- Email: [email protected]
Microchemical urinalysis
MICROCHEMICAL
JOURNAl.15,
673-676
(1970)
Microchemical VII.
Determination
of Citric
Urinalysis Acid
in Microliter
Quantities
of Urine ’ W.
BENJAMIN
Departmetrt
GRUNBAUA4
.4ND
PACE
NELLO
of Physiolo~g-A~~~~f~t)~?‘, Uni,,ersity BcrkeleJ. California 94720
of Californirl,
Received June 13. 1970
Citric acid is an important urinary constituent. Its quantity in the urine, or its excretion rate, is a reflection of a varied number of metabolic activities (4, 5, 6), as well as exogenous sources. In a diet free of citric acid, an enzyme, citrogenase, present in heart, liver, and kidney will catalyze the condensation of pyruvic and oxalacetric acid (3). suggesting an endogenous origin. It is not known to what degree synthesis of citric acid will occur in an organism on a diet plentiful in citric acid. In our own studies, urinary citrate contributes substantially as a negative ion in the overall electrolyte balance, as well as to the general physiological profile. Citric acid excretion is closely related to calcium excretion. A large part of total body citrate is present in bones as a superficial deposit on the crystal lattice of apatite. It forms soluble and poorly ionized complexes with calcium and can effectively remove calcium ions from solution. A local increase in citric acid concentration as a result of cellular activity might possibly be a factor in promoting dissolution of bone salt. Since our last paper in this series (2) dealt with quantitation of calcium in urine, it is appropriate to consider now the quantitation of urinary citric acid. In this communication a simple and convenient procedure is described for the quantitative estimation of urinary citric acid in both man and monkeys. Our method is a modification of that described by Beutler and Yeh (I), who made a thorough investigation of the various steps involved in the analysis, as previously published by others. MATERIALS
AND
METHODS
Reagents 1. Citric acid. Monohydrate crystals (mol wt, 210.14) from Baker * This
study
was
supported
by NASA
Grant
673
NGL-05-003-024.
674
GRUNBAUM
AND
PACE
and Adamson. Weigh 100.0 mg and transfer into loo-ml volumetric flask, Dissolve and adjust volume with 1 N sulfuric acid. This standard stock solution is then diluted 1: 10, 1:4, and 1:2 to yield three working standards of 10, 25, and 50 pg/lOO d, respectively. These standards were kept in the refrigerator at 4°C and were found to be stable for many months. 2. Sulfuric acid. Reagent grade, Baker and Adamson. Prepare 50% solution by slowly adding while mixing 1 vol of concentrated sulfuric acid to 1 vol of water. 3. Potassium bromide. Analytical grade (mol wt, 119.02) from Baker and Adamson. Prepare 1 M solution, by dissolving 11.9 g with water in a loo-ml volumetric flask. 4. Potassium permanganate. Saturated and weak solutions. J. T. Baker crystals (mol wt, 158.03) were used to prepare a saturated solution (approx 12 g/100 ml of water). The weak potassium permanganate was prepared by mixing 1 ml of saturated solution with about 30 ml of water. 5. Hydrogen peroxide. A 2% solution is prepared just before use from a 30% solution (J. T. Baker); i.e., a 1: 15 dilution. 6. n-Heptane. Eastman-Kodak Catalog No. 2215. 7. Thiourea-borax. Sodium borate, decahydrate crystals (mol wt, 381.42) J. T. Baker. Thiourea (mol wt, 76.12) Eastman-Kodak. Two g of sodium borate were dissolved with water in a loo-ml volumetric flask. The dissolution can be aided through use of warm water or shaking in a water bath. Four g of thiourea are then added, dissolved in more water up to the loo-ml mark. Thiourea-borax should be prepared on the day of intended use. Equipment
1. Pyrex screw-cap test tubes, 16 X 100 mm. 2. Repipets, 1 and 5 ml. 3. GrunbaumcR) pipette, 100 ~1. Items 2 and 3 are available from Labindustries, 1802-2nd Street, Berkeley, California 94710. 4. Disposable glass transfer pipettes, 5% inches long. 5. Vortex mixer. 6. Mechanical shaker. 7. Volumetric pipettes, 2 ml. 8. Spectrophotometer with a capability of registering absorbance in cuvettes of IO-mm light path and less than 2-ml volume. Procedure
1. Oxidation. water, working
Into a 16 X loo-mm test tube, place 100 ~1 of either standards, citric acid, or urine sample. Then add in
MICROCHEMICAL
URINALYSIS.
VII
675
succession 100 PI of 50% sulfuric acid and 1 ml of water, and mix contents. Next add 50 ,J of 1 M potassium bromide and follow with 0.2 ml of saturated potassium permanganate. After mixing, allow to stand for 5 minutes. Then chill the samples in an ice water bath. Decoiorize the permanganate by adding, dropwise. 2% hydrogen peroxide while mixing the tubes on a vortex mixer. Avoid adding excess of hydrogen peroxide. Again. while mixing on the vortex mixer, add, dropwise. weak potassium permangannte solution until a faint violet color appears and persists for more than 5 seconds. 2. Extmction. Add exactly 3.0 ml of n-heptane with a Repipct. Screw tops on tightly and shake on a mechanical shaker for 5 minutes. Transfer 2 ml of upper layer, which is the n-heptane extract, into a clean 16 X loo-mm screw-cap tube. 3. Color devrlopment. To the 2 ml of n-heptane extract, add 2.0 ml of freshly prepared thiourca-borax solution. Shake the tubes on a mechanical shaker for 5 minutes. Color development is complete at this time and remains stable for several hours. The n-heptane phase is aspirated with water suction. The chromophorc is formed in the thiourea-borax phase. The latter is transferred into an appropriate cuvette with a lo-mm light path and the absorbance is measured at 445 m/i,. A blank and three different concentrations of standard citric acid wcrc prepared by the same procedure as outlined above. The blanks should have a negligible absorbance.
4. Calculation. OD unknown X concentration
of standard
= /!,g of citric acid/100
,L*I
OD standard RESULTS
AND
DISCUSSION
The absorbance was a linear function only up to 50 rtg of citric acid/loo-PI sample. Under the conditions of the above described procedure, the range of linearity extended to about 0.3 optical density. The citric acid concentration in either human or monkey urine rarely exceeded an absorbance of 0.3 when 100 ~1 of urine was used. Human urines generally had a considerably higher citric acid content, compared to monkey urine. Perhaps this is a reflection of a higher intake of exogenous citrate by humans. By the method described, the reliable range for citric acid determination is between 5 and 50 pg. However. it is possible to quantitate I IJ-g of citric acid with an absorbance of 0.03 if instead of 2 ml of thiourea-borax only 0.4 mI arc used in a IO-mm light path cuvette with a lOO- to 200-~1 total volume requirement. Table 1 shows the reproducibility achieved from day to day and the absorbance of about 0.3 at
676
GRUNBAUM TABLE REPLICATE
Citric acid (P8/100 PI) 10
25 50 75 100
AND PACE 1
DETERMINATIONS OF STANDARD IN 2 CONSECUTIVE DAYS
First day OD a 0.059 0.148 0.285 0.428 0.552
0.060 0.148 0.281 0.428 0.548
CITRIC
ACID
Second day OD 0 0.050 0.151 0.287 0.428 0.555
0.063 0.155 0.286 0.434 0.565
0 Absorbance of duplicate samples.
which the relationship of concentration to optical density begins to deviate from a straight line. In this laboratory, 30 urine specimens were conveniently analyzed by 1 technician in 1 day’s work. REFERENCES 2. Beutler, E., and Yeh, M. K. Y., A simplified method for the determination of citric acid. J. Lab. Clin. Med. 54, 125-131 (1959). 2. Grunbaum, B. W., and Pace, N., Microchemical Urinalysis. VI. Determination of sodium, potassium, calcium, and magnesium by atomic absorption spectrophotometry in the microliter range of urine. Microchem. J. 15, 666-672 (1970). 3. Oder, B. L., ed. “Hawk’s Physiological Chemistry,” 14th ed. p. 1180. McGrawHill, New York, 1965. 4. Ostberg, O., Studien iiber die Zitronens&rreausscheidung der Menschenniere in normalen und pathologischen ZustHnden. Skand. Arch. Physiol. 62, 81-222 (1931). 5. Shorr, E., Bernheim, A. R., and Taussky, H., The relation of urinary citric acid excretion to the menstrual cycle and the steroidal reproductive hormones. Science 95, 606-607 (1942). 6. Shot-r, E., The possible usefulness of estrogens and aluminum hydroxide gels in the management of renal stone. J. Ural. 53, 507-520 (1945).
JOURNAl.15,
673-676
(1970)
Microchemical VII.
Determination
of Citric
Urinalysis Acid
in Microliter
Quantities
of Urine ’ W.
BENJAMIN
Departmetrt
GRUNBAUA4
.4ND
PACE
NELLO
of Physiolo~g-A~~~~f~t)~?‘, Uni,,ersity BcrkeleJ. California 94720
of Californirl,
Received June 13. 1970
Citric acid is an important urinary constituent. Its quantity in the urine, or its excretion rate, is a reflection of a varied number of metabolic activities (4, 5, 6), as well as exogenous sources. In a diet free of citric acid, an enzyme, citrogenase, present in heart, liver, and kidney will catalyze the condensation of pyruvic and oxalacetric acid (3). suggesting an endogenous origin. It is not known to what degree synthesis of citric acid will occur in an organism on a diet plentiful in citric acid. In our own studies, urinary citrate contributes substantially as a negative ion in the overall electrolyte balance, as well as to the general physiological profile. Citric acid excretion is closely related to calcium excretion. A large part of total body citrate is present in bones as a superficial deposit on the crystal lattice of apatite. It forms soluble and poorly ionized complexes with calcium and can effectively remove calcium ions from solution. A local increase in citric acid concentration as a result of cellular activity might possibly be a factor in promoting dissolution of bone salt. Since our last paper in this series (2) dealt with quantitation of calcium in urine, it is appropriate to consider now the quantitation of urinary citric acid. In this communication a simple and convenient procedure is described for the quantitative estimation of urinary citric acid in both man and monkeys. Our method is a modification of that described by Beutler and Yeh (I), who made a thorough investigation of the various steps involved in the analysis, as previously published by others. MATERIALS
AND
METHODS
Reagents 1. Citric acid. Monohydrate crystals (mol wt, 210.14) from Baker * This
study
was
supported
by NASA
Grant
673
NGL-05-003-024.
674
GRUNBAUM
AND
PACE
and Adamson. Weigh 100.0 mg and transfer into loo-ml volumetric flask, Dissolve and adjust volume with 1 N sulfuric acid. This standard stock solution is then diluted 1: 10, 1:4, and 1:2 to yield three working standards of 10, 25, and 50 pg/lOO d, respectively. These standards were kept in the refrigerator at 4°C and were found to be stable for many months. 2. Sulfuric acid. Reagent grade, Baker and Adamson. Prepare 50% solution by slowly adding while mixing 1 vol of concentrated sulfuric acid to 1 vol of water. 3. Potassium bromide. Analytical grade (mol wt, 119.02) from Baker and Adamson. Prepare 1 M solution, by dissolving 11.9 g with water in a loo-ml volumetric flask. 4. Potassium permanganate. Saturated and weak solutions. J. T. Baker crystals (mol wt, 158.03) were used to prepare a saturated solution (approx 12 g/100 ml of water). The weak potassium permanganate was prepared by mixing 1 ml of saturated solution with about 30 ml of water. 5. Hydrogen peroxide. A 2% solution is prepared just before use from a 30% solution (J. T. Baker); i.e., a 1: 15 dilution. 6. n-Heptane. Eastman-Kodak Catalog No. 2215. 7. Thiourea-borax. Sodium borate, decahydrate crystals (mol wt, 381.42) J. T. Baker. Thiourea (mol wt, 76.12) Eastman-Kodak. Two g of sodium borate were dissolved with water in a loo-ml volumetric flask. The dissolution can be aided through use of warm water or shaking in a water bath. Four g of thiourea are then added, dissolved in more water up to the loo-ml mark. Thiourea-borax should be prepared on the day of intended use. Equipment
1. Pyrex screw-cap test tubes, 16 X 100 mm. 2. Repipets, 1 and 5 ml. 3. GrunbaumcR) pipette, 100 ~1. Items 2 and 3 are available from Labindustries, 1802-2nd Street, Berkeley, California 94710. 4. Disposable glass transfer pipettes, 5% inches long. 5. Vortex mixer. 6. Mechanical shaker. 7. Volumetric pipettes, 2 ml. 8. Spectrophotometer with a capability of registering absorbance in cuvettes of IO-mm light path and less than 2-ml volume. Procedure
1. Oxidation. water, working
Into a 16 X loo-mm test tube, place 100 ~1 of either standards, citric acid, or urine sample. Then add in
MICROCHEMICAL
URINALYSIS.
VII
675
succession 100 PI of 50% sulfuric acid and 1 ml of water, and mix contents. Next add 50 ,J of 1 M potassium bromide and follow with 0.2 ml of saturated potassium permanganate. After mixing, allow to stand for 5 minutes. Then chill the samples in an ice water bath. Decoiorize the permanganate by adding, dropwise. 2% hydrogen peroxide while mixing the tubes on a vortex mixer. Avoid adding excess of hydrogen peroxide. Again. while mixing on the vortex mixer, add, dropwise. weak potassium permangannte solution until a faint violet color appears and persists for more than 5 seconds. 2. Extmction. Add exactly 3.0 ml of n-heptane with a Repipct. Screw tops on tightly and shake on a mechanical shaker for 5 minutes. Transfer 2 ml of upper layer, which is the n-heptane extract, into a clean 16 X loo-mm screw-cap tube. 3. Color devrlopment. To the 2 ml of n-heptane extract, add 2.0 ml of freshly prepared thiourca-borax solution. Shake the tubes on a mechanical shaker for 5 minutes. Color development is complete at this time and remains stable for several hours. The n-heptane phase is aspirated with water suction. The chromophorc is formed in the thiourea-borax phase. The latter is transferred into an appropriate cuvette with a lo-mm light path and the absorbance is measured at 445 m/i,. A blank and three different concentrations of standard citric acid wcrc prepared by the same procedure as outlined above. The blanks should have a negligible absorbance.
4. Calculation. OD unknown X concentration
of standard
= /!,g of citric acid/100
,L*I
OD standard RESULTS
AND
DISCUSSION
The absorbance was a linear function only up to 50 rtg of citric acid/loo-PI sample. Under the conditions of the above described procedure, the range of linearity extended to about 0.3 optical density. The citric acid concentration in either human or monkey urine rarely exceeded an absorbance of 0.3 when 100 ~1 of urine was used. Human urines generally had a considerably higher citric acid content, compared to monkey urine. Perhaps this is a reflection of a higher intake of exogenous citrate by humans. By the method described, the reliable range for citric acid determination is between 5 and 50 pg. However. it is possible to quantitate I IJ-g of citric acid with an absorbance of 0.03 if instead of 2 ml of thiourea-borax only 0.4 mI arc used in a IO-mm light path cuvette with a lOO- to 200-~1 total volume requirement. Table 1 shows the reproducibility achieved from day to day and the absorbance of about 0.3 at
676
GRUNBAUM TABLE REPLICATE
Citric acid (P8/100 PI) 10
25 50 75 100
AND PACE 1
DETERMINATIONS OF STANDARD IN 2 CONSECUTIVE DAYS
First day OD a 0.059 0.148 0.285 0.428 0.552
0.060 0.148 0.281 0.428 0.548
CITRIC
ACID
Second day OD 0 0.050 0.151 0.287 0.428 0.555
0.063 0.155 0.286 0.434 0.565
0 Absorbance of duplicate samples.
which the relationship of concentration to optical density begins to deviate from a straight line. In this laboratory, 30 urine specimens were conveniently analyzed by 1 technician in 1 day’s work. REFERENCES 2. Beutler, E., and Yeh, M. K. Y., A simplified method for the determination of citric acid. J. Lab. Clin. Med. 54, 125-131 (1959). 2. Grunbaum, B. W., and Pace, N., Microchemical Urinalysis. VI. Determination of sodium, potassium, calcium, and magnesium by atomic absorption spectrophotometry in the microliter range of urine. Microchem. J. 15, 666-672 (1970). 3. Oder, B. L., ed. “Hawk’s Physiological Chemistry,” 14th ed. p. 1180. McGrawHill, New York, 1965. 4. Ostberg, O., Studien iiber die Zitronens&rreausscheidung der Menschenniere in normalen und pathologischen ZustHnden. Skand. Arch. Physiol. 62, 81-222 (1931). 5. Shorr, E., Bernheim, A. R., and Taussky, H., The relation of urinary citric acid excretion to the menstrual cycle and the steroidal reproductive hormones. Science 95, 606-607 (1942). 6. Shot-r, E., The possible usefulness of estrogens and aluminum hydroxide gels in the management of renal stone. J. Ural. 53, 507-520 (1945).