Pulse Pressure Correlates in Humans With a Proscillaridin A Immunoreactive Compound

Materials

Gelatin was obtained from Bio-Rad, heptafluorobutyric acid from Pierce Chemical Co, and the silicon oils AR 200 and AR 20 from Wacker Chemie. N-Acetylglucosaminyl-(β1-4)-N-acetylmuramyl-l-alanyl-d-isoglutamin (GMDP) was from Gerbu Biotechnik, the protein A column MemSep 1010 from Millipore, ⁸⁶RbCl from Amersham/Buchler, and biotinylated goat anti-rabbit IgG as well as streptavidin conjugated to alkaline phosphatase from Jackson ImmunoResearch Dianova. All other chemicals were of the highest purity available and were obtained from Merck, Sigma Chemical Co, Roth, Boehringer, and Serva. Marinobufagenin was a kind gift from Dr A.Y. Bagrov, Laboratory of Pharmacology, Sechenov Institute of Evolutionary Physiology and Biochemistry, St Petersburg, Russia.

Patients

Plasma for use in this study was obtained from 22 essential hypertensive patients, 60 normotensive control subjects, and 19 patients with chronic renal failure. The characteristics of the patients are given in Table 1⇓. Patients were randomly recruited from among outpatients. All were free of major medical illness and did not have significant pain, elevated temperature, or any acute condition. Individuals with major medical illness and those who had taken prescription medications (including antihypertensive medication, nonsteroidal anti-inflammatory drugs, aspirin, or cardiac glycosides) were excluded. After subjects had rested for 10 minutes, seated BP measurements were obtained with conventional sphygmomanometric methods by the same individual. Subjects were considered normotensive if their BP was less than 140/90 mm Hg. A diagnosis of hypertension was based on a BP greater than or equal to 160/90 mm Hg or a diastolic BP of 95 mm Hg or higher. Diagnosis of chronic renal failure was based on serum creatinine concentration greater than or equal to 106 μmol/L. Chronic renal failure was due to chronic glomerulonephritis (n=11), interstitial nephritis (n=6), and polycystic kidney disease (n=2). None of the patients was undergoing hemodialysis.

[Na_i]_P was determined fluorimetrically by use of the sodium-sensitive fluorescent indicator sodium-binding benzofuran isophthalate.¹⁴ All other serum values—sodium (Na⁺), potassium (K⁺), calcium (Ca²⁺), glucose, creatinine, uric acid, and urea—were obtained by routine clinical chemical methods.

Production and Purification of Antibodies Against Proscillaridin A

Specific polyclonal antibodies against the bufadienolide proscillaridin A were raised in four New Zealand White rabbits. Proscillaridin A was coupled to bovine serum albumin according to a procedure similar to the one of Terano et al¹⁵ and Harris et al.¹⁶ Proscillaridin A (100 μg) coupled to bovine serum albumin in 0.5 mL of 0.9% NaCl solution was emulsified with 0.5 mL Freund’s complete adjuvant. This mixture was injected intradermally at five to six sites along the spinal column. Booster injections of 100 μg antigen dissolved in 0.5 mL of 0.9% NaCl solution and also in either 0.5 mL incomplete Freund’s adjuvant or 10 μg GMDP were administered at 4-week intervals as described above. Rabbits were bled 40 weeks after the start of the immunization procedure. The globulin fraction of antiserum in 1 mol/L glycine and 0.5 mol/L NaCl (pH 8.9) was applied to a protein A MemSep 1010 column. IgG was eluted with 0.1 mol/L citric acid (pH 3.0); the pH of the eluate was adjusted to 7 with Tris base and concentrated by ultrafiltration. The concentrated IgG fraction was stored in Tris-NaCl buffer (50 mmol/L Tris, 150 mmol/L NaCl, pH 7.8) containing 0.02% NaN₃ and 50% glycerol at −20°C.

Preparation of Plasma for Measurement of Proscillaridin A Immunoreactivity by Indirect ELISA and for Detection of Sodium Pump Inhibition With ⁸⁶Rb⁺ Uptake Into Human Red Blood Cells

The extraction was performed as described previously.¹⁷ The water-soluble extract of 2 mL plasma after ethanol precipitation was applied to a LiChrospher 250-5 RP18 HPLC column and separated by a propanol/isopropanol gradient in 0.1% heptafluorobutyric acid according to Hamlyn et al.¹⁸ Three fractions, ED₁, ED₂, and ED₃, with increasing hydrophobicity were collected and measured by an indirect competitive ELISA. Each of the ED₁, ED₂, and ED₃ fractions was dissolved in 500 μL PBS, and volumes between 10 and 100 μL were included in the ELISA test system. All measurement was done in duplicate. Only those values showing a concentration dependency were included in this study.

For testing of the ability of the ED₁, ED₂, and ED₃ fractions to inhibit the sodium pump, the residue was taken up in 250 μL distilled water after lyophilization.

Indirect ELISA for Proscillaridin A Immunoreactivity

Microtiter plates were coated with 100 μL antigen per well (proscillaridin A coupled to gelatin with a variation of the procedures of References 15 and 16^{15 16} ) in coating buffer (15 mmol/L Na₂CO₃, 35 mmol/L NaHCO₃, pH 9.6) with a concentration of 100 μg/mL for 1 hour. Free binding sites were blocked with 1% gelatin in PBS for 30 minutes. Thereafter, 10 to 100 μL of the HPLC-purified plasma sample (0.04 to 0.4 mL plasma equivalents) was added and incubated with 10 μL proscillaridin A–IgG (1:1000 diluted in PBS) for 1 hour. Bound proscillaridin A–IgG was detected with 100 μL biotinylated goat anti-rabbit IgG (1:5000 in PBS for 1 hour) and 100 μL alkaline phosphatase coupled to streptavidin (1:500 in PBS for 30 minutes). Phosphatase activity was measured by addition of 100 μL p-nitrophenylphosphate (1 mg/mL) as a substrate in 0.5 mmol/L MgCl₂ and 10 mmol/L diethanolamine (pH 9.5). The phosphatase reaction proceeded for 30 minutes and was stopped by addition of 50 μL of 3 mol/L NaOH. The yellow p-nitrophenolate formed was measured by a microtiter photometer (Multiscan MCC/340) at 405 nm. Between the incubation steps, washing was performed three times for 3 minutes with 200 μL of 150 mmol/L PBS, pH 7.4 (140 mmol/L NaCl, 3 mmol/L KCl, 1.5 mmol/L KH₂PO₄, and 5.5 mmol/L Na₂HPO₄).

This assay system was also used for measurement of the serum titer for proscillaridin A antibodies and testing of cross-reactivity of the purified anti–proscillaridin A–IgG with other cardiac glycosides and steroid hormones.

Quantification of Sodium Pump Inhibitor by Measurement of Ouabain-Sensitive ⁸⁶Rb⁺ Uptake Into Human Red Blood Cells

To examine whether HPLC fractions cross-reacting with proscillaridin A antibodies inhibit the sodium pump, we tested their potency to inhibit ouabain-sensitive ⁸⁶Rb⁺ uptake into human red blood cells by a procedure described previously.¹⁹ For the quantification of the inhibitor concentration, it was assumed that the endogenous inhibitor binds with the same affinity as ouabain binds to the sodium pump of human red blood cells. Half-maximal inhibition of the sodium pump of human red blood cells was seen at 4×10⁻⁸ mol/L ouabain. Concentrations of the HPLC-purified plasma extract giving half-maximal inhibition were assumed to contain 4×10⁻⁸ mol/L inhibitor units. Half-maximal inhibition was extrapolated after subtraction of the ouabain-independent ⁸⁶Rb⁺ uptake from all values from a double logarithmic plot (% inhibition/100−% inhibition) against the concentration of the inhibitor.¹⁹

Statistics

Statistical analysis was done by the BMDP computer programs²⁰ and GraphPad Prism (GraphPad Software). Because the statistical distribution of the ED₁, ED₂, and ED₃ concentrations were nearly log normal, their values are represented as geometric mean with dispersion factor. All other data are represented as mean value and SE. The geometric means of the proscillaridin A immunoreactivities ED₁, ED₂, and ED₃ of the three groups of patients (normotensive, hypertensive, and patients with chronic renal failure) were compared by the nonparametric Kruskal-Wallis test as well as multiple comparison (Nemenyi test) using the BMDP-3S program. Regression analysis of log ED₁, log ED₂, and log ED₃ was tested as a function of systolic, diastolic, and mean arterial BP values as well as of pulse pressure, [Na_i]_P, age, body mass index, and serum concentrations of Na⁺, K⁺, Ca²⁺, glucose, creatinine, uric acid, and urea with GraphPad Prism. Mean values of parameters other than ED₁, ED₂, and ED₃ were compared in a two-sided Student’s t test or Mann-Whitney test with comparison related significance level. The two-tailed level of significance was set at a value of P<.05.

Development of an Indirect ELISA for Proscillaridin A and Bufadienolide-Like Substances

To develop an immunological assay for the bufadienolide proscillaridin A and bufadienolide-like immunoreactive substances, we immunized rabbits with proscillaridin A coupled to bovine serum albumin. Rabbits produced antibodies with a titer of 1:10³ after 40 weeks and a K_d value of 2×10⁻⁹ mol/L. This titer and affinity remained constant from the 25th to the 120th week after immunization. When the specificity of the antibodies was tested, only bufadienolides such as proscillaridin A and bufalin were recognized (marinobufagenin was almost not recognized); cardenolides such as ouabain and digoxin or other steroid hormones were not recognized (Table 2⇓). With these antibodies, concentrations as low as 100 fmol per well or 10⁻⁹ mol/L could be detected. The range for determinations of proscillaridin A was 10⁻⁷ to 10⁻¹⁰ mol/L. The interassay coefficient of variation of the method was 18.6%, and the intra-assay coefficient was 8.8%. The curve of proscillaridin A immunoreactivity as a function of plasma volume was superimposed over the proscillaridin A standard curve (Fig 1⇓).

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Figure 1.

Dilution curve obtained from extracted and HPLC-purified plasma (ED₁, reported as mL equivalents of plasma per well) compared with the proscillaridin A standard curve.

Characterization of Patients

All three groups of patients—normotensive subjects, patients with hypertension, and patients with chronic renal failure—had normal and equal serum levels of Na⁺, K⁺, Ca²⁺, and glucose. Patients with chronic renal failure had significantly elevated levels of creatinine (P<.001), uric acid (P<.05), and urea (P<.001) compared with normotensive subjects and hypertensive patients (Table 1⇑). Body mass index of all groups did not differ. The hypertensive patients had a significantly higher (P<.05) diastolic BP compared with normotensive subjects and patients with chronic renal failure (Table 3⇓). Patients with chronic renal failure had a significantly (P<.05) higher [Na_i]_P compared with normotensive subjects and hypertensive patients (Table 3⇓).

Determination of Proscillaridin A Immunoreactivity in Serum of Healthy Normotensive Subjects and Patients With Essential Hypertension and Chronic Renal Failure

Before measurement of immunoreactivity, we separated the ethanolic extracts by reverse-phase C18 HPLC using a propanol/isopropanol gradient as described by Hamlyn et al.¹⁸ Chromatograms of the ethanolic plasma extracts are shown in Fig 2⇓. It is evident that three peaks of proscillaridin A immunoreactive material with different polarities and retention times were eluted from the C18 reverse-phase column (Fig 2B⇓). These three peaks also inhibited the cardiac glycoside–sensitive ⁸⁶Rb⁺ uptake into human red blood cells (Fig 2A⇓). The peak with a retention time of 20 minutes and almost the same polarity as ouabain was denoted as ED₁ in all further investigations. In addition, more hydrophobic fractions with proscillaridin A immunoreactivity were seen. The peak with a retention time of approximately 40 minutes was called ED₂, and the most lipophilic fraction with a retention time of 60 minutes and a polarity similar to that of proscillaridin A was named ED₃ (Fig 2⇓).

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Figure 2.

HPLC chromatograms of the ethanol extract of human plasma. A, Effect of the fractions on cardiac glycoside–sensitive ⁸⁶Rb⁺ uptake into human red blood cells. Sodium pump inhibitory activity was expressed as nmol/L inhibitor units as defined in “Methods.” B, Determination of proscillaridin A immunoreactivity of individual fractions. Means±SD of 10 different sera are shown. Arrows indicate the positions where ouabain and proscillaridin A are eluted. Dotted lines show the increase of propanol concentration in 5 to 10 minutes from 0% to 10% and increase of isopropanol in 10 to 60 minutes from 10% to 30%. Gradients always contained 0.1% heptafluorobutyric acid.

An analysis of the plasma concentrations of proscillaridin A immunoreactivity of 60 normotensive subjects revealed a non-gaussian distribution of the individual values. However, it showed a logarithmic distribution curve like other hormones. Therefore, geometric means and dispersion factors were calculated for each fraction of ED₁, ED₂, and ED₃ proscillaridin A immunoreactivity. When the concentrations of ED₁, ED₂, and ED₃ were compared, for all groups of patients, ED₁<ED₂<ED₃ (Table 3⇑). Although inspection of the ED₁, ED₂, and ED₃ values of proscillaridin A immunoreactivity indicates a tendency for higher values in hypertensive patients compared with normotensive subjects for the geometric mean (Table 3⇑), statistical analysis by the Kruskal-Wallis test and Tukey comparison was unable to detect any significant differences between the patient groups. However, we cannot exclude the possibility that a higher number of investigations may reveal such differences.

No correlations were found between the ED₂ and ED₃ concentrations of proscillaridin A immunoreactivity and the individual systolic, diastolic, and mean BP values of their plasma donors; pulse pressure; body mass index; and age of the patients. There also was no correlation with [Na_i]_P and other plasma concentrations such as Na⁺, K⁺, Ca²⁺, glucose, creatinine, uric acid, and urea. The ED₁ values of proscillaridin A immunoreactivity also did not correlate with these plasma concentrations, the [Na_i]_P of their donors, body mass index, or age. However, the ED₁ concentration of proscillaridin A immunoreactivity showed a significantly positive correlation with systolic BP (P<.01, Fig 3A⇓), mean BP (P<.05, Fig 3B⇓), and pulse pressure (P<.0001, Fig 3C⇓), but it did not correlate with diastolic BP.

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Figure 3.

Regression analysis between polar ED₁ and BP parameters. A, Positive correlation between log ED₁ and systolic BP. The line calculated by linear regression analysis is given by log ED₁ [nmol/L]=0.010×mm Hg−2.23 (r=.32, P<.01). B, Positive correlation between log ED₁ and mean arterial BP. The line calculated by linear regression analysis is described by log ED₁ [nmol/L]=0.013×mm Hg−2.17 (r=.25, P<.05). C, Positive correlation between log ED₁ and pulse pressure. The line calculated by linear regression analysis corresponds to log ED₁ [nmol/L]=0.019×mm Hg−1.80 (r=.38, P<.0001).