Ouabain-like factor quantification in mammalian tissues and plasma: Comparison of two independent assays

Mara Ferrandi, Paolo Manunta, Silvana Balzan, John M. Hamlyn, Giuseppe Bianchi, Patrizia Ferrari

Research output: Contribution to journalArticle

115 Citations (Scopus)

Abstract

The resolution of controversies that concern the detectability of an endogenous ouabain-like factor (OLF) in mammalian tissues and plasma was approached by the application of a standardized method for its extraction and quantification. Two independent assays were used to quantify the OLF: (1) a radioimmunoassay, which used a polyclonal anti-ouabain antiserum, and (2) a radioenzymatic assay based on the inhibition of dog kidney Na+,K+-ATPase. Plasma and tissues were obtained from the Milan hypertensive strain (MHS) and the Milan normotensive strain (MNS) of rats and from healthy human volunteers. Results indicate that (1) a single high-performance liquid chromatography (HPLC) fraction identical to that of ouabain was identified by both assay methods in the rat hypothalamus and hypophysis and in both rat and human plasma; (2) dilution curves of OLF and standard ouabain were parallel and with a similar K(d), both in radioimmunoassay (3 nmol/L) and ATPase assay (14 nmol/L); (3) after HPLC, OLF was similarly quantified by the two methods in the hypothalamus, hypophysis, adrenals, and plasma of rats and in human plasma; (4) OLF was present in larger amounts in the hypothalamus, hypophysis, and plasma of MHS rats than that of MNS rats; (5) the HPLC fraction of human plasma was quantified similarly by both assays (range, 60 to 150 pmol/L); (6) recovery of standard ouabain in pre-HPLC plasma extracts was approximately 90%; and (7) pre-HPLC OLF concentrations in human plasma ranged between 0.05 and 0.75 nmol/L. Rat cerebral tissues and both rat and human plasma contained measurable amounts of OLF, which were quantified similarly by radioimmunoassay and ATPase assay, both before and after HPLC fractionation. The increased MHS tissue and plasma levels of OLF are in keeping with the pathogenetic role of this factor in MHS hypertension.

Original languageEnglish
Pages (from-to)886-896
Number of pages11
JournalHypertension
Volume30
Issue number4
Publication statusPublished - Oct 1997

Fingerprint

High Pressure Liquid Chromatography
Ouabain
Pituitary Gland
Hypothalamus
Radioimmunoassay
Adenosine Triphosphatases
digoxin-like factors
Immune Sera
Healthy Volunteers
Dogs
Hypertension
Kidney

Keywords

  • Human
  • Na,K-ATPase
  • Ouabain
  • Plasma
  • Rats
  • Tissue

ASJC Scopus subject areas

  • Internal Medicine

Cite this

Ferrandi, M., Manunta, P., Balzan, S., Hamlyn, J. M., Bianchi, G., & Ferrari, P. (1997). Ouabain-like factor quantification in mammalian tissues and plasma: Comparison of two independent assays. Hypertension, 30(4), 886-896.

Ouabain-like factor quantification in mammalian tissues and plasma : Comparison of two independent assays. / Ferrandi, Mara; Manunta, Paolo; Balzan, Silvana; Hamlyn, John M.; Bianchi, Giuseppe; Ferrari, Patrizia.

In: Hypertension, Vol. 30, No. 4, 10.1997, p. 886-896.

Research output: Contribution to journalArticle

Ferrandi, M, Manunta, P, Balzan, S, Hamlyn, JM, Bianchi, G & Ferrari, P 1997, 'Ouabain-like factor quantification in mammalian tissues and plasma: Comparison of two independent assays', Hypertension, vol. 30, no. 4, pp. 886-896.
Ferrandi, Mara ; Manunta, Paolo ; Balzan, Silvana ; Hamlyn, John M. ; Bianchi, Giuseppe ; Ferrari, Patrizia. / Ouabain-like factor quantification in mammalian tissues and plasma : Comparison of two independent assays. In: Hypertension. 1997 ; Vol. 30, No. 4. pp. 886-896.
@article{9c285ad642c24d26ba3b2880ba9123a8,
title = "Ouabain-like factor quantification in mammalian tissues and plasma: Comparison of two independent assays",
abstract = "The resolution of controversies that concern the detectability of an endogenous ouabain-like factor (OLF) in mammalian tissues and plasma was approached by the application of a standardized method for its extraction and quantification. Two independent assays were used to quantify the OLF: (1) a radioimmunoassay, which used a polyclonal anti-ouabain antiserum, and (2) a radioenzymatic assay based on the inhibition of dog kidney Na+,K+-ATPase. Plasma and tissues were obtained from the Milan hypertensive strain (MHS) and the Milan normotensive strain (MNS) of rats and from healthy human volunteers. Results indicate that (1) a single high-performance liquid chromatography (HPLC) fraction identical to that of ouabain was identified by both assay methods in the rat hypothalamus and hypophysis and in both rat and human plasma; (2) dilution curves of OLF and standard ouabain were parallel and with a similar K(d), both in radioimmunoassay (3 nmol/L) and ATPase assay (14 nmol/L); (3) after HPLC, OLF was similarly quantified by the two methods in the hypothalamus, hypophysis, adrenals, and plasma of rats and in human plasma; (4) OLF was present in larger amounts in the hypothalamus, hypophysis, and plasma of MHS rats than that of MNS rats; (5) the HPLC fraction of human plasma was quantified similarly by both assays (range, 60 to 150 pmol/L); (6) recovery of standard ouabain in pre-HPLC plasma extracts was approximately 90{\%}; and (7) pre-HPLC OLF concentrations in human plasma ranged between 0.05 and 0.75 nmol/L. Rat cerebral tissues and both rat and human plasma contained measurable amounts of OLF, which were quantified similarly by radioimmunoassay and ATPase assay, both before and after HPLC fractionation. The increased MHS tissue and plasma levels of OLF are in keeping with the pathogenetic role of this factor in MHS hypertension.",
keywords = "Human, Na,K-ATPase, Ouabain, Plasma, Rats, Tissue",
author = "Mara Ferrandi and Paolo Manunta and Silvana Balzan and Hamlyn, {John M.} and Giuseppe Bianchi and Patrizia Ferrari",
year = "1997",
month = "10",
language = "English",
volume = "30",
pages = "886--896",
journal = "Hypertension",
issn = "0194-911X",
publisher = "Lippincott Williams and Wilkins",
number = "4",

}

TY - JOUR

T1 - Ouabain-like factor quantification in mammalian tissues and plasma

T2 - Comparison of two independent assays

AU - Ferrandi, Mara

AU - Manunta, Paolo

AU - Balzan, Silvana

AU - Hamlyn, John M.

AU - Bianchi, Giuseppe

AU - Ferrari, Patrizia

PY - 1997/10

Y1 - 1997/10

N2 - The resolution of controversies that concern the detectability of an endogenous ouabain-like factor (OLF) in mammalian tissues and plasma was approached by the application of a standardized method for its extraction and quantification. Two independent assays were used to quantify the OLF: (1) a radioimmunoassay, which used a polyclonal anti-ouabain antiserum, and (2) a radioenzymatic assay based on the inhibition of dog kidney Na+,K+-ATPase. Plasma and tissues were obtained from the Milan hypertensive strain (MHS) and the Milan normotensive strain (MNS) of rats and from healthy human volunteers. Results indicate that (1) a single high-performance liquid chromatography (HPLC) fraction identical to that of ouabain was identified by both assay methods in the rat hypothalamus and hypophysis and in both rat and human plasma; (2) dilution curves of OLF and standard ouabain were parallel and with a similar K(d), both in radioimmunoassay (3 nmol/L) and ATPase assay (14 nmol/L); (3) after HPLC, OLF was similarly quantified by the two methods in the hypothalamus, hypophysis, adrenals, and plasma of rats and in human plasma; (4) OLF was present in larger amounts in the hypothalamus, hypophysis, and plasma of MHS rats than that of MNS rats; (5) the HPLC fraction of human plasma was quantified similarly by both assays (range, 60 to 150 pmol/L); (6) recovery of standard ouabain in pre-HPLC plasma extracts was approximately 90%; and (7) pre-HPLC OLF concentrations in human plasma ranged between 0.05 and 0.75 nmol/L. Rat cerebral tissues and both rat and human plasma contained measurable amounts of OLF, which were quantified similarly by radioimmunoassay and ATPase assay, both before and after HPLC fractionation. The increased MHS tissue and plasma levels of OLF are in keeping with the pathogenetic role of this factor in MHS hypertension.

AB - The resolution of controversies that concern the detectability of an endogenous ouabain-like factor (OLF) in mammalian tissues and plasma was approached by the application of a standardized method for its extraction and quantification. Two independent assays were used to quantify the OLF: (1) a radioimmunoassay, which used a polyclonal anti-ouabain antiserum, and (2) a radioenzymatic assay based on the inhibition of dog kidney Na+,K+-ATPase. Plasma and tissues were obtained from the Milan hypertensive strain (MHS) and the Milan normotensive strain (MNS) of rats and from healthy human volunteers. Results indicate that (1) a single high-performance liquid chromatography (HPLC) fraction identical to that of ouabain was identified by both assay methods in the rat hypothalamus and hypophysis and in both rat and human plasma; (2) dilution curves of OLF and standard ouabain were parallel and with a similar K(d), both in radioimmunoassay (3 nmol/L) and ATPase assay (14 nmol/L); (3) after HPLC, OLF was similarly quantified by the two methods in the hypothalamus, hypophysis, adrenals, and plasma of rats and in human plasma; (4) OLF was present in larger amounts in the hypothalamus, hypophysis, and plasma of MHS rats than that of MNS rats; (5) the HPLC fraction of human plasma was quantified similarly by both assays (range, 60 to 150 pmol/L); (6) recovery of standard ouabain in pre-HPLC plasma extracts was approximately 90%; and (7) pre-HPLC OLF concentrations in human plasma ranged between 0.05 and 0.75 nmol/L. Rat cerebral tissues and both rat and human plasma contained measurable amounts of OLF, which were quantified similarly by radioimmunoassay and ATPase assay, both before and after HPLC fractionation. The increased MHS tissue and plasma levels of OLF are in keeping with the pathogenetic role of this factor in MHS hypertension.

KW - Human

KW - Na,K-ATPase

KW - Ouabain

KW - Plasma

KW - Rats

KW - Tissue

UR - http://www.scopus.com/inward/record.url?scp=0030828005&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0030828005&partnerID=8YFLogxK

M3 - Article

C2 - 9336389

AN - SCOPUS:0030828005

VL - 30

SP - 886

EP - 896

JO - Hypertension

JF - Hypertension

SN - 0194-911X

IS - 4

ER -