Get documents about "Automated Simultaneous Determination ofp-Aminohippurate
Document Sample
CLIN. CHEM. 20/3,348-352 (1974)
Automated SimultaneousDeterminationof p-Aminohippurate
and Creatinine in Plasma or Urine
C. K. Parekh, J. Kirpan, A. Peterson, G. L. Hassert, and B. F. Murphy
Methods have been developed for using the Beck- methods, all of which require several steps before the
man “Discrete Sample Analyzer” (DSA-560), for au- development of color. In the extensively used method
tomated simultaneous determinations of p-aminohip- of Bratton and Marshall (2) the PAH is diazotized,
purate (PAH) and creatinine in the same 50- or and the diazotized product is coupled with N-(1-
10-al samples of plasma or urine, respectively, at naphthyl)-ethylenediamine dihydrochloride. Related
the r,ate of 80 samples per hour. In determinations of methods include that of Brun (3), in which p-di-
PAH, a single reagent, p-dimethylaminocinnamal-
methylaminobenzaldehyde is used to form a colored
dehyde in 0.1 mol/liter HCI, was added to a protein-
free filtrate from plasma or urine. The intensity of the product, and those of Sakai et al. (4) and of Shiraka-
color, measured at 550 nm, obeyed Beer’s law for ta et al. (5), in which an alcoholic solution of p-di-
PAH concentrations from 0.005 to 5.0 g/liter. Data methylaminocinnamaldehyde is used.
obtained for the same plasma and urine samples by A simple method that is adapted easily to auto-
the manual method, were, in general, within ±5% of mated analytical systems has been devised for deter-
the results obtained by the automated method. Cre- mining PAH in plasma or urine. An acidic solution
atinine was simultaneously determined by adapting of p-dimethylaminocinnamaldehyde is added to a
the method of Taussky to the DSA-560 instrument; the protein-free filtrate of either body fluid to form a col-
intensity of the color, measured at 510 nm, obeyed ored product. With dual-channel automated equip-
Beer’s law for creatinine concentrations ranging ment, creatinine may be determined simultaneously
from 0.005 to 5.0 g/liter. Data obtained for the same
in the same filtrates of plasma or urine by a modifi-
plasma and urine samples by the manual method,
cation of the method of Taussky (6). To establish the
were, in general, within ±5% of the results obtained
by the automated method. validity of the procedure, we analyzed the same sam-
ples of plasma or urine for PAH and creatinine by
Additional Keyphrases: renal function #{149} effective both the automated methods described here and the
renal plasma flow #{149}glomerular filtration rate pro-
#{149} standard manual procedures (2, 7).
Beckman OSA
tein-free filtrate #{149}
Materials and Methods
Methods commonly used to evaluate renal func-
We used the “Discrete Sample Analyzer” (DSA-
tion include estimations of effective renal plasma
560) (Beckman Instruments, Inc., Fullerton, Calif.
flow and glomerular filtration rate from the clearan-
92634).
ces of PAH1 and creatinine, respectively (1). The
amounts of PAH in plasma and urine, from which Reagents
effective renal plasma flow may be calculated, are
p-Dimethylammnocinnamaldehyde was purchased
usually estimated by one of several colorimetric
from Aldrich Chemical Co. Inc., Cedar Knolls, N. J.
All other chemicals used in the procedure were of an-
Department of Toxicology, The Squibb Institute for Medical alytical grade, obtained from standard chemical-sup-
Research, New Brunswick, N. J. 08903.
This paper was presented at the 8th International Congress on ply houses.
Clinical Chemistry, Copenhagen, Denmark, June 18-23, 1972. For PAH analysis: Dissolve 300 g of TCA and di-
1 Nonstandard abbreviations used: PAH, p-aminohippurate; lute to 1000 ml with distilled water. Dissolve 330 mg
TCA, trichloroacetic acid; PFF, protein-free filtrate; and DACA,
p-dimethylaminocinnamaldehyde. of DACA and dilute to 100 ml with 0.1 mol/liter
Received April 25, 1973; accepted Dec. 14, 1973. HC1. Prepare the solution daily.
348 CLINICAL CHEMISTRY, Vol. 20, No. 3, 1974
For creatinine analysis: Saturated aqueous solu- Analytical Procedure for DSA-560
tion of picric acid. Sodium hydroxide, 2.5 mol/liter. Summary: Before intravenous administration of
PAH and creatinine for clearance studies in dogs,
Standards
obtain control samples of plasma and urine for use as
For PAH in plasma: Stock solution: To 100 ml of blanks. After the DSA-560 has been programmed
p-aminohippuric acid add 100 ml of distilled water (Figure 1), standardized as indicated above, and
and 5 ml of 1 mol/liter NaOH. Dilute the solution started, automatically pipet an aliquot of plasma or
(obtained by warming) to 1 liter with distilled water urine into a plastic reaction cup (“Q cup”). After
(0.1 g of PAH/liter). From this stock solution, pre- deproteinization has been completed, transfer an ali-
pare the following working standards: 5, 10, 20, 30, quot of PFF to another cup, and add the reagent for
and 50 mg/liter, by diluting 5-, 10-, 20-, 30-, and color development to the PFF at designated stops in
50-ml volumes of stock solution to 100 ml with dis- a 35-station sequence. After color develops, pump
tilled water. the solutions through a precalibrated dual-beam col-
For PAH in urine: Stock solution: To 10 g of p- orimeter, determine the absorbances vs. a water
aminohippuric acid add 100 ml of distilled water and blank, and print out the concentrations of PAH or
5 ml of 1 mol/liter NaOH. Dilute the solution (dis- creatinine by “Teletype.” Samples can be analyzed
solve by warming) to 1 liter with distilled water (10 g at the rate of 80/h.
of PAH per liter). From this stock solution prepare Detailed procedure: 1. Pipet an aliquot of the
0.5, 1.0, 3.0, and 5.0 g/liter working standards by sample (50 tl of plasma or 10 il of urine) into cup A
diluting 5-, 10-, 30-, and 50-ml volumes of stock so- and dilute with 250 tl of distilled water at station 1.
lution to 100 ml with distilled water. 2. Add 250 tl of a 300 g/liter solution of TCA to
For creatinine in plasma: Stock solution: Dissolve cup A with 300 sl of distilled-water rinse at station
1 g of creatinine (dry, “certified” grade) and dilute 3.
to 1 liter with 0.1 mol/liter HC1. Dilute a 10-ml ali- 3. Obtain the PFF by reverse filtration: Drop a
quot of this solution to 100 ml with distilled water to “hat,” the top made of filter paper, upside down into
give a diluted stock solution (0.1 g of creatinine per cup A (station 7), and then apply a vacuum, 3 to 4
liter). From this diluted stock solution, prepare the mm Hg, to the hat (station 8).
following working standards: 5, 10, 20, 30, and 50 4. Transfer 200-tl aliquots of the clear filtrate,
mg/liter, as described above for PAH in plasma. collected in the hat, from cup A to cup C with a
For creatinine in urine: Stock solution: Dissolve 10 50-.tl distilled-water rinse at station 9 and to cup D
g of creatinine (dry, “certified” grade) and dilute to with a 450-z1 distilled-water rinse at station 11.
1 liter with 0.1 mol/liter HC1 (10 g of creatinine per 5. For PAH determinations, add 1000 tl of
liter). From this stock solution prepare working stan- DACA reagent to cup C without a distilled-water
dards of 0.5, 1.0, 3.0, and 5.0 g/liter, as described rinse at station 13 (pipet the DACA reagent with the
above for PAH in urine. diluent pump).
6. For creatinine determination, add 150 zl of
Standardization of DSA-560 picric acid with a 150-izl distilled-water rinse and 200
For plasma samples: The DSA-560 was calibrated izl of 2.5 mol/liter NaOH with a 200-z1 distilled-
vs. water with standards containing either PAH or water rinse to cup D at stations 15 and 16, respec-
creatinine, 50 mg/liter. tively.
For urine samples: The DSA-560 was calibrated 7. The solutions in cups C and D develop color
against water with standards containing either PAH while moving from stations 13 and 16, respectively,
or creatinine, 1.0 g/liter. to station 29.
Fig. 1. Diagram of arrange-
ment for simultaneous deter-
mination of creatinine and
PAH (p-aminohippurate) in
urine or plasma with the
Beckman “Discrete Sample
Analyzer” (DSA-560)
Symbols: * The first and second nu-
merals refer to the volume of sam-
ple (10 l of urine and 50 zl of plas-
ma) or of reagent and distilled
water, respectively. Pump No. 5 de-
livered DACA reagent only, with no
distilled-water rinse. ** Filtration
module at stations 7 and 8. t The
sensitivity setting for urine samples
was Xl for both channels, but for
plasma samples it was Xl for chan-
nel 1 and X3 for channel 2. Absor-
bances of the colored solutions were
measured vs. water
CLINICAL CHEMISTRY, Vol. 20, No.3, 1974 349
8. At station 29, measure the absorbances of the
colored solutions vs. water in a pair of dual-beam
flow-cell colorimeters at 510 nm (for creatinine) and 0
‘0
550nm (for PAH).
9. After every six samples, analyze the calibra-
5.0-
tion standards to check for drift in the instrument. If
the values for the standards deviate from their true
4.0
concentrations by more than ±5%, recalibrate the
DSA-560 and re-test the plasma or urine samples. 3.0
10. Calculate the true concentrations of PAH and
creatinine in samples of plasma and urine by sub- 2.0
tracting the appropriate
parent
“Teletype.”
PAH and creatinine
blank values from the ap-
values recorded on the
:‘; 1.0
.01 .02 .03 .04 .05 .10
Tests of the Automated Procedures
.0 2.0 3.0 4.0 5.0 .-,
Compliance with Beer’s law. Both automated pro- Concentration of Creatinine Standards g/Ilter
cedures were checked for their compliance with
Fig. 3. Concentrations of creatinine obtained from DSA-
Beer’s law. The four series of aqueous PAH and cre- 560 vs. prepared aqueous standards
atinine standards that were used (described pre- The lower range of concentrations (5 to 100 mg/liter) was used for plas-
viously) covered the ranges of concentrations found ma samples; the higher range of concentrations (0.5 to 5.0 g/Iiter) was’
used for urine samples
in both plasma and urine samples.
Recoveries of PAH and creatinine added to human
plasma and or creatinine
urine samples. PAH was
added to pooled samples of human plasma and of
human urine in concentrations from 5 to 100 mg/ Table 1. Recoveries, by Automated (DSA)
liter and 0.5 to 5.0 g/liter, respectively. All samples Procedures, of PAH and Creatinine Added to
Samples of Human Plasma and Urine
were analyzed by the automated procedures for their Amount of PAH Amount found
PAH and creatinine contents. or creatinine
added PAH Creatinine
Reproducibility of the automated procedures. An
(guitar)
anesthetized dog was given intravenous priming
doses of PAH, 10 mg/kg body wt, and of creatinine, Plasma
60 mg/kg body wt, after which both compounds were Blank (0.010) (0.022)
administered by constant infusion at 0.3 mg/kg per 0.005 0.006 0.005
minute. Urine was collected via ureteral catheters, 0.010 0.010 0.009
and plasma was obtained from a heparinized sample 0020 0.020 0.019
of venous blood. PAH and creatinine were determined 0.030 0.028 0.029
0.050 0.050 0.052
0.100 0.100 0.098
Urine
Blank (0.06) (1,15)
0 0.50 0.51 051
‘0
U-)
.10 1.00 0.98 0.96
/ 3.00 3.01 2.97
5.0 5.00 4.96 4.98
J 4.0’
oEach sample value has been corrected for the blank value,
and is the mean of four determinations. The blank value rep-
3.0
resents endogenous creatinine
ent in normal plasma and urine.
or interfering chromogens pres-
-
2.0
a
‘1 .0’ .01
C
10 successive times by the automated procedures, for
0 one urine sample and one plasma sample.
.01 .02 .03 .04 .05 .10 a-e
#{149} Comparison of results by the automated and man-
1.0 2.0 3.0 4.0 5.0 .-.
ual procedures. A second anesthetized dog was
Concentration of PAH Standards g/liter
primed and infused with PAH and creatinine, and
Fig. 2. Concentrations of PAH obtained by DSA-560 vs urine and plasma samples were obtained as de-
prepared aqueous standards scribed above. Venipunctures were made midway
The lower range of concentrations (5 to 100 mg/liter) was used for plas-
ma samples: the higher range of concentrations (0.5 to 5.0 g/liter) was through consecutive 15-mm urine-collection periods
used for urine samples during a 3-h study. The urine and plasma samples
350 CLINICAL CHEMISTRY, Vol. 20, No.3, 1974
were analyzed by the automated and manual meth-
ods for PAH (2) and creatinine (7) concentrations. Table 3. Determination, by Automated (DSA)
and Manual Procedures, of PAH Concentration
Results in 13 Samples of Plasma and Urine from a
Dog Infused Intravenously with PAH
Compliance with Beer’s law. Arithmetic plots of Plasma Urine
PAH and creatinine concentrations, obtained for the DSA Manual DSA Manual
four series of standards by the automated proce- g/liter
dures, were linear from 0.005 to 0.1 and from 0.5 to
(0.011) plasma blank (0.04) urine blank
5.0 g/liter (Figures 2 and 3).
0.022 0.021 1.11 1.16
Recoveries of PAH and creatinine added to human 0.020 0.019 1.12 1.14
plasma and urine samples. Although the recoveries 0.021 0.018 1.05 1.05
of PAH and creatinine added to human plasma and 0.020 0.019 0.90 0.89
urine samples ranged from 90 to 120% (Table 1), the 0.018 0.019 0.75 0.75
absolute values for plasma and urine did not deviate 0.018 0.020 0.65 0.66
by more than 2 and 40 mg/liter, respectively. 0.021 0.019 0.74 0.74
Reproducibility of the automated In 10
procedure. 0.021 0.021 0.63 0.62
successive determinations, by the automated proce- 0.023 0.019 0.77 0.75
dures for PAH and creatinine concentrations in the 0.020 0.021 1.26 1.26
0.022 0.021 1.17 1.14
same samples of plasma and urine from a dog, the
0.021 0.021 0.80 0.79
coefficients of variation for plasma were 5.0 and oThedog was maintained on a continuous infusion of creati-
3.5%, respectively, and for urine were 3.6 and 1.2%, nine and PAH, after priming doses. For details, see text. The
respectively, demonstrating good reproducibility blank value represents endogenous interfering chromogens
(Table 2). present in plasma and urine. Each sample value has been cor-
Comparison of results by the automated and the rected for the blank value, and is the mean of two determina-
tions.
manual procedure. PAH and creatinine were deter-
mined in samples of dog plasma and urine by the au-
tomated procedure described above and by the man-
ual methods. The results, generally, agreed within Table 4. Determination, by Automated (DSA)
±5% (Tables 3 and 4). The differences for absolute and Manual Procedures, of Creatinine
values of PAH ranged from -2 to +4 mg/liter in Concentration in 13 Samples of Plasma and Urine
plasma, and from -50 to +30 mg/liter in urine. The from a Dog Infused Intravenously with
differences for absolute values of creatinine ranged Creatininea
Plasma Urine
from -10 to +3 mg/liter in plasma, and from -20 to
DSA Manual OSA Manual
+100mg/liter in urine.
g/liter
(0.006) plasma blank (0.14) urine blank
0.069 0.067 2.08 2.06
0.068 0.066 2.07 1.97
Table 2. Reproducibility, by the Automated 0.065 0.062 2.03 2.00
Procedure (DSA-560), of PAH and Creatinine 0.063 0.061 1.97 1.95
Values’ for the Same Sample of Plasma or 0.063 0.061 1.90 1.84
Urine (10 Samples) 0.063 0.062 1.82 1.76
Plasma Urine
0.062 0.062 1.79 1.77
PAH Creatinine PAH Creatinine 0.062 0.063 1.75 1.72
(g/liter) 0.062 0.062 1.72 L74
0.019 0.060 0.55 0.82 0.062 0.062 1.70 1.65
0.020 0.056 0.58 0.82 0.063 0.061 1.66 1.66
0.022 0.059 0.57 0.83 0.063 0.060 1.67 1.66
0.021 0.059 0.55 0.82 The dog was maintained on a continuous infusion of cre-
0.020 0.056 0.58 0.84 atinine and PAH, after priming doses. For details, see text. The
0.021 0.056 0.55 0.85 blank value represents endogenous creatinine or interfering
chromogens present in plasma and urine. Each sample value
0.021 0.056 0.56 - 0.85 has been corrected for the blank value, and is the mean of two
0.020 0.055 0.56 0.82 determinations.
0.020 0.058 0.54 0.84
0.019 0.058 0.54 0.84
Mean 0.020 0.057 0.56 0.83 Discussion
± SD ±0.001 ±0.002 ±0.02 ±0.01
In the automatedmethod of Harvey and Brothers
Coefficient
of (8) for determination of PAH, an adaptation of the
variation, method of Bratton and Marshall (2), a fractional di-
% 5.0 3.5 3.6 1.2 alysis of plasma is done, followed by (a) diazotiza-
CEach sample value has been corrected for the blank value. tion of the PAH with sodium nitrite, (b) destruction
of excess nitrite by sulfamate, and (c) color develop-
CLINICAL CHEMISTRY, Vol. 20, No. 3. 1974 351
ment by coupling with N-(1-naphthyl) ethylenedi- treated with drugs that contain the para aromatic
amine hydrochloride. Addition of each reagent re- amine group.
quires mixing and a suitable time delay. The authors
indicate that the most troublesome aspect of the We thank Mr. Peter Arnow for assistance in preparation of the
method is that gas bubbles form, which may stick to manuscript.
the colorimeter cell. Our method requires the addi-
tion of only one reagent to the PFF, there are no
References
other time delays, and it is not troubled by the for-
1. Smith, H. W., Principles of Renal Physiology. Oxford Univer-
mation of bubbles. sity Press, New York, N. Y., 1956, pp 196-202, 208-214.
In the method of Shirakata et al. (5), the DACA 2. Bratton, A. C., and Marshall, B. K., Jr., A new coupling com-
reagent is prepared in absolute ethanol, which is ponent for sulfanilamide determination. J. Biol. Chem. 128, 537
used as the protein precipitant. This technique is (1939).
3. Brun, C., A rapid method for the determination of para-ami-
less satisfactory for use with the DSA-560 because nohippuric acid in kidney function test. J. Lab. Clin. Med. 37,
alcohol is lost during filtration under reduced pres- 955 (1951).
sure, as well as during the period of color develop- 4. Sakai, S., Suzuki, K., Mori, H., and Fujino, M., Amine color
ment after the 0.1 mol/liter HC1 is added to the development by the use of p-dimethylaminocinnamaldehyde.
Bunseki Kagaku 9,862 (1960).
PFF. This potential source of error has been elimi-
5. Shirakata, T., Sota, N., Toyota, C., and Chikatsune, M., A
nated by use of an acidic aqueous DACA reagent simple method for the determination of p-aminohippuric acid in
that is added to the PFF in one step and is less the renal function test. Igaku To Seibutsugaku 76, 246(1968).
subject to loss by evaporation. Color was most in- 6. Taussky, H. H., A microcolorimetric determination of creati-
tense when the DACA reagent had been prepared in nine in urine by the Jaffb reaction. J. . Biol. Chem. 208, 853
(1954).
0.1 mol/liter HC1. Plasma and urine blank values 7. Bonsnes, R. W., and Taussky, H. H., On the colorimetric de-
must be determined to correct for the presence of en- termination of creatinine by the Jaff#{233}
reaction. J. Biol. Chem.
dogenous chromogens that may also react with the 158, 581 (1945).
DACA reagent to form colored products. Neither this 8. Harvey, R. B., and Brothers, A. J., Renal extraction of para-
aminohippurate and creatinine measured by continuous in vivo
method nor others that use DACA in the determina- sampling of arterial and renal-vein blood. Ann. N. Y. A cad. Sci.
tion of PAH is suitable for samples from subjects 102, 46 (1962).
352 CLINICAL CHEMISTRY, Vol. 20, No. 3, 1974
Related docs
