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Articles |
Department of Pediatrics, University of Vienna, Währinger Gürtel 18-20, A-1090 Vienna, Austria.
a Author for correspondence. Fax 43 1 40400 3238; e-mail franz.waldhauser{at}akh-wien.ac.at.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Determination of plasma AVP is useful for both investigation of physiological water metabolism and diagnosis of pathological conditions such as syndrome of inappropriate secretion of antidiuretic hormone, diabetes insipidus, psychogenic water intoxication, and chronic hyponatremia (2). Measurement of AVP in plasma is still difficult, as indicated by the considerable diversity in adult AVP reference values reported by different laboratories and the rarity of such data for children (3). Published plasma AVP mean values for endocrinologically healthy humans range from 0.7 to >10 ng/L (4)(5)(6)(7)(8).
Interfering factors and low plasma AVP concentrations necessitate extraction and concentration of AVP before RIA. Several extraction and RIA procedures have been reported (9)(10)(11)(12)(13). However, there is still a lack of methods combining high sensitivity, small sample volume, and an appropriate physiological detection range. We sought to develop a procedure that optimizes AVP measurement, to identify possible causes for the variations in reported AVP mean values (i.e., whether the premeasurement treatment of blood samples, such as storage and separation, had an influence on plasma AVP concentrations), and to provide reference values for children. We therefore developed an AVP measurement procedure that included certain premeasurement steps, extraction and concentration of AVP from plasma with a 100-mg Isolute C18 column, and the use of a commercially available antigen and antiserum for the RIA. This method combines a very low detection limit and small sample volumes. In addition, we demonstrated the influence of blood storage and centrifugation speed on plasma AVP and provided reference values for children close to those published recently for adults.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Sample extraction.
Isolute C18 columns (100 mg;
Jones Chromatography) were attached to a vacuum manifold (Vac Elut SPS
24; Analytichem International), and were activated with 2 mL of
methanol (>99.8%, analytical grade) and equilibrated with 2 mL of
deionized water to prevent the columns from running dry. The vacuum
manifold allowed us to use as many as 24 columns simultaneously. Plasma
(0.5 mL) was acidified with 50 µL of 1 mol/L HCl to pH 3.5. A 0.5-mL
aliquot of this acidified plasma was loaded onto a column and allowed
to pass through at a rate of 50 µL/min. The columns were then washed
with 3 mL of acetic acid (0.67 mol/L) and allowed to run dry by means
of suction for 15 min. Elution was carried out by leaving 0.5 mL of
methanol containing 1.0 g/L trifluoracetic acid in contact with the
sorbent for at least 6 min. The eluates were evaporated to dryness by a
vacuum centrifuge (Univapo 150 H; Uniequip).
AVP RIA.
Residues were reconstituted in 250 µL of 0.05 mol/L
phosphate buffer (pH 7.5) containing 2.5 g/L bovine serum albumin, 0.01
mol/L Na2EDTA, and 1 g/L neomycin sulfate. Calibrators were
assayed in triplicate; samples were assayed in duplicate. Polyclonal
AVP antiserum (25 µL) in an eightfold higher final dilution than that
recommended by the manufacturer (Amersham) was added to 100 µL of
calibrator or sample. All samples and calibrators were incubated in
polyethylene tubes for 24 h at 4 °C. Diluted
125I-labeled AVP (25 µL; 1500 cpm/25 µL; specific
activity, 74 TBq/mmol), purchased from Amersham, was then added, and
the mixture was incubated for 16 h at 4 °C. To separate the
AVP, 0.5 mL of 2.5 g/L activated charcoal coated with 0.25 g/L dextran
dissolved in 0.05 mol/L phosphate buffer (pH 7.5) was added to the
calibrators and samples, and the mixture was centrifuged immediately
(3600g at 4 °C for 30 min). Supernatants were removed,
and the radioactivity of the pellets was measured for 20 min (Cobra
II-Counting-Systems; Packard Instruments; >74% counting efficiency).
All values obtained were corrected for recovery.
premeasurement sample treatment
Effect of centrifugation on plasma platelet counts and plasma AVP
concentrations.
A 20-mL blood sample was taken from each of five
healthy adult volunteers (ages, 23–50 years; all nonsmokers) and
divided into five aliquots. One aliquot per volunteer was centrifuged
at 200g, 1000g, 1850g, or
6200g (at 4 °C), and one aliquot per volunteer was
centrifuged once at 1250g and then twice at 2100g
(15 min at 4 °C each time). The resulting plasma AVP concentrations
and plasma platelet counts, measured by a platelet counter (Sysmex
2000; TOA Medical Electronics), were determined.
Effect of delayed blood preparation on plasma AVP
concentrations.
A 30-mL blood sample was collected from each of
six healthy adult volunteers (ages, 22–32 years) and divided into 16
aliquots. Individual aliquots were stored at either 4 or 25 °C for
time periods ranging from 0.5 to 48 h. The plasma was subsequently
separated, and plasma AVP concentrations were determined.
avp reference values for children
A total of 203 children and adolescents (105 males and 98 females;
ages, 1 day to 18 years) were studied after routine physical activity
and unrestricted food and water intake. Only subjects in good physical
condition without any obvious disturbance of water or electrolyte
metabolism, nausea, or vomiting were included. Neonates studied during
the first days and weeks of life had a mild degree of
hyperbilirubinemia. The majority of the older children were patients
who were either consulting our general outpatient unit for minor
disorders, reexamination after minor illness, or basic evaluation
before intended surgery, or coming to our specialized unit for
disturbances of sexual development, such as constitutional delay of
growth and puberty, gynecomastia, obesity, or idiopathic precocious
puberty.
Five patients with clinically diagnosed nephrogenic diabetes insipidus (ages, 1.5–2.5 years), and two patients with clinically diagnosed neurogenic diabetes insipidus (ages, 1.5 and 13 years) were studied by way of comparison. Sixteen healthy adults (ages, 23–40 years) served as controls. Blood (1.0–1.5 mL) was obtained from each subject by venipuncture in combination with routine blood collection between 0800 and 1200. Informed consent was obtained from all subjects and/or their parents, and the guidelines of the Helsinki Declaration of 1975 were followed.
statistics
Data are given as means ± SD. Within- and between-run
coefficients of variation (CV) and SD were calculated as described by
Krouwer and Rabinowitz (14). The slope of the calibration
curve was calculated after logit transformation of B/B0 as
described by Rodbard (15). The Student t-test was
used for comparison. P <0.05 was considered significant.
The correlation coefficient (r) was determined, and linear
regression analysis was performed for determining relations.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. The average SD over the entire curve was 3.4%. The slope of
the logit-log transformed calibration curve was -2.1 ± 0.1. The
range of the 80% effective dose (ED80) to the 20%
effective dose (ED20) was 0.045–0.93 pg AVP/tube, which
was equal to 0.25–5.1 ng AVP/L plasma when the 0.5-mL samples were and
extraction procedure described in Materials and Methods were
used. The 50% intercept (ED50) was at 0.2 ± 0.017 pg
AVP/tube (1.1 ± 0.1 ng AVP/L plasma).
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Limit of detection and nonspecific binding.
The minimum
detectable concentration of the assay, defined as the concentration
corresponding to a signal 3 SD above the mean for a calibrator free of
AVP, was 0.06 ng/L when a 0.5-mL sample was extracted. Nonspecific
binding, determined by performing the RIA without antibody, was
1.8% ± 0.3% of the total counts (n = 30).
Within- and between-assay precision.
AVP-free plasma was made
by treating outdated plasma from the blood bank with activated charcoal
(16). Three plasma pools were prepared by adding 0.4, 1.1,
and 3.1 ng/L synthetic AVP (Sigma Chemical Co.) to AVP-free plasma.
Intra- and interassay CVs were assessed by repeated analysis (n =
16) of samples from these plasma pools. The intraassay CV was 10.2%,
5.8%, and 8.5%, and the interassay CV was 11.7%, 6.5%, and 6.6%
for the three plasma pools.
Recovery.
Recovery of cold (unlabeled) AVP in low, medium, and
high physiological concentrations was determined in 16 consecutive
assays. The recovery averaged 83.3% at 0.4 ng/L, 82.5% at 1.1 ng/L,
and 75.2% at 3.1 ng/L. When 1.25 ng of 125I-labeled AVP
was added to 0.25, 0.5, 1, and 2 mL of AVP-free plasma, the recovery
was 86.7%, 86.8%, 86.3%, and 74.5%, respectively.
Dilution test.
Outdated plasma from a blood bank was pooled
and supplemented with 4 mg/L synthetic AVP. Subsequently, it was
further diluted with AVP-free plasma and assayed. As depicted in Fig. 1
, the dilution curve obtained paralleled the calibration curve.
premeasurement sample treatment
Effect of centrifugation on resulting plasma platelet and plasma
AVP concentrations.
The negative relationships between
centrifugation and plasma AVP concentration, and centrifugation and
platelet count, respectively, are shown in Fig. 2
. The significant linear correlation between platelets and AVP
concentration is shown in Fig. 3
(r = 0.899; P <0.001). However, it
is noteworthy that the slopes of the individual correlation curves
differ considerably (Fig. 3
).
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Effect of delayed blood preparation on measured plasma AVP
concentrations.
Storage of blood at ambient temperature (25 °C)
led to an increase in plasma AVP that was significant after 2 h and
reached virtually 100% after 24 h. In contrast, plasma AVP
concentrations of blood stored at 4 °C did not change significantly
within a 48-h storage period (Fig. 4
).
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avp reference values for children
In 203 fully hydrated infants, children, and adolescents, plasma
AVP averaged 1.1 ± 0.6 ng/L. There was no correlation with age
and no significant difference associated with sex. AVP plasma
concentrations in 16 adult volunteers averaged 1.0 ± 0.5 ng/L and
did not differ significantly from those in children and adolescents.
Plasma AVP of five patients with nephrogenic diabetes insipidus was
substantially increased (7.7 ± 4.5 ng/L). Plasma AVP of two
patients suffering from hypophysial diabetes insipidus was below the
detection limit of this RIA (Fig. 5
).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The extraction procedure with the 100-mg Isolute C18 columns that we used is technically simple. With sample volumes of 0.25–1 mL, the recovery was comparable to that reported for Sep-Pak C18 columns (Waters Corp.) loaded with two- or fourfold larger sample volumes (17)(18)(19)(20). However, when loaded with just 0.5 mL of plasma, 360-mg C18 Sep-Pak columns showed markedly lower and more variable recoveries than Isolute C18 columns (data not shown) in our laboratory. These findings are consistent with reports by Ysewijn Van Brussel and De Leenheer (17) of clearly decreased extraction efficiency in Sep-Pak C18 columns when smaller sample volumes (1 mL instead of 2 mL) were processed. One reason for this may be a disproportion between the sorbent and sample volumes. In our laboratory, the extraction efficiency of 100-mg Isolute C18 columns was higher at low volumes and did not differ significantly from 0.25 to 1 mL.
To improve the detection range and assay sensitivity, we tested
nonequilibrium conditions and different dilutions of antiserum with
different quantities of tracer. When extracted plasma was incubated
with antiserum for 24 h before being incubated with antigen for an
additional 16 h, the detection limit was 0.06 ng/L when 0.5 mL of
plasma was extracted, as is done routinely in our laboratory. This, to
our knowledge, is lower than any detection limit reported previously
(Table 1
). In addition, the steep, stable calibration curve, as
indicated by its low SD value, facilitated low intra- and interassay
CVs. The most precise part of the detection range, between
ED80 and ED20 (~0.25–5.1 ng/L),
coincided excellently with physiological AVP plasma concentrations
(Fig. 5
).
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It is well known that contamination of plasma by platelet-bound AVP is considerably greater in platelet-rich than in platelet-poor plasma (5)(21)(22)(23)(24). We ascertained that plasma platelet counts and plasma AVP concentrations are linearly correlated (r = 0.899; P <0.001) and thus confirmed earlier findings by Preibisz et al. (21), Bichet et al. (23) and Inaba et al. (24). We also demonstrated the crucial role of centrifugation speed during blood preparation in the final outcome of AVP measurement. These results emphasize the need for careful plasma preparation because platelets present in plasma cause overestimation of plasma AVP and might be one of the most important reasons for the great differences in basal AVP concentrations observed in different laboratories (21). To produce virtually platelet-free plasma for the assay described, we centrifuge blood samples at 3600g for 20 min at 4 °C.
When blood was stored at 25 °C before separation, plasma AVP concentrations increased significantly; however, they did not change significantly after storage at 4 °C. The increase in AVP at room temperature cannot be explained by alterations in platelet counts because it is known that platelets are stable at room temperature (25); in addition, there was no change in platelet counts during our study of sample storage temperature (data not shown). Therefore, AVP probably was released from blood cells in vitro during storage at room temperature. Anfossi et al. (26) reported a release from platelets after aggregation. Thus, a possible explanation for the increase in AVP is aggregation of platelets caused by the release of platelet aggregation-inducing factors (such as adenosine 5'-pyrophosphate or arachidonate) from hemolyzed red blood cells.
AVP concentrations in our children (1.1 ± 0.6 ng/L) were very similar to those reported recently by other groups (5)(21)(22)(23)(24) in adults when platelet-free plasma was extracted (0.7–1.7 ng/L). However, the AVP concentrations in our children are substantially lower than the values reported by Rascher et al. (3) in 145 children, and their study was the only one on reference values in a large number of children up to now. We were not able to confirm the tendency toward higher plasma AVP concentrations in infants (ages, 1–12 months) reported by Rascher et al. (3). In addition to the influence of platelets on measured AVP concentrations, the hydration status of patients or characteristics of the antisera used may differ, and thus may be additional reasons for diverging results.
In conclusion, this optimized procedure for determining AVP in plasma provides an extremely low detection limit even when small sample volumes are extracted, a detection range adapted to physiological AVP concentrations, and a high degree of reproducibility. Therefore, it may serve as a tool for determining AVP in infants and children. The simple extraction procedure, short incubation periods, and the commercial availability of reagents permit the easy establishment of this method in any suitably equipped laboratory.
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Acknowledgments |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) generated from 16
consecutive calibration curves and dilution curve (
) from pooled
plasma supplemented with synthetic AVP and diluted with AVP-free
plasma.
), and two
patients with central diabetes insipidus (