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Articles |
1
Research Laboratory of Nephrology and Hypertension, Aarhus University Hospital, Skejby Hospital, Aarhus 8200, Denmark.
2
Research Laboratory of Molecular Pathology, Institute of
Pathology, Aarhus Kommunehospital, Aarhus, Denmark.
a Author for correspondence. Fax +45 89 49 60 03.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Key Words: indexing terms: atrial natriuretic peptide • sodium • diuresis • circadian rhythm
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Studies have shown that urinary excretion of URO correlates closely with variations in salt excretion in humans (6)(7), and that urinary excretion of URO may have a closer correlation with urinary sodium loss than plasma ANP (8). URO seems to be an important natriuretic peptide, but numerous questions about the role of URO in human physiology and pathophysiology are still open. Therefore, a reliable specific assay for measuring URO in urine is needed to investigate further the biological role of URO. Drummer et al. (9) developed a URO-specific RIA involving a polyclonal URO antibody specific for the four N-terminal residues of URO. This specific URO antibody was raised in rabbits by immunizing them with the short N-terminal fragment of human URO containing the four amino acids by which URO differs from ANP. This URO antibody is not commonly available and the immune respose that is correlated to immunogens with low molecular mass often is poor; therefore we report here the development and purification of a URO antibody raised in rabbits immunized with the whole 32-amino acid URO peptide containing the immunogenic ring structure.
The aims of the present study were (a) to raise a URO antibody in rabbits by immunization with the synthetic URO peptide of 32 amino acids and to purify the resulting URO antiserum with CNBr-activated Sepharose 4B affinity chromatography to a degree without cross-reactivity with ANP analogs, (b) to develop a specific and sensitive RIA for measuring URO-like immunoreactivity (irURO) in human urine, and (c) to investigate if the excretion rate of URO in urine has a circadian variation in healthy volunteers.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-ANP (human, 99–126), pro ANP (human, 1–30), pro ANP
(human, 31–67), and C-type natriuretic peptide-22 (human CNP-22) from
Peninsula Labs. Europe, St. Helens, Merseyside, UK; brain natriuretic
peptide (BNP) from Clinalfa, Lävjeljingen, Switzerland; arginine
vasopressin (AVP) from Ferring, Malmö, Sweden; aldosterone
(Aldo), bovine thyroglobulin (BTG), bovine serum albumin (BSA),
1,3,4,6-tetrachloro-3-,6--diphenylglycouracil (Iodo-Gen), and pig
-globulins from Sigma Chemical Co., St. Louis, MO; cGMP and
Na125I from Amersham, Bucks, UK;
N-3-(dimethylaminopropyl)-N'-ethylcarbodiimide
hydrochloride (CD1) from Fluka, Buchs, Switzerland; Freund's
incomplete adjuvant from Statens Seruminstitut, Copenhagen, Denmark;
CNBr-activated Sepharose-4B and DEAE-Sephadex A-25 from Pharmacia
Biotech, Uppsala, Sweden; human serum albumin (HSA) from
Hoechst-Behring, Marburg, Germany; Triton X-100, EDTA, Tween 20,
methanol, ethanol, triflouroacetic acid (TFA), and acetonitrile from
Merck, Darmstadt, Germany; and polyethylene glycol 6000 (PEG) from
Merck, Hohenbruun, Germany.
preparation of immunogen
URO (95–126) (MW 3506) was covalently conjugated
to BTG. URO (2.4 mg) dissolved in 3.2 mL of demineralized water was
coupled to 7.4 mg of BTG with 0.25 mg of CD1 as a coupling agent. The
molar ratio of URO:BTG:CD1 was 60:1:112. The solution was kept for
24 h at 20 °C with constant stirring. The mixture was then
dialyzed against demineralized water for 48 h at 4 °C.
immunization procedure
Aliquots of 160 µg each (100 µL) of the URO-thyroglobulin
complex were emulsified with 100 µL of incomplete Freund's adjuvant
and injected subcutaneously into the backs of white rabbits.
Immunizations were performed every 2 weeks, and after day 42, a booster
injection was given at 4-week intervals. Blood was drawn 12 days after
each booster injection (10).
affinity chromatography
As the resulting polyclonal URO antibodies cross-reacted with ANP,
CNBr-activated Sepharose 4B affinity chromatography was used for
purification of the rabbit antiserum (11). In short, three
different columns of CNBr-activated Sepharose-4B were coupled to BSA,
ANP, and URO, respectively. Freeze-dried CNBr-activated Sepharose-4B
was suspended in ice-cold HCl (1 mmol/L). The gel was washed and
reswelled three times. It was then immediately transferred to a
solution of the ligand, 30 mg of BSA, 1 mg of ANP, and 2 mg of URO
dissolved in coupling buffer (0.1 mol/L NaHCO3 containing
0.5 mol/L NaCl, pH 7–8). The three protein–gel suspensions were
rotated end-over-end for 2 h at room temperature. After coupling,
the solutions were transferred to a 0.2 mol/L glycine buffer, pH 8.0,
standing overnight at 4 °C, to block remaining active groups on the
gel. The three ligand–Sepharose conjugates were packed in rheodex
columns. To remove excess uncoupled ligand, the absorbent was washed
alternately with high- and low-pH buffer solutions (coupling buffer
followed by 0.1 mol/L acetate buffer, pH 4, containing 0.5 mol/L NaCl).
Finally, 10 mmol/L Tris buffer, pH 7.5, was used to wash away the
blocking agent. The rabbit serum was applied to the BSA column with
free flow; the unbound fraction of the serum was transferred to the ANP
column, and subsequently the nonadsorbed amount was applied to the URO
column. A glycine buffer (0.1 mol/L, pH 2.5) was used as the eluting
agent on the URO column. The eluted fractions were collected in tubes
containing a small amount of 1 mol/L Tris buffer, pH 8.0. The fraction
with the highest protein content measured at wavelength 260 nm was
selected, stored at -20 °C, and investigated for URO-specific
antibodies.
iodination procedure
URO was iodinated by the Iodo-Gen method according to Salacinski
et al. (12) with minor modifications (13). In
short, Iodo-Gen was dissolved in dichloromethane/trichloromethane (40
mg/L). Aliquots (150-µL) of the Iodo-Gen solution were evaporated by
dry nitrogen atmosphere in cryotubes. Ten microliters of a solution
containing 0.2 mg of synthetic URO per mL of 0.1 mol/L acetic acid was
transferred to the Iodo-Gen tube and mixed with 100 µL of 0.5 mol/L
sodium phosphate buffer, pH 7.4, and 7 µL of Na125I (25.9
MBq). The iodination reaction was allowed to run for 10 min. The
solution was then applied to a DEAE-Sephadex A-25 column equilibrated
with 0.1 mol/L sodium phosphate buffer, pH 7.4, which also was the
eluting buffer. The fraction with peak radioactivity was selected and
diluted with assay buffer. Aliquots of the URO tracer were stored at
-20 °C for up to 6 weeks.
sample extraction
Human urine samples were extracted by addition of absolute ethanol
(1:1.5 dilution of samples) with subsequent centrifugation and
lyophilization of the supernatant. Dried extracts were resuspended in
assay buffer.
radioimmunoassay
A 0.04 mol/L sodium phosphate solution (pH 7.4) containing 0.5 g/L
sodium azide, 12 g/L EDTA, 1 mL/L Triton X-100, and 2 g/L HSA was used
as assay buffer. The URO calibrators (range 0–128 fmol/tube) were
prepared from a stock solution (10 mmol/L) and performed in triplicate.
One hundred microliters of calibrator or urine extract (duplicates)
were incubated with 100 µL of URO antibody for 24 h at 4 °C.
One hundred microliters of [125I]URO (~3000 cpm)
was added and the mixture was incubated for a further 24 h. PEG (2
mL, 200 g/L) and 100 µL of pig -globulins (15 g/L per tube) were
used for separation.
high-performance liquid chromatography
The identity of the immunoreactive material in human urine was
studied by HPLC. A HPLC system from Pharmacia LKB was used.
Reversed-phase HPLC was performed on a C18 column
(250 x 4.6 mm, 5 µm) (The Separations Group, Hesperia, CA).
Eluent A consisted of water with 1 g/L TFA and eluent B was a mixture
of water:acetonitrile (20:80 by vol) with 1 g/L TFA. Eluent A (1000
mL/L) was applied for 2 min followed by 30 min of a linear gradient to
1000 mL/L eluent B with a resulting content of acetonitrile in the
eluent ranging from 0 to 800 mL/L. The flow was 1 mL/min. Samples with
synthetic URO and extracted human urine were dissolved in eluent A
before injection.
healthy volunteers
Twenty four healthy volunteers, all men, with a mean age of 44
years, range 23–66 years, were studied during a 24-h period in the
hospital. The inclusion criteria were: (a) healthy male
volunteer, (b) age between 20 and 70 years, and
(c) written informed consent to undergo the study. The
exclusion criteria were: (a) clinical and (or) laboratory
evidence of renal, hepatic, cardiovascular, endocrinological, allergic,
infectious, or neoplastic disease; (b) history of bladder
dysfunction; and (c) alcohol abuse. They received a standard
hospital diet and a fluid intake of 1500 mL/m2 per 24
h. The meals were given at 0730, 1200, and 1800 and there was no fluid
intake during the night. The subjects were only in the supine position
during the night, and only minimal activity was allowed during the
daytime. Urine was collected in the following seven periods:
1500–1800, 1800–2100, 2100–2400, 0000–0700, 0700–1000, 1000–1300,
and 1300–1500. The subjects gave written informed consent, and the
study was approved by the local Ethics Commitee.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The polyclonal URO antibody was purified by CNBr-Sepharose affinity
chromatography. The fraction eluting from the URO column with the
highest protein concentration contained a highly specific URO antibody
with a cross-reactivity of <0.1% with human ANP (99–126) and human
pro-ANP (31–67) (Fig. 1
). No cross-reactivity was observed with pro-ANP (1–30), BNP,
CNP, Aldo, AVP, and cGMP. The purified antiserum was used at a final
dilution of 1:4000. Scatchard plot analysis (14) of the
purified antiserum exposed a homogeneous antibody population. The
antibody is characterized by a K of 1.05 x
1011 L/mol.
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iodination of uro
Twenty three percent of the radioactive iodine was incorporated in
the final tracer preparation. The specific activity of the iodized URO
substance was 15.84 MBq/µg, calculated by the self-displacement
ability as described by Morris (15). Approximately 0.47
mol of iodide/mol of URO was available during the iodination process.
Assuming a specific activity of 647.5 GBq/mg Na125I and
monoiodination of URO, it could be calculated that ~64% of the URO
molecules in the tracer solution were labeled.
calibration curve
The RIA for URO was optimized. Reproducible calibration curves
were obtained and a typical zero calibrator binding, 50% inhibitory
dose (ID50), and nonspecific binding was ~50%, 19
fmol/tube, and 7%, respectively. Nonspecific binding of
[125I]URO in human urine was ~4%, indicating that
interference by substances in urine was not significant.
lower limit of detection
The lower limit of detection was 0.5 fmol/tube (P
<0.05, paired test, n = 12) with a 95% level of confidence,
corresponding to a urine concentration of 7.5 pmol/L URO if 400 µL of
urine was extracted and resuspended in 600 µL of assay buffer, using
100 µL for assay.
precision
The imprecision for analysis of irURO in human urine was assessed
from measurements of internal quality-control pools in 12 consecutive
assays over a period of 3 months. The intraassay CV was 6.7% and the
interassay CV 14.1% at a concentration of 200 pmol/L.
extraction
The recovery of [125I]URO added to human urine
samples before extraction was 89.9% ± 2.8% in 10 consecutive assays.
The recovery of 30 fmol and 60 fmol of unlabeled synthetic URO added to
morning urine samples from 12 healthy adults was 112.1% ± 20.7% and
104.9% ± 16.7% (mean ± SD), respectively.
The amount of URO measured is a linear function of the volume of urine
extract assayed in RIA. The y-axis intercept is not
significantly different from zero as seen in Fig. 2
. The measurable irURO in urine extract diluted parallel to the
calibration curve.
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The immunoreactivity of URO in extracts (1:1.5 dilution with ethanol) from 300, 500, 750, and 1500 µL of morning urine from five healthy subjects was the same (55.9 ± 25.2, 52.6 ± 23.7, 58.9 ± 25.9, 54.8 ± 28.5 pmol/L) (mean ± SD). The proportion between the volume of urine and ethanol used in the extraction procedure seems to have important influence on the measured concentration of irURO.
Varying the ethanol volume between 0 and 500 µL in the extraction of 500 µL of urine (n = 4) showed that irURO graduallly decreased from 114.5 pmol/L to 93.5 pmol/L.
stability
Urine samples (n = 8) kept at room temperature for 24 h
to 72 h before extraction did not show a decay of endogenous URO
immunoreactive material. Storage of urine samples at -20 °C for up
to 3 months or repeated cycles (n = 4) of freezing and thawing did
not change the measured immunoreactivity of URO.
hplc identification of iruro from human urine extract
HPLC tracing of 1 pmol of URO injected directly to the column
eluted with a major peak after 17 min. HPLC tracing of 0.5 mL of
ethanol-extracted human urine supplemented with 1 pmol of URO showed a
major peak after 17 min and a small peak after 4 min. If human urine
supplemented with 1 pmol of URO was stored at room temperature for
24 h, the elution profile showed that the majority of irURO eluted
after 4 min and a minor part after 17 min. This early peak of irURO
could represent a decomposition product of URO. The eluting pattern of
ethanol-extracted human urine consisted of a major peak after 4 min and
in addition two minor peaks after 12 min and 17 min. Thus, the identity
of irURO in human urine extracts seems to have an elution profile
identical to URO and a degradation product of URO.
reference values in healthy volunteers
The urinary URO excretion in 24 healthy adults during a 24-h study
period with seven urine collections is illustrated in Fig. 3
. The excretion of URO did not change significantly during the
24 h, but there was a tendency towards a lower URO excretion rate
in the urine collected from 0000 to 0700.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The immunoextracted rabbit antiserum contains one homogeneous population of antibodies against URO, indicated by linearity of the Scatchard plot. The affinity of the purified antibody population is approximately the same as the affinity of the URO-specific antibody developed by Drummer et al. (Kd = 9.5 pmol/L compared with Kd = 15 pmol/L). The sensitivities of the assays were almost identical, with detection limits in the range 7–7.5 pmol/L.
Problems associated with reduced sensitivity and nonspecific absorption
have been described for ANP assays involving the charcoal procedure for
separation of bound and free ligand (16). In the present
assay, the lowest nonspecific bound values (~7%) were obtained when
using a mixture of PEG and porcine -globulin as carrier instead of
PEG alone or dextran-coated charcoal. This is a simple, fast, cheap,
and reproducible precipitation method (17).
The Iodo-Gen method was chosen as Rasmussen et al. (13) reported about this simple and succesful iodination method for preparation of an ANP tracer without loss of immunoreactivity during iodination as described by Gutkowska et al. with the chloramine T method (18). The method was reproducible in the present study.
The irURO in urine proved to be stable at room temperature for 72 h. Likewise, the present data showed no loss of irURO after prolonged storage at -20 °C or by repeated cycles of freezing and thawing. In contrast, Drummer et al. (9) observed a significant decay of immunoreactive material if human urine samples were stored at room temperature. As well, conflicting data exist about the stability of ANP (19)(20)(21)(22). An explanation of the unchanged concentration of irURO could be that the purified URO antibody cross-reacted also with a degradation product of URO generated during storage. This is supported by HPLC analyses that showed that the irURO in stored human urine had an elution profile identical to URO and a degradation product of URO.
The circadian study showed that the excretion rates of URO in urine were almost unchanged during day and night. Despite a tendency towards a lower URO excretion rate during the night, we did not find a significant circadian 24-h rhythm with minimal excretion rates during the night as indicated by Drummer et al. in their study with a small number of subjects (6). The URO excretions, determined with the use of our specific RIA, were higher than indicated by Drummer et al. (6) and Kentsch et al. (23), but in all three studies the excretion rates were in the range of 20–200 fmol/min. The proportion between the amount of urine and ethanol used in the extraction procedure might have an important influence on the measured concentration of irURO.
In conclusion, the present combination of immunization of rabbits with the URO (95–126) peptide and subsequent purification of the resulting URO antiserum with CNBr-activated Sepharose affinity chromatography was a simple way of producing a URO-specific antibody without cross-reactivity with ANP analogs. The RIA for URO demonstrated specificity and sensitivity for URO in human urine. The usefulness of the assay has been presented in the circadian study, and it seems to be convenient when a large number of clinical samples are to be assayed. Further studies are needed to elucidate the physiological significance of URO in urine under various physiological and pathological conditions.
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Acknowledgments |
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Footnotes |
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, 6--diphenylglycouracil; TFA,
trifluoroacetic acid; and PEG, polyethylene glycol. 
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-diphenyl glycoluril (Iodogen). Anal Biochem 1981;117:136-146.
[Web of Science][Medline]
[Order article via Infotrieve]
-human atrial natriuretic peptide (-hANP) in urine using combination of HPLC with RIA strongly indicates non-immunoreactive metabolites. J Biochem Biophys Methods 1990;20:113-121.
[Web of Science][Medline]
[Order article via Infotrieve]
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