Automated Simultaneous Determination ofp-Aminohippurate by obr18219


									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-
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,
                                                                      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   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
550nm (for PAH).
     9. After every six samples, analyze the calibra-
tion standards    to check for drift in the instrument.   If
the values for the standards      deviate from their true
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
           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
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

                                                                                                                     Blank                 (0.06)                                    (1,15)
       0                                                                                                             0.50                       0.51             051
                             .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-

                                                                                                         resents endogenous creatinine
                                                                                                         ent in normal plasma and urine.
                                                                                                                                          or interfering    chromogens  pres-
       ‘1       .0’          .01
                                                                                                         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-
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.

                                                                                      (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
     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
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
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

To top