Rapid microplate method for PAH estimation
Rajiv
Agarwal
(With the Technical Assistance of Shawn D. Chase)
Division of Nephrology, Department of Medicine, Indiana
University and Richard L. Roudebush Veterans Affairs Medical
Center, Indianapolis, Indiana 46202
 |
ABSTRACT |
Evaluation of renal
hemodynamics requires estimation of effective renal plasma flow, which
is commonly measured by the renal clearance of
p-aminohippuric acid (PAH). There are many existing methods
for PAH assay that are complicated, expensive, or time consuming. We
describe a rapid, precise, and accurate microplate-based assay of PAH
using p-dimethylaminocinnamaldehyde, which produces a red
color on reaction with PAH, and compare it with a reference HPLC
method. Renal PAH clearances were measured in 10 volunteers, and
clearances were calculated by using the new and HPLC methods. There was
excellent agreement between the HPLC and the microplate method of PAH
assay. The average ratio of microplate to HPLC method was nearly 1.0, and the limits of agreement (2 SD) for plasma, urine, and clearances
were 17.2, 19.3, and 25.5%, respectively. Intraday coefficient of
variation for urine and plasma was <7%; interday coefficient of
variation was <6% for urine and plasma samples. The microplate method
is a reliable alternative to a reference HPLC method and can be
performed for a fraction of the cost, time, and reagents.
high-performance liquid chromatography; colorimetry; renal plasma
flow; p-aminohippuric acid
 |
INTRODUCTION |
EFFECTIVE RENAL PLASMA
flow, a major determinant of glomerular filtration rate, is
required for evaluating renal hemodynamics and is traditionally
measured by p-aminohippuric acid (PAH) clearance (9,
10, 20). PAH estimation using a modified Bratton-Marshall reaction is cumbersome (19), whereas performance of
measurements with radioactive compounds (12, 16, 21)
creates additional problems, such as disposal of waste and radiation
exposure of subjects and workers. Techniques such as HPLC require large
capital investment and establishment of methodology, which can take
several months. Additional procedures that include extensive and
arduous extractions may be involved when PAH is estimated by means of HPLC (5). Microplate methods have not been reported for
PAH quantification assays. Herein is reported a microplate method allowing rapid, accurate, and precise estimation of PAH compared with
the established standard of HPLC.
 |
METHODS |
Reagents.
p-Dimethylaminocinnamaldehyde (DACA) was purchased from
Sigma (St. Louis, MO). PAH was purchased from Merck (West Point, PA). HPLC-grade ethanol and tricholoroacetic acid were purchased from Fisher
Scientific (Fair Lawn, NJ). A 1% solution of DACA was made in ethanol
(stable if stored at 4°C for several weeks), and a 15% solution of
trichloroacetic acid was made in distilled deionized water (DDW).
Methods described by Newman et al. (14) were used to
prepare N-acetyl-PAH for interference studies. In brief,
10.4 M acetic anhydride was added to a 10% solution of PAH in a molar
ratio of 2:1. The mixture was gently rotated for 1 h at room
temperature. A thick white amorphous mass formed; this was vacuum
filtered with a 0.22-µm filter (Millipore, Bedford, MA), and the
paste was dried in a light-protected desiccating chamber overnight. Other drugs used for the interference studies were obtained from the
hospital pharmacy.
Standards and quality controls.
A stock solution of PAH was prepared as 10 mg/ml PAH in DDW. Standard
curve and quality control (QC) samples were prepared, aliquoted, and
stored at
85°C until analysis. These standards were then used for
all experiments.
Eight standards were made for plasma and urine. Drug-free blank urine
was diluted 1:10 with DDW and used in all standards. Dilutions of the
stock PAH solution were made to yield concentrations of 1,000, 800, 600, 500, 400, 300, 200, and 100 µg/ml PAH in diluted urine. The
plasma standard curve was prepared with expired fresh-frozen plasma
obtained from the blood bank. PAH stock solution was diluted with blank
donor plasma to 30 µg/ml. This was added in varying volumes to blank
plasma to yield concentrations of 30, 24, 18, 15, 12, 9, 6, and 3 µg/ml PAH.
Nominal concentrations of 21 and 9 µg/ml in serum and 700 and 300 µg/ml in urine were used to assess low and high QCs, respectively.
Fifty samples from study patients that contained unknown amounts of PAH
were analyzed in duplicate on the same day to assess the intraday
coefficient of variation (CV). Plasma QC samples were analyzed on 13 separate days and urine QCs on 14 separate days to assess interday CV.
Sample preparation.
Urine from research subjects and standards were diluted 1:10 with DDW.
One hundred microliters of diluted urine were transferred to
microcentrifuge tubes. To this were added 100 µl of 15%
trichloroacetic acid to precipitate proteins. The tubes were vortexed
briefly, then centrifuged at 14,000 g for 4 min. The clear
supernatant (50 µl) was transferred to a 96-well ELISA plate
(Echostar, Corning, NY), to which were added 150 µl of ethanolic 1%
DACA solution. The plate was incubated for 20 min at room temperature
and read at 550 nm with a Spectramax 190 plate reader with the
PathCheck feature turned on (Molecular Devices, Sunnyvale, CA). Turning on the PathCheck feature yields absorbances equivalent to 1-cm path
length despite considerably shorter path lengths. Plasma samples were
prepared identically, except that samples were not diluted and were
centrifuged for 8 min. All analyses were performed in duplicate.
Calculations and statistical methods.
Standard curves were created by linear regression of optical density of
PAH vs. nominal concentrations of PAH. Concentrations of QC and unknown
samples were estimated by applying the standard curve linear regression
equation to the sample optical density. Recovery was determined by
calculating the mean difference between expected and observed
concentrations of QCs expressed as a percentage of expected
concentration as well as its 95% confidence intervals (CIs). Precision
of the assay was assessed over two concentrations, 9 and 21 µg/ml in
plasma and 300 and 700 µg/ml in urine. Interday and intraday CV were
calculated by one-way ANOVA with the method described by Chinn
(8). The lower limit of detection was calculated as
described by Anderson (2). Commonly used drugs that may interfere with the colorimetric assay were tested. These included acetyl-p-aminophenol (acetaminophen),
p-aminobenzoic acid, sulfadiazine, sulfanilamide, and
sulfacetamide. In addition, because PAH is metabolized to
N-acetyl-PAH in the kidney, interference was also tested.
Finally, to determine the limits of agreement, bias, and precision, a
Bland-Altman analysis (4) was performed by using a
previously established HPLC method as a reference standard
(1).
PAH clearance studies in volunteers.
The study was approved by the Institutional Review Board for Human
Studies of Indiana University. Written informed consent was obtained
from each volunteer. A water load of 10 ml/kg body wt was given orally,
and 5 ml/kg water was given every hour to maintain urine flow. A
loading dose of 10 mg/kg of 20% PAH was administerd intravenously.
This was followed by infusion of a solution of PAH in normal saline at
a rate calculated to give a serum PAH concentration between 10 and 20 µg/ml. Infusion rate was <1 ml/min to match insensible losses. Urine
and blood were sampled every 30 min for four consecutive periods after
1 h of PAH infusion. Each urine collection period was bracketed by
serum sample collection. Each volunteer was studied on 2 consecutive days. The clearances of PAH were calculated by the traditional UV/P
method whereby U is the urinary concentration of PAH, P is the
geometric mean of the bracketing PAH plasma concentrations, and V is
the urine flow rate. All samples were stored at
85°C until analyzed.
 |
RESULTS |
Analyzing PAH over a range of clinically relevant concentrations
in urine and plasma demonstrated a time-dependent development and
deepening of a red color. However, on testing a range of concentrations of DACA between 1 and 10% in ethanol, we found that the lowest concentration was the least sensitive to the time-dependent changes in
color but produced a deep color at 20-min incubation at room temperature. Thus the assay was performed with 1% DACA solution in
ethanol and incubated with protein-free, tricholoracetic
acid-precipitated plasma or urine samples. Although the color
tended to deepen over time, because all samples (standards and unknown)
underwent a similar change, no substantial differences were seen in
results for plates read between 15 and 25 min.
Figure 1 shows that for urine and
plasma standard curves, the coefficient of determination was 0.99 or
better. The standard error of estimate of PAH concentration using this
standard curve was between 1.5 and 3.6 µg/ml. None of the intercepts
was significantly different from zero. The lower limits of detection
for plasma and urine samples were 1 and 3.3 µg/ml, respectively. The
small positive intercept is likely due to primary amines that react to
give a low level of nonspecific chromogens.

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Fig. 1.
Plasma (A) and urine (B) standard
curves using the p-dimethylaminocinnamaldehyde
(DACA) assay show a nearly perfect correlation.
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Intraday CV was 3.8% for plasma and 6.3% for urine. Table
1 shows the interday precision (% of CV)
and accuracy (% of recovery) for high and low concentrations of PAH in
urine and plasma. Accuracy was >95% and precision was within 6% for
all analyses. Studies performed with six compounds demonstrated no
interference with acetaminophen, N-acetyl-PAH, or
sulfasalazine (all <1%). Interference was noted with
p-aminobenzoic acid (120%), sulfacetamide (121%), and
sulfisoxazole (43%). Additional experiments were conducted in which
plasma and urine (diluted 1:10) were each supplemented with urea (200 mg/dl) or creatinine (13.5 mg/dl urine, 7.5 mg/dl plasma). PAH
was added in concentrations of 20 and 50 µg/ml, respectively. No
interference was seen with creatinine in urine or plasma at baseline or
after PAH was added. However, blank plasma supplemented with urea
yielded concentrations of PAH shown in Table
2.
Ten volunteers [8 men and 2 women, aged 69 ± 10 (SD) yr] were
recruited from the renal clinic for the clearance study. Calculated creatinine clearances (Cockcroft-Gault) ranged from 22 to 72 ml/min (mean, 48; SD, 18 ml/min).
Figure 2 shows the correlation between
HPLC and DACA techniques for plasma, urine, and clearance results.
Coefficients of determination (r2) for all three
methods were excellent, and the standard error of estimates was small.

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Fig. 2.
Plasma (A) and urine (B)
p-aminohippuric acid (PAH) concentrations and renal PAH
clearances (PAH Clr; C) calculated by using the DACA method
vs. the HPLC method. SEE, standard error of estimate; n,
number of samples or clearances.
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Figure 3 shows the ratio plot of PAH in
plasma with the two methods. The x-axis shows average PAH
concentration with the two techniques, and the y-axis shows
the ratio of PAH concentration obtained with DACA/HPLC methods. The
average ratio was 1.019 (95% CI; 1.002, 1.036; P = 0.04), indicating that there was between 0.2 and 3.6% overestimation
of plasma PAH with the new method. The limits of agreement shown by the
dotted line were within 20% (2 SD = 17.2%). Only 4 of 98 samples
are outside these limits of agreement. Overall, the CV between the two
methods was 5.98%.

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Fig. 3.
Bland-Altman plot of average plasma PAH concentration
with DACA and HPLC methods and DACA/HPLC ratio of plasma PAH
concentration. Horizontal solid line, bias, which in this case is
between 0.2 and 3.6% (horizontal dotted lines); horizontal dashed
lines, limits of agreement are within 20% (±2 SD).
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Figure 4 shows the ratio plot of PAH in
urine with the two methods. The average ratio was 1.0 (95% CI; 0.981, 1.189; P > 0.2), indicating that there was no bias in
the results obtained with the new method. The limits of agreement shown
by the dotted line were within 20% (2 SD = 19.3%). Only 6 of 99 samples are outside these limits of agreement. Overall, the CV between
the two methods was 7.52%.

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Fig. 4.
Bland-Altman plot of average urine PAH concentration with
DACA and HPLC methods and DACA/HPLC ratio of urine PAH concentration.
Horizontal solid line, no bias (1.0); horizontal dashed lines, limits
of agreement are within 20% (±2 SD).
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|
Figure 5 shows the ratio plot of PAH
clearances obtained with the two methods. The average ratio was 1.0 (95% CI; 0.972, 1.028; P > 0.2), indicating that
there was no bias in the results obtained with the new method. The
limits of agreement shown by the dotted line are ~25% (2 SD = 25.5%). Only 4 of 78 clearances are outside these limits of agreement.
Overall, the CV between the two methods was 9.66%.

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Fig. 5.
Bland-Altman plot of average renal PAH clearances
calculated with DACA and HPLC methods and DACA/HPLC ratio of renal PAH
clearances. Horizontal line, no bias (1.0); horizontal dashed lines,
limits of agreement are within 25% (±2 SD).
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|
 |
DISCUSSION |
Bratton and Marshall (6), more than 60 years ago,
reported a colorimetric technique for the assay of sulfanilamide in
urine and blood. In this technique, a trichloroacetic acid filtrate of
the sample was mixed in a stepwise fashion with sodium nitrite, ammonium sulfamate, and N-(1-naphthyl)-ethylenediamine to
yield a colored dye, the intensity of which was read at 545 nm. Smith et al. (19) modified this procedure by producing a cadmium
sulfate filtrate of plasma or urine that was first acidified by 0.2 volume of 1.2 N-hydrochloric acid before the sequential
steps noted above. It was several decades later that it was realized
that chloride and temperature were critical for color development in
the above assays (22). The above assay is not specific for
p-aminohippuric acid. Free primary o- and
m-aminoaromatic compounds (19), phenols, tryptophan, and indican also give color reactions (23).
Interference from some sulfa drugs, such as sulfamethoxazole, can be
overcome with isoamyl acetate extraction (17), and this
assay has even been automated (11, 13). Nevertheless,
fresh preparation of reagents (nitrite, sulfamate), sequential addition
of three reagents, the timing of which is critical, and temperature
dependence of these assays make these techniques cumbersome.
Some simplification of the PAH assay technique was obtained when Brun
(7) created a two-step procedure. In this technique, plasma protein is precipitated by Somogyi's zinc sulfate and sodium hydroxide, followed by addition of
p-dimethylaminobenzaldehyde (Ehrlich's reagent) in
acid alcohol to form a yellow dye read at 465 nm in a colorimeter.
However, different analytical procedures are required for high and low
plasma concentrations. More recently, Waugh and Beall (23)
reported a two-step procedure in which buffered 1.0 M dichloroacetate
and 0.3 M p-toluenesulfonate reagent are used for
deproteinization and acidification of the sample; a yellow product is
then obtained by adding 57% ethanolic 1%
p-dimethylaminobenzaldehyde. Even in this assay, blank
plasma chromogen ranges between 21 and 73%, and urea and other
sulfonamides containing free p-amino radical interfere with
the assay.
Initially described by Japanese investigators, DACA has been used for
simplified determination of PAH using DACA in 0.17 mmol hydrochloric
acid (24), and even this method has been automated with an
autoanalyzer by Parekh et al. (15). Others have reported that substituting hydrochloric acid with ethanol gives a deeper and
more persistent color and improves the assay performance
(18). Because of the simplicity of this assay, we adapted
it to the microtiter plate.
The established standard for estimation of PAH is HPLC. Baranowski and
Westenfelder (3) found an excellent correlation between an
HPLC method and a colorimetric technique. Our laboratory also reported
an HPLC method that simultaneously assays PAH and iothalamate, which
does not require the micropartition system described by Baranowski and
Westenfelder (1). However, HPLC requires a large capital
investment and is cumbersome and time-consuming. Therefore, our
laboratory developed a rapid colorimetric method and compared it with
our reference HPLC method.
Our results show that the new method using DACA in microplates for
measurement of PAH is rapid, accurate, precise, and less expensive and
compares favorably with the HPLC method. The limits of sensitivity for
our assay is at least 50 ng/well for plasma and 165 ng/well for urine.
As expected, some sulfonamides with the free p-amino group
give color reactions, but not substituted amino groups such as
acetaminophen or N-acetyl-p-aminohippuric acid, a
metabolite of PAH. Our data demonstrate ~10-15% interference with urea, at most, when the plasma urea concentrations are in the
uremic range. Because there are numerous compounds that can potentially
give color reactions with DACA, it is recommended to run blank samples
to screen for these potentially interfering compounds. The interday
reproducibility of the new assay was superior to the HPLC method.
Moreover, excellent agreement exists between the reference and new
methods for plasma and urine concentrations as well as calculated PAH
clearances performed in nearly 100 samples. We speculate that wider
adoption of such simple and precise methods for estimation of renal
plasma flow may improve our ability to determine the pathophysiology
and better characterize the progression of renal diseases.
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ACKNOWLEDGEMENTS |
The nursing assistance of Don Jasinski and Rebecca R. Lewis is acknowledged.
 |
FOOTNOTES |
Address for reprint requests and other correspondence: R. Agarwal, Div. of Nephrology, Dept. of Medicine, Indiana Univ. and Richard L. Roudebush Veterans Affairs Medical Ctr., 1481 West 10th St.,
111N, Indianapolis, IN 46202 (E-mail:
ragarwal{at}iupui.edu).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
January 8, 2002;10.1152/ajprenal.00336.2001
Received 8 November 2001; accepted in final form 7 January 2002.
 |
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