Printer Friendly

Radioiodinated tyrosyl-ouabain and measurement of a circulating ouabain-like compound.

A sodium-potassium pump inhibitor has been isolated and chemically characterized from human plasma (1, 2), bovine hypothalamus (3-5), and bovine adrenals (6, 7). Several studies, especially one using nanogram-scale chemical derivatization (4), have indicated that the compound isolated from human plasma and bovine hypothalamus is an isomer of ouabain, rather than ouabain itself. However, in recent studies using microgram amounts of purified material (5, 7), molecular mass and proton nuclear magnetic resonance ([sup.1]H NMR) [4] spectroscopic measurements have demonstrated that the adrenal and hypothalamic sodium pump inhibitors are indistinguishable from authentic ouabain. Because cardiac glycosides, such as ouabain, inhibit [Na.sup.+],[K.sup.+]-ATPase (8) and thus increase intracellular sodium, they might have a physiological role (e.g., during pregnancy) as endogenous natriuretic and vasoactive substances. Increased concentrations of immunoreactive ouabain have been observed in hypertensive patients, indicating that the measurement of an endogenous ouabain-like compound might have clinical interest (9, 10), especially because there are studies showing an endogenous, i.e., adrenocortical, origin of this compound (6, 11).

According to data available, plasma immunoreactive ouabain concentrations are laboratory-specific (12), indicating that reliable measurement is not without problems. Cardiac glycosides conjugated to proteins are good immunogens, and several groups have been able to establish enzyme and [3sup.H]-immunoassays for the measurement of ouabain immunoreactivity in body fluids (13-18). RIAs and ELISAs are notoriously prone to nonspecific interference when used to measure picomolar, i.e., low endogenous concentrations of biologically active substances. This may be a reason for the different plasma ouabain equivalents reported previously, varying from <5 pmol/L (15) to 100-300 pmol/L (14,17,18) to [greater than or equal to] 1000 pmol/L (13) for human plasma, and from [less than or equal to] 50 pmol/L (19) to [greater than or equal to] 200 pmol/L for rat plasma (18) in solid-phase-extracted samples. It has even been reported that the immunoreactive ouabain in human plasma is not authentic ouabain because in some HPLC analyses of plasma extracts, no immunoreactivity was observed at the elution position of authentic ouabain (15, 20). These discrepant results indicate the need for further development of ouabain assay methods suitable for physiological and clinical studies.

In the present study, we raised in rabbits high-affinity polyclonal antisera against ouabain and synthesized a new [sup.125]I-labeled tyrosyl-ouabain derivative for use as a tracer of high specific activity. The new RIA could be used, in conjunction with solid-phase extraction, for routine measurement of an endogenous ouabain-like compound present in biological samples.

Materials and Methods

MATERIALS

Ouabain (G-strophanthin) octahydrate, ouabagenin, dihydroouabain, digoxin, digitoxin, digoxigenin, and other related steroids (aldosterone, bufalin, cortisone, hydrocortisone, progesterone, [beta]-estradiol, estrone, testosterone, and dehydroepiandrosterone), bovine thyroglobulin, and sodium cyanoborohydride were obtained from Sigma. Freund's incomplete and complete adjuvants were purchased from Difco Laboratories, L-tyrosine and polyethylene glycol 6000 were from Fluka AG, [[sup.3]H]ouabain ([21,22-[sup.3]H]ouabain) and Na [sup.125]I (IMS 300) were from Amersham Pharmacia Biotech, and Sephadex G-50F was from Pharmacia Fine Chemicals. NaI[O.sub.4], Chloramine-T, and all other chemicals were obtained from E. Merck AG.

PREPARATION OF OUABAIN ANTIGEN AND ANTISERA

Ouabain was coupled with bovine thyroglobulin as described by Masugi et al. (13). Ouabain (29 mg) was allowed to react with NaI[O.sub.4] (11 mg) in distilled water overnight at 4[degrees]C. Ethanol (1 mL) was then added, and the reaction solution was adjusted to pH 8 with 1 mol/L NaOH. Finally, bovine thyroglobulin (20 mg dissolved in 1 mL of 0.05 mol/L sodium phosphate, pH 9) was added dropwise to 1 mL of the reaction solution (14.5 mg of ouabain and 5.5 mg of NaI[O.sub.4]). After vortex-mixing and incubation for 2 h, sodium cyanoborohydride (2.5 mg in 200 [micro]L of distilled water) was added and the reaction solution was applied to a gel filtration column (Sephadex G-50F in 9 g/L NaCl) for fractionation. The ouabain-thyroglobulin conjugate, eluting in the void volume, was emulsified in Freund's complete adjuvant and injected into five rabbits (1 mg of conjugate per rabbit). Booster injections of 0.5 mg per rabbit, emulsified in Freund's incomplete adjuvant, were given at monthly intervals. As measured by the absorbance at 219 nm, the hapten-carrier ratio of the conjugate was 160:1.

PREPARATION OF RADIOIODINATED OUABAIN TRACER

Ouabain was first coupled with L-tyrosine with a method analogous to the one described above. After the reaction of ouabain (3.1 mg) with NaI[O.sub.4] (1.4 mg) in distilled water (0.1 mL), ethanol was added to the reaction solution, and the pH was adjusted to 8. L-Tyrosine (0.8 mg in 0.4 mL of 0.05 mol/L sodium phosphate, pH 9) was then added dropwise, followed by incubation for 2 h. Sodium cyanoborohydride (0.25 mg in 0.2 mL of distilled water) was added, and the reaction solution was fractionated by reversed-phase HPLC [Vydac [C.sub.18] column; 30-min linear 5-50% methanol gradient in 0.5 mL/L trifluoroacetic acid (TFA); flow-rate, 1.0 mL/min]. The main peak with ultraviolet (UV) absorbance in the HPLC profile of the tyrosylating solution (23 min) was used for radioiodination as follows: Tyrosyl-ouabain (1 [micro]g) was allowed to react with Na125I (19 MBq) in the presence of Chloramine-T (10 [micro]g); after 30 s, sodium metabisulfite (10 [micro]g) was added, and the reaction solution was applied into the HPLC column (Vydac [C.sub.18]) for reversed-phase fractionation (30-min 10-40% acetonitrile gradient in 1 mL/L TFA; flow-rate, 1.0 mL/min).

MASS SPECTROMETRIC ANALYSES OF TYROSYL-OUABAIN

The main peak (23 min), as detected by UV absorbance, in the HPLC separation of the tyrosylating reaction products of ouabain was further purified in another HPLC using a Waters SymmetryShield[TM]1 R[P.sub.8] column and a 30-min gradient from 0% to 30% methanol in distilled water with a flow rate of 1.0 mL/min. The product (0.2-10 [micro]g) was analyzed with a quadrupole time-of-flight tandem instrument (Micromass Ltd) equipped with electrospray ionization and atmospheric pressure chemical ionization (APCI) sources. The sample was infused at a 1:1000 dilution in acetonitrile-water (50:50 by volume) containing 2 mL/L formic acid into the electrospray source, using a syringe pump (Harward Apparatus).

SepPak SOLID-PHASE EXTRACTION OF PLASMA SAMPLES

Human blood from healthy adults taking no medication was collected from an antecubital vein into EDTA tubes at 9-10 h. Rat blood was obtained by decapitation of adult Sprague-Dawley rats of both sexes at 9-10 h. The blood samples were immediately centrifuged, and the plasmas were stored at -20[degrees]C. For ouabain measurements, the plasma samples were extracted using SepPak [C.sub.18] cartridges (Waters) and an automated Gilson Aspec system. Briefly, the cartridges were preconditioned with 2-propanol and 1 mL/L TFA. The 1-mL plasma samples were acidified with 0.2 mL of 1 mol/L HCl containing 16 g/L glycine and passed through the cartridges. After a 2-mL wash with 1 mL/L TFA, ouabain was eluted with 3 mL of 400 mL/L acetonitrile in 1 mL/L TFA. After evaporation in a SpeedVac (Savant Instruments), the extracts were reconstituted in 250 [micro]L of RIA buffer.

RIA PROCEDURE

The calibrators and samples were pipetted in duplicates of 100 [micro]L. The antiserum (a-ouabain-199-13-4-95) and tracer were added simultaneously (total volume added, 100 [micro]L). After an overnight incubation, the bound and free fractions were separated by double-antibody precipitation for 15 min at room temperature. Precipitation was accelerated by the addition to the assay tubes of polyethylene glycol 6000 in a final concentration of 57 g/L. The precipitates were counted in a C1iniGamma gamma counter (Wallac). For [sup.3]H measurements, the precipitates were resuspended in 250 [micro]L of distilled water, transferred into counting vials containing scintillation fluid, and counted in a Wallac beta counter (16).

RIA VALIDATION

Serial dilutions of SepPak extracts of human and rat plasma were assayed against the ouabain calibrator. Plasma extracts were also analyzed by reversed-phase HPLC, using a 4.6 x 150 mm Vydac [C.sub.18] 218 TP column (Separations Group) and a 30-min linear gradient from 5% to 50% acetonitrile in 1 mL/L TFA with a flow rate of 1.0 mL/min. The fractions were dried in a SpeedVac, redissolved in RIA buffer, and subjected to the ouabain RIA. From a selected chromatographic run, one-third of the peak fraction with ouabain immunoreactivity was reanalyzed in another HPLC system with different selectivity, using a 4.6 x 250 mm Vydac [C.sub.8] 228TP pH-stable column and a 30-min linear gradient from 0% to 30% acetonitrile in 1 mL/L TFA with the flow rate of 1.0 mL/min. Blank runs, using water as the sample, were carried out for both HPLC systems to control for background immunoreactivity.

Results

ANTISERUM TITER

All five rabbits produced ouabain antisera against the ouabain-thyroglobulin conjugate. After the first booster, the titers were 1:3000-1:7000 with the tritiated tracer and 1:300 000-1:1000 000 with the radioiodinated tracer. The titer could be maintained or even increased for at least 1 year with monthly boosters (Table 1). The antiserum bleed selected for RIA use (see below) bound 30% of the [sup.125]I-labeled tyrosyl-ouabain tracer (estimated specific activity, 2100 Ci/ mmol) at a final dilution of 1:2 000 000 (Table 1). By comparison, a dilution of 1:14 000 of the same antiserum was required to bind 30% of [[sup.3]H]ouabain tracer (specific activity reported by the manufacturer, 20 Ci/mmol).

RADIOIODINATED OUABAIN TRACER

The coupling of tyrosine to ouabain yielded several products separable by HPLC. As seen in Fig. 1, the main peak, as detected by UV absorbance, in the first HPLC purification step eluted at 23 min, 5 and 15 min later than ouabain and tyrosine, respectively. Radioiodination of the main peak and subsequent purification by reversed-phase HPLC yielded a radioactive product that showed high binding affinity to the ouabain antisera (Table 1). The [sup.125]I-labeled tyrosyl tracer was relatively stable and could be used in the RIA for 4 months when stored at [less than or equal to] 10[degrees]C.

MASS SPECTRUM OF TYROSYL-OUABAIN

The APCI mass spectrum of the main HPLC peak of the coupling reaction solution is shown in Fig. 2. The mass of the suggested protonated molecule was 702 Da. Furthermore, the exact mass measurements done using the electrospray mode showed that the [[M-H].sup.+] ion of tyrosylouabain was 702.3510 Da, corresponding to the molecular formula of [C.sub.37][H.sub.52][O.sub.12]N. Tandem mass spectrometric measurement revealed two main fragments at m/z 439.2352 (C.sub.23][H.sub.35][O.sub.8]) and at m/z 264.1236 ([C.sub.14][H.sub.12][O.sub.4]N), corresponding to cleavage of the glycosidic bond with a proton left on either side. Fig. 3 shows the postulated reaction mechanism of ouabain and tyrosine based on the schedule of Harris et al. (14). The schedule leads to a tyrosyl-ouabain conjugate with a formula mass of 702 Da.

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]

ASSAY CHARACTERISTICS

As seen in Table 1, the assay detection limit (defined at 5% displacement of tracer) for each antiserum was [less than or equal to] 1.8 fmol/tube ([less than or equal to] 1.3 pg/tube), which corresponded to a plasma concentration of [less than or equal to] 5 pmol/L ([less than or equal to] 3 ng/L). The 50% displacement was achieved at 6-14 fmol (4-10 pg). Fig. 4 demonstrates that the assay detection limit is 0.6 fmol/ tube (0.4 pg/tube) with the [sup.125]I-labeled tyrosyl tracer and 44 fmol/tube (32 pg/tube) with the [sup.3]H tracer, using the antiserum a-ouabain-199 selected for RIA use. The displacement was linear between 3 and 50 fmol. The specificities of the different antisera were tested with ouabain, digitalis, and related compounds as well as with several steroids. The cross-reactivity data of a-ouabain-199 are presented in Table 2. Ouabagenin showed the highest cross-reactivity (52%), followed by digoxin (1.7%) and digitoxin (0.6%), whereas the cross-reactivity of all steroids was <0.001%. The intraassay CVs for low (6.5 pmol/L), medium (37 pmol/L), and high (168 pmol/L) ouabain concentrations were 8.5%, 3.7%, and 4.3%, respectively (n = 10). The interassay CVs for low (15 pmol/L), medium (42 pmol/L), and high (167 pmol/L) ouabain concentrations were 13%, 9.4% and 10%, respectively (n = 10). Nonspecific binding was 1-2%.

[FIGURE 3 OMITTED]

RIA VALIDATION

Different amounts of plasma solid-phase extracts displaced [sup.125]I-labeled tyrosyl-ouabain tracer in parallel with the authentic ouabain calibrator (Fig. 4). Immunoreactive ouabain in human and rat plasma extracts coeluted with authentic ouabain in two different reversed-phase HPLC conditions (at 14 min in the first and 12 min in the second HPLC system; see Fig. 5). The recovery of added ouabain in a HPLC test run was 89.6%. No ouabain immunoreactivity was detected in the blank samples.

[FIGURE 4 OMITTED]

PLASMA CONCENTRATIONS OF IMMUNOREACTIVE OUABAIN IN HEALTHY HUMANS AND RATS

The recovery of ouabain added to human plasma samples (1 mL) from solid-phase extraction with SepPak [C.sub.18] cartridges was 102.8% [+ or -] 2.5% (mean [+ or -] SE; n = 5) for a low dose (34 fmol added) and 93.6% [+ or -] 1.3% for a high dose (103 fmol added). In accordance with these results, the recovery in acidic extraction conditions with respect to neutral conditions (HCl and TFA replaced with distilled water) was 97.5% [+ or -] 2.1% (mean [+ or -] SE; n = 6). Plasma samples (1 mL) from healthy adult women and men and from adult rats of both sexes were extracted by the solid-phase cartridges and measured in the ouabain RIA. The plasma concentrations were 10.0 [+ or -] 1.3 pmol/L (mean [+ or -] SE) in women 22-45 years of age (n = 10), 12.0 [+ or -] 0.9 pmol/L in men 23-55 years of age (n = 10), and 41.2 [+ or -] 9.6 pmol/L in rats.

[FIGURE 5 OMITTED]

Discussion

In the present study, we developed a [sup.125]I-RIA for ouabain measurements with a detection limit almost 100-fold lower than that of our corresponding [sup.3]H-RIA. The key components in our method are the new [sup.125]I-labeled tyrosyl-ouabain tracer and the high-affinity ouabain antiserum. The work was based on our previous results involving the synthesis and use of a radioiodinated tracer in melatonin RIA (21, 22). We adopted a modification of the method of Masugi et al. (13) to make the ouabain-thyroglobulin conjugate, which was used as an immunogen, and to make the ouabain-tyrosine conjugate, which was used to prepare radioiodinated tracer for the RIA. In both cases, the insertion probably takes place in the sugar moiety of ouabain. This is supported by the cross-reactivity profile of the ouabain antiserum (a-ouabain-199) raised against the ouabain-thyroglobulin conjugate, which demonstrated the highest cross-reactivity (52%) to the aglycone ouabagenin. The steroid nucleus with its hydroxyl groups and apparently also the lactone ring thus form the antigenic determinant. Previously, Takemura et al. (23) used periodate to cleave the sugar ring of digoxin. However, they used 2-hydroxy-3-methylbenzoyl-hydrazide, instead of sodium cyanoborohydride, in the preparation of a derivative for radioiodination. This derivatization did not eliminate the biologic or immunologic activity of digoxin. In some digoxin assays, radioiodinated tracers have also been prepared by coupling digoxigenin to tyrosine via succination without periodation (24).

We characterized the structure of the synthesized putative tyrosyl-ouabain by mass spectrometry. The product was first purified to homogeneity by reversed-phase HPLC. APCI analysis gave a molecular ion mass of 702 Da. Interestingly, this is consistent with the reaction mechanism proposed by Harris et al. (14) for ouabain and bovine serum albumin. In this proposed two-phase reaction mechanism, the rharnnose moiety of ouabain is first opened by periodation, losing a carbon atom, a water molecule, and two hydrogen atoms. Subsequently, in the coupling phase, the side chain of the tyrosine molecule is attached to the opened rharnnose moiety of ouabain when two more oxygen atoms are lost. The mechanism would provide a molecular mass of exactly 702 Da to the product, tyrosyl-ouabain.

Several studies have shown that human plasma contains a cardiac glycoside, i.e., a ouabain-digitalis-like factor, the structure of which is indistinguishable from plant-derived ouabain as judged from various HPLC, mass spectrometric, and RIA analyses (1, 2, 13, 14, 17, 18,25-27). However, a mass spectrometric analysis by Mathews et al. (27) raised the possibility of slight differences in hydroxylation positions of the steroid nucleus of the human plasma compound. Initially, this review was reinforced by studies using nanoscale fluorescent naphthoylate derivatives of the human and bovine compounds (3, 4). However, recent NMR studies (5) showed that the bovine compounds formed complexes with borate under isolation conditions and NMR processes carried out in borosilicate glassware (4,5). Therefore, although not confirmed by direct observation, it was inferred that the bovine hypothalamic compound as isolated would have been indistinguishable from ouabain. Furthermore, another recent study based on mass spectrometric and [sup.1]H NMR analyses showed that the compound isolated from the bovine adrenal glands was also indistinguishable from ouabain (7). Thus, it now seems that the endogenous bovine adrenal and hypothalamic ouabain-like material is ouabain (circulating compound remains to be resolved).

Circulating ouabain or ouabain-like activity has been measured by many techniques, including bioassays, radioreceptor assays using membrane fractions from several tissues (28), and ouabain and digoxin immunoassays. The bioassay methods, which are based on [Na.sup.+],[K.sup.+]-ATPase inhibition (ATP hydrolysis by the sodium pump or the inhibition of the in-trans location activity of the [Na.sup.+],[K.sup.+]-ATPase) are ouabain-specific and have been used to verify the authenticity of isolated endogenous compounds. These methods are not ideal for routine measurements because they are cumbersome and have lower sensitivities than immunoassays.

The available ouabain immunoassays have recently been compared in reviews by Semra et al. (16) and by us (12). Although the best enzyme immunoassays and RIAs are believed to possess detection limits (10-30 pmol/L) suitable for physiological studies, widely varying plasma concentrations, ranging from 0.04 to 1.1 nmol/L, have been reported for healthy adults in different studies (13-18). Moreover, no circulating ouabain immunoreactivity was detected in some studies (15, 20). These laboratory-specific results might be related to differences in antiserum specificity and sensitivity; the use of different tracers, generally [[sup.3]H]ouabain and enzyme-linked ouabain analogs; the use of different sample volumes, causing variable matrix effects; and/or the presence of potential interfering factor(s) in plasma, leading to over-estimation of concentrations (17, 28). Because of the laboratory-specific differences, a reference interval of normal concentrations cannot be reliably inferred, especially when sufficient validation has not been carried out in all studies regarding the substance measured in the assay. Thus, at present, ouabain equivalents are preferred over ouabain, meaning that from a methodological perspective, plasma measurements are lacking a laboratory routine.

Therefore, it was necessary for us to carry out several validation tests giving special attention to the identity of plasma immunoreactivity measured in our RIA. We found that the extraction of plasma samples is essential for valid measurements. The SepPak [C.sub.18] reversed-phase extraction method provided necessary purification and a nearly complete recovery of ouabain added to human plasma and rat samples. Validation of quantification of plasma immunoreactive ouabain was demonstrated by the parallel dilution of plasma extracts with the ouabain calibrator. Furthermore, additional support for the analytical validity of the measurements was obtained by the finding that immunoreactive ouabain in human plasma extracts coeluted with authentic ouabain in two reversed-phase HPLC systems with different selectivities. Moreover, our assay detected the previously isolated ouabainlike compound from human plasma (endogenous ouabain discovered by Hamlyn) with high cross-reactivity (12).

The results obtained with our new sensitive RIA support previous findings that ouabain itself or ouabain-like compound is present in normal human plasma (1, 2,13, 14, 18, 26,27). In the present study, we were able to detect immunoreactive ouabain both before and, unlike in the study of Lewis et al. (15), after HPLC fractionation. According to our plasma measurements, however, immunoreactive ouabain concentrations are low in healthy adults, 1.5- to 3-fold lower than those reported in the most sensitive enzyme immunoassay-based and [sup.3]H-RIA studies (19, 29, 30).

In conclusion, we have synthesized and characterized by mass spectrometry a tyrosyl-ouabain conjugate suitable for radioiodination. The new molecule made possible development of a ouabain [sup.125]I-RIA with superior sensitivity. Coupled with solid-phase extraction of plasma samples, the assay reliably detects immunoreactive ouabain concentrations of 5 pmol/L. In our HPLC and other validation tests, this immunoreactive ouabain behaved like authentic ouabain. Thus, our RIA allows measurement of the proposed low endogenous ouabain equivalents in both physiological and pathophysiological situations. The new method provides an analytical tool with which it is possible to establish the potential homeostatic and clinical significance of endogenous ouabain.

We thank Tuula Lumijarvi and Micromass Ltd application laboratory for expert assistance and support. Sighvatur S. Arnason was supported by the Icelandic University Research Fund.

Received January 24, 2000; accepted October 27, 2000.

References

(1.) Hamlyn JM, Blaustein MP, Bova S, DuCharme DW, Harris DW, Mandel F, et al. Identification and characterization of a ouabain-like compound from human plasma. Proc Natl Acad Sci U S A 1991;88:6259-63.

(2.) Ludens JH, Clark MA, DuCharme DW, Harris DW, Lutzke BS, Mandel F, et al. Purification of an endogenous digitalis-like factor from human plasma for structural analyses. Hypertension 1991;17:923-9.

(3.) Tymiak AA, Norman JA, Bolgar M, DiDonato GC, Lee H, Parker WL, et al. Physicochemical characterization of a ouabain isomer isolated from bovine hypothalamus. Proc Natl Acad Sci U S A 1993;90:8189-93.

(4.) Zhao N, Lo LC, Berova N, Nakanishi K, Tymiak A, Ludens JH, Haupert GT. Na-K ATPase inhibitors from bovine hypothalamus and human plasma are different from ouabain: nanogram scale CD structural analysis. Biochemistry 1995;34:9893-6.

(5.) Kawamura A, Guo J, Itagaki Y, Bell C, Wang Y, Haupert GT Jr, et al. On the structure of endogenous ouabain. Proc Natl Acad Sci U S A 1999;96:6654-9.

(6.) Laredo J, Hamilton BP, Hamlyn JM. Ouabain is secreted by bovine adrenocortical cells. Endocrinology 1994;135:794-7.

(7.) Schneider R, Wray V, Nimtz M, Lehmann WD, Kirch U, Antolovic R, Schoner W. Bovine adrenals contain in addition to ouabain, a second inhibitor of the sodium pump. J Biol Chem 1998;273:784-92.

(8.) Goto A, Yamada K, Yagi N, Yoshioka M, Sugimoto T. Physiology and pharmacology of endogenous digitalis-like factors. Pharmacol Rev 1992;44:377-99.

(9.) Hamlyn JM, Lu ZR, Manunta P, Ludens JH, Kimura K, Shah JR, et al. Observations of the nature, biosynthesis, secretion and significance of endogenous ouabain. Clin Exp Hypertens 1998;20:523-33.

(10.) Doris PA, Bargrov AY. Endogenous sodium pump inhibitors and blood pressure regulation: an update on recent progress. Proc Soc Exp Bio Med 1998;218:156-67.

(11.) Perrin A, Brasmes B, Chambaz EM, Defayne G. Bovine adrenocortical cells in culture synthesize an ouabain-like compound. Mol Cell Endocrinol 1997;126:7-15.

(12.) Vakkuri O, Arnason SS, Pouta A, Vuolteenaho O, Leppaluoto J. Radioimmunoassay of plasma ouabain in healthy and pregnant subjects. J Endocrinol 2000;165:669-77.

(13.) Masugi F, Ogihara T, Hasegawa T, Tomii A, Nagano M, Higashimori K, et al. Circulating factor with ouabain-like immunoreactivity in patients with primary aldosteronism. Biochem Biophys Res Commun 1986;135:41-5.

(14.) Harris DW, Clark MA, Fisher JD, Hamlyn JM, Kolbasa KP, Ludens JH, DuCharme DW. Development of an immunoassay for endogenous digitalis-like factor. Hypertension 1991;17:936-43.

(15.) Lewis LK, Yandle TG, Lewis JG, Richards AM, Pidgeon GB, Kaaja RJ, Nicholls MG. Ouabain is not detectable in human plasma. Hypertension 1994;24:549-55.

(16.) Semra YK, Butt AN, Swaminathan R. Effect of salt intake on excretion of endogenous ouabain-like substance, measured by RIA. Clin Chem 1996;42:1949-54.

(17.) Harwood S, Little JA, Gallacher G, Perrett D, Edwards R, Dawnay A. Development of enzyme immunoassay for endogenous ouabain-like compound in human plasma. Clin Chem 1997;43:715-22.

(18.) Ferrandi M, Manunta P, Balzan S, Hamlyn JM, Bianchi G, Ferrari P. Ouabain-like factor quantification in mammalian tissues and plasma: comparison of two independent assays. Hypertension 1997;30:886-96.

(19.) Gomez-Sanchez EP, Foeking MF, Sellers D, Blankenship MS, Gomez-Sanchez CE. Is the circulating ouabain-like compound ouabain? Am J Hypertens 1994;7:647-50.

(20.) Doris PA, Jenkins LA, Stocco DM. Is ouabain an authentic endogenous mammalian substance derived from the adrenal? Hypertension 1994;23:632-8.

(21.) Vakkuri O, Leppaluoto J, Vuolteenaho O. Development and validation of a melatonin radioimmunoassay using radioiodinated melatonin as tracer. Acta Endocrinol 1984;106:152-7.

(22.) Vakkuri O, Lamsa E, Rahkamaa E, Ruotsalainen H, Leppaluoto J. Iodinated melatonin: preparation and characterization of the molecular structure by mass and 1H NMR spectroscopy. Anal Biochem 1984;142:284-9.

(23.) Takemura Y, Fujibayashi Y, Dote N, Taniuchi H, Iijima N, Konishi J, Yokoyama A. Development of glycoside-bound radiopharmaceuticals: novel radioiodination method of digoxin. Biol Pharm Bull 1994;17:97-101.

(24.) Barbieri U, Gandolfi C. Digoxin radioimmunoassay: a comparison of the assay performances using tritiated and [sup.125]I radioiodinated tracers. Clin Chim Acta 1977;77:257-67.

(25.) Bernini G, Paci A, Sgro M, Moretti A, Salvetti A. Endogenous digitalis-like factor and ouabain immunoreactivity in adrenalectomized patients and normal subjects after acute and prolonged salt loading. Am J Hypertens 1998;11:1-7.

(26.) Butt AN, Semra YK, Lane SJ, Lee T, Swaminathan R. Endogenous ouabain secretion in man is not regulated by ACTH. J Steroid Biochem Mol Biol 1998;66:151-7.

(27.) Mathews WR, DuCharme DW, Hamlyn JM, Harris DW, Mandel F, Clark MA, Ludens JH. Mass spectral characterization of an endogenous digitalis-like factor from human plasma. Hypertension 1991;17:930-5.

(28.) Paci A, Ledda A, Ciarimboli G, Pascal B, Bernini GP. Commercial enzyme immunoassay reagent pack for ouabain compared with human placenta radioreceptor assay. Clin Chem 1996;42:648-50.

(29.) Naruse K, Naruse M, Tanabe A, Yoshimoto T, Watanabe Y, Kurimoto F, et al. Does plasma immunoreactive ouabain originate from the adrenal gland? Hypertension 1994;23(Suppl 1):I102-5.

(30.) Balzan S, Montali U, DiBartolo V, Ghione S. Further evidence for an endogenous digitalis-like compound in newborn and adult plasma detected by antiouabain antiserum. Life Sci 1997;60:893-8.

[4] Nonstandard abbreviations: NMR, nuclear magnetic resonance; TFA, trifluoroacetic acid; UV, ultraviolet; and APCI, atmospheric pressure chemical ionization.

Olli Vakkuri, [1] * Sighvatur S. Arnason, [2] Paivi Joensuu, [3] Jorma Jalonen, [3] Olli Vuolteenaho, [1] and Juhani Leppaluoto [1]

[1] Department of Physiology, University of Oulu, PO Box 5000, 90401 Oulu, Finland.

[2] Department of Physiology, University of Iceland, Vatnsmyrarvegi 16, IS-101 Reykjavik, Iceland.

[3] Department of Chemistry, University of Oulu, PO Box 333, 90571 Oulu, Finland.

* Author for correspondence. Fax 358-08-5375320; e-mail olli.vakkuri@ oulu.fi.
Table 1. Titer (a) and detection limit (b) values for ouabain
antisera after three boosters.

Ouabain Detection limit,
antiserum Titer fmol/tube

a-ouabain-199 1:2 000 000 0.6
a-ouabain-200 1:10 000 000 0.4
a-ouabain-201 1:3 000 000 0.7
a-ouabain-202 1:1500 000 0.8
a-ouabain-203 1:500 000 0.4

(a) Titer was determined as the antiserum dilution that bound 30%
of the radioiodinated tracer.

(b) Detection limit was calculated as fmol/tube at the antiserum
dilution that bound 95% of the radioiodinated tracer.

Table 2. Cross-reactivity of various analogs and other
related compounds of ouabain with ouabain antiserum
a-ouabain-199. (a)

Compound Cross-reactivity,

Ouabain 100
Ouabagenin 52.2
Dihydroouabain 0.7
Digoxin 1.7
Digitoxin 0.6
Digoxigenin 0.5
Bufalin 1.0
Aldosterone <0.001
Hydrocortisone <0.001
Cortisone <0.001
Progesterone 0.00017
Testosterone <0.001
Dehydroepiandrosterone <0.001
3[beta]-Hydroxy-5-androsten-17-one-S[O.sub.4] <0.001
[beta]-Estradiol 0.00006
Estrone <0.001

(a) Cross-reactivity was determined at 50% displacement of
[sup.125]Habeled tyrosylouabain on weight basis.
COPYRIGHT 2001 American Association for Clinical Chemistry, Inc.
No portion of this article can be reproduced without the express written permission from the copyright holder.
Copyright 2001 Gale, Cengage Learning. All rights reserved.

Article Details
Printer friendly Cite/link Email Feedback
Title Annotation:Endocrinology and Metabolism
Author:Vakkuri, Olli; Arnason, Sighvatur S.; Joensuu, Paivi; Jalonen, Jorma; Vuolteenaho, Olli; Leppaluoto,
Publication:Clinical Chemistry
Date:Jan 1, 2001
Words:4695
Previous Article:Correlation of mycophenolic acid pharmacokinetic parameters with side effects in kidney transplant patients treated with mycophenolate mofetil.
Next Article:Use of free radical chemistry in an immunometric assay for 17[beta]-estradiol.
Topics:


Related Articles
High-pressure hormone: mysterious blood compound may plant seeds of hypertension.
Cardiovascular effects of the aqueous extract of Gynostemma pentaphyllum Makino.
Digoxin-like immunoreactive factors induce apoptosis in human acute T-cell lymphoblastic leukemia.
Human adrenal cells in culture produce both ouabain-like and dihydroouabain-like factors.
Sodium pump isoforms in xenotransplantation: importance of biochemical compatibility.
Biosynthesis of endogenous cardiac glycosides by mammalian adrenocortical cells: three steps forward.
De novo biosynthesis and radiolabeling of mammalian digitalis-like factors.
Isolation and characterization of a digoxin-like immunoreactive substance from human urine by affinity chromatography.
Development of enzyme immunoassay for endogenous ouabain-like compound in human plasma.

Terms of use | Copyright © 2018 Farlex, Inc. | Feedback | For webmasters