A. Clerico

M.G. Del Chicca

S. Balzan

S. Ghione

CLERICO, ALDO

M. G. Del Chicca

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37305#{128}525300- 2735’2502 225usPOOL A VARIABILITY000000020000 00 200 000 400 500 400TIME, doysPOOL B VARIABILITYS S340 CLINICALCHEMISTRY. Vol. 33, No. 2, 1987parathyroid gland, medullothyroidgland, and pancreas f3 cells), l,25-dihy-droxyvitamin D, and enzymes are in-volved (3,4). Moreover, arteriosclerosisand atherosclerosis are calcium-depen-dent (3, 5). In IDD women, the signifi-cant negative correlation between P1-Ca and total cholesterol might be ac-counted for by the internalization ofthe calcium or even the cholesterol inthe artery wall at a younger age, de-pending on whether cholesterol or cal-cium increased in their plasma. Thus,an increase in plasma cholesterol couldallow P1-Ca to penetrate into the ar-tery intima, to form atheroscleroticplaques in patients for whom athero-sclerosis is very common and occursearly (4). The Bogalusa Heart Study(Framingham Junior survey) has justprovided additional proof that choles-terol is one, if not the major, cause ofatherosclerosis (6). Could calcium bethe missing piece in this puzale ofatherosclerotic plaque formation?Once again, biological results arenot the same for both sexes (2-4). Theepidemiologic, clinical, ergometric, andtherapeutic particularities of womenpresenting with coronary insufficiencyhave just been described (7). Our re-sults raise questions that were notresolved in that study, and suggestthat much work remains to be done.References1. Speich M. More on the relation betweenmagnesium and calcium in serum and plas-ma [Letter]. Clin Chem 1986;32:221-2.2. Speich M. Correlation between calciumand zinc in plasma [Letter]. Clin Chem1986;32:1427-.8.3. Speich M, Gelot S, Arnaud P, Van Goc N,Robinet N, Pineau A. Multiple and simplecorrelations between magnesium, calcium,zinc, potassium, total- and HDL-cholesterolin 111 reference subjects. Magnesium-Bull1984;6:137-41.4. Speich M, Gelot S, Arnaud P, Murat A.Plasma and erythrocyte magnesium in in-sulin-dependent and non-insulin-dependentdiabetics: correlations between twelve vari-ables. Magnesium-Bull 1985;7:140-5.5. Speich M, Metayer C, Arnaud P, NguyenVG, Boiteau IlL. Concentrations of lead,magnesium, calcium, zinc and cadmium intwenty rabbit tissues after exposure to lowlead doses and atherogemc diet. Ann NutrMetab 198327:531-41.6 Newman WP, Freedman DS, Voors AW,et al. Relation of serum lipoprotein levelsand systolic blood pressure to early athero-sclerosis. The Bogalusa Heart Study. NEngl J Med 1986;314:138-44.7. Broustet JP, Douard H, Mora B. Insuffi-sance coronaire chez Ia femme: particular-it#{234}sepid#{233}miologiques,cliques, ergom#{233}tri-ques et th#{233}rapeutiques.Rev Prat (Paris)1986;36:2043-8.Michelle SpeichLab. de Biochimie PharmaceutiqueFacult#{233}de Pharmacie1, rue Gaston VeilF-44035 Nantes Cedar, FranceProblems in Standardization ofDigitalis-like Substance Assays byMeans of CompetitiveImmunological MethodsTo the Editor:Although several studies have beenrecently published concerning assay ofendogenous digoxin-like immunoreac-tive substance(s) (Dus) in human bodyfluids (1-6), and the putative endoge-nous substance has been almost par-tially purified and identified (7), thereis no accord on the nomenclature (ous,DLF, OLF, Endoxin, Endaline, etc.)and the best procedure(s) of samplecollection, storage, and assay. Thislack of standardization not only gener-ates confusion, but also can be thecause of discordant results and errone-ous findings. The aim of this Letter isto discuss some methodological pointsof mis assay, adding personal contribu-tions, in order to stimulate a moregeneral discussion among the groupsinterested in studies concerning endog-enous cardiac glycosides-like sub-stances.Sample preparation-Several au-thors (8-13) used deproteinized (byboiling) and concentrated plasma ex-tracts for their studies in animals orhumans. We have recently used theextraction with C18 reversed-phasecartridges (Sep Pak, Waters Asso-ciates) for the concentration and pre-purification of biological samples (5,14). This procedui-e seems preferablewhen radioreceptor assay (RRA) and(or) transmembrane ion-transportstudies are used with the ifiA for de-tection of cardiac glycosides-like com-pounds. In fact, by loading the samplethrough the column, washing it with40 mL of distilled water, and elutingthe plasma extracts with methanol it ispossible to remove the ions and pro-teins and avoid this kind of interfer-ences in RRA or transmembrane ion-transport methods (14). This procedureis rapid and the mean recovery was82% (14).On the other hand, we think that adirect assay (without sample extrac-tion and concentration) should be usedwhen plasma samples from newborns,pregnant women, and patients withrenal or liver disease are assayed, be-cause of their higher mis concentra-tions in plasma in comparison with000 00 200 000 500 0.00TIME. doUsFig. 1.nusstabibty of two unnaly pools collect-ed fromnewborns in the first day (Pool A) andin the thirdand fourth days of extrauterine life(Pool B)The two pools were dispensed in several 1.mLaliquotsarid stored at -20 ‘C until AlA.Only pool Ashowed a signWucantlydecreased immunoreactlvityduring the study (r = 0.405, n = 50,0.005< p <0.0001, y = 294.6 - 0.12xnormal adult subjects (2,3,5). In addi-tion, urine samples from adults andnewborns have DLIS concentrationsfour to six times higher than those inplasma samples, so also in this case thedirect RIA seems preferable, as wehave previously reported (5, 15).Sample storage-There are no datain the literature on stability of DLISduring storage. In our laboratory, twopools (A and B) of urine from newbornswere stored in various 1-mL aliquots at-20 #{176}Cand assayed by RIA for 17months. Only pool A demonstrated asignificant decrease in immunoreactiv-ity during this interval (Figure 1). Thisdiscrepancy was probably ascribable tothe higher imprecision for assay of poolB (CV about 22% vs 18%). The decay ofdigoxin-like immunoreactivity was,however, very small (about 1% permonth) and, also for pool A, a signifi-cant decrease of mis was not detectablebefore eight months of storage at-20 #{176}C.Evidently DLIS can be validlymeasured in urine samples stored forsome months at -20 #{176}C.Unfortunate-ly, at present we have no definitivedata on the stability of mis in serum(plasma) pools. We directly (i.e., with-out extraction) assayed, during 10months, a pool of plasma from blooddonors, but the very low DLIS concen-tration, about 15 ng/L, did not permit asignificant statistical analysis becauseof the high imprecision of the assayCLINICAL CHEMISTRY, Vol. 33, No. 2, 1987 341(CV >40%). Pooled plasma from new-borns, from pregnant women, or frompatients with renal diseases, withhigher than normal DLIS concentra-tions, could be used to test the stabilityof digitalis-like immunoreactivity inplasma.Preparation of standard curve-Mostauthors who have measured DLIS withRIA or enzyme immunoassay methodshave used commercial kits and per-formed the assay as suggested by themanufacturers. In commercial kits fordigoxin assay, digoxin added to humanplasma (or serum) is generally used asstandard. We have previously demon-strated (5, 11, 15) that, in normal plas-ma, low but measurable amounts ofdigoxin-like substance(s) are present.Using a RIA kit with serum standardsfor DLIS assay (SPAC Digoxin; Mallinck-rodt, Dietzenbach, F.R.G.) we havefound that most (>70% in two differentassays) plasma samples (direct assay)with mis concentrations within thenormal range (0-35 ngIL) have abound radioactivity higher than that ofthe B0 standard. Therefore, it is impor-tant to test for the presence of signifi-cant amounts of DLIS in the serum-basal standards used for the calibra-tion curve. To avoid this kind ofinterference we recommend the use ofbuffer solutions containing standarddigoxin and human serum albumin(11, 15) or low-molecular-mass-de-prived human serum as standard forcalibration curves.Immunological and biological activi-ty-Because the chemical nature ofDUS is not yet known, it is not possibleto perform specific RIA assays for misby use of specific antisera against DLIS,with DLIS as standard. The relativelypoor specificity of measurement of DLISwith digoxin RIA kits makes it possi-ble that substances with biological ac-tivity similar to cardiac glycosides arenot detected by digoxin RIA methods.For example, ouabain, a compoundthat strongly inhibits Na/K ATPaseactivity, does not significantly inter-fere in our digoxin RIA system (11). Onthe other hand, it is also possible thatseveral compounds interfering in theRIA system do not share any biologicalactivity (12, 16, 17). However, we haveobtained a significant correlation (r =0.72, n = 21, p <0.001) between DLISvalues obtained by RIA method or ra-dioreceptor assay for 21 chromato-graphed extracts (made by use of C15Sep Pak cartridges) of plasma fromnormal adults, pregnant women, andneonates (14). Therefore, we think thatdigoxin RIA or enzyme immunoassaymethods can be useful for DUB determi-nation because they generally aremore sensitive, precise, and simplethan other techniques used to detectendogenous cardiac-glycosides-likecompounds. However, immunologicalmethods should be used only as ascreening or preliminary test to detectthe possible presence of carcliac-glyco-sides-like substances in human biologi-cal fluids; for confirmation, more spe-cific methods, used after chromato-graphic purification, are required.References1. McCarthy RC. Minimizing the effect ofdigoxin-like immunoreactive substances inimmunoassays for digoxin in neonatal se-rum [Letter]. Clin Chem 1985;31:1240-1.2. Valdes R Jr. Endogenous digoxin-likeimmunoreactive factors: impact on digoxinmeasurements and potential physiologicalimplications. Clin Chem 1985;31:1525-32.3. Soldin SJ. Digoxin-issues and contro-versies [Review]. Clin Chem 1986;32:5-12.4. Scherrmann JM, Sandoux P, GuedeneyX. Specific interaction between antidigoxinantibodies and cligoxin-ilke immunoreac-five substances in cord serum. Clin Chem1986;32:97-100.5 Clerico A, Ghione S, Balzan 8, DelChicca MG. Digoxin-like immunoreactivesubstance: non-specific interference or anew hormone? J Nuci Med Allied Sci1984;28:297-301.6. De Wardener HE, Clarkson EM. Conceptof natriuretic hormone [Review]. PhysiolRev 185;65:658-759.7. Cloix JF, Crabox M, Wainer 1W, et al.High-yield purification of urinary Na-pump inhibition. Biochem Biophys ResComm 1985;131:1234-40.8. Gruber KA, Buckalew VM Jr. Furthercharacterization and evidence for a precur-sor in the formation of plasma antinatrifericfactor. Proc Soc Exp Biol Med 1978;159:463-7.9. Gruber KA, Whitaker JA, Buckalew VMJr. Endogenous digitalis-like substance inplasma of volume-expanded dogs. Nature(London) 1980;287:1-3.10. Ghione S, Balzan S, Montali U, et al.Plasma digoxin-like immunoreactivity inessential hypertension: relation to plasmaresin activity. J Hypertension 1983;1(suppl2):142-4.1L Ba]zan S, Clerico A, Del Chicca MG, etal. Digoxin-like immunoreactivity in nor-mal human plasma and urine, as detectedby a solid-phase radioimmunoassay. ClinChem 1984;30:450-1.12. Kelly RA, O’Hara DS, Canesaa ML, etal. Characterization of digitalis-like factorsin human plasma. Interactions with NaK-ATPase and cross-reactivity with cardiacglycoside-specific antibodies. J Biol Chem1985;260:11396-405.13. Valdes R Jr, Graves SW, Becker SLProtein binding of endogenous digoxin-im-munoactive factors in human serum and itsvariation with clinical condition. J ClinEndocrinol Met.ab 1985;60:1135-43.14. Balzan S, Ghione 5, Clerico A, MontaliU. Correlation between digoxin like iminu-noreactivity and 3H-ouabain displacementon erythrocyte membranes in extracts ofhuman plasma. Clin Biochem (in press,1986).15. Clerico A, Del Chicca MG, MontereggiA, et al. Digoxin-like immunoreactivity inhuman body fluids. J Nucl Med Allied Sci198529:305-12.16. Hnatowich M, LaBella F. Endogenousdigitalis-like factors: in vitro comparison ofbiological and immunological activities ofpeptide and steroid candidates. Eur J Phar-macol 1984;106:567-75.17. Kramer HJ, Heppe M, Weiler E, et al.Further characterization of the endogenousnatriuretic and digoxin-like immunoreact-ing activities in human urine: effects ofchange in sodium intake. Renal Physiol1985;8:80-9.A. ClericoS. GhioneM. G. Del ChiccaS. BaizanCR.R. Institute of din Physiol.University of PisaVia Savi 856100 Pisa, ItalyMeasurement of Stable GlycatedHemoglobinTo the Editor:In view of the concern regarding theeffect of the labile Schiff base adduct(pre-Hb A1) on the true values for HbA1 as measured by cation-exchangechromatography (1, 2), we report thesimple method used routinely in ourlaboratory to remove pre-Hb A1.Hb A1 was measured in one normalsubject and eight diabetic patients byuse of a medium-sized chromatograph-ic column containing Bio-Rex 70 cat-ion-exchange resin (3). Blood was col-lected with EIYFA as anticoagulant,and samples were variously treated asbelow before the hemolysates were pre-(a) Hb A1 was measured immediate-ly after the blood was drawn.(b) Whole anticoagulated blood (1.5mL) was pipetted into 8.5 mL of isoton-ic saline in a tube, mixed several timeswith gentle inversion, then centrifuged(10 mm, 3000 rpm) and the supernatewas discarded. A second 8.5-mL all-quot of isotonic saline was added to thesedimented erythrocytes, mixed bygentle inversion until all the cells werein suspension. The tube was then cen-trifuged and the supernatant fluid wasagain discarded. We then added 8.5 mLof saline to the washed erythrocytes,mixed well by gentle inversion until allthe cells were in suspension, then incu-bated at 37 #{176}Cfor 6 h in a shaking

Problems in standardization of digitalis-like substance assays by means of competitive immunological methods

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