buffer, pH 7.0 (100 pmole/ml): (a) 13.6 g of KH,POJliter, (b) 17.4 g of K,HPOJliter. Combine 390 ml of (a) with 610 ml of (b). Adjust pH to 7.0 with 5 N HCl. (ii) NADH (45 pmolelml): 600 mg of NADH (Boehringer Mannheim) in 20 ml of 1% NaHCO,. (iii) p-OHB dehydrogenase: 15 pmole/ml (Boehringer Mannheim). (iv) HgSO,: 7.3 g of red mercuric oxide in 100 ml of 4 N H,SO,. (v) 50% H,SO, (v/v). (vi) Toluene-phosphor scintillation fluid: 5 g of 2.5-diphenyloxazole and 0.3 g of 2.2~p-phenylene bis-5phenyloxazole in 1 liter of toluene. (vii) Bray’s scintillation fluid: 120 g of naphthalene, 8 g of PPO (Packard), 0.4 g of POPOP (Packard), 40 ml of ethylene glycol, 200 ml of methanol. Bring to total volume of 2000 ml with dioxane. (viii) Color reagent: Place 400 ml of 4.8 N (exact) KOH in a beaker. While mixing on a magnetic stirrer, slowly add 5 ml of salicylaldehyde.
310
HAFF AND REICHARLI
FIG. 2. Distillation apparatus consisting of: (A) heating mantle, controlled by rheostat: (B) 250 ml-boiling flask with microseparatory funnel attached to side arm; (C) distillation trap; (D) West condenser, “no hold-up”; (E) angled distillation spout (special order from Lab Glass, N. J.): (F) 25-ml volumetric flask packed in ice.
Allow to stand at room temperature for 1 hr, then filter through No. 12 folded filter paper. This reagent should be prepared fresh daily. (ix) Stock acetone standard (1 @mole/ml): Working acetone standards to cover a range of 0.02-I pmole/ml. All glassware and water used in the preparation of standards must be chilled. Standards should be prepared immediately before use. Conversion of AcAc to POHB Blood samples are collected in heparinized tubes and centrifuged at 4°C. A plasma or urine aliquot (containing not more than 10 pmole of AcAc) is added to a two-necked 250-ml boiling flask containing 50 ml of deionized water, 2 ml of phosphate buffer, 1 ml of NADH, and 0.2 ml of P-OHB dehydrogenase. The microseparatory funnel attached to the side neck contains 2 ml of water. The boiling flask is immediately connected to the distillation apparatus and all joints are water sealed. The tip of the distillation spout is immersed in 5 ml of deionized water contained in a 2%ml volumetric flask packed in ice. Conversion of AcAc to /3-OHB takes place at room temperature over a 2thr incubation period. At the end of the incubation period, 2 ml of H,SO, are layered under the water in the separatory funnel. The stopcock is opened to allow the addition of the sulfuric acid, but the water head is maintained in the funnel. This system has been found to stoichiometri-
ACETONE
CONCENTRATION
IN
BLOOD
AND
URINE
311
tally convert 10 pmole of AcAc to fi-OHB in 29 hr. Following conversion, the mixture is distilled for 20 min at a rate of about 0.8 ml/min. The distillate is brought to a final volume of 25 ml with deionized water. Estimation of Radioactivity in Acetone Twenty milliliters of distillate are placed in a tared, 50-m& screwcapped centrifuge tube and 30 pmole of carrier AcAc are added. Acetone is precipitated as the Deniges salt when 6 ml of HgSOd and 3 ml of 50% H&SO., are added and the sealed tube is boiled for 45 min. The precipitate is washed twice with iced deionized water and then with acetone, and finally dried in vacua over silica gel. We have consistently observed complete recovery of acetone as the Deniges salt with a ratio of 1.16 mg of salt/pmole of AcAc or acetone, a value almost exactly that originally reported by Van Slyke (6). A known amount of Denigbs salt is transferred to a counting vial and suspended in 10 ml of toluene-phosphor scintillation fluid and 0.5 g of thixcin (Baker Castor Oil Co., Bayonne, N. J.). The sealed vials are shaken on a mechanical shaker for 2 hr before counting. Automated
Assay for Acetone
The remaining distillate is analyzed for acetone concentration in accordance with the flow diagram outlined in Fig. 2. Acetone concentrations of the distillates should fall within the optimal range of the color determination of 0.02-l pmole/ml. A freshly prepared set of working standards covering this range is used in each run. Samples are aspirated at the rate of 30/hr, using a cam with a 1:2 sample-to-wash ratio (Technicon Corp.). They are mixed with color reagent and passed into the 40-ft time-delay coil of a heating bath thermostatically controlled at 60°C. Intensity of color is measured photometrically at 480 nm. RESULTS
The validity of the enzymatic conversion of AcAc to P-OHB was substantiated as follows. [3-14C]AcAc was added to acetone-free plasma. Varying amounts of AcAc were added to aliquots of this plasma to give AcAc concentrations of 1,2,5,7, and 10 pmole/ml. The plasma standards were then enzymatically converted under the conditions described. Two milliliters of distillate were counted in Bray’s solution and 20 ml of the distillate were used to prepare a Deniges salt. The procedure was repeated on numerous occasions. Consistently, the absence of radioactivity in the Deniges salt and the distillate indicated that conversion of up to 10 pmole of AcAc was complete after 2) hr of incubation at room temperature. A similar set of standards, containing in addition [2-14Cl acetone and carrier acetone to give a concentration of 2 pmole/ml, was treated in the
312
HAFF
AND
REICHARD
same manner. Recovery of acetone counts from the distillate counted both in Bray’s solution and in the Deniges salt was 96-1040/o. When this experiment was repeated using aqueous standards, essentially the same percentage of counts was recovered. Distillates from the same standards were assayed for acetone concentration using the automated method previously described. The mean recovery for both aqueous and plasma standards was 95%. Color development agrees with Beer’s law over a range of acetone concentration from 0.02 to 1 @mole (Fig. 3): there is little experimental deviation. Noise levels are consistently low, and reproducibility of standard curves on a day-to-day basis has proved to be good. The suspension of Deniges salt in a toluene-PPO-POPOP-thixcin scintillation fluid results in a decrease in counting efficiency associated with counting radioactivity in heterogenous systems (7, 8). Correction for this loss of efficiency can be made arithmetically. An aqueous standard of
7- r I FIG.
3.
Recorder
tracing
of standard
curve
for acetone
determination
ACETONE
CONCENTRATlON
IN BLOOD AND URINE
313
TABLE 1 CORRECTION FOR DECREASE IN COUNTING EFFICIENCY IN HETEROGENOUS SYSTEM dpm [Wlacetone added 6,397 8,739 61,360 32,147 8,607 8,631
dpm recovered from Denigks salt
dpm recovered in distillate 6,419 8,679 65,413 3 1,475 8,588 8,506
4,095 7,149 54,166 23,682 5.970 6,602
Correction factor 1.30 1.21 1.21 1.33 1.44 1.29
[2-*4C] acetone and 30 pmoles of carrier AcAc is distilled and an aliquot of the distillate is counted in Bray’s solution. The acetone in a second aliquot is precipitated as the Denigis salt and counted in the scintillation gel described above. A factor which will correct for the loss in counting efficiency is arrived at by comparison of the two sets of counts. The consistency of this number has been repeatedly demonstrated (Table l), but to eliminate possible error this procedure is carried out with each group of experimental samples. In fed or overnight-fasted humans, plasma acetone concentrations were found to be less than 0.1 mM. Values found in other groups studied are outlined in Table 2. Using a chromatographic technique, Sulway and Malins (9) report similar findings in diabetic ketoacidotic patients. DISCUSSION
In the automated procedure described, the normality of the KOH solution, used in the preparation of the color reagent, is critical. When solutions of KOH with normalities of 4.0,4.4,4.6,4.8, and 5.0 were used in its preparation, color reaction increased markedly with normality. The reagent prepared with 5 N KOH, however, crystallized out at room temperature. To maintain uniformity of standards from day to day, it is TABLE 2 PLASMA ACETONE (@mole/ml)
Nonobese, 3-day fasted Obese, 3-day fasted Obese, 21-day fasted Ketotic diabetic Ketoacidotic diabetic
N
Mean
Range
6 6 3 10 12
0.8 0.3 1.4 0.4 5.0
(0.5-1.4) (0.2-0.5) (1.0-1.7) (0.1-0.9) (1.7-12.4)
314
HAFF AND REICHARD
mandatory that the normality of the KOH solution be exact. In order to ensure constant baseline values, the color reagent should be allowed to stand for I hr at room temperature before use. When used immediately following its preparation. an upward drift in baseline is occasionally observed. SUMMARY
A method is described for the measurement of acetone radioactivity blood and urine. An automated procedure allows for the determination acetone concentration in an aliquot of the same sample.
in of
REFERENCES 1. Peden, V. H.. J. Lab. Clin. Med. 63, 332 (1964). 2. Bates, M. W., Krebs, H. A., and Williamson, D. H., Biochem J. 110, 655 (1968). 3. McGarry, J. D., Guest, M. J., and Foster, D. W., J. Biol. Chem. 245, 4382 (1970). 4. Procos, J., Cfin. Chem. 7, 97 (1961). 5. Weichselbaum, T. E., and Somogyi. M., J. Biol. Chem. 140, 5 (1941). 6. Van Slyke, D. D., J. Biol. Chem. 32, 455 (1917). 7. White, C. G., and Helf, S., Nucleonics 14, 46 (1956). 8. Nathan, D. G., Davidson, J. D., Waggoner, J. C., and Berlin, N. I., J. Lab. Clin. Med. 52, 915 (1958). 9.
Sulway, M. J., and Malins, J. M.. Lancer 2, 736 (1970).
