ANALYTICAL
67, 192- 197 (1975)
BIOCHEMISTRY
Comparison
of Automated
of Estimation SUSAN Department
Manual
of Methylmalonic
M. OACE’ of Nutrition,
and
AND SHIRLEY University
Methods Acid
CHIH-HSUAN
of California,
Dn\G,
CHEN~
California
95616
Received November 4. 1974; accepted January 24, I975 An automated method for estimation of methylmalonic acid in urine is described that compares well with results obtained by a manual method. The temperature at which color is developed can be set anywhere between ambient and 90°C without loss of linearity over a certain range of concentrations. The higher temperatures have greater sensitivity but a smaller linear range than do lower incubation-temperatures.
Urinary methylmalonic acid (MMA)-excretion is elevated in pernicious anemia in man (1,2) as well as in experimental vitamin Blz deficiency in rats (3-5). A quantitative calorimetric determination based on combination of MMA with diazotized p-nitroaniline was proposed by Giorgio and Plaut (6) as more convenient for routine use than paper (7) or gas chromatographic (1) techniques. Williams er ~11.(4) introduced a modification of this method to improve linearity and stability and which somewhat alleviated the interference by atmospheric carbon dioxide. Oace and Abbott (5) further modified this reaction by allowing color to develop at 37°C for 30 min rather than at 94°C for 3 min because of the difficulty in standardizing the time and temperature for large numbers of tubes. A major improvement in the assay was reported by Gawthorne et al. (8) who adapted the original Giorgio and PIaut procedure to automated analysis. In this closed system, atmospheric carbon dioxide was excluded completely, and reagent additions as well as heating and cooling times were controlled precisely. In our hands, the setup described by Gawthorne er al. resulted in a poor bubble pattern, incomplete mixing and occasional flocculant precipitates after heating. Minor modification of the flow diagram improved mixing, and reduction of temperature eliminated the occurence of precipitation. Gawthorne et al. (8) warned that full color-development did not occur at lower temperatures. This would not detract from the valid’ Present address: Department of Nutritional Sciences, University of California, Berkeley, CA 94720. * Present address: Division of Nutritional Sciences, Cornell University, Ithaca, NY 14850. 192 Copyright @ 1975 by Academic Press. Inc. All rights of reproduction in any form reserved.
AUTOMATED
METHYLMALONATE
ASSAY
193
ity of the assay if color development were linear. We therefore investigated the effect of different temperatures on linearity and sensitivity of the procedure. Since we previously have published data on MMA excretion of rats utilizing a manual method with color development at 37°C we reanalyzed 157 rat-urine samples with the new procedure in order to determine the comparability of results. MATERIALS
AND
METHODS
Preparation of rut-urine sumple. Identical procedures are used to prepare urine samples for automated and manual color-development. Twenty-four-hour urine samples are collected in flasks containing about 2 milliequivalents (meq) of H&SO, (usually 6 ml of 0.36 N H,SO,) as a preservative and to maintain an acid pH for maximum stability of methylmalonic acid. Samples are filtered through Whatman No. 1 filter paper into graduated cylinders and are diluted to a convenient volume (usually 20-30 ml). After adjustment to pH 6.5 with NaOH, one-tenth the volume is applied to a column (1 -cm diameter and containing a 50-ml reservoir) which has been plugged with glass wool and packed with 1 g of anion exchange resin (AG-3A X4, 200-400 mesh, chloride form: BioRad Laboratories, Richmond, CA). The sample is rinsed twice with 50-ml portions of deionized water, and then the methylmalonic acid is eluted with 30 ml of 0.1 N HCl. One milliliter of this eluate represents l/300 of the original urine sample. This acid eluate or solutions of standard MMA in 0.1 N HCl are appropriate for manual or automated colordevelopment and quantification of MMA. Methylmalonic acid standards. A stock standard solution containing 10 Fmoleslml (10 mM) is prepared by dissolving 1,18 1 mg of methylmalonic acid in 1,000 ml of 0.1 N HCl. Appropriate dilutions are made to provide working standards ranging from 0.05-5.0 pmoleslml (0.05 to 5.0 mM) in 0.1 N HCl. Standards are stable for several months at 4°C. Reagents. Acetate buffer (1.0 M. pH 4.3) and diazo reagent (0.5% sodium nitrite, 0.075% p-nitroaniline, 0.2 M sodium acetate, 4 : 15 : 4) are prepared according to Gawthorne et al. (8). Carbonate-free sodium hydroxide (4 N) is prepared by dilution of 5 N carbonate-free sodium hydroxide (DILUT-IT, J. T. Baker Chem. Co., Phillipsburg, NY) in boiled, deionized water. Solution is stored in a polypropylene flask fitted with a two-hole stopper containing a CO, trap (Mallcosorb, indicating carbon dioxide absorbent packaged in polyethylene tubes, Mallinckrodt, St. Louis, MO). The flow diagram for the manifold used for automated calorimetric determination of MMA in this study is presented in Fig. 1. The differences in this design from that described by Gawthorne et al. (8) are: 1) Introduction of two small air bubbles rather than one large bubble and
194
OACE AND CHEN PUMP CHANNELS
SAMPLE/WASH
SAMPLER l50/HOUR)
(2:l
ratio)
FIG. 1. Flow diagram for automated methylmalonic acid assay apparatus. Sampler, pump, tubing, connectors (Dl and C5) and coils (8. 15, 23 and 400 cm, 2.4 mm inside diameter) were purchased from Technicon Instruments Corp., Tarrytown, NY; spectrophotometer and recorder from Beckman Instruments, Inc., Fullerton, CA; flow cell from A. H. Thomas Co., Philadelphia. PA; constant-temperature circulator for oil bath from Bronwill Scientific. Rochester, NY; CO? trap from Mallinckrodt, St. Louis, MO. Standard manifold tubing for pump channels 1-8 were purchased from Gradko Scientific, Yonkers, NY. Inside diameters (inches) were as follows: 1, 0.040; 2. 0.056; 3, 0.040; 4, 0.065; 5. 0.056; 6, 0.045: 7, 0.065; 8, 0.081.
use of longer mixing-coils to improve bubble pattern; 2) use of a timedelay coil; 3) increased sample concentration and flow-cell length allowing greater sensitivity; and 4) reduction of the oil-bath temperature from 94 to 90°C eliminating occasional precipitates. Manual colordevelopment was carried out according to Oace and Abbott (5). Standard MMA solutions were analyzed at several oil-bath temperatures, ranging from ambient to 95”C, to check on linearity of color development. Recovery experiments also were carried out. Methylmalonic acid concentration was measured in 157 urine samples from vitamin B,,-deficient rats by manual and by automated color-development. RESULTS AND
DISCUSSION
Eflect of Temperature
Standard solutions containing from 0.05-5.0 pmoles of MMA per ml of 0.1 N HCl were analyzed by the automated apparatus with the oilbath temperature set at each of six temperatures from ambient to 95 “C. At 25, 37 and 55°C the response was linear throughout the range of
AUTOMATED
SENSITIVITY
Temperature (“0 ?5
37 55 75 90 95
OF THE
AUTOMATED
METHYLMALONATE
TABLE MMA
195
ASSAY
1 ASSAY
Column eluate or standard n (~moleiml) 0.65 0.35 0.09 0.03 0.025 0.023
AT VARIOUS
TEMPERATURES
Rat urine 2%hr excretion” (pmoleiday) 168 105 21 9 1.5 6.9
” The concentration of MMA in column eluate or standard solutions that would produce a peak height of 0.03 OD, the smallest response quantifiable with our equipment. * Using our procedure for sample preparation (1110 of daily urine sample applied to column and elution of MMA in 30-ml volume), the lower limit of quantification of daily MMA excretion is 300 times the minimum amount of MMA standard detectable per milliliter.
standard concentrations. Color development deviated from linearity at concentrations above 1.0 pmole/ml at 75, 90 and 90°C. Slopes of standard curves derived from these data were progressively steeper with increasing temperatures. Because of the width of the pen and a slight lag in pen response, we considered 0.03 OD to be the limit of sensitivity for quantitative purposes. The sensitivities of the automated procedure at each temperature are presented in Table I. Reports of MMA excretion of vitamin B,,-sufficient rats have been variable probably due to somewhat different diets, ages and methods of MMA determination. However, all investigators (3-5) have reported less than 15 pmoles of MMA excreted per 24 hr among vitamin B,,-sufficient rats. It is important to have the upper limit of normal within the quantifiable range of the assay so that excretions indicative of deficiency are identifiable. Using 15 pmoles/day as the upper limit of normal, it is apparent from the calculations in Table 1 that temperatures of 55°C and below are not sufficiently sensitive to detect this daily excretion rate. It is possible that sensitivity could be improved by prolonging incubation time, but this was not tested. The 95°C incubation-temperature resulted in occasional precipitation of diazotized MMA and is therefore not recommended. Recovery
MMA concentration was measured in a rat-urine sample which, after elution from the ion-exchange column contained 1.20 pmole of MMA per ml of eluate. This urine eluate was mixed with a standard solution of 0.25 pmole of MMA per ml in proportions of 2: 1, 1: 1 and 1: 2. The
196
OACE AND CHEN
recovery of MMA added to the urine sample ranged from 102-l 04%, indicating that the urine eluate did not contain substances that enhanced or depressed color development of the standard MMA solution. Comparison of Marural and Automated Analysis
Since previous work from our laboratory (5) had been conducted using the manual color-development method for MMA estimation, it was important to determine whether or not the new method would introduce quantitative differences. Figure 2 displays a plot of 157 samples of urine from vitamin B,,-deficient rats analyzed by the manual method (5) and by the automated apparatus described in this paper. Samples ranged from 0.03 to 2.0 mmoles of methylmalonic acid per day (0.1-6.7 ~moles/ml of column effluent). The more concentrated samples (i.e., those in excess of 1.0 ~mole/ml after column purification) were diluted with 0.1 N HCI so that they were within the range of the assay. Results obtained by the two methods agreed very closely; the correlation coefficient (0.988) indicated that linearity was highly significant (P < 0.001) and the y-intercept of the regression line (-0.02) deviated only slightly from the origin. In general, when MMA concentration was high (greater than 1.0 mmole/day) the manual method yielded slightly higher results than did the automated method. We attribute this to interference by
miltimoles MMA / day automated method FIG. 2. Comparison of MMA content of 157 rat urine samples as measured by the manual method and the automated method.
AUTOMATED
METHYLMALONATE
197
ASSAY
atmospheric CO, in the case of the manual method. At lower concentrations of MMA (less than 0.4 mmole/day), the automated method yielded slightly higher results than did the manual method. This may be due to some carryover from concentrated samples because of imperfect washout in the automated apparatus. These differences are minor, however, and we consider data determined by the two methods to be comparable. Investigation of the stability of MMA was not a specific objective of this study. However, the automated analyses were carried out on column eluates (in 0.1 N HCl) that had been frozen at - 16°C for 6 months after analysis by the manual method. The similarity of the data derived from the two analyses indicate that MMA is quite stable under these storage conditions. ACKNOWLEDGMENTS The authors thank Candace Sousa and Ronald Simpson for assistance with the assays and Joseph Watson for advice in modifying the manifold design.
REFERENCES 1. COX, E. V., and White, A. M. (1962) Lancet 2, 853. 2. Brozovic, M., Hoffbrand, A. V., Dimitriadou, A., and Mollin. Huematol.
D. L. (1967)
Brit.
J.
13, 1021.
3. Bamess, L. A., Young, D. G., and Nacho, R. (1963) Science 140, 76. 4. Williams, D. L., Spray, G. H., Newman, G. E., and O’Brien, J. R. P. (1969) Brit. J. Nrrtr.
23, 343.
5. Oace, S. M., and Abbott, J. M. (1972) .I. Nufr. 102, 17. 6. Giorgio, A. J., and Plaut, G. W. E. (1965) J. C/in. Lob. Med. 66, 667. 7. Barness, L. A., Young, B. S., Wellman, W. J., Kahn, S. B.. and Williams, W. J. (1963) N. Engl. .I. Med. 268, 144. 8. Gawthome, J. M.. Watson, J., and Stokstad. E. L. R. (1971) Anal. Biochem. 42, 555.
67, 192- 197 (1975)
BIOCHEMISTRY
Comparison
of Automated
of Estimation SUSAN Department
Manual
of Methylmalonic
M. OACE’ of Nutrition,
and
AND SHIRLEY University
Methods Acid
CHIH-HSUAN
of California,
Dn\G,
CHEN~
California
95616
Received November 4. 1974; accepted January 24, I975 An automated method for estimation of methylmalonic acid in urine is described that compares well with results obtained by a manual method. The temperature at which color is developed can be set anywhere between ambient and 90°C without loss of linearity over a certain range of concentrations. The higher temperatures have greater sensitivity but a smaller linear range than do lower incubation-temperatures.
Urinary methylmalonic acid (MMA)-excretion is elevated in pernicious anemia in man (1,2) as well as in experimental vitamin Blz deficiency in rats (3-5). A quantitative calorimetric determination based on combination of MMA with diazotized p-nitroaniline was proposed by Giorgio and Plaut (6) as more convenient for routine use than paper (7) or gas chromatographic (1) techniques. Williams er ~11.(4) introduced a modification of this method to improve linearity and stability and which somewhat alleviated the interference by atmospheric carbon dioxide. Oace and Abbott (5) further modified this reaction by allowing color to develop at 37°C for 30 min rather than at 94°C for 3 min because of the difficulty in standardizing the time and temperature for large numbers of tubes. A major improvement in the assay was reported by Gawthorne et al. (8) who adapted the original Giorgio and PIaut procedure to automated analysis. In this closed system, atmospheric carbon dioxide was excluded completely, and reagent additions as well as heating and cooling times were controlled precisely. In our hands, the setup described by Gawthorne er al. resulted in a poor bubble pattern, incomplete mixing and occasional flocculant precipitates after heating. Minor modification of the flow diagram improved mixing, and reduction of temperature eliminated the occurence of precipitation. Gawthorne et al. (8) warned that full color-development did not occur at lower temperatures. This would not detract from the valid’ Present address: Department of Nutritional Sciences, University of California, Berkeley, CA 94720. * Present address: Division of Nutritional Sciences, Cornell University, Ithaca, NY 14850. 192 Copyright @ 1975 by Academic Press. Inc. All rights of reproduction in any form reserved.
AUTOMATED
METHYLMALONATE
ASSAY
193
ity of the assay if color development were linear. We therefore investigated the effect of different temperatures on linearity and sensitivity of the procedure. Since we previously have published data on MMA excretion of rats utilizing a manual method with color development at 37°C we reanalyzed 157 rat-urine samples with the new procedure in order to determine the comparability of results. MATERIALS
AND
METHODS
Preparation of rut-urine sumple. Identical procedures are used to prepare urine samples for automated and manual color-development. Twenty-four-hour urine samples are collected in flasks containing about 2 milliequivalents (meq) of H&SO, (usually 6 ml of 0.36 N H,SO,) as a preservative and to maintain an acid pH for maximum stability of methylmalonic acid. Samples are filtered through Whatman No. 1 filter paper into graduated cylinders and are diluted to a convenient volume (usually 20-30 ml). After adjustment to pH 6.5 with NaOH, one-tenth the volume is applied to a column (1 -cm diameter and containing a 50-ml reservoir) which has been plugged with glass wool and packed with 1 g of anion exchange resin (AG-3A X4, 200-400 mesh, chloride form: BioRad Laboratories, Richmond, CA). The sample is rinsed twice with 50-ml portions of deionized water, and then the methylmalonic acid is eluted with 30 ml of 0.1 N HCl. One milliliter of this eluate represents l/300 of the original urine sample. This acid eluate or solutions of standard MMA in 0.1 N HCl are appropriate for manual or automated colordevelopment and quantification of MMA. Methylmalonic acid standards. A stock standard solution containing 10 Fmoleslml (10 mM) is prepared by dissolving 1,18 1 mg of methylmalonic acid in 1,000 ml of 0.1 N HCl. Appropriate dilutions are made to provide working standards ranging from 0.05-5.0 pmoleslml (0.05 to 5.0 mM) in 0.1 N HCl. Standards are stable for several months at 4°C. Reagents. Acetate buffer (1.0 M. pH 4.3) and diazo reagent (0.5% sodium nitrite, 0.075% p-nitroaniline, 0.2 M sodium acetate, 4 : 15 : 4) are prepared according to Gawthorne et al. (8). Carbonate-free sodium hydroxide (4 N) is prepared by dilution of 5 N carbonate-free sodium hydroxide (DILUT-IT, J. T. Baker Chem. Co., Phillipsburg, NY) in boiled, deionized water. Solution is stored in a polypropylene flask fitted with a two-hole stopper containing a CO, trap (Mallcosorb, indicating carbon dioxide absorbent packaged in polyethylene tubes, Mallinckrodt, St. Louis, MO). The flow diagram for the manifold used for automated calorimetric determination of MMA in this study is presented in Fig. 1. The differences in this design from that described by Gawthorne et al. (8) are: 1) Introduction of two small air bubbles rather than one large bubble and
194
OACE AND CHEN PUMP CHANNELS
SAMPLE/WASH
SAMPLER l50/HOUR)
(2:l
ratio)
FIG. 1. Flow diagram for automated methylmalonic acid assay apparatus. Sampler, pump, tubing, connectors (Dl and C5) and coils (8. 15, 23 and 400 cm, 2.4 mm inside diameter) were purchased from Technicon Instruments Corp., Tarrytown, NY; spectrophotometer and recorder from Beckman Instruments, Inc., Fullerton, CA; flow cell from A. H. Thomas Co., Philadelphia. PA; constant-temperature circulator for oil bath from Bronwill Scientific. Rochester, NY; CO? trap from Mallinckrodt, St. Louis, MO. Standard manifold tubing for pump channels 1-8 were purchased from Gradko Scientific, Yonkers, NY. Inside diameters (inches) were as follows: 1, 0.040; 2. 0.056; 3, 0.040; 4, 0.065; 5. 0.056; 6, 0.045: 7, 0.065; 8, 0.081.
use of longer mixing-coils to improve bubble pattern; 2) use of a timedelay coil; 3) increased sample concentration and flow-cell length allowing greater sensitivity; and 4) reduction of the oil-bath temperature from 94 to 90°C eliminating occasional precipitates. Manual colordevelopment was carried out according to Oace and Abbott (5). Standard MMA solutions were analyzed at several oil-bath temperatures, ranging from ambient to 95”C, to check on linearity of color development. Recovery experiments also were carried out. Methylmalonic acid concentration was measured in 157 urine samples from vitamin B,,-deficient rats by manual and by automated color-development. RESULTS AND
DISCUSSION
Eflect of Temperature
Standard solutions containing from 0.05-5.0 pmoles of MMA per ml of 0.1 N HCl were analyzed by the automated apparatus with the oilbath temperature set at each of six temperatures from ambient to 95 “C. At 25, 37 and 55°C the response was linear throughout the range of
AUTOMATED
SENSITIVITY
Temperature (“0 ?5
37 55 75 90 95
OF THE
AUTOMATED
METHYLMALONATE
TABLE MMA
195
ASSAY
1 ASSAY
Column eluate or standard n (~moleiml) 0.65 0.35 0.09 0.03 0.025 0.023
AT VARIOUS
TEMPERATURES
Rat urine 2%hr excretion” (pmoleiday) 168 105 21 9 1.5 6.9
” The concentration of MMA in column eluate or standard solutions that would produce a peak height of 0.03 OD, the smallest response quantifiable with our equipment. * Using our procedure for sample preparation (1110 of daily urine sample applied to column and elution of MMA in 30-ml volume), the lower limit of quantification of daily MMA excretion is 300 times the minimum amount of MMA standard detectable per milliliter.
standard concentrations. Color development deviated from linearity at concentrations above 1.0 pmole/ml at 75, 90 and 90°C. Slopes of standard curves derived from these data were progressively steeper with increasing temperatures. Because of the width of the pen and a slight lag in pen response, we considered 0.03 OD to be the limit of sensitivity for quantitative purposes. The sensitivities of the automated procedure at each temperature are presented in Table I. Reports of MMA excretion of vitamin B,,-sufficient rats have been variable probably due to somewhat different diets, ages and methods of MMA determination. However, all investigators (3-5) have reported less than 15 pmoles of MMA excreted per 24 hr among vitamin B,,-sufficient rats. It is important to have the upper limit of normal within the quantifiable range of the assay so that excretions indicative of deficiency are identifiable. Using 15 pmoles/day as the upper limit of normal, it is apparent from the calculations in Table 1 that temperatures of 55°C and below are not sufficiently sensitive to detect this daily excretion rate. It is possible that sensitivity could be improved by prolonging incubation time, but this was not tested. The 95°C incubation-temperature resulted in occasional precipitation of diazotized MMA and is therefore not recommended. Recovery
MMA concentration was measured in a rat-urine sample which, after elution from the ion-exchange column contained 1.20 pmole of MMA per ml of eluate. This urine eluate was mixed with a standard solution of 0.25 pmole of MMA per ml in proportions of 2: 1, 1: 1 and 1: 2. The
196
OACE AND CHEN
recovery of MMA added to the urine sample ranged from 102-l 04%, indicating that the urine eluate did not contain substances that enhanced or depressed color development of the standard MMA solution. Comparison of Marural and Automated Analysis
Since previous work from our laboratory (5) had been conducted using the manual color-development method for MMA estimation, it was important to determine whether or not the new method would introduce quantitative differences. Figure 2 displays a plot of 157 samples of urine from vitamin B,,-deficient rats analyzed by the manual method (5) and by the automated apparatus described in this paper. Samples ranged from 0.03 to 2.0 mmoles of methylmalonic acid per day (0.1-6.7 ~moles/ml of column effluent). The more concentrated samples (i.e., those in excess of 1.0 ~mole/ml after column purification) were diluted with 0.1 N HCI so that they were within the range of the assay. Results obtained by the two methods agreed very closely; the correlation coefficient (0.988) indicated that linearity was highly significant (P < 0.001) and the y-intercept of the regression line (-0.02) deviated only slightly from the origin. In general, when MMA concentration was high (greater than 1.0 mmole/day) the manual method yielded slightly higher results than did the automated method. We attribute this to interference by
miltimoles MMA / day automated method FIG. 2. Comparison of MMA content of 157 rat urine samples as measured by the manual method and the automated method.
AUTOMATED
METHYLMALONATE
197
ASSAY
atmospheric CO, in the case of the manual method. At lower concentrations of MMA (less than 0.4 mmole/day), the automated method yielded slightly higher results than did the manual method. This may be due to some carryover from concentrated samples because of imperfect washout in the automated apparatus. These differences are minor, however, and we consider data determined by the two methods to be comparable. Investigation of the stability of MMA was not a specific objective of this study. However, the automated analyses were carried out on column eluates (in 0.1 N HCl) that had been frozen at - 16°C for 6 months after analysis by the manual method. The similarity of the data derived from the two analyses indicate that MMA is quite stable under these storage conditions. ACKNOWLEDGMENTS The authors thank Candace Sousa and Ronald Simpson for assistance with the assays and Joseph Watson for advice in modifying the manifold design.
REFERENCES 1. COX, E. V., and White, A. M. (1962) Lancet 2, 853. 2. Brozovic, M., Hoffbrand, A. V., Dimitriadou, A., and Mollin. Huematol.
D. L. (1967)
Brit.
J.
13, 1021.
3. Bamess, L. A., Young, D. G., and Nacho, R. (1963) Science 140, 76. 4. Williams, D. L., Spray, G. H., Newman, G. E., and O’Brien, J. R. P. (1969) Brit. J. Nrrtr.
23, 343.
5. Oace, S. M., and Abbott, J. M. (1972) .I. Nufr. 102, 17. 6. Giorgio, A. J., and Plaut, G. W. E. (1965) J. C/in. Lob. Med. 66, 667. 7. Barness, L. A., Young, B. S., Wellman, W. J., Kahn, S. B.. and Williams, W. J. (1963) N. Engl. .I. Med. 268, 144. 8. Gawthome, J. M.. Watson, J., and Stokstad. E. L. R. (1971) Anal. Biochem. 42, 555.
