Endogenous sodium pump inhibitors in human urine further identification of inhibitors of Na-K-ATPase

AJH 1995; 8:753-760

Endogenous Sodium Pump Inhibitors in Human Urine Further Identification of Inhibitors of Na-K-ATPase Herbert J. Kramer, Gottfried Krampitz, Angela Bdcker, Harald Michel, Georg Krampitz, and Harald Meyer-Lehnert We investigated the presence of endogenous Na-KATPase inhibitor(s), ie, ouabain-like factors (OLFs), in the urine of salt-loaded healthy subjects. For this purpose 24-h urine was collected on days 3, 4, and 5 of high sodium intake (>30 g NaC1/day). The samples then were lyophilized. Redissolved urine concentrates were acidified (pH 3.5) and subjected to gelchromatography on a Sephadex G-25 column where the OLFs eluted in the post-salt fraction IV. When lyophilized fraction IV was rechromatographed on Sephadex G-10, OLFs with molecular mass (M,) of approximately 400 eluted in a late fraction IV/8 separate from added ouabain, ouabagenin (or digoxin), which eluted shortly after void volume. With the subsequent reverse-phase HPLC of fraction IV/8 a polar OLF-1 eluted in fraction IV/8a after the void volume in the water phase and a more apolar OLF-2 eluted at 20% acetonitrile in fraction IV/8d. Only the more apolar OLF-2 cross-reacted with a digoxin

antibody. By preparative thin-layer chromatography OLF-1 and OLF-2 were purified as single compounds with potent dose-dependent Na-K-ATPase inhibition and Ki-values approximating 1.5 x 10 -5 mol/L and 1.5 x 1 0 - 4 mol/L, respectively. Massspectroscopy (MS) showed M, of 391 and 1H-NMR characterized the endogenous urinary apolar OLF-2 as a compound that is structurally totally unrelated to ouabain; infrared (IR) spectroscopy of OLF-1 and OLF-2 also revealed no similarity with ouabain. In contrast, data from MS, NMR-, and IRspectroscopy show structural resemblance with ascorbic acid derivatives, some of these having strong inhibitory effects on Na-K-ATPase. A m J Hypertens 1995;8:753-760

w e n t y - f i v e years ago we first d e m o n s t r a t e d the p r e s e n c e of a fraction in the p l a s m a of acutely saline-loaded a n d v o l u m e - e x p a n d e d animals w h i c h w a s natriuretic a n d inhibited

the N a - K - A T P a s e e n z y m e in vitro in a m a n n e r similar to t h a t of t h e c a r d i a c g l y c o s i d e o u a b a i n (gs t r o p h a n t h i n ) . l It w a s therefore t e r m e d ouabain-like factor (OLF), w h i c h is a s s u m e d to be vasoconstrictive a n d natriuretic. T h e c r u d e n a t r i u r e t i c a n d N a - K ATPase inhibiting plasma fraction eluted from a S e p h a d e x G-25 c o l u m n after the inorganic salts. 2 We also detected this fraction w i t h similar p r o p e r t i e s in the p l a s m a 3 a n d urine 4 f r o m salt-loaded h e a l t h y subjects a n d in the p l a s m a 3 f r o m patients w i t h p r i m a r y aldosteronism. The OLF w a s s u b s e q u e n t l y incorporated into a p a t h o g e n e t i c s c h e m e of arterial h y p e r t e n sion. 5 Later, this fraction in the p l a s m a of v o l u m e e x p a n d e d dogs w a s f o u n d to contain t w o inhibitors of the N a - K - A T P a s e e n z y m e w h i c h c r o s s - r e a c t e d with a digoxin antibody. 6 W e h a v e s h o w n , h o w e v e r ,

T

Received August 5, 1994. Accepted February 28, 1995. From the Renal Section (HJK, AB, HM, HM-L), Medical Policlinic, Department of Medicine, Medical Faculty, and Division of Biochemistry (GK, GK), Institute of Anatomy and Physiology, Faculty of Agricultural Sciences, University of Bonn, Bonn, Germany. Part of this study was presented at the 8th Scientific Meeting of the American Society of Hypertension, May 19 to 22, 1993 (Abstract No. 1226). The studies were supported in part by grants from the Deutsche Forschungsgemeinschaft, Bonn (Kr 433/8-1), and the Ministerium ffir Wissenschaft und Forschung des Landes Nordrhein-Westfalen, D~isseldorf (Kra IV A6-403 046 87), Germany. Address correspondence and reprint requests to Herbert J. Krarner, MD, Professor of Medicine and Nephrology, Med. Univ. Poliklinik, Wilhelmstrasse 35-37, D-53111 Bonn, Germany. © 1995 by the American Journal of Hypertension, Ltd.

KEY WORDS: Sodium pump inhibitors, human, urine, ouabain-like factors, ascorbic acid derivatives.

0895-7061/95/$9.50 0895-7061 (95)00125-9

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KRAMERET AL

AJH-JULY 1995-VOL. 8, NO. 7

that such cross-reaction is unspecific and cannot be used to identify endogenous Na-K-ATPase inhibitors or natriuretic factors, respectively. 7 Recently, one such inhibitor was isolated from human plasma and was identified as endogenous ouabain 8 that is secreted by the mammalian adrenal glands. 9 This latter postulate, however, is not supported by recent findings in hypertensive rats 1° and humans. 11 In the present study we attempted to further characterize and identify endogenous OLFs after their isolation from the urine of healthy salt-loaded subjects. MATERIALS AND METHODS Dietary Regimen and Urine Collections Ten healthy volunteers, four female and six male medical students and laboratory personnel aged 21 to 27 years, who had not taken any medication prior to the experiments, participated in the studies. Twentyfour-hour urine samples were collected daily for 5 days during a low salt intake of 2 g sodium chloride daily and subsequently during 5 days of high salt intake of more than 30 g sodium chloride daily. This 10-day dietary regimen was repeated twice. Urinary sodium excretion averaged 31 + 8 mmol/24 h and 431 +- 40 mmol/24 h on the 5th day of low and on the 5th day of high sodium intake, respectively. For the present study, 24-h urine samples from days 3, 4, and 5 on high sodium intake were pooled so that a total of approximately 100 L of urine was available. Urines were kept at refrigerator temperature until individual daily collections were completed. After determination of urine volume and sodium concentration all urines were lyophilized to dryness, resulting in approximately 100 g powder which was stored frozen at -18°C until further processing. C h r o m a t o g r a p h i c P r o c e d u r e s Gel Chromatography. The desalted (post-salt) urine fraction IV, obtained by t • ,

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gel chromatography on a Sephadex G-25 column as previously described, 7 was lyophilized and rechrom a t o g r a p h e d on a Sephadex G-10 column. 3Ho u a b a i n ( 3 H - g - s t r o p h a n t h i n , M r 585) a n d 3Houabagenin (3H-g-strophanthidin, M r 439), purchased from Amersham (Braunschweig, Germany) were also chromatographed on this column (Figure 1). The eluate collected from the Sephadex G-10 column was subdivided into fractions IV/1 to IV/8 according to UV254 patterns (Figure 1), and were then lyophilized. All redissolved fractions were assayed for Na-K-ATPase inhibition and cross-reaction with a digoxin antibody (see below). The active Na-KATPase inhibiting fraction IV/8 underwent further chromatographic purification by reverse-phase (RP)HPLC using water-acetonitrile with 0.1% trifluoroacetic acid (TFA) as eluent. After 10 min of elution with water, acetonitrile at a gradient from 0 to 50% followed. The eluates were again subdivided arbitrarily according to UV226 patterns into seven subfractions, ie, fractions IV/8a-g (Figure 2). Thin Layer Chromatography (TLC). After lyophilization the active fractions IV/8a and IV/8d were redissolved in 10% aequous dilution of formic acid and were then subjected to preparative multiple TLC on silica gel plates. The samples were applied as lines to the plates and chromatographed first in the solvent system: n-butanol-acetic acid-water (v/v/v: 5:4:1). The chromatograms were inspected under UV light (265 and 280 nm) and the UV-positive zone with strong Na-K-ATPase inhibition, which was also the most prominent one, was selected for further purification. This zone from at least 15 plates was removed and extracted with methanol. The extracts were dried and rechromatographed in a different solvent system (pyridine-isoamyl alcohol-water Iv/v/v: 3:1:2]). Again the most prominent Na-K-ATPase inhibiting zone

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NA-K-ATPASE INHIBITORS IN HUMAN URINE

AJH-JULY 1995-VOL. 8, NO. 7

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was extracted and tested for homogeneiety in at least 10 further solvent systems. Usually the samples were sufficiently pure after the second chromatographic step. Only material that was vigorously controlled for purity was assayed for Na-K-ATPase inhibition, as well as for spectroscopic analyses. The preparation of ascorbic acid derivatives will be described elsewhere. Assay for Na-K-ATPase Inhibition. Each fraction obtained at the various chromatographic steps as well as ascorbic acid were assayed for their inhibiting effect on the Na-K-ATPase enzyme derived from hog cerebral cortex (Sigma Co, St. Louis, MO), as described previously. 4 Na-K-ATPase activity was calculated as micromoles Pi liberated/milligram protein/hour. Data are presented as percent inhibition from control in the presence of the solvent solution. Radioimmunoassay for Digoxin-Like Immunoactivity (DLIA). Fractions obtained from RP-HPLC were assayed for DLIA by radioimmunoassay as previously described. 7 A commercially available specific rabbit digoxin antibody prereacted with an antiserum to rabbit gammaglobulin was used, which was purchased from N e w England Nuclear (NEN) Co., North Billerica, MA. DLIA was quantitated as ng digoxin equivalents per mL. Concentrations of sodium and potassium in urine and in single fractions previously eluted from the Sephadex G-25 column were determined by flame photometry.

755

Spectroscopic and Nuclear Magnetic Resonance (NMR) Analyses. Ouabain and the u n k n o w n c o m p o u n d s were analyzed by mass spectroscopy (EAB [ + ]) using a Krator instrument. They were also subjected to 1HNMR characterization at 200 MHz. Since NMR spectra alone cannot solve the problem of identification of the structure of OLFs, infrared spectroscopy (IR), in addition, has been applied. Infrared spectroscopy was performed as FT-IR spectroscopy (Perkin-Elmer) using the potassium bromide technique.

RESULTS Chromatographic Separation, Cross-reaction with Digoxin Antibody, and Na-K-ATPase Inhibition Gel Chromatography. From the Sephadex G-10 column 3H-ouabain elutes at a VE/V0 ratio of 1.5 and 3H-ouabagenin at a VE/V0 ratio of 1.9 (fractions IV/23; Figure 1). When the post-salt fraction IV from the Sephadex G-25 column is rechromatographed on the Sephadex G-10 column, strong Na-K-ATPase inhibiting activity appears in late fractions (fraction IV/8; Figure 1). Thus, this Na-K-ATPase inhibiting activity elutes apart from ouabain or ouabagenin (Figure 1). Reverse-phase HPLC and Digoxin-like Immunoactivity. When fraction IV/8 from the Sephadex G-10 column is subjected to RP-HPLC, two fractions inhibit Na-KATPase, namely fraction IV/8a, which elutes in the water phase immediately after void volume, and fraction IV/8d which elutes at an acetonitrile concentration of approximately 20% (Figure 2). Fractions IV/8 a-c show no cross-reaction with the digoxin antibody, whereas fraction IV/8d and the subsequent fractions IV/8e-g react with the digoxin antibody to a degree which roughly parallels the rise in acetonitrile concentration (Figure 2). Thin-layer Chromatography (TLC). When all fractions obtained by two-dimensional TLC are assayed for Na-K-ATPase inhibition, from each fraction IV/8a and fraction IV/8d one single fraction is obtained with strong enzyme inhibition. They are termed OLF-1 and OLF-2, respectively, and inhibit the Na-KATPase enzyme almost completely, whereas other fractions reveal unspecific enzyme inhibition between 5% and 20%. The active fractions do not react positively with ninhydrin reagent. The spot of OLF-1 shows UV-positive fluorescence; the spot of OLF-2 is also UV-positive with bluish fluorescence. OLF-2 has its UV maximum at a wavelength of 265 nm, which is compatible with a heterocyclic compound. Dose Response of Na-K-ATPase Inhibition. The doseresponse curves for inhibition of Na-K-ATPase from hog cerebral cortex by ouabain, ouabagenin, and by

KRAMERET AL

756

AIH-JULY 1995-VOL. 8, NO. 7

100

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tones (Figure 6). The spectra lack peaks corresponding to aromatic structures and others already mentioned (see NMR spectroscopy). In addition, there are no indications for aliphatic structures carrying methyl, ethyl, and other groups, for peptide bonds, amides, etc. Ouabain (Figure 6) and ouabagenin, or glycyrrhetinic acid (see Discussion section), show no spectroscopic patterns identical to those of OLF-2.

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FIGURE 3. In vitro inhibition of hog cerebral Na-K-ATPase. Dose-response curves for inhibition by ouabain (solid circles) and ouabagenin (open circles), and by OLF-1 (solid triangles) and OLF-2 (open triangles), based on an approximate M r of 400 (see text). the purified OLF-1 and OLF-2 are shown in Figure 3. Ouabain and ouabagenin reveal K i values of 4.1 x 10 -6 mol/L and 2.1 x 10 -6 mol/L, respectively. OLF-1 and OLF-2 show K i values of approximately 1.5 x 10 -s and 1.5 x 10 -4 tool/L, respectively, ie, their dose-response curves are shifted to the right and they are much steeper than those of ouabain or ouabagenin (Figure 3). Ascorbic acid did not inhibit Na-K-ATPase in this assay system. The kinetics of enzyme inhibition by its derivatives will be reported elsewhere. Mass Spectroscopy (MS). The assumption of a relative mass (Mr) of the OLFs of approximately 400, as derived from the elution patterns of the samples with gel permeation (see Discussion section), is supported by signals on mass spectra obtained from OLF-1 and OLF-2. Actually, there are peaks around 400, but none at 585 or 439 (Figure 4). They thus indicate the absence of ouabain (585) and ouabagenin (439). MS signals between 126 and 181 indicate fragments of the analyzed sample. Structural Criteria

Nuclear Magnetic Resonance (NMR) Spectroscopy. 1HNMR spectra of OLFs show only a few peaks, most of which are difficult to identify because of their unspecific character (Figure 5). There are indications for hydroxyl groups, but, in turn, several structures can be excluded because of the absence of corresponding signals. Thus, aromatic structures and heterocyclic structures such as purines, pyrimidines, imidazoles, pyrrols, etc. are not present in the unknown substances. Infrared (IR) Spectroscopy. When OLF-1 and OLF-2 are studied by IR spectroscopy, there are signals for hydroxyl and carbonyl groups and for esters or lac-

Endogenous inhibitors of the cell membrane-bound Na-K pump are assumed to play an important role in the pathogenesis of arterial hypertension. 12 Since these factors inhibit the Na-K-ATPase enzyme similar to ouabain, including displacement of 3H-ouabain bound to the enzyme in vitro, and some of them were found to also cross-react with digoxin antibodies, they were termed ouabain-like factors (OLF), or, eg, digoxin-like immunoactivity (DLIA), respectively. It should be emphasized, however, that cross-reaction with a digoxin antibody is not necessarily associated with Na-K-ATPase inhibitory activity and, therefore, the term DLIA or similar terms should not be used any more as markers for the presence of OLF. In fact, it was shown, for example, that more than 70% of DLIA in the plasma of a uremic patient on regular hemodialysis treatment was due to dehydroepiandrosterone sulfate (DHEAS) and its glucuronide or other derivatives. 13 In the past, various groups of investigators isolated endogenous OLFs as inhibitors of the cell sodium pump, which they claimed to have either peptidic, lipid, steroidal, or other structures yet unidentified (for review see references 12, 14). More recently, Weinberg et al is found two digitalis-like substances structurally related to digoxin and ouabain and Mathews et al s isolated ouabain as the endogenous Na pump inhibitor from human plasma. Subsequently, this latter group of investigators d e t e c t e d o u a b a i n also in the p l a s m a of o t h e r mammals, s'9 apparently secreted by the adrenal glands, 9'16 but these findings have been challenged recently. 11,17 The OLF-1 and OLF-2, on which we report here, do not relate to ouabain or digoxin, which both elute from the Sephadex G-25 is and G-10 (Figure 1) columns, respectively, in early fractions after the void volume. In contrast, the presently described OLFs elute from the Sephadex G25 column in the early part of the post-salt fraction at a VE/V0 ratio of approximately 2.3 and from the Sephadex G-10 column in a late fraction, respectively. Provided the compounds are separated on these Sephadex columns according to their molecular size (and not retained by adsorptive forces), the Mr of OLFs of approximately 400 is estimated from their elution to be close to those of aldosterone (Mr 360) and PGF2a (Mr 369) from the

AJH-JULY 1995-VOL. 8, NO. 7

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characteristics certainly do not confirm its nature as a cardiac glycoside. Recently, structurally related glycyrrhetinic acid-like factors (GALFs) were detected in human urine, 19 but their spectroscopic patterns are not compatible with those of our OLFs. The MS signals of OLF-2 between 126 and 181 indicate fragments of the analyzed sample, which are not helpful in further explaining the particular structure of the unknown compound. NMR spectra alone also cannot solve the problem of identification of the structure of OLFs. Therefore, IR spectroscopy, in addition, has been applied, which reveals much more information on the structural character of OLFs than does the NMR technique itself.

758

KRAMERET AL

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AJH-JULY 1995-VOL. 8, NO. 7

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Our results from spectroscopic analyses of OLF-2 suggest a chemical structure which resembles that of a sugar or a sugar derivative. We arrived at that conclusion by elimination of all those structures not existing in OLF-2. See Results section for NMR and IR spectroscopy. In general, however, sugars do not exhibit any kind of fluorescence, while OLFs were detected on TLC by UV light. This property requires the 00 existence of an II II arrangement within the structure -c-cof the compound. One candidate substance that would fit in this working hypothesis is ascorbic acid, but ascorbic acid has a M r of only 176 and not one of approximately 400, as has the unknown compound. Furthermore, ascorbic acid does not inhibit Na-KATPase. Nevertheless, additional information suggests that the u n k n o w n compound(s) are ascorbic acid derivatives. Their IR spectra resemble those found for OLF-2 in the present study. Moreover, in confirming this assumption we could demonstrate that ascorbic acid derivatives, which we synthesized, show strong dose-dependent inhibitory effects on Na-K-ATPase. We will report on these compounds and their kinetics of enzyme inhibition elsewhere. This type of structure of an endogenous inhibitor of Na-K-ATPase would offer an easier understanding of its presence and physiologic role in the mammalian organism than the postulated endogenous synthesis of ouabain or structurally related compounds. In the presence of our own findings, it is of interest to note that Bricker et al 2° suggested recently that the natriuretic hormone isolated from the urine of uremic subjects contains carbonyl and hydroxymethyl groups. With respect to the potential physiologic or pathophysiologic roles, it should be emphasized that we found that the activity of OLF-2 does not change with

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large changes in sodium intake, 12 an observation in accordance with the findings of Goto et al. 21 These authors reported that the activity of a more apolar inhibitor of Na-K-ATPase, which they isolated from the urine of healthy subjects and which eluted at 30% acetonitrile under similar conditions as those applied in our present study, did not change significantly with changes in sodium intake, while a more polar inhibitor eluting at 18% acetonitrile did rise with high sodium intake. Therefore, a role for the more apolar factor in body fluid volume regulation seems unlikely, although a potential role in blood pressure regulation cannot be excluded. However, Weiler et a122 showed that no major difference in the activities of a similarly apolar factor was observed in the urine from hypertensive as compared to that from normotensive subjects. In this context, it is also of interest to note

NA-K-ATPASE INHIBITORS IN HUMAN URINE 759

AJH-JULY 1995-VOL. 8, NO. 7

that Boulanger et a116 f o u n d that the plasma concentration of the e n d o g e n o u s ouabain changed inversely with v o l u m e loading or v o l u m e depletion. In contrast to the m o r e apolar OLF-2, OLF-1 is a polar c o m p o u n d that does not cross-react with the digoxin antibody. Its IR spectrum is different from those of ouabain and OLF-2. This c o m p o u n d is app r o x i m a t e l y t e n f o l d s t r o n g e r in inhibiting Na-KATPase in vitro than OLF-2. Its activity in the urine changes in parallel with changes in s o d i u m intake. 12 The present d o s e - r e s p o n s e curves of Na-K-ATPase inhibition by OLF-1 and OLF-2, w h e n c o m p a r e d to ouabain, are m u c h steeper and shifted to the right. T h e y m a y look similar to those published for lysop h o s p h o l i p i d s and free fatty acids (for review see reference 12). H o w e v e r , a l t h o u g h we estimated their M r as described above, shift and steepnes of these curves should be i n t e r p r e t e d cautiously since overestimation of their absolute w e i g h t by contamination with material from the TL plate, which dissolved in methanol, cannot be completely ruled out. Increasing evidence suggests a potential role for these Na-K-ATPase inhibitors in the pathogenesis of various forms of v o l u m e - d e p e n d e n t e x p e r i m e n t a l and h u m a n h y p e r t e n s i o n (for review see reference 12). This is s u p p o r t e d by their m o d e of action on (Ca2÷)i in isolated cultured vascular s m o o t h muscle cells, which w e have described previously. 12'23 The biologic properties of the OLFs isolated in the present s t u d y are well compatible with the findings of others, 2°'21 and make t h e m excellent candidates for hypertensinogenic factors. H o w e v e r , as long as the exact nature and source of the various c o m p o u n d s isolated from the plasma and urine of h u m a n subjects and other m a m m a l s are not k n o w n , and the finding of ouabain as e n d o g e n o u s OLF s still awaits confirmation, their identity remains speculative. 24 Therefore, further studies are required to identify the endogen o u s Na-K-ATPase inhibitors isolated in this and other studies and to clarify their potential physiologic and pathologic significance. Final identification and characterization of the two Na-K-ATPase inhibitors isolated from h u m a n urine in this s t u d y are currently in progress.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

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ACKNOWLEDGEMENTS We are grateful to the Department of Organic Chemistry, University of Bonn, for performing the IR spectra and mass spectroscopy, as well as NMR and IR spectroscopy. We also gratefully acknowledge the excellent technical assistance of Mrs. Gudrun Djajakusuma, Division of Biochemistry, Institute of Anatomy and Physiology, Faculty of Agricultural Sciences, University of Bonn. REFERENCES

1.

Kramer HJ, Gonick HC, Paul W, Lu E: Third factor: Inhibitor of Na-K-ATPase? Presented at the IVth In-

15.

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